Polypeptides targeting cxcr2 and uses

Abstract

The application discloses a polypeptide targeting CXCR2 and application. The polypeptide has an amino acid sequence as shown in any one of SEQ ID NO: 1 and SEQ ID NO: 2. The polypeptide provided by the application can specifically bind to CXCR2, and can effectively inhibit macrophage chemotactic migration mediated by CXCR2, with an inhibition rate of 50% or above (for example, 50%-70%), and can efficiently block downstream signal transduction of CXCR2, thereby providing a powerful and precise action target for treatment of inflammatory diseases and tumors.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically involving peptides targeting CXCR2 and their applications. Background Technology

[0002] Chemokine receptor protein CXCR2, a core member of the G protein-coupled receptor (GPCR) superfamily, is a key target for regulating inflammatory responses and tumor progression. By binding to ligands such as CXCL1, CXCL2, and CXCL8 (IL-8), it mediates the chemotactic infiltration of immune cells, including macrophages, and activates downstream signaling pathways such as PI3K / Akt and calcium mobilization. CXCR2 directly participates in the pathological processes of inflammatory diseases such as chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, and psoriasis, as well as malignant tumors such as lung cancer, pancreatic cancer, and renal cell carcinoma. Therefore, CXCR2-targeted therapy has clear clinical value.

[0003] Domestically and internationally, peptide-based targeting technologies for CXCR2 have formed multiple research directions. The core implementation schemes and representative results are as follows: (1) Natural chemokine derivatives: Using human CXCL8 (IL-8) as the natural parent, by site-directed mutation of its N-terminal key functional motif "ELR" (Glu) 4 -Leu 5 -Arg 6 This involves disrupting receptor activation while retaining binding activity, resulting in a competitive CXCR2 antagonist. A typical mutant is CXCL8-Glu4AlaLeu5AlaArg6His, which achieves specific blockade of CXCR2 by replacing three key amino acids in the ELR motif, with an IC50 value of [missing information]. 50 The values ​​are mostly in the range of 18-92 μM, and CXCR1 / CXCR2 cross-binding phenomenon is common. (2) Dual complementary site binding peptides: By tandem two antigen-binding domains that recognize different epitopes of CXCR2 (such as the domains that recognize the N-terminal F11 / F14 / W15 epitope and the outer ring W112 / G115 epitope), dual complementary site nanobodies or peptides are formed. The representative patent is Chinese CN104487456B (disclosing dual complementary site nanobodies 163d2 / 127d1, 54b12 / 163e3, etc.). This type of peptide enhances targeting through synergistic binding and can inhibit CXCL8-induced GTPγS release, but the structure depends on the linker region to connect two functional domains, the molecular weight is large (usually >20kDa) and the synthesis process is complicated. (3) Phage display screening peptides: Phage display technology was used to screen targeted peptides from combinatorial peptide libraries. Representative sequences included linear hexapeptides MSRAKE and CAKELR, and disulfide bond-bound peptides CLRSGRFC and CLPWKENC. These peptides can inhibit calcium mobilization and cell migration by binding to CXCR2 by mimicking the C-terminal spatial structure of CXCL8. However, they are mostly short linear peptides and lack structural designs to resist protease degradation. Brazilian patent BR-112014027697-B1 discloses a pharmaceutical composition of this type of peptide for the treatment of inflammation and tumors, but the stability problem has not been solved. (4) Extracellular domain blocking peptides: Long-chain linear peptides (such as EDFNMESDSFEDFWKGED) are designed based on the extracellular domain sequence of CXCR2 (such as amino acid residues 106-120 and 184-208) to achieve blocking by covering the ligand binding region. These peptides have shown certain anti-inflammatory activity in in vitro experiments, but their molecular weight is mostly >15kDa. Folding errors are prone to occur during preparation, and the purification yield is less than 30%, which limits large-scale production. (5) Peptide modification technology: To address the lack of stability of natural peptides, chemical modifications (PEGylation, esterification, cyclization) are used to extend the in vivo half-life and improve bioavailability. For example, palmitic acid chains are linked to the N-terminus of the peptide (esterification modification) or PEG2000-5000 (PEGylation modification). Partial cyclization modification enhances structural stability by introducing disulfide bonds, but the increased molecular weight after modification may reduce tissue penetration and binding affinity. (6) Antibody-based targeted molecules: CXCR2 monoclonal antibodies (such as IgG18 and IgG56) developed in recent years achieve specific binding by recognizing the N-terminal domain, which can inhibit the proliferation and migration of pancreatic cancer cells and show tumor suppressor activity in the xenograft model. However, antibody molecules have complex preparation processes, high production costs, and immunogenicity risks, and the clinical translation cycle is long.

