Polypeptides targeting gm-csf r and uses thereof

Abstract

This application discloses a polypeptide targeting GM-CSFR and its applications. The polypeptide has the amino acid sequence shown in SEQ ID NO:1. The polypeptide of this application can specifically bind to GM-CSFR, increasing white blood cell count without promoting tumor proliferation, posing no risk of tumor growth, and possessing high safety. It can be used to treat chemotherapy side effects, especially neutropenia.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical technology, specifically relating to peptides targeting GM-CSFR and their applications. Background Technology

[0002] Chemotherapy-induced neutropenia (CIN) is a dose-limiting side effect with an incidence rate of 40%-80% in cancer chemotherapy. Its severity is classified into grades 1-4 according to the Common Adverse Event Evaluation Criteria (CTCAE), with grade 3 (neutrophil count 0.5 × 10⁻⁶) being the lowest. 9 / L~<1.0×10 9 / L), Level 4 (<0.5×10 9 / L) indicates severe / extremely severe myelosuppression. Patients are prone to fatal infections (such as sepsis), which can lead to chemotherapy interruption, increased treatment costs, or even death, seriously affecting treatment adherence and long-term survival rates in cancer patients.

[0003] For chemotherapy-induced neutropenia, the core implementation plans in the current clinical and research stages can be mainly divided into three categories, as follows: (1) Recombinant cytokine drugs Represented by G-CSF (such as ibegase α) and GM-CSF, human cytokines are recombinantly expressed through genetic engineering technology and administered intravenously or subcutaneously (clinical dose of G-CSF 5-10 μg / kg, GM-CSF 3-5 μg / kg). By specifically binding to the hematopoietic stem cell surface receptor (G-CSFR / GM-CSFR), they activate downstream proliferation and differentiation signaling pathways, accelerate the generation and release of neutrophils, and are used for the treatment and prevention of grade 2-4 neutropenia. (2) Oral adjuvant drugs Including leukocyte-stimulating factor, vitamin B4, and squalene, these substances exert a mild white blood cell-boosting effect by participating in purine and pyrimidine nucleic acid metabolism (vitamin B4) or protecting the stability of hematopoietic stem cell membranes (squalene). The oral dosages are leukocyte-stimulating factor 20 mg / time (3 times daily) and vitamin B4 10-20 mg / time (3 times daily), mainly used as adjunctive treatment for grade 1-2 mild leukopenia. (3) Traditional Chinese medicine compound preparations Represented by Compound Donkey-hide Gelatin Oral Solution (FEJ) and Ginseng and Astragalus Injection, based on the TCM theory of "tonifying both qi and blood", FEJ is administered orally (the clinically routine dose is 20mL / time, 3 times a day). It is speculated that it increases white blood cell count by protecting the bone marrow hematopoietic microenvironment and regulating the levels of hematopoietic-related cytokines (such as IL-3 and EPO). It is mainly used as an adjunct therapy for bone marrow suppression after radiotherapy and chemotherapy.

[0004] However, mainstream clinical drugs for increasing white blood cell count (recombinant cytokine drugs, such as G-CSF and GM-CSF) have issues such as tumor-promoting risks (G-CSF binding to G-CSFR can promote malignant tumor behavior, and GM-CSF may be associated with bone metastasis risk) and insufficient safety. G-CSF, after binding to its receptor G-CSFR, can activate pro-cancer signaling pathways such as STAT3 and AKT, accelerating tumor cell proliferation and metastasis. Although there is no direct evidence of GM-CSF promoting cancer, animal experiments have shown that it may increase the risk of bone metastasis by regulating the tumor microenvironment, limiting its applicability to cancer patients. Furthermore, these drugs rely on injection, leading to poor patient compliance. Long-term use can easily produce neutralizing antibodies (occurring in approximately 5%-10%), resulting in decreased efficacy. They may also cause adverse reactions such as musculoskeletal pain and allergies, and are contraindicated in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia. Moreover, existing drug administration methods are limited (G-CSF is mainly administered by injection, which is inconvenient) and have a single therapeutic effect (single-target action).

[0005] Regarding the limited efficacy and slow onset of oral small molecule adjuvant drugs: these drugs have a mild effect, with an efficacy rate of only 40%-70% for grade 3-4 severe myelosuppression, and a slow onset of action (requiring 1-2 weeks), making it impossible to quickly control the risk of infection; long-term use also requires monitoring of liver and kidney function, and there are tolerance issues such as gastrointestinal irritation.

[0006] The traditional Chinese medicine compound FEJ suffers from problems such as unclear active ingredients and poor targeting: FEJ has a complex component system (containing 5 medicinal materials such as donkey-hide gelatin and red ginseng, as well as hundreds of bioactive substances), and the material basis of the core white blood cell-boosting active ingredient is not clear, resulting in efficacy differences of more than 20% between different batches; its mechanism of action is only described as "protecting the hematopoietic microenvironment" in a macroscopic way, lacking clear specific targets and molecular pathways, making it impossible to achieve precise drug delivery and limiting the upgrade from adjuvant therapy to targeted drug.

[0007] Therefore, there is an urgent need to find a new type of drug that targets GM-CSFR. Summary of the Invention

[0008] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a GM-CSFR-targeting peptide for treating chemotherapy-related neutropenia.

[0009] This application is based on the following discoveries of the inventors: Compound Donkey-hide Gelatin Oral Liquid (FEJ) is a classic Chinese medicine compound composed of donkey-hide gelatin (principal ingredient), red ginseng, codonopsis, rehmannia glutinosa, hawthorn, and other medicinal materials. In traditional Chinese medicine theory, it is mainly used to regulate the deficiency of both qi and blood. Modern clinical research and animal experiments have confirmed its clear value in the adjuvant treatment of bone marrow suppression after radiotherapy and chemotherapy. It can effectively increase the number of peripheral blood leukocytes and neutrophils by protecting the bone marrow hematopoietic microenvironment and promoting the proliferation of hematopoietic-related cells, thereby reducing the risk of infection caused by bone marrow suppression in patients. However, due to its complex composition system, the core active substances, specific targets, and specific molecular mechanisms of its white blood cell-increasing effect have not been systematically elucidated. This leads to the clinical application relying on whole-body administration, insufficient stability of efficacy, and the lack of precise target support in its clinical application, making it difficult to achieve precise regulation of efficacy and optimization of the drug.

