Peptides specific to transferrin receptor 1

Abstract

This invention relates to a polypeptide specific to transferrin receptor 1 (TfR1), specifically, the polypeptide comprising the structure shown below: X1-A1-A2-A3-A4-A5-X2, where A3 is Chg; X1 and X2 are each independently an absent, signal, or transmembrane peptide; A1, A2, A4, and A5 are each independently selected from the group consisting of Chg, Leu, Trp, Ile, Nle, Phe(Br), or Tic; wherein two or more of A1, A2, A4, and A5 are Chg. The polypeptide of this invention exhibits strong binding affinity to transferrin receptor 1, with a KD ≤ 400 nM. The polypeptide of this invention can penetrate cells overexpressing TfR1, possessing the potential for drug delivery.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology or biomedical technology, and more specifically to a polypeptide specific to transferrin receptor 1. Background Technology

[0002] Transferrin receptor 1 (TfR1) is a transmembrane protein widely expressed in almost all cell and tissue types in the human body, primarily responsible for intracellular iron uptake and transport. Due to its significant upregulation on the surface of cancer cells, TfR1 has become an important target in cancer treatment and diagnosis.

[0003] TfR1-specific peptides are small molecule peptides designed and synthesized using biotechnology that specifically bind to TfR1. These peptides possess high specificity and affinity, enabling them to precisely recognize and bind to TfR1, thereby regulating TfR1 function. This specific binding characteristic makes TfR1-specific peptides promising for broad applications in disease treatment, drug delivery, and molecular imaging. In disease treatment, TfR1-specific peptides can serve as carriers for targeted drugs, precisely delivering drugs to the lesion site, thereby improving therapeutic efficacy and reducing side effects. Furthermore, these peptides can also be used to regulate TfR1 function, influencing cellular iron uptake and transport, providing new strategies for treating iron metabolism-related diseases. In drug delivery, TfR1-specific peptides can bind to TfR1 on the cell membrane surface, promoting drug entry into the cell and improving drug bioavailability and therapeutic efficacy. In molecular imaging, TfR1-specific peptides can be used to label and detect TfR1 expression at lesion sites, providing strong support for early diagnosis and efficacy evaluation of diseases.

[0004] Therefore, there is an urgent need in this field for peptides with high specificity and high affinity for transferrin receptor 1. Summary of the Invention

[0005] The purpose of this invention is to provide a peptide with high specificity and high affinity for TfR1.

[0006] In a first aspect, the present invention provides a polypeptide specific to transferrin receptor 1, said polypeptide comprising a structure as shown in formula (I):

[0007] X1-A1-A2-A3-A4-A5-X2(I)

[0008] In the formula, A3 represents Chg;

[0009] X1 and X2 are independently either non-signal peptides, signal peptides, or membrane-penetrating peptides;

[0010] A1, A2, A4, and A5 are each independently selected from the following groups: Chg, Leu, Trp, Ile, Nle, Phe(Br), or Tic;

[0011] Among them, A1, A2, A4 and A5 contain two or more Chg;

[0012] Chg represents cyclohexylglycine.

[0013] In another preferred embodiment, at least two cyclohexylglycine residues are adjacent in the polypeptide.

[0014] In another preferred embodiment, the cyclohexylglycine is of type D.

[0015] In another preferred embodiment, all amino acids in the polypeptide are D-type amino acids.

[0016] In another preferred embodiment, A1 is selected from the group consisting of: Chg, Leu, Trp, Nle, Tic, or Phe