Polypeptides targeting the epidermal growth factor receptor

Abstract

This invention relates to the field of biotechnology, specifically to polypeptides targeting the epidermal growth factor receptor (EGFR). The amino acid sequence of the polypeptide comprises any one of the following (i) to (iii): (i) a sequence as shown in any one of SEQ ID NO. 1 to 4; (ii) a sequence in which one or more amino acids are substituted, deleted, or added to a sequence as shown in any one of SEQ ID NO. 1 to 4, and which has EGFR-targeting activity; (iii) a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with a sequence as shown in any one of SEQ ID NO. 1 to 4, and which has EGFR-targeting activity. The polypeptide has a relatively small molecular weight, is stable, and exhibits a strong affinity for EGFR, greatly aiding in the development of drugs targeting EGFR.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202410754232.X, filed on June 11, 2024, entitled "Polypeptide Targeting Epidermal Growth Factor Receptor". Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically to polypeptides that target the epidermal growth factor receptor. Background Technology

[0003] The human epidermal growth factor receptor (HER) family includes four members: HER1 (ErbB1, EGFR), HER2 (ErbB2, NEU), HER3 (ErbB3), and HER4 (ErbB4). These family members play important regulatory roles in cellular physiological processes. Among them, the epidermal growth factor receptor (EGFR) is the receptor for epidermal growth factor (EGF)-induced cell proliferation and signal transduction, and belongs to the tyrosine kinase type receptor. EGFR is a transmembrane glycoprotein composed of three parts: an extracellular domain (N-terminus), a transmembrane domain (a hydrophobic α-helix structure), and an intracellular domain (C-terminus), with a molecular weight of approximately 170 kDa.

[0004] EGFR is activated by binding to ligands, including EGF and transforming growth factor α (TGFα). The ligands bind to the extracellular domains of EGFR, forming homodimers or heterodimers (e.g., from monomers to dimers, or by polymerizing with other members of the HER family such as ErbB2 / HER2 / NEU). Following EGFR dimerization, receptor autophosphorylation occurs at sites including Y992, Y1045, Y1068, Y1148, and Y1173. Autophosphorylation at these sites can guide downstream phosphorylation and activate intracellular kinase pathways, including the MAPK, Akt, and JNK pathways, inducing cell proliferation.

[0005] EGFR is expressed on the surface of normal epithelial cells. Studies have shown that EGFR is highly or abnormally expressed in many solid tumors, and EGFR overexpression is associated with tumor cell proliferation, metastasis, invasion, and poor prognosis. Therefore, developing drugs targeting EGFR is of great significance for cancer treatment. Currently, the most widely used EGFR-targeting peptide on the market is GE11, and research has shown that it can serve as an effective active targeted agent in cancer treatment.

[0006] Furthermore, compared to proteins with larger molecular weights, peptides, with their smaller molecular size, offer advantages such as ease of synthesis, modification, and optimization, high product purity, stable properties, and specific biological activities, making them a hot research topic in the biopharmaceutical field. Currently, peptide-based drugs include peptide vaccines, anti-tumor peptides, peptide-guided drugs (or peptide-targeted drugs), cytokine mimic peptides, antibacterial active peptides, and diagnostic peptides. Peptide-guided drugs, in particular, fuse peptides with target-binding capabilities with cytotoxins or cytokines, directing them to the lesion site to exert a therapeutic effect while reducing toxic side effects. The development of peptide-guided drugs is currently a major research focus.

[0007] Currently, methods for obtaining peptides include searching for naturally occurring unknown peptides in nature or screening from artificially constructed peptide libraries. Screening methods include, for example, phage display technology. Phage display technology involves inserting a foreign gene encoding a peptide or protein into an appropriate position in the structural gene of the phage capsid protein using genetic engineering techniques. This causes the foreign peptide or protein to form a fusion protein on the phage capsid protein, which is then presented on the phage surface during the reassembly of progeny phages, thus forming a phage display library. Phage display technology can maintain the relative spatial structure and biological activity of the foreign peptide or protein. Then, using a target molecule and appropriate washing methods, non-specifically bound phages can be washed away. Phages bound to the target molecule can then be eluted using acids, bases, or competing molecules. The neutralized phages are then used to infect *E. coli* for amplification. After 3-5 rounds of screening and enrichment, peptides or proteins that recognize the target molecule can finally be obtained. Phage display technology is an operating system for expressing exogenous genes. It has the advantages of being simple, effective, and easy to control, and can achieve the unification of phenotype and genotype, and has broad application prospects. Summary of the Invention