[0004] However, no related peptide drugs have been approved for marketing in the past 30 years. The core bottleneck lies in the multiple technical defects of existing CXCR2-targeting peptides: First, the structural design is highly homogeneous, with more than 80% relying on ELR motifs, modifications of natural chemokines, or double complementary site structures, resulting in a single binding mode and a lack of innovation; second, receptor specificity is poor, with peptides based on ELR motifs generally exhibiting CXCR1 / CXCR2 cross-binding activity, easily leading to non-targeted side effects; third, the preparation process is complex, with the synthesis of long-chain peptides or double complementary site molecules involving cumbersome steps and low yields (mostly below 30%), making large-scale production difficult; fourth, application scenarios are limited, with administration methods mostly limited to intravenous injection, resulting in insufficient adaptability and difficulty in covering multiple indications. Therefore, it is crucial to find a novel CXCR2-targeting drug. Summary of the Invention

[0005] 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 polypeptide targeting CXCR2.

[0006] This application is based on the following discoveries of the inventors: This application employs a standardized process of "screening-preparation-dual verification-functional confirmation" to screen for two CXCR2-targeting peptides (Peptide25 and Peptide3) with novel amino acid sequences and unique structural features (Gly-Pro-Val enrichment, no dependence on traditional functional motifs). This approach specifically addresses the core technical challenges of existing technologies, such as structure dependence on traditional motifs, insufficient specificity, high preparation difficulty, and limited application scenarios. Through targeted screening, dual binding verification, and cell function experiments, it demonstrates efficient and specific binding to CXCR2 and regulation of its bioactivity. Simultaneously, it simplifies the preparation process, expands application boundaries, and provides new technical means for the treatment of inflammatory diseases and tumors.

[0007] Therefore, in a first aspect of this application, a polypeptide is provided. According to embodiments of this application, the polypeptide has an amino acid sequence as shown in either SEQ ID NO:1 or SEQ ID NO:2.

[0008] 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.

[0009] According to embodiments of this application, the recombinant protein further includes at least one of a bioactive protein or a fragment thereof, a bioactive polypeptide or a fragment thereof.

[0010] According to embodiments of this application, the bioactive protein or fragment thereof is selected from at least one of protein tags, reporter proteins, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof, and Fc fragments.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] According to embodiments of this application, the pharmaceutical composition further includes pharmaceutically acceptable excipients, carriers, or mediators.

[0016] 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 CXCR2-related diseases is provided.

[0017] According to embodiments of this application, the CXCR2-related diseases include inflammatory diseases or cancer.

[0018] In an eighth 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 the CXCR2 targeting peptide is proposed.