[0010] Based on this, this application focuses on the material basis and molecular targets of FEJ's white blood cell-boosting activity, breaking through the limitation of "ambiguous effects of traditional Chinese medicine compound formulas." Through experiments, it clarifies for the first time the clinical value and research gaps of FEJ in boosting white blood cell count, providing a logical starting point for subsequent screening of single active ingredients from compound formulas and elucidating targeting mechanisms, thus solving the problem that existing traditional Chinese medicine compound formulas cannot precisely regulate efficacy. Furthermore, as... Figure 1 As shown, this application develops a novel short peptide targeting GM-CSFR for increasing white blood cell count through a technical route of "FEJ white blood cell activity verification - FEJ active ingredient screening and target confirmation - targeted binding verification - in vivo and in vitro functional verification".

[0011] Therefore, in a first aspect of this application, a polypeptide is provided. According to an embodiment of this application, the polypeptide has an amino acid sequence as shown in SEQ ID NO:1.

[0012] In a second aspect of this application, a recombinant protein is proposed. According to embodiments of this application, it includes the polypeptide described in the first aspect.

[0013] According to embodiments of this application, the recombinant protein further includes at least one of a protein tag, a reporter protein, serum albumin or a fragment thereof, and an Fc fragment.

[0014] In a third aspect, this application provides a nucleic acid molecule. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect or the recombinant protein described in the second aspect.

[0015] In a fourth aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the third aspect.

[0016] In a fifth aspect, this application provides a recombinant cell. According to an embodiment of this application, the recombinant cell comprises: Carrying the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or Expressing the polypeptide described in the first aspect or the recombinant protein described in the second aspect.

[0017] In a sixth aspect, this application provides a pharmaceutical composition. According to embodiments of this application, the pharmaceutical composition comprises: The polypeptides described in the first aspect, the recombinant proteins described in the second aspect, the nucleic acid molecules described in the third aspect, the expression vectors described in the fourth aspect, or the recombinant cells described in the fifth aspect.

[0018] According to embodiments of this application, the pharmaceutical composition further includes pharmaceutically acceptable excipients, carriers, or mediators.

[0019] In a seventh aspect of this application, the use of the polypeptide described in the first aspect, the recombinant protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a medicament for the prevention and / or treatment of chemotherapy side effects is provided.

[0020] According to an embodiment of this application, the chemotherapy side effect is neutropenia.

[0021] According to embodiments of this application, the drugs causing the neutropenia are selected from docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, doxorubicin, pentorubicin, and pharmaceutically acceptable salts thereof.

[0022] Beneficial effects: 1. The peptide in this application can specifically bind to GM-CSFR, posing no risk of tumor promotion, significantly improving safety, and avoiding the core risks of existing drugs. Specifically: 1) No risk of tumor promotion: In Apc Min / + In a spontaneous colorectal cancer mouse model, the number of intestinal adenomas after Peptide 13 treatment was not significantly different from that in the saline control group. P>0.05), clearly demonstrating that it increases white blood cell count without promoting tumor proliferation, comprehensively addressing the core deficiency of G-CSF drugs—"increasing white blood cell count with the risk of tumor promotion"—from target selection to in vivo validation. 2) Protecting hematopoietic organ function: In vivo experiments confirmed that Peptide 13 can significantly reduce chemotherapy-induced splenomegaly in mice ( P <0.05), reducing damage to hematopoietic organs and compensating for the shortcomings of existing drugs that only focus on white blood cell increase efficacy while neglecting the protection of the hematopoietic microenvironment. 3) Solid safety foundation: The target peptide was verified by HPLC to have a purity of ≥95%, and its stability was confirmed by solubility experiments. No obvious cytotoxicity was observed in in vitro experiments, providing a reliable safety guarantee for subsequent clinical applications.

[0023] 2. The peptide in this application has definite therapeutic effects and covers key clinical needs. Specifically, it is demonstrated that: 1) its white blood cell-boosting effect is comparable to that of clinical drugs: In in vitro experiments, Peptide 13's ability to promote the formation of mouse bone marrow granulocyte colonies (CFU-G) and CFU-GM was not significantly different from that of G-CSF and GM-CSF. P >0.05), confirming that its regulatory activity on the proliferation and differentiation of hematopoietic progenitor cells reached the clinical drug level; after two rounds of in vivo treatment, the white blood cell and neutrophil counts of chemotherapy mice significantly recovered ( P <0.01), which can effectively improve severe myelosuppression. 2) Specific action and rapid onset of action in vivo: Peptide 13 directly targets GM-CSFR to specifically regulate the proliferation and differentiation of granulocyte hematopoietic progenitor cells. The target of action is clear and there is no off-target effect, avoiding the problem of unclear mechanism of action of existing drugs. In vivo experiments show that after treatment with Peptide 13, peripheral blood indicators of chemotherapy mice showed significant improvement after the first round of treatment. Compared with the characteristic of oral adjuvant drugs that "require 1-2 weeks to take effect", it can relieve myelosuppression more quickly and reduce the risk of infection. It is especially suitable for emergency intervention scenarios of severe myelosuppression.