[0008] The problem the invention aims to solve

[0009] EGFR overexpression is closely related to tumor occurrence, development, and prognosis. In response to the market demand for developing peptide-guided drugs that target EGFR, this application provides a peptide with high affinity for EGFR, which can help in the development of EGFR-targeted drugs.

[0010] Solution for solving the problem

[0011] [1] A polypeptide, characterized in that the amino acid sequence of the polypeptide comprises any one of the sequences shown in (i) to (iii) below:

[0012] (i) A sequence as shown in any one of SEQ ID NO. 1 to 4;

[0013] (ii) A sequence having one or more amino acids substituted, deleted or added in any of the sequences shown in SEQ ID NO. 1 to 4 and having activity targeting the epidermal growth factor receptor;

[0014] (iii) A sequence having at least 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any of the sequences shown in SEQ ID NO. 1 to 4 and having activity targeting the epidermal growth factor receptor.

[0015] [2] The polypeptide according to [1] is characterized in that the amino acid sequence of the polypeptide comprises any one of SEQ ID NO. 1 to 4.

[0016] [3] The polypeptide according to [1] or [2] is characterized in that the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO. 1 to 4.

[0017] [4] A polynucleotide, characterized in that the polynucleotide encodes a polypeptide according to any one of [1] to [3].

[0018] [5] A recombinant expression vector, characterized in that the recombinant expression vector comprises the polynucleotide according to [4];

[0019] Preferably, the recombinant expression vector includes a plasmid vector and a viral vector;

[0020] More preferably, the plasmid vector includes pET series vectors, and / or, more preferably, the viral vector includes bacteriophages.

[0021] [6] A recombinant host cell, characterized in that the recombinant host cell comprises the recombinant expression vector according to [5];

[0022] Preferably, the recombinant host cells include eukaryotic cells and prokaryotic cells;

[0023] More preferably, the eukaryotic cells include yeast, and / or, more preferably, the prokaryotic cells include Escherichia coli.

[0024] [7] A method for preparing a polypeptide according to any one of [1] to [3], characterized in that the method includes the step of culturing a recombinant host cell according to [6] and expressing the polypeptide.

[0025] [8] A drug, characterized in that the drug comprises a polypeptide according to any one of [1] to [3];

[0026] Preferably, the drug targets the epidermal growth factor receptor;

[0027] More preferably, the drug targeting the epidermal growth factor receptor is used for the prevention and / or treatment of tumors;

[0028] Even more preferably, the tumor includes at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer, and prostate cancer; more preferably, the tumor is non-small cell lung cancer.

[0029] [9] A detection or diagnostic kit containing any one of the polypeptides described in [1] to [3].

[0030]

[10] Use of the polypeptide according to any one of [1] to [3] in the preparation of a drug;

[0031] Preferably, the drug targets the epidermal growth factor receptor;

[0032] More preferably, the drug targeting the epidermal growth factor receptor is used for the prevention and / or treatment of tumors;

[0033] Even more preferably, the tumor includes at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer, and prostate cancer; more preferably, the tumor is non-small cell lung cancer.

[0034] The effects of the invention

[0035] This invention provides a polypeptide that targets the epidermal growth factor receptor, which has a relatively small molecular weight, stable properties, and a strong affinity for EGFR, thus greatly assisting in the development of drugs targeting EGFR. Detailed Implementation

[0036] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0037] The first aspect of this invention provides a polypeptide that can target the epidermal growth factor receptor. Specifically, this invention utilizes phage display technology to screen a polypeptide with extremely strong affinity for EGFR from an artificially constructed phage library (Ph.D.™-12 Phage Display Peptide Library Kit v2, NEB#E8210-1) through multiple rounds of screening.