[0019] Beneficial effects: (1) This application is the first to discover novel amino acid sequences of Peptide25 and Peptide3, which do not have ELR motifs or double complementary site structures dependent on CXCR2-targeting peptides. With “Gly-Pro-Val enrichment” as the core structural feature, they belong to a novel type of CXCR2-binding peptide, filling the gap of such structural peptides in the prior art, and the structural innovation is significant. (2) Verified by both ELISA and BLI experiments, Peptide25 and Peptide3 of this application have clear targeted binding characteristics to CXCR2. From a structural perspective, these two peptides bind to CXCR2 through a unique "Gly-Pro-Val enrichment" structural mode, which significantly reduces the adaptability of the binding epitope to the homologous receptor, and the risk of cross-binding is much lower than that of existing technologies. This can minimize interference from non-target signaling pathways and provide higher safety assurance for clinical treatment. (3) Cell migration experiments confirmed that Peptide25 and Peptide3 of this application can effectively inhibit CXCR2-mediated macrophage chemotaxis and migration, with an inhibition rate of 50% or more (e.g., 50%-70%), and can efficiently block downstream signal transduction of CXCR2, providing a powerful and precise target for the treatment of inflammatory diseases and tumors. (4) The Peptide25 and Peptide3 of this application are rich in Gly-Pro rigid peptide bond structure. This structure is often associated with the ability to resist protease degradation in natural peptides. Therefore, the peptides of this application have the potential advantage of resisting protease hydrolysis. Theoretically, they can avoid the problem of easy degradation caused by the lack of rigid structural barrier of existing linear peptides, and provide structural possibilities for prolonging the in vivo action time and reducing the frequency of administration. (5) The polypeptides of this application are prepared by solid-phase synthesis process, which is simple, has good product uniformity, low production cost, and a total yield of over 75%, making it easy to achieve large-scale production. Moreover, the polypeptides have excellent biocompatibility and can be developed into various administration methods such as injections, inhalants, and topical preparations according to clinical needs, which are suitable for various indications such as inflammation and tumors. They also have the potential to be modified into diagnostic probes, taking into account both treatment and diagnosis scenarios, and have a wide range of applications.

[0020] 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

[0021] 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 The results show the binding activity of Peptide25 and Peptide3 to CXCR2 protein in Examples 1 and 2 of this application; where A: bioinformatics prediction results; B: ELISA quantitative results, and the vertical axis represents absorbance OD. 450 Value (reflecting the amount of "peptide-CXCR2 binding complex" generated), the horizontal axis is the peptide concentration gradient; C: Biomembrane interference (BLI) detection kinetics spectrum, the vertical axis is the change in interference signal (nm), and the horizontal axis is the detection time (sec). Figure 3This document describes the inhibitory effects of Peptide25 and Peptide3 on macrophage migration in Example 2 of this application. A: The left image shows the morphology of uninduced THP-1 cells (suspension growth), and the right image shows the morphological characteristics of macrophages after 24 h of PMA (25 ng / mL) induction (adherent growth, cell enlargement, pseudopodia extension). B: The left image shows representative microscopic images (10×) of cell migration from the control group, Peptide25 group, and Peptide3 group, and the right image shows a histogram of the number of migrating cells analyzed quantitatively by ImageJ. Data are expressed as "mean ± standard deviation". Detailed Implementation

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 CXCR2. In some specific embodiments, it is determined, for example, according to ELISA and BLI techniques.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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).

[0034] 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.

[0035] 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 CXCR2.

[0036] 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.

[0037] 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.

[0038] In this application, the term "diagnosis" refers to the identification of a pathological state, disease, or condition, such as the identification of a CXCR2-related disease, or the identification of a subject with a CXCR2-related disease who may benefit from a specific treatment regimen. In some embodiments, diagnosis includes identifying abnormal levels or activity of CXCR2. In some embodiments, diagnosis refers to identifying cancer or an autoimmune disease in a subject.

[0039] 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.

[0040] 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.

[0041] 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 a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to the disease but not yet diagnosed with it; (b) inhibition of disease, such as blocking disease progression; or (c) alleviation of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or a peptide or recombinant protein that specifically binds to CXCR2 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 CXCR2 as described herein to an individual in need.

[0042] This application proposes a peptide targeting CXCR2 and its applications, which will be described in detail below.

[0043] polypeptide In a first aspect, this application provides a polypeptide. According to embodiments of this application, the polypeptide has an amino acid sequence as shown in either SEQ ID NO:1 or SEQ ID NO:2.