[0024] 3. The peptides in this application possess clear clinical translational potential and application advantages. Specifically: 1) Strong targeting and low off-target risk: ELISA and BLI dual verification show that Peptide 13 binds specifically and with high affinity to GM-CSFR, precisely regulating the hematopoietic pathway and avoiding the off-target effects that may occur with existing non-specific white blood cell-boosting drugs, providing a material basis for precision treatment. 2) High formulation development potential: Peptide 13 is a chemically synthesized short peptide with a purity ≥95%, and its solubility and stability in experimental systems have been confirmed. Compared to G-CSF-like recombinant protein drugs, it is easier to optimize the formulation process (e.g., exploring oral and long-acting formulations), potentially solving the problem of poor adherence to injection administration of existing drugs and reducing clinical usage costs. 3) Wide applicable population: Peptide 13 has no tumor-promoting risk and also promotes the proliferation and differentiation of human hematopoietic progenitor cells. Compared to the limitations of G-CSF in the use of MDS and acute myeloid leukemia patients, its applicable population has greater potential for expansion, and its clinical application scenarios are broader.

[0025] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0026] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is an experimental flowchart illustrating peptide screening, targeted binding verification, and functional activity detection in the embodiments of this application. Figure 2 This is the detection result of FEJ in vivo reversing chemotherapy-induced neutropenia in Example 1 of this application; Figure 3 The results of screening and target binding verification of FEJ-derived active short peptides in Examples 1 and 2 of this application; Figure 4 This is the detection result of Peptide 13 in Example 3 of this application activating the downstream STAT5 / ERK1 / 2 signaling pathway of GM-CSFR in vitro and promoting the formation of hematopoietic progenitor cell colonies; Figure 5 This is the result of testing the effect of Peptide 13 in vivo on reversing chemotherapy-induced neutropenia and protecting the spleen, as described in Example 3 of this application. Detailed Implementation

[0027] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0028] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0029] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0030] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0031] In this application, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0032] In this application, the terms "identity," "homology," or "similarity" are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined using conventional methods. The percentage refers to the degree to which the amino acids in two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. The alignment of amino acid sequence identity percentages can be performed using various methods within the art, such as software well-known in the art, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine appropriate parameters for the aligned sequences, including any algorithms required to achieve maximum alignment of the full length of the compared sequences.

[0033] In this application, the term "specific binding" or "specifically bound" refers to a non-random binding reaction between two molecules, such as the reaction between a peptide and GM-CSFR. In some specific embodiments, it is determined, for example, according to ELISA and BLI techniques.

[0034] In this application, the term "affinity" or "bondability" refers to the strength of the non-covalent interaction between a peptide and a receptor. The strength of the binding interaction, or affinity, can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates higher affinity. KD can be determined using any conventional method known in the art, including but not limited to ELISA and BLI.

[0035] In this application, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0036] In optional embodiments, the nucleic acid molecule is RNA or DNA, and can be single-stranded or double-stranded, preferably double-stranded DNA. When a nucleic acid molecule is placed in a functional relationship with another nucleic acid sequence, the nucleic acid molecule is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence. DNA is preferably used when it is ligated into a vector.

[0037] In this application, the term "vector" or "expression vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. For example, vectors include plasmids, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vector (e.g., expression vector) provided in this application contains a nucleic acid sequence encoding a polypeptide or recombinant protein as described in this application, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one selection marker.

[0038] In this application, the terms "purified" or "isolated" associated with polypeptides or nucleic acids mean that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Therefore, the term "isolated" includes polypeptides or nucleic acids removed from their original environment, such as if they are naturally occurring. Associated with nucleic acids, the terms "isolated" or "purified" indicate, for example, that the nucleic acid is not in its natural genomic background (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

[0039] In this application, the term "recombinant cell" refers to a cell into which exogenous polynucleotides and / or vectors can be or have been introduced. The exogenous polynucleotides may or may not be integrated into the genome of the "recombinant cell." When the recombinant cell contains a vector, the vector can be introduced into mammalian cells to construct recombinant cells, which are then used to express the polypeptides or recombinant proteins provided in this application. The corresponding polypeptides or recombinant proteins can be obtained by culturing the recombinant cells. Suitable mammalian cells include CHO cells, etc.

[0040] In this application, the term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition is administered. In some specific embodiments, the polypeptides or recombinant proteins contained in or expressed in the pharmaceutical composition are capable of specifically targeting and binding to GM-CSFR.

[0041] In this application, "pharmaceutically acceptable carrier" may include any solvent, carrier, excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients that are incompatible with the polypeptide or recombinant protein of this application, such as any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this application.

[0042] In this application, the terms "subject" or "patient" refer to a mammalian subject or patient. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, alpacas, poultry, goats, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject is a person suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0043] In this application, the term "diagnosis" refers to the identification of a pathological state, disease, or condition, such as the identification of chemotherapy side effects, or the identification of a subject suffering from chemotherapy side effects who may benefit from a particular treatment regimen.

[0044] In this application, the term "effective dose" refers to a therapeutic dose sufficient to reduce or improve the severity and / or duration of a condition or one or more of its symptoms; prevent disease progression; cause disease remission; prevent recurrence, development, or progression of one or more disease-related symptoms; detect disease; or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). The therapeutically effective dose of the polypeptide or recombinant protein described in this application depends on a variety of factors known in the art, such as weight, age, medical history, current treatment, the subject's health status and potential for cross-infection, allergies, hypersensitivity, and side effects, as well as the route of administration and the extent of tumor development. Those skilled in the art (e.g., physicians or veterinarians) may proportionally reduce or increase the dose based on these or other conditions or requirements.

[0045] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The polypeptides, recombinant proteins, or pharmaceutical compositions of this application may be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but this application is not limited to these exemplified routes of administration. Preferably, the compositions of this application are administered via intravenous or subcutaneous injection.

[0046] In this document, the term "treatment" refers to the administration of a drug to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) alleviation of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or a peptide or recombinant protein that specifically binds to GM-CSFR to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a peptide or recombinant protein that specifically binds to GM-CSFR as described herein to an individual in need.

[0047] This application proposes a polypeptide targeting GM-CSFR and its applications, which will be described in detail below.