[0038] In this invention, the terms "polypeptide," "protein," and "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone. Furthermore, the polypeptides described in this invention can be linear or cyclic.

[0039] In some embodiments, the amino acid sequence of the polypeptide provided by the present invention comprises any one of the sequences shown in (i) to (iii) below:

[0040] (i) A sequence as shown in any one of SEQ ID NO. 1 to 4;

[0041] (ii) A sequence having one or more amino acids substituted, deleted or added in any of the sequences shown in SEQ ID NO. 1 to 4 and having activity targeting the epidermal growth factor receptor;

[0042] (iii) A sequence having at least 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any of the sequences shown in SEQ ID NO. 1 to 4 and having activity targeting the epidermal growth factor receptor.

[0043] In this invention, the term "amino acid" can include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers. The amino acids and their abbreviations and English abbreviations in this invention are as follows: Histidine (His, H); Serine (S); Glutamine (Gln, Q); Glycine (Gly, G); Threonine (Thr, T); Phenylalanine (Phe, F); Aspartic acid (Asp, D); Tyrosine (Tyr, Y); Isoleucine (Ile, I); Arginine (Arg, R); Alanine (Ala, A); Glutamine (Gln, Q); Valine (V); Tryptophan (Trp, W); Leucine (Leu, L); Methionine (Met, M); Asparagine (Asn, N); Cysteine ​​(Cys, C); Lysine (Lys, K); Proline (Pro, P).

[0044] In this invention, "addition" of an amino acid refers to adding an amino acid at any position, such as the C-terminus, N-terminus, or a position between the C-terminus and N-terminus, of an amino acid sequence. The added amino acids may be all or partially adjacent to each other, or none of them may be adjacent to each other. "Deletion" of an amino acid refers to the deletion of one, two, or three or more amino acids from an amino acid sequence. "Replacement" of an amino acid refers to the substitution of an amino acid at a certain position in an amino acid sequence by another amino acid, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence. Amino acid substitution can be a conserved amino acid substitution, where several amino acids are replaced by amino acids with similar or related properties to form a peptide (conserved variant peptide). Amino acid substitution can also be a non-conservative amino acid substitution.

[0045] In this invention, "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0046] In some specific embodiments, the amino acid sequence of the polypeptide provided by the present invention comprises the sequence shown in any one of SEQ ID NO. 1 to 4. In some specific embodiments, the amino acid sequence of the polypeptide provided by the present invention comprises the sequence shown in any one of SEQ ID NO. 1 to 3.

[0047] In some more specific embodiments, the amino acid sequence of the polypeptide provided by the present invention comprises the sequence shown in SEQ ID NO. 1. In some more specific embodiments, the amino acid sequence of the polypeptide provided by the present invention comprises the sequence shown in SEQ ID NO. 2. In some more specific embodiments, the amino acid sequence of the polypeptide provided by the present invention comprises the sequence shown in SEQ ID NO. 3. In some more specific embodiments, the amino acid sequence of the polypeptide provided by the present invention comprises the sequence shown in SEQ ID NO. 4.

[0048] In some preferred embodiments, the amino acid sequence of the polypeptide provided by the present invention is as shown in any one of SEQ ID NO. 1 to 4. In some specific embodiments, the amino acid sequence of the polypeptide provided by the present invention is as shown in any one of SEQ ID NO. 1 to 3.

[0049] In some preferred embodiments, the amino acid sequence of the polypeptide provided by the present invention is as shown in SEQ ID NO.1. In some preferred embodiments, the amino acid sequence of the polypeptide provided by the present invention is as shown in SEQ ID NO.2. In some preferred embodiments, the amino acid sequence of the polypeptide provided by the present invention is as shown in SEQ ID NO.3. In some preferred embodiments, the amino acid sequence of the polypeptide provided by the present invention is as shown in SEQ ID NO.4.

[0050] The peptide targeting the epidermal growth factor receptor provided by this invention has a small molecular weight, consisting of only 12 amino acid residues, exhibiting stable properties and good EGFR affinity and targeting. In some embodiments, the peptide can form a cyclic peptide by forming a disulfide bond through the thiol group in the cysteine ​​residues within the molecule.