[0044] This application is the first to discover that the peptides (Peptide25 and / or Peptide3) of this application can specifically bind to the chemokine receptor protein CXCR2, and exert targeted anti-inflammatory and anti-tumor effects by precisely blocking CXCR2-mediated pathological signal transduction. 1. Anti-inflammatory effect: As a core regulatory target of the inflammatory response, CXCR2 can mediate the chemotactic infiltration of macrophages to the site of inflammation by binding to ligands such as CXCL1, CXCL2, and CXCL8 (IL-8), thereby releasing inflammatory factors, aggravating tissue damage, and amplifying the inflammatory effect. The peptide of this invention competitively binds to the CXCR2 ligand binding site, inhibiting the macrophage chemotactic migration mediated by it, blocking the infiltration of inflammatory cells and the pathological cascade reaction from the source, and achieving a targeted anti-inflammatory effect. 2. Anti-tumor effect: CXCR2 plays a key role in the formation of the tumor microenvironment. It can recruit macrophages into the tumor site and transform them into tumor-associated macrophages (TAMs). As pro-cancer macrophages, TAMs can accelerate tumor proliferation, migration and invasion through secretion of growth factors, promotion of angiogenesis and inhibition of anti-tumor immunity. The peptide of this invention can effectively inhibit the chemotactic recruitment of macrophages to the tumor microenvironment by specifically binding to CXCR2, reduce the formation of TAMs and their pro-cancer function, and thus block CXCR2-mediated tumor progression signals to achieve a targeted anti-tumor effect.

[0045] Therefore, the peptide of this application can specifically bind to CXCR2 and effectively inhibit CXCR2-mediated macrophage chemotaxis and migration with an inhibition rate of 50% or more (e.g., 50%-70%). It can efficiently block downstream signal transduction of CXCR2, providing a potent and precise target for the treatment of inflammatory diseases and tumors.

[0046] In one optional embodiment of this application, such as Figure 1 As shown, this application clarifies the binding characteristics and biological functions of Peptide25 and Peptide3 with CXCR2 through three core experimental steps: peptide screening, targeted binding verification, and functional activity detection.

[0047] According to embodiments of this application, the polypeptide has an amino acid sequence as shown in either SEQ ID NO:1 or SEQ ID NO:2, or an amino acid sequence having at least 80% homology with it, or an amino acid sequence differing from it by one or two amino acids.

[0048] 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%.

[0049] 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 either SEQ ID NO:1 or SEQ ID NO:2, 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 polypeptides with amino acid sequences as shown in either SEQ ID NO:1 or SEQ ID NO:2, and having the same or substantially similar properties or functions as the polypeptides identified in this application, particularly a good binding affinity for human CXCR2. The amino acid sequences mentioned in this application are shown from the N-terminus to the C-terminus.

[0050] 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.

[0051] 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 bioactive protein or a fragment thereof, a bioactive polypeptide or a fragment thereof.

[0052] According to embodiments of this application, the bioactive protein or fragment thereof is selected from at least one of protein tags, reporter proteins, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof, and Fc fragments.

[0053] 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.

[0054] 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.

[0055] In this document, "toxin" generally refers to substances toxic to the host, including protein toxins and non-protein toxins, particularly suitable for targeting and killing tumor cells or pathological neutrophils that highly express CXCR2. Protein toxins include, but are not limited to, abrin, ricin A, Pseudomonas exotoxin, and diphtheria toxin. In this application, the protein toxin is preferably an enzymatic protein toxin.

[0056] In this article, "tumor necrosis factor" generally refers to substances that can cause hemorrhagic necrosis in various tumors, including but not limited to TNF-α and TNF-β.

[0057] In this article, "interferon" generally refers to a glycoprotein that has the ability to directly kill or inhibit viruses. This includes, but is not limited to, IFN-α, IFN-β, and IFN-γ.

[0058] In this article, "biological response modulators" generally refers to a class of protein substances that enhance the body's anti-tumor effects directly or indirectly through the immune system. These include, but are not limited to, lymphokines, IL-2, IL-15, IL-6, IL-10, and GM-CSF.

[0059] 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.

[0060] 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.

[0061] According to embodiments of this application, the nucleic acid includes DNA or RNA.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] In some optional embodiments of this application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0067] In some optional embodiments of this application, the expression vector is a plasmid expression vector or a lentiviral expression vector.

[0068] 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.

[0069] According to embodiments of this application, the cells are obtained by introducing the above-described expression vector into cells or a host.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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, achieves a total yield of over 75%, and is easily scalable for mass production.