[0048] polypeptide In a first aspect, this application provides a polypeptide. According to embodiments of this application, the polypeptide has the amino acid sequence shown in SEQ ID NO:1. The polypeptide of this application can specifically bind to GM-CSFR, increasing white blood cell count without promoting tumor proliferation, posing no risk of tumor promotion, and has the advantages of high safety. It can be used to treat chemotherapy side effects, especially neutropenia and myelosuppression.

[0049] In one optional embodiment of this application, such as Figure 1 As shown, this application clarifies the binding characteristics and biological functions of Peptide13 and GM-CSFR through four core experimental steps: verification of the white blood cell-boosting activity of the traditional Chinese medicine compound FEJ, screening of FEJ active ingredients and target confirmation, targeted binding verification, and in vitro and in vivo functional verification.

[0050] According to embodiments of this application, the polypeptide has an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 80% homology with it, or an amino acid sequence differing from it by 1 or 2 amino acids.

[0051] In this paper, the term "at least 80% homology" refers to at least 80% homology with each reference sequence, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. The term "at least 85% homology" refers to at least 85% homology with each reference sequence, which can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. The term "at least 90% homology" means that the homology with each reference sequence is at least 90%, and can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0052] Another aspect of this application covers functionally conserved variants of peptides. Functionally conserved variants are those variants in which a given amino acid residue in the peptide has been altered without changing the overall conformation and function of the peptide, including (but not limited to) substitutions of amino acids with amino acids having similar properties (e.g., polarity, hydrogen binding potential, acidity, basicity, hydrophobicity, aromaticity, and similar properties). Proteins may differ in the amino acids other than those indicated as conserved, such that the percentage of protein or amino acid sequence similarity between two proteins with similar functions, as determined by an alignment scheme (such as clustering), can vary and may be, for example, from 80% to 99%, where the similarity is based on the MEGALIGN algorithm. "Functionally conserved variants" also include polypeptides with amino acid sequences as shown in SEQ ID NO:1, having at least 80% amino acid homology, as determined by BLAST or FASTA algorithms, or being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide with amino acid sequences as shown in SEQ ID NO:1, and having the same or substantially similar properties or functions as the polypeptides identified in this application, particularly a good binding affinity for human GM-CSFR. The amino acid sequences mentioned in this application are shown from the N-terminus to the C-terminus.

[0053] Recombinant protein In a second aspect of this application, a recombinant protein is proposed. According to embodiments of this application, it includes the polypeptide described in the first aspect.

[0054] According to embodiments of this application, the recombinant protein may further include at least one of the following technical features: According to embodiments of this application, the recombinant protein further includes at least one of a protein tag, a reporter protein, serum albumin or a fragment thereof, and an Fc fragment.

[0055] In this article, "protein tag" generally refers to a polypeptide or protein fused together with a target protein (peptide) for expression, detection, detection, or purification of the target protein. Examples include, but are not limited to, His tags (also known as His-Tag, sequence HHHHHH), Flag tags (also known as Flag-Tag, sequence DYKDDDDK), GST tags (also known as GST-Tag, glutathione thiotransferase tag), MBP tags (also known as MBP-Tag, maltose-binding protein tag), SUMO tags, and C-Myc tags.

[0056] In this article, "reporter protein" generally refers to a polypeptide or protein expressed in fusion with a target protein (peptide), which can be used for protein detection, thereby indirectly reflecting the expression level of the target gene, cellular state, or molecular events. This includes, but is not limited to, fluorescent proteins (e.g., GFP, eGFP, RFP, mCherry, FRET, etc.), luciferases (e.g., Fluc, RLuc, BRET, etc.), and colorimetric reporter proteins.

[0057] In this article, "serum albumin" usually refers to In this document, "Fc fragment" generally refers to the Fc region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM, including CH2, CH3 regions and optionally hinge regions. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM are derived from mouse, human, primate, or alpaca sources.

[0058] Nucleic acid molecules, expression vectors, recombinant cells In a third aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect or the recombinant protein described in the second aspect. The nucleic acid according to embodiments of this application can encode the aforementioned polypeptide or recombinant protein.

[0059] According to embodiments of this application, the nucleic acid includes DNA or RNA.

[0060] It should be noted that, for the nucleic acids mentioned herein, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases herein, the other complementary strand is also disclosed. Furthermore, the molecular sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0061] In a fourth aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the third aspect. When the nucleic acid molecule is linked to the expression vector, it can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the nucleic acid molecule and the control elements only need to be operably linked.

[0062] In this document, "operably ligated" refers to ligating a foreign gene to an expression vector, enabling the control elements within the expression vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the expression vector is introduced into suitable recipient cells, the aforementioned polypeptides or recombinant proteins can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro production of polypeptides or recombinant proteins.

[0063] According to embodiments of this application, the expression vector may refer to a cloning vector, which can be obtained by operatively ligating the nucleic acid to a commercially available expression vector (such as a plasmid or viral vector). The expression vector in this application is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0064] In some optional embodiments of this application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0065] In some optional embodiments of this application, the expression vector is a plasmid expression vector or a lentiviral expression vector.

[0066] In a fifth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell comprises: carrying the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expressing the polypeptide described in the first aspect or the recombinant protein described in the second aspect. The recombinant cell according to embodiments of this application carries the aforementioned nucleic acid molecule or the aforementioned expression vector; or, the recombinant cell expresses the polypeptide described in the first aspect or the recombinant protein described in the second aspect. Using this cell or host under suitable conditions, the aforementioned polypeptide or recombinant protein can be effectively expressed within the cell or host.

[0067] According to embodiments of this application, the cells are obtained by introducing the above-described expression vector into cells or a host.

[0068] It should be noted that the cells or hosts used in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0069] In one optional embodiment of this application, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, but do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0070] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the polypeptide or recombinant protein described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the polypeptide or recombinant protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the conditions for the polypeptide or recombinant protein according to the specific environment of their laboratory.

[0071] It should be noted that the use of nucleic acid molecules, expression vectors, and recombinant cells to prepare peptides in this application is only one example of the preparation method in this application.