[0051] A second aspect of the present invention provides a polynucleotide capable of encoding the polypeptide described in the first aspect of the present invention.

[0052] In this invention, the terms "polynucleotide," "nucleic acid molecule," "polynucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or similar compounds. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Furthermore, the nucleotides described in this invention can be linear or circular. The polynucleotides of this invention can be in DNA or RNA form. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0053] The polynucleotide encoding the polypeptide of the present invention includes: a coding sequence that encodes only the polypeptide; a coding sequence of the polypeptide and various additional coding sequences; a coding sequence of the polypeptide (and optional additional coding sequences) and a non-coding sequence. Wherein, "polynucleotide encoding the polypeptide" may include a polynucleotide encoding the polypeptide, or it may include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0054] A third aspect of the present invention provides a recombinant expression vector comprising the polynucleotides described in the second aspect of the present invention.

[0055] In this invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, Cosmids, etc. Furthermore, the term "recombinant expression vector" refers to an expression vector into which a foreign gene has been introduced.

[0056] In some embodiments, the recombinant expression vector provided by the present invention further includes a suitable promoter or control sequence, which can be used to transform a suitable host cell so that it can express a protein.

[0057] In some embodiments, the recombinant expression vector of the present invention includes a plasmid vector or a viral vector; for example, the plasmid vector includes pET series vectors, and the viral vector includes bacteriophages.

[0058] A fourth aspect of the present invention provides a recombinant host cell which is introduced with or contains the recombinant expression vector described in the third aspect of the present invention.

[0059] In this invention, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; Bacillus family members such as *Bacillus subtilis*; *Streptococcus pneumoniae*; *Streptococcus*; and *Haemophilus influenzae*. Suitable microorganisms include *Saccharomyces cerevisiae* and *Pichia pastoris*. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells. Further, the term "recombinant host cell" refers to a host cell that differs from the parent cell after the introduction of exogenous polynucleotides or recombinant expression vectors; specifically, recombinant host cells can be obtained through transformation.

[0060] In some embodiments, the host cell described in this invention includes eukaryotic cells or prokaryotic cells; for example, the eukaryotic cell includes yeast, and the prokaryotic cell includes Escherichia coli.

[0061] A fifth aspect of the present invention provides a method for preparing the polypeptide described in the first aspect of the present invention, comprising the steps of culturing the recombinant host cells described in the fourth aspect of the present invention and expressing the polypeptide. The preparation method of the present invention may further include the step of isolating and purifying the polypeptide from the culture.

[0062] A sixth aspect of the present invention provides a medicine comprising the polypeptide described in the first aspect of the present invention.

[0063] In some implementations, the drug is an EGFR-targeting drug; for example, the drug is a peptide-guided drug.

[0064] In some implementations, the EGFR-targeting drug is used for the prevention and / or treatment of tumors.

[0065] In this invention, "treatment" means that, prior to the onset of a disease, the subject is exposed to (e.g., administered) a drug containing the polypeptide of this invention, thereby delaying or reducing the progression of the disease or condition, including alleviating, relieving or reducing one or more symptoms of the condition or symptom, but does not mean that the symptoms of the disease must be completely suppressed.

[0066] In this invention, "prevention" means that before a subject develops a disease, by exposing the subject to (e.g., administering medication) a drug containing the polypeptide of this invention, the symptoms of the disease are reduced compared to when the subject is not exposed, and does not mean that the disease must be completely suppressed.

[0067] In some implementations, the tumor includes at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer, and prostate cancer.

[0068] In some preferred embodiments, the tumor is non-small cell lung cancer.

[0069] A seventh aspect of the present invention provides a detection or diagnostic kit comprising the polypeptide described in the first aspect of the present invention.

[0070] The kit provided by this invention, by utilizing the EGFR targeting properties of the peptide, can be used to detect EGFR or to diagnose diseases related to high EGFR expression or to detect lesions.