[0075] Pharmaceutical Composition In a sixth aspect of this application, a pharmaceutical composition is provided. 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 cell described in the fifth aspect. As is known, the polypeptide described in the first aspect can specifically bind to CXCR2 and effectively inhibit CXCR2-mediated macrophage chemotaxis and migration, with an inhibition rate of 50% or higher (e.g., 50%-70%), efficiently blocking downstream signal transduction of CXCR2, providing a potent and precise target for the treatment of inflammatory diseases and tumors. Therefore, a pharmaceutical composition containing the above-mentioned polypeptide can be used to treat CXCR2-related diseases.

[0076] 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, and mediators.

[0077] 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.

[0078] In one alternative embodiment of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field.

[0079] 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).

[0080] 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.

[0081] In some alternative embodiments, the pharmaceutical composition of this application may also contain other active ingredients for treatment.

[0082] 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.

[0083] In one optional embodiment of this application, the polypeptide of this application, with its advantages of short molecular weight (814.92-1143.29 Da) and high penetrability, is suitable for diverse routes of administration and multiple disease treatment scenarios. Pharmaceutical compositions containing the above-mentioned polypeptide are suitable for diverse routes of administration and multiple disease treatment scenarios.

[0084] 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.

[0085] 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 CXCR2-related diseases is provided.

[0086] 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.

[0087] As previously known, the aforementioned peptides (Peptide25 and Peptide3) can specifically bind to the chemokine receptor protein CXCR2, exerting targeted anti-inflammatory and anti-tumor effects by precisely blocking CXCR2-mediated pathological signal transduction. Therefore, the peptides of this application can be used for the prevention and / or treatment of inflammatory diseases and cancer.

[0088] According to embodiments of this application, the CXCR2-related diseases include inflammatory diseases and cancer.

[0089] In this article, "cancer" includes, but is not limited to, lung cancer, pancreatic cancer, renal cell carcinoma, non-small cell carcinoma, colon cancer, esophageal cancer, ovarian cancer, breast cancer, melanoma, and hepatocellular carcinoma.

[0090] In an eighth 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 the CXCR2 targeting peptide is proposed.

[0091] Methods for preventing and / or treating CXCR2-related diseases In a ninth aspect of this application, a method for preventing and / or treating CXCR2-related diseases is proposed. According to embodiments of this application, the method comprises 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.

[0092] In one alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0093] 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.

[0094] 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.

[0095] As previously known, the aforementioned peptides (Peptide25 and Peptide3) can specifically bind to the chemokine receptor protein CXCR2, exerting targeted anti-inflammatory and anti-tumor effects by precisely blocking CXCR2-mediated pathological signal transduction. Therefore, the peptides of this application can be used for the prevention and / or treatment of inflammatory diseases and cancer.

[0096] According to embodiments of this application, the CXCR2-related diseases include inflammatory diseases and cancer.

[0097] In this article, "cancer" includes, but is not limited to, lung cancer, pancreatic cancer, renal cell carcinoma, non-small cell carcinoma, colon cancer, esophageal cancer, ovarian cancer, breast cancer, melanoma, and hepatocellular carcinoma.

[0098] 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.

[0099] Example 1: Screening and Preparation of Peptides This embodiment abandons the traditional approach of phage random peptide library screening and site-directed mutagenesis of natural chemokines in existing technologies. Instead, it adopts a targeted screening strategy of "natural product enzymatic digestion-mass spectrometry enrichment-target docking," eliminating the need to construct artificial peptide libraries or modify natural protein sequences. The peptide preparation method in this application simplifies the complex preparation process of double complementary site peptides and long-chain extracellular domain blocking peptides using solid-phase synthesis + HPLC purification, solving the problems of low yield (<30%) and poor uniformity in existing technologies, ensuring stable peptide quality and scalable production.