[0072] In an optional embodiment of this application, the polypeptide can also be prepared using a solid-phase synthesis process. This method is simple, produces products with good uniformity, has low production costs, and is easily scalable.

[0073] Pharmaceutical Composition In a sixth aspect of this application, a pharmaceutical composition is proposed. According to embodiments of this application, the pharmaceutical composition comprises: the polypeptide described in the first aspect, the recombinant protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cells described in the fifth aspect. As is known, the polypeptide described in the first aspect can specifically bind to GM-CSFR, increasing white blood cell count without promoting tumor proliferation, posing no risk of tumor promotion, and possessing the advantages of high safety. Therefore, a pharmaceutical composition containing the aforementioned polypeptide can be used to treat chemotherapy side effects, especially neutropenia.

[0074] According to embodiments of this application, the above-described pharmaceutical composition may further include at least one of the following technical features: According to embodiments of this application, the pharmaceutical composition further includes pharmaceutically acceptable excipients, carriers, or mediators.

[0075] In one optional embodiment of this application, pharmaceutically acceptable excipients refer to pharmaceutical excipients that are conventional in the pharmaceutical field, such as absorption enhancers, isotonic agents, stabilizers, regulators, etc.

[0076] In one alternative embodiment of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field.

[0077] In one alternative embodiment of this application, pharmaceutically acceptable mediators refer to pharmaceutical mediators conventional in the pharmaceutical field, such as solutions (e.g., water).

[0078] In one alternative embodiment of this application, examples of suitable pharmaceutically acceptable carriers, excipients, and mediators are well known in the art. Pharmaceutical compositions comprising such carriers, excipients, and mediators can be formulated using known conventional methods.

[0079] In some alternative embodiments, the pharmaceutical composition of this application may also contain other active ingredients for treatment.

[0080] The pharmaceutical composition of this application can be administered via various routes (e.g., orally or intravenously). Preferably, the pharmaceutical composition of this application is in solution form. Clinical dosing regimens are determined by the attending physician and clinical factors. As is known in the medical field, the dosage for any given patient depends on many factors, including patient size, body surface area, age, the drug to be administered, sex, time and route of administration, general health, and other concurrently administered medications. The pharmaceutical composition of this application can be administered topically or systemically. Preferably, it can be administered intravenously or subcutaneously. The pharmaceutical composition of this application can also be administered directly to the target site, for example, by targeted delivery to internal or external target sites.

[0081] In this article, the dosage forms of the above-mentioned peptides and pharmaceutical compositions can be prepared according to clinical needs, including but not limited to injections, inhalations, topical preparations, capsules, powders, tablets, granules, pills, oral liquids, patches, nano-preparations, biphasic preparations, or sustained-release preparations.

[0082] use In a seventh aspect of this application, the use of the polypeptide described in the first aspect, the recombinant protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a medicament for the prevention and / or treatment of chemotherapy side effects is provided.

[0083] The terms “treatment” and “prevention” as used herein, and words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, different degrees of treatment or prevention exist, and those skilled in the art will recognize that such treatment or prevention has potential benefit or therapeutic effect. Furthermore, the treatment or prevention provided in this application may include treatment or prevention of one or more diseases, such as cancer, or symptoms of a patient. Additionally, for the purposes of this document, “prevention” may encompass delaying the onset of a disease or its symptoms or the patient's condition.

[0084] According to embodiments of this application, the main side effect of chemotherapy is neutropenia.

[0085] According to embodiments of this application, the drugs causing the neutropenia are selected from docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, doxorubicin, pentorubicin, and pharmaceutically acceptable salts thereof.

[0086] Methods to treat chemotherapy side effects In an eighth aspect of this application, a method for treating chemotherapy side effects is provided. According to embodiments of this application, the method includes administering to a subject a pharmaceutically acceptable dose of the polypeptide described in the first aspect, the recombinant protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cells described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect.

[0087] In one alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0088] The effective amount of the polypeptide, recombinant protein, or pharmaceutical composition described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0089] The polypeptides, recombinant proteins, or pharmaceutical compositions of this application may be incorporated into suitable pharmaceuticals, which may be prepared in various forms, such as liquids. Various routes of administration of the polypeptides, recombinant proteins, pharmaceutical compositions, or pharmaceuticals of this application are contemplated, including intravenous, intramuscular, and subcutaneous injection, but this application is not limited to these exemplified routes of administration.

[0090] According to an embodiment of this application, the chemotherapy side effect is neutropenia.

[0091] According to embodiments of this application, the drugs causing the neutropenia are selected from docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, doxorubicin, pentorubicin, and pharmaceutically acceptable salts thereof.

[0092] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0093] Example 1: Validation of FEJ white blood cell-boosting activity, screening of active ingredients and determination of target sites 1. Verification of FEJ's white blood cell-boosting activity: 1.1 Establishment of a chemotherapy-induced neutropenia model Existing technologies mostly use healthy mice to establish bone marrow suppression models, which can only verify the efficacy of drugs in increasing white blood cell count, but cannot assess the impact on tumors. This embodiment uses a spontaneous colorectal cancer mouse model to integrate "efficacy verification" and "safety assessment," avoiding the potential risk of existing drugs promoting tumor growth while increasing white blood cell count. See the schematic diagram of the chemotherapy and FEJ treatment experimental design. Figure 2 a. The specific steps are as follows: Select 12-14 week old C57BL / 6J Apc Min / + Spontaneous colorectal cancer mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.), this model is due to... APC Gene mutations readily lead to intestinal adenomas, and the tumor microenvironment of clinical cancer patients is simulated, making it an ideal model for assessing the safety of white blood cell-boosting drugs (without tumor-promoting risk). A neutropenia model was established by inducing bone marrow suppression through intraperitoneal injection of cisplatin (2.5 mg / kg, once daily for 4 days as one cycle, for a total of 3 cycles). Peripheral blood was collected from the orbital venous plexus before and after each chemotherapy cycle, and white blood cell (WBC) and neutrophil (neutrophil) counts were measured using a fully automated blood analyzer (Mindray BS-240 Vet) to confirm the model's success (the chemotherapy group showed significantly lower WBC and neutrophil levels compared to the control group). P <0.05).