[0071] The eighth aspect of the present invention provides a method for detecting EGFR, the method comprising contacting the polypeptide described in the first aspect of the present invention with a test sample and detecting the binding between the two, thereby determining whether EGFR is present in the test sample.

[0072] In some embodiments, the method for detecting EGFR is for non-diagnostic or non-therapeutic purposes. In other embodiments, the method for detecting EGFR is for diagnostic or therapeutic purposes.

[0073] The ninth aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention in the preparation of pharmaceuticals, particularly pharmaceuticals targeting EGFR.

[0074] In some implementations, the EGFR-targeting drug is used for the prevention and / or treatment of tumors.

[0075] In some implementations, the tumor includes at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer, and prostate cancer.

[0076] In some preferred embodiments, the tumor is non-small cell lung cancer.

[0077] The tenth aspect of the present invention provides a treatment method for tumors, comprising the step of administering an effective amount of a drug to an individual in need, said drug comprising the polypeptide described in the first aspect of the present invention.

[0078] In this invention, "application" means administration by a nurse, healthcare provider, patient, or any other individual, including self-application. The application includes not only delivery into the body but also prescription, distribution, or any other means of facilitating delivery.

[0079] In this invention, "individual," "patient," or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0080] In some implementations, the drug is a drug that targets EGFR.

[0081] In some implementations, the tumor includes at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer, and prostate cancer.

[0082] In some preferred embodiments, the tumor is non-small cell lung cancer.

[0083] Example

[0084] The present invention is further illustrated below by way of examples, but is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or function as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available.

[0085] Example 1: Screening for EGFR-binding peptides using phage display technology

[0086] Take 10 μL of phage library (12 peptide library (2 × 10) 13pfu / mL, Ph.D.™-12 Phage DisplayPeptide Library Kit v2, NEB#E8210-1)) (2×10 11 PFU was added to 790 μL of 2% Milk-PBS (PBS with 2% milk added) for blocking, and incubated at room temperature for 1 h. 100 μL of streptavidin magnetic beads (Dynabeads™ M-280, Invitrogen, catalog number 11206D) was washed three times with PBS (1 mL / wash), then blocked with 2% Milk-PBS and incubated at room temperature for 2 h. Biotin-EGFR (Yiqiao 10001-H27H-B) was added to the blocked phage-milk mixture and incubated at room temperature for 1.5 h. The 2% Milk-PBS used for blocking was removed from the magnetic beads, and the beads were resuspended in 200 μL of 0.1% PBST-2% Milk (i.e., 0.1% PBST, 2% milk). The resuspended magnetic beads were added to the incubated phage-antigen mixture and incubated at room temperature for 30 min. Place the above mixture into a magnetic rack. After the magnetic beads adhere to the tube wall, remove the liquid from the tube and then wash the magnetic beads 10 times with 0.1% PBST. Allow the magnetic beads to adhere to the tube wall and remove the PBST from the tube. Add 1 mL of elution buffer (0.2 M Gly-HCl, 1 mg / mL BSA, pH 2.2), and incubate at room temperature for 30 min. Immediately add 150 μL of neutralization buffer (1 M Tris-HCl, pH 9.1, filtered sterile) to neutralize. Reserve 5–10 μL of the elution buffer for titration, and immediately amplify the remaining elution buffer.

[0087] Inoculate *E. coli* K12 ER2738 (ER2738 (NEB #E4104S)) into 20 mL LB medium and shake at 37°C and 250 rpm for 1 hour. Add phage elution buffer and continue shaking at 37°C and 250 rpm for 4.5–5 hours. Centrifuge at 5000g for 10 minutes at 4°C. Resuspend the supernatant in PEG / NaCl (20% (w / v) PEG-8000, 2.5M NaCl), mix gently, and allow the phage to precipitate at 4°C for 2 hours or overnight. Centrifuge, discard the supernatant, resuspend in PBS, centrifuge again, transfer the supernatant to a fresh microcentrifuge tube, add 200 μL of PEG / NaCl (the solution will quickly become turbid), and incubate on ice for 0.5–1 hour. Centrifuge, discard the supernatant, and resuspend the precipitate in 0.5 mL of LB medium. In PBS, centrifuge for 1 minute to precipitate any remaining insoluble matter. Transfer the supernatant to a fresh tube. This is the eluent after amplification.