[0100] 1. Screening of peptides: The core component of Compound Donkey-hide Gelatin Oral Liquid, donkey-hide gelatin, was hydrolyzed with collagenase to produce polypeptide fragments of different molecular weights. Mass spectrometry analysis was used to screen the top 30 polypeptides by enrichment. Bioinformatics target docking technology was employed to simulate the interaction between each polypeptide and the chemokine receptor protein CXCR2, calculating binding free energy and other binding strength correlation coefficients. Potential high-affinity binding polypeptides were preliminarily screened: Peptide25 (amino acid sequence GQAGVMGFPGPK (SEQ ID NO:1)) containing 12 amino acid residues with a molecular weight of 1143.29 Da, and Peptide3 (amino acid sequence GVVGPQGAR (SEQ ID NO:2)) containing 9 amino acid residues with a molecular weight of 814.92 Da. The bioinformatics prediction results for Peptide25 and Peptide3 with CXCR2 are shown in [link to relevant documentation]. Figure 2 A shows the potential binding sites, intermolecular interaction patterns, and key amino acid residue docking of Peptide25 and Peptide3 with the CXCR2 protein.

[0101] 2. Preparation of polypeptides: The target peptides (Peptide25 and Peptide3) were synthesized by GenScript, and their purity was verified to be ≥95% by high performance liquid chromatography (HPLC). Their solubility stability in the experimental system was confirmed by solubility experiments, which met the requirements of subsequent experiments.

[0102] Example 2: CXCR2 Binding Activity Verification Experiment 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 The BLI precisely quantifies the binding specificity and stability (KD value), providing complementary dual data verification to address the issues of insufficient binding activity verification and single parameters in existing technologies, ensuring the specific binding ability of the peptide to CXCR2.

[0103] 1. ELISA detection method: Recombinant CXCR2 protein (purchased from MCE, HY-P700536) 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 seven concentration gradients of Peptide 25 and Peptide 3 were set up (see Table 1). Each group was used in duplicate, and the Peptide 25 was added to the plates 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 in each well using a microplate reader. 450 The binding affinity of the peptide to CXCR2 protein was assessed based on changes in OD values. For ELISA results of Peptide25 and Peptide3, please refer to [link to ELISA results]. Figure 2B. The curve trend shows that the binding activity of the two peptides increases significantly with increasing concentration, while the blank control group (concentration 0 μg / mL) shows no obvious binding signal.

[0104] Table 1: Peptide concentration gradient design

[0105] 2. BLI detection method: Biomembrane interference (BLI) technology was used to further verify the binding activity of the peptide to CXCR2. The experiment was performed at 25°C using a GatorPrime label-free biomolecular interaction analyzer with SA XT probes. The specific operation is as follows: First, 1 μg / ml of Peptide25 or Peptide3 was biotinylated (Peptide25 was labeled with a biotin tag at the N-terminus, and Peptide3 was labeled with a biotin tag at the C-terminus) and immobilized on the surface of an SA XT probe for 2 min. Then, the probe was washed with 0.02% PBST buffer for 1 min. Next, the probe was immersed in reaction wells containing different concentrations of recombinant CXCR2 protein for 5 min of binding reaction, followed by a 5 min buffer dissociation step. The binding rate constant K was obtained by real-time monitoring of changes in the biomembrane interference signal and analysis of the data using the standard Global 1:1 binding model with GraphPad PRISM 10 software. on Dissociation rate constant K off Equilibrium dissociation constant K D Kinetic parameters directly reflect the specific binding ability and affinity of the peptides to the CXCR2 protein. The kinetic maps of supplemental biomembrane interference (BLI) detection for Peptide25 and Peptide3 are shown in [reference needed]. Figure 2 C, the curve reflects the real-time binding-dissociation process of the two peptides with CXCR2 protein, and the binding rate constant (K). 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.

[0106] 3. Results Analysis: Both ELISA and BLI assays confirmed that Peptide 25 and Peptide 3 specifically bind to CXCR2 protein, exhibiting strong binding affinity, and the binding activity was concentration-dependent. The affinity K for Peptide 25 to CXCR2 is [not specified in the original text]. D The value is 2.26 μM, and the binding constant (K) on The value is 6.18 × 10 3 M-1 s -1 dissociation constant (K) off The value is 1.39 × 10 -2 s -1 The affinity K for Peptide 3 to CXCR2 D The value is 335 nM, combined with the constant (K) on The value is 3.90 × 10 4 M -1 s -1 dissociation constant (K) off The value is 1.31 × 10 -2 s -1 .