[0094] The dynamic changes in peripheral blood leukocyte and neutrophil counts during 3 cycles of cisplatin chemotherapy (n=10) are shown in the following results. Figure 2 b. The results showed that with the increase of chemotherapy cycles, the white blood cell and neutrophil counts in mice showed a progressive decreasing trend, reaching the lowest value after the end of the 3rd chemotherapy cycle, and the difference compared with the pre-chemotherapy values ​​was statistically significant. P <0.001), confirming the successful establishment of the neutropenia model.

[0095] 2.2 Whitening Activity Experiment of FEJ Existing technologies only verify the white blood cell-raising effect of FEJ, without focusing on its impact on tumors. This embodiment is the first to demonstrate that FEJ does not promote tumor proliferation while raising white blood cell count, thus overcoming the deficiency in existing evidence regarding the safety of traditional Chinese medicine compound formulas. The specific steps are as follows: The model mice were randomly divided into an FEJ treatment group and an H2O control group (n=5 in each group). The FEJ treatment group was given 32 mL / kg FEJ (Dong-E-E-Jiao Co., Ltd., batch number Z20083345) by gavage daily, while the control group was given an equal volume of sterile pure water. The treatment was continued for 2 weeks. The mice were euthanized on day 30 of the experiment. Peripheral blood indicators were dynamically monitored during the period, and the intestinal adenomas of the mice were analyzed by image analysis and the number of adenomas was quantitatively analyzed.

[0096] For the results of peripheral blood marker recovery after FEJ treatment, please refer to [link / reference needed]. Figure 2 c. Compared with the control group (H2O gavage), the white blood cell and neutrophil counts of mice in the FEJ treatment group were significantly increased after treatment and recovered to near pre-chemotherapy levels, while the control group showed no significant recovery. P >0.05), confirming the whitening effect of FEJ.

[0097] Representative images of intestinal adenomas and quantitative analysis results of the number of adenomas can be found in [link to image]. Figure 2 d. The number of adenomas in mice in the cisplatin chemotherapy group was significantly reduced compared with the control group without chemotherapy, while there was no significant difference in the number of adenomas between the FEJ treatment group and the H2O control group, confirming that FEJ does not promote tumor proliferation while increasing white blood cell count, and has good safety.

[0098] The results in summary indicate that the white blood cell and neutrophil counts in the FEJ-treated mice were significantly restored compared to before treatment. P <0.05, and the difference was statistically significant compared with the control group; after euthanasia, the intestinal tissue of the mice was dissected and the number of adenomas was systematically counted, confirming that there was no significant difference between the FEJ treatment group and the control group ( P >0.05), indicating that FEJ does not promote tumor proliferation while increasing white blood cell count.

[0099] 2. Target selection: Existing literature indicates that G-CSF binding to G-CSFR carries a risk of tumor promotion, while GM-CSFR, as the target of GM-CSF, a mainstream clinical drug for increasing white blood cell count, has no clear association with tumor promotion. Therefore, the white blood cell-enhancing functional region of GM-CSFR was selected as the target.

[0100] 3. Short peptide design and screening: The core component of Compound Donkey-hide Gelatin Oral Solution, donkey-hide gelatin, was hydrolyzed using collagenase to produce polypeptide fragments of different molecular weights. Mass spectrometry analysis was used to screen the top 30 polypeptides by enrichment level. The sequence list of the top 30 candidate peptides in the FEJ polypeptide library is as follows: Figure 3 As shown in a.

[0101] Based on the results of peptidomics analysis, potential peptides with white blood cell-boosting activity were screened from the above peptides. Then, de novo protein design technology was used to design six candidate short peptides targeting the GM-CSFR spatial structure. Molecular docking simulations were used to predict the binding modes and key interacting residues between the donkey-hide gelatin-derived peptides and the designed short peptides with GM-CSFR. The molecular docking simulation results of the binding modes and key interacting residues of the candidate peptides with GM-CSFR protein are shown below. Figure 3 As shown in b, the results showed that Peptide 13 (sequence: PCPIGPAGAR (SEQ ID NO:1)) derived from donkey-hide gelatin had the lowest binding free energy and the best binding stability with GM-CSFR. Therefore, this peptide was selected for further verification.

[0102] The target peptide was synthesized by GenScript and its purity was verified to be ≥95% by high performance liquid chromatography (HPLC). Its solubility stability in the experimental system was confirmed by solubility experiments, which met the requirements of subsequent experiments.

[0103] Example 2: Verification of binding affinity of Peptide 13 This embodiment breaks through the existing single-validation mode (such as ELISA or SPR only), and adopts a dual validation system of "ELISA quantification of binding activity + BLI quantification of kinetic parameters". BLI technology is simpler to operate and more resistant to interference than SPR, and is suitable for detecting short peptide-protein interactions, requiring no complex chip pretreatment. Therefore, ELISA can directly reflect the amount of binding complex generated (OD). 450 BLI precise quantification combined with specificity and stability (K value), D The dual data complementarity verification (value) solves the problems of insufficient binding activity verification and single parameters in existing technologies, ensuring the specific binding ability of peptides to GM-CSFR.