[0088] The amplified eluent was titrated using LB / IPTG / Xgal plates (LB medium + 15 g / L agar powder. Autoclaved, cooled to below 70°C, then 1 ml IPTG / Xgal was added, mixed, and poured onto plates) according to the titration method. The titer was determined by the number of blue spots on the counting plate.

[0089] Repeat the above steps for the second, third, and fourth rounds of panning. After four rounds of panning, do not amplify the eluent; instead, titrate the eluent to obtain single plaques for phage sequencing reactions. Measure the titer of the unamplified eluent obtained in the fourth round of panning on LB / IPTG / Xgal plates. The phage plaques obtained during titer determination can be used for sequencing.

[0090] Example 2: Determination of phage titer

[0091] Inoculate a single ER2738 clone into 5–10 mL of LB medium and incubate with shaking until mid-logarithmic growth (OD600 = 0.5). During cell growth, microwave to melt the top agarose gel and aliquot into sterile culture tubes, 3 mL per tube. Maintain at 45°C. Preheat LB / IPTG / Xgal culture plates to 37°C. Serially dilute the phage with LB medium 10-fold. Once the ER2738 culture reaches mid-logarithmic growth, aliquot into microcentrifuge tubes, 200 μL per tube. Add the serially diluted phage supernatant to the microcentrifuge tube containing the bacterial culture, adding only 10 μL of one dilution per microcentrifuge tube, vortex rapidly, and incubate at room temperature for 1–5 min. Transfer to tubes containing a 45°C top agarose gel, vortex rapidly to mix, and immediately pour onto the preheated LB / IPTG / Xgal plate, gently shaking to distribute the top gel evenly. Cool the plates for 5 minutes and incubate them upside down at 37°C overnight. Plaque counts are based on plates with approximately 100 plaques. Multiplying the plaque count by the dilution yields the titer-plaque-forming units (pfu) per 10 μL of phage.

[0092] Example 3: Amplification of Plaques

[0093] Overnight ER2738 culture was diluted 1:100 and inoculated onto LB medium, 200 μl / well in a 96-well bacterial culture plate. Using a sterile toothpick or pipette tip, blue plaques were picked and transferred to the 96-well plate, one plaque per well. Note: Picking should be done from plates with fewer than 100 plaques to ensure each plaque contains only one DNA sequence. Incubate at 37°C with shaking for 4.5–5 hours, then store at 4°C for later use.

[0094] Example 4: Detection of the binding of selected peptides to target molecules using ELISA

[0095] Antigen coating with 0.1M NaHCO3 (pH 8.6): Coat 100µL of the target molecule Recombinant Human EGFR Protein (ECD, His Tag) (Sino, Cat: 10001-H08H) at a concentration of 10µg / mL into an ELISA plate, 100µl / well, and incubate overnight at 4°C. Phage treatment: Centrifuge the phage-packaged plates at 4°C and 3000g for 10 min. Transfer 100 μl of supernatant to a new 96-well plate (pre-added with 100 μl / well of 2% milk-PBS) and incubate for 1 h. Wash the antigen-coated 96-well ELISA plate three times with 1×PBS / 1‰ Tween (prepared with Biosharp PBS buffer (ready-to-use dry powder) to a 10 mM concentration, pH 7.2–7.4, catalog number BL601A; Tween20, Sigma, catalog number P9416-100ml). Block with 2% milk-PBS for 30 min, 350 μl / well. Wash three times with PBST. Add the blocked phage to the ELISA plate and incubate at room temperature for 1 h. Wash three times with PBST. Dilute the HRP-labeled anti-M13 antibody (Sino, 11973-MM05T-H) at a ratio of 1:2000. Add 100 µl of diluted antibody to each well and incubate at room temperature with shaking for 1 h. Wash 6 times with PBST; for color development: add 100 μl of TMB chromogenic solution and develop for 2 min. For termination: add 100 μl / well of TMB stop solution to stop the reaction. For reading: measure OD at 450 nm using a microplate reader. Select positive monoclonal cultures for sequencing analysis and peptide synthesis.