[0107] Example 3: Verification Experiment of Cell Migration Inhibition Function This embodiment specifically selects macrophages (core effector cells in inflammatory infiltration and the tumor microenvironment) and uses a standardized process of "induction of differentiation - group intervention - quantitative counting" to directly verify the correlation between binding activity and biological function. This allows for precise targeting of the core pathological process of CXCR2-mediated macrophage chemotaxis and migration, directly confirming the functional activity of the peptide, solving the problem of "disconnect between binding activity and physiological function" in existing technologies, and providing direct cellular-level evidence for the clinical application of the peptide.

[0108] The effect of peptides on macrophage migration was detected using the Transwell assay. The specific steps are as follows: Human acute monocytic leukemia THP-1 cells were induced to differentiate into macrophages for 24 h with phorbol-12-myristate-13-acetate (PMA). In the experimental group, the upper layer of the Transwell chamber was supplemented with fresh medium containing a working concentration of 25 ng / mL PMA and a final concentration of 400 μM Peptide 25 or Peptide 3, while the control group received an equal volume of serum-free DMEM. The lower chamber contained 700 μL of fresh medium containing a working concentration of 25 ng / mL PMA as a chemotactic signal source. After incubation at 37°C and 5% CO2 for 24 h, the chambers were washed with PBS buffer, cells were fixed with 4% paraformaldehyde solution for 15 min, washed with PBS, stained with 0.1% crystal violet for 15 min, washed again to remove unbound dye, and then the unmigrated cells inside the upper chamber membrane were gently wiped away with a sterile cotton swab. Migrating cells on the surface of the lower membrane layer of the chamber were observed and photographed under a 10× optical microscope. The number of migrating cells was quantitatively analyzed using ImageJ software, and the migration inhibition rate between the experimental group and the control group was statistically analyzed. (See results below.) Figure 3 ,in Figure 3A: The left image shows the morphology of uninduced THP-1 cells (suspension growth), and the right image shows the morphological characteristics of macrophages after 24 h of PMA (25 ng / mL) induction (adherent growth, cell enlargement, pseudopodia extension). Figure 3 B: The left side shows representative microscopic images (10×) of cell migration in the control group, Peptide25 group, and Peptide3 group. The right side shows the histogram of the number of migrating cells analyzed by ImageJ. The data are expressed as "mean ± standard deviation".

[0109] Figure 3 A. Macrophage induction was confirmed to be successful. Figure 3 B. Statistical analysis showed a significant difference between the experimental group and the control group. P The result (<0.05) indicates that both peptides significantly inhibit macrophage migration. The experimental results show that, compared with the control group, the number of migrating macrophages in the Peptide25 and Peptide3 treatment groups was significantly reduced, confirming that both peptides can effectively inhibit CXCR2-mediated macrophage chemotactic migration, demonstrating clear biological activity.

[0110] 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.

[0111] 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 an amino acid sequence as shown in either SEQ ID NO:1 or SEQ ID NO:

2.

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, It further includes at least one of a bioactive protein or a fragment thereof, a bioactive polypeptide or a fragment thereof.

4. The recombinant protein according to claim 3, characterized in that, The bioactive protein or its fragment is selected from at least one of protein tags, reporter proteins, protein toxins or their fragments, tumor necrosis factor or its fragments, interferons or their fragments, biological response regulators or their fragments, and Fc fragments.

5. 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 4.

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

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

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

9. Use of the polypeptide of claim 1, the recombinant protein of any one of claims 2 to 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the recombinant cell of claim 7, or the pharmaceutical composition of claim 8 in the preparation of a medicament for the prevention and / or treatment of CXCR2-related diseases; Optionally, the CXCR2-related diseases include inflammatory diseases or cancer.

10. Use of the polypeptide of claim 1, the recombinant protein of any one of claims 2 to 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the recombinant cell of claim 7, or the pharmaceutical composition of claim 8 in the preparation of the CXCR2 targeting peptide.

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