[0104] 1. ELISA detection method: Recombinant GM-CSFR protein (purchased from MCE) was used to coat ELISA plates at a concentration of 2000 ng / mL and incubated overnight at 4°C. The next day, the plates were washed three times with 0.05% PBST buffer, and then blocked with PBS containing 0.25% Tween 20 and 1% bovine serum albumin (BSA) at 37°C for 2 h to block non-specific binding. After blocking, the plates were washed, and Peptide 13 was added to seven concentration gradients (see Table 1) in two replicates per group, in descending order of concentration. The plates were incubated at 37°C for 1 h. After washing, horseradish peroxidase (HRP)-labeled secondary antibody was added and incubated in the dark for 30 min. After washing again, TMB chromogenic solution was added and the plates were incubated at room temperature in the dark for 15-20 min. The reaction was terminated by adding stop solution, and the absorbance (OD) at 450 nm was measured using a microplate reader. 450 The binding affinity of the peptide to GM-CSFR protein was assessed based on changes in OD values. For Peptide 13, see [link to ELISA results]. Figure 3 c (The vertical axis represents the absorbance (OD) at 450 nm) 450 The graph shows the amount of peptide-GM-CSFR binding complex generated (the horizontal axis represents the concentration gradient of peptide 13), and the trend of the curve shows that the binding activity of the peptide increases significantly with increasing concentration, while the blank control group (concentration 0 μg / mL) showed no obvious binding signal.

[0105] Table 1: Peptide concentration gradient design

[0106] 2. BLI detection method: The binding kinetics of GM-CSFR protein and peptide were detected using biomembrane interference (BLI) technology. The experiment was performed at 30°C using a GatorPlus label-free biomolecular interaction analyzer with SA XT probes. The specific procedures are as follows: GM-CSFR protein was biotinylated (labeling ratio 0.6:1), diluted to 10 μg / mL with 0.05% PBST buffer, and immobilized on the surface of an SA XT probe biosensor for 82 min. The probe was then washed with 0.05% PBST buffer for 1 min, equilibrated in HBSP buffer (10 mM HEPES, 150 mM NaCl, and 0.05% v / v surfactant P20) for 2 min, and then immersed in reaction wells containing different concentrations of peptides (diluted with HBSP buffer) for a 60 s binding reaction. A subsequent 50 s buffer dissociation step was performed in HBSP buffer. The experimental data were analyzed using GraphPad Prism 9 software and a standard 1:1 binding model by real-time monitoring of the biomembrane interference signal, and the binding rate constant K was calculated. on Dissociation rate constant K off and equilibrium dissociation constant K D Kinetic parameters are used to intuitively characterize the specific binding ability and affinity of GM-CSFR protein to peptides.

[0107] The kinetics of the supplemental biomembrane interference (BLI) detection for Peptide13 can be found in [link to relevant data]. Figure 3 d (the vertical axis represents the change in interference signal (nm), and the horizontal axis represents the detection time (sec)). The curve reflects the real-time binding-dissociation process of the two peptides with GM-CSFR protein, and the binding rate constant (K) is also shown. on ), dissociation rate constant (K) off and equilibrium dissociation constant (K) D Further quantitative analysis confirmed the existence of a stable and efficient specific interaction between the two.

[0108] Therefore, this embodiment confirms that Peptide 13 can bind with GM-CSFR with a specific high affinity, demonstrating that its targeting is superior to existing non-specific white blood cell-boosting drugs, and can solve the problems of poor targeting and easy off-target effects of existing drugs.

[0109] Example 3: In vitro and in vivo functional validation of Peptide 13 Existing technologies only focus on the effect of increasing white blood cell count, without paying attention to chemotherapy-induced damage to hematopoietic organs (such as splenomegaly). Therefore, this embodiment simultaneously verifies the effect of Peptide 13 in increasing white blood cell count, its spleen-protective effect, and its lack of tumor-promoting risk, providing a more comprehensive functional assessment.

[0110] 1. In vitro experiments - Western blotting and bone marrow cell colony formation assay (CFU): 1.1 Wild-type mouse bone marrow mononuclear cells (BMNCs) were used in the experiment. First, the activation status of the GM-CSFR downstream signaling pathway was detected by Western blotting: the concentration of BMNCs was adjusted to 1×10⁻⁶. 6 Cells were incubated at 37°C in a 5% CO2 incubator. Different concentration intervention groups were established (Peptide 13: 10–300 ng / mL, GM-CSF: 5–50 ng / mL, and a blank control group was treated with an equal volume of PBS). Total protein was extracted from the cells and analyzed by Western blotting after drug administration. Simultaneously, the effect of BMNCs on the proliferation and differentiation of granulocyte-macrophage progenitor cells was assessed using an in vitro colony-forming assay (CFU). The concentration of BMNCs was adjusted to 1 × 10⁻⁶. 5 CFU-G were inoculated at a concentration of 100 ng / mL on methylcellulose semi-solid medium (MethoCult M3434). Two groups were established: a plasma intervention group (mice plasma administered via gavage for 2 weeks with H2O or FEJ, diluted 1:100) and a peptide and protein intervention group (equal volumes of PBS, negative control Peptide 2, target peptide Peptide 13, and positive control G-CSF were added, all at a final concentration of 100 ng / mL). After drug administration, the cells were incubated at 37°C in a 5% CO2 incubator for 7 days, and the number of CFU-G cells was counted under an inverted microscope.

[0111] See Western Blot results. Figure 4 a. The results showed that Peptide 13 significantly upregulated the phosphorylation levels of p-STAT5 and p-ERK1 / 2 in a concentration-dependent manner.

[0112] The effect of FEJ-treated serum on CFU-G in mouse bone marrow cells can be found in [the original text]. Figure 4 b (Left image is a representative microscopic image (×4), right image is the quantitative analysis), the results showed that the number of CFU-G in the serum-treated group of FEJ gavage mice was significantly increased compared with the H2O serum group ( P <0.01).

[0113] The effects of different treatment groups on CFU-G in mouse bone marrow cells are shown in the following results. Figure 4c (Left image is a representative microscopic image (×4), right image is the quantitative analysis). The results showed that the number of CFU-G in the PBS group and Peptide 2 group was lower; the number of CFU-G in the Peptide 13 group and G-CSF group was significantly increased. P <0.01), and there was no significant difference between the two groups.