[0096] Positive monoclonal bacterial cultures were selected for sequencing analysis and peptide synthesis.

[0097] Example 5: Determination of peptide affinity

[0098] The binding affinity between peptides and EGFR antigens was obtained by surface plasmon resonance (SPR) analysis using a surface plasmon resonance spectrometer.

[0099] Antigen fixation: Recombinant human EGFR protein (ECD, His Tag), Sino, Cat: 10001-H08H was diluted to 25 μg / mL with 10 mM sodium acetate solution (pH 4.0) and coupled to a dextran surface chip (manufacturer Reichert, catalog number 13206066) to achieve a final antigen binding amount of 1500 μRIU.

[0100] Sample preparation: Dilute the prepared peptide ligand stock solution (5mM) 100-fold with PBST (0.05% Tween 20) + 1% DMSO to an initial concentration of 50 μM. Then perform serial dilutions of 2-fold with PBST (0.05% Tween 20) + 1% DMSO, resulting in seven concentration gradients: 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 μM. Insert a PBST (0.05% Tween 20) + 1% DMSO sample as a blank control every three samples.

[0101] Procedure: Inject the test samples with the following parameters: binding time 3 min, dissociation time 5 min, regeneration reagent (10 mM Gly-HCl pH 3.0) binding time 30 s, regeneration reagent dissociation time 1 min, loop repetition number set according to sample quantity, flow rate 25 μL / min, run buffer PBST (0.05% Tween 20) + 1% DMSO. All experiments were performed at room temperature (25°C), and samples were kept at 4°C before flowing through the chip. Steady-state fit was determined using Langmuir 1:1 in conjunction with TraceDrawer software (Reichert Technologies).

[0102] Table 1 shows the binding affinity of the EGFR polypeptide ligands screened by phage display in this invention and the known GE11 polypeptide (SEQ ID NO. 5) to human EGFR protein. The experimental results show that the EGFR polypeptide ligands obtained by this invention all have considerable affinity, with dissociation equilibrium constants (KD) around 2.80 × 10⁻⁶. -5 ~1.69×10 -4 The M-M pair can meet the requirements for developing coupled ligands.

[0103] The affinity test results are shown in Table 1 below:

[0104] Table 1. Affinity test results between peptides and target molecules

[0105]

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide includes any one of the sequences shown in (i) to (iii) below: (i) A sequence as shown in SEQ ID NO.1; (ii) A sequence having one or more amino acids substituted, deleted, or added in the sequence shown in SEQ ID NO.1 and having activity targeting the epidermal growth factor receptor; (iii) A sequence having at least 85%, 90%, 95%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO.1 and having activity targeting the epidermal growth factor receptor.

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide includes the sequence shown in SEQ ID NO.

1.

3. The polypeptide according to claim 1 or 2, characterized in that, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.

1.

4. A polynucleotide, characterized in that, The polynucleotide encodes a polypeptide according to any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide according to claim 4; Preferably, the recombinant expression vector includes a plasmid vector and a viral vector; More preferably, the plasmid vector includes pET series vectors, and / or, more preferably, the viral vector includes bacteriophages.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the recombinant expression vector according to claim 5; Preferably, the recombinant host cells include eukaryotic cells and prokaryotic cells; More preferably, the eukaryotic cells include yeast, and / or, more preferably, the prokaryotic cells include Escherichia coli.

7. The method for preparing the polypeptide according to any one of claims 1 to 3, characterized in that, The method includes the steps of culturing the recombinant host cells according to claim 6 and expressing the polypeptide.

8. A drug, characterized in that, The drug comprises a polypeptide according to any one of claims 1 to 3; Preferably, the drug targets the epidermal growth factor receptor; More preferably, the drug targeting the epidermal growth factor receptor is used for the prevention and / or treatment of tumors; Even more preferably, the tumor includes at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer, and prostate cancer; more preferably, the tumor is non-small cell lung cancer.

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