[0114] In summary, Peptide 13 can effectively promote the proliferation and differentiation of mouse granulocyte-macrophage progenitor cells by activating the downstream STAT5 / ERK1 / 2 signaling pathway of GM-CSFR.

[0115] 1.2 Aseptic isolation of rhG-CSF-mobilized human peripheral blood mononuclear cells (PBMCs) was performed first, followed by Western blotting: the PBMC concentration was adjusted to 1×10⁻⁶. 6 Cells were incubated at 37°C in a 5% CO2 incubator. Different concentration intervention groups were set up (Peptide 13: 100, 300 ng / mL; GM-CSF: 5 ng / mL; blank control group with an equal volume of PBS). Total protein was extracted from the cells and analyzed by Western blotting after drug administration. Simultaneously, in vitro colony formation experiments were conducted, with PBMCs concentration adjusted to 1×10⁻⁶. 5 Cells / mL were inoculated into methylcellulose semi-solid medium (MethoCult H4434); a plasma intervention group (containing peripheral blood plasma collected from volunteers before and after drinking FEJ for 2 weeks, diluted 1:100) and a peptide and protein intervention group under the same conditions as mouse cells were set up. After culturing under the same conditions for 7-14 days, CFU-GM was counted.

[0116] See Western Blot results. Figure 4 d. Results showed that Peptide 13 upregulated the phosphorylation levels of p-STAT5 and p-ERK1 / 2. The effects of serum on bone marrow CFU-GM levels before and after FEJ consumption in volunteers are shown in [reference needed]. Figure 4 e (Left image is a representative microscopic image (×4), right image is the quantitative analysis), the results showed that the serum CFU-GM level in the FEJ treatment group was significantly increased after 2 weeks of drinking FEJ compared with before drinking FEJ. P <0.01).

[0117] The results of the effects of different treatment groups on human bone marrow cell CFU-GM are shown in [link to relevant documentation]. Figure 4f (Left image is a representative microscopic image (×4), right image is the quantitative analysis). The results showed that the number of CFU-GM in the Peptide 13 and G-CSF groups was significantly increased compared with the PBS group and the Peptide 2 group. P <0.01).

[0118] In summary, FEJ can produce hematopoietic components in the human body. Its core active peptide, Peptide13, can promote the proliferation and differentiation of human granulocyte-macrophage hematopoietic progenitor cells by activating the downstream STAT5 / ERK1 / 2 signaling pathway of GM-CSFR, thus possessing clear clinical translational value.

[0119] 2. In vivo experiments See the schematic diagram of the chemotherapy and Peptide 13 treatment trial design. Figure 5 a. The specific steps are as follows: Chemotherapy-induced myelosuppression mouse model Apc Min / + Mice were randomly divided into three groups (n=5) after three cycles of cisplatin induction: saline group, Peptide 13 group (200 mg / kg, tail vein injection, once daily for two rounds), and G-CSF group (100 μg / kg, tail vein injection, once daily for two rounds). Peripheral blood parameters were measured before treatment, after the first round of treatment, and after the second round of treatment. Mice were euthanized on day 23 of the experiment, and intestinal adenomas were image-analyzed and the number of adenomas was quantitatively analyzed.

[0120] Changes in peripheral blood leukocyte and neutrophil counts (n=5 per group) are shown in the results below. Figure 5 b. The results showed that there were no significant differences among the three groups before treatment; after the first round of treatment, the white blood cell and neutrophil counts in the Peptide 13 group and the G-CSF group were significantly increased; after the second round of treatment, the indicators in both groups further recovered, and the differences compared with the saline control group were statistically significant. P <0.01), and there was no significant difference between the Peptide 13 group and the G-CSF group.

[0121] Representative images of intestinal adenomas and quantitative analysis of the number of adenomas (n=5 per group) are shown in the attached image. Figure 5 c. The results showed that there was no significant difference in the number of adenomas between the Peptide 13 group, the G-CSF group, and the saline control group after chemotherapy. P >0.05), and none of them showed adenoma proliferation.

[0122] In summary, it can be concluded that the white blood cell and neutrophil counts in both the Peptide 13 group and the G-CSF group were significantly restored. P <0.01), no significant change was observed in the saline group; intestinal adenoma count showed no significant difference between the Peptide 13 group and the saline group after chemotherapy ( P >0.05), confirming no risk of tumor promotion.

[0123] Therefore, the results of this embodiment confirm that Peptide 13 can reverse chemotherapy-induced neutropenia in vivo, protect hematopoietic organ function, and has no risk of promoting tumors, thus comprehensively solving the problems of existing drugs having "single efficacy and insufficient safety".

[0124] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0125] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A polypeptide, characterized in that, The polypeptide has the amino acid sequence shown in SEQ ID NO:

1.

2. A recombinant protein, characterized in that, Includes the polypeptide described in claim 1.

3. The recombinant protein according to claim 2, characterized in that, Further includes at least one of a protein tag, a reporter protein, serum albumin or a fragment thereof, and an Fc fragment.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1 or the recombinant protein of any one of claims 2 to 3.

5. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 4.

6. A recombinant cell, characterized in that, include: Carrying the nucleic acid molecule of claim 4 or the expression vector of claim 5; or Express the polypeptide of claim 1 or the recombinant protein of any one of claims 2 to 3.

7. A pharmaceutical composition, characterized in that, include: The polypeptide of claim 1, the recombinant protein of any one of claims 2-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant cell of claim 6, and Optional pharmaceutically acceptable excipients, carriers, or mediators.

8. Use of the polypeptide of claim 1, the recombinant protein of any one of claims 2-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant cell of claim 6, or the pharmaceutical composition of claim 7 in the preparation of a medicament for the prevention and / or treatment of chemotherapy side effects.

9. The use according to claim 8, characterized in that, The chemotherapy side effect was neutropenia.

10. The use according to claim 9, characterized in that, The drugs that cause the aforementioned neutropenia are selected from docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, doxorubicin, pentorubicin, and drug-acceptable salts thereof.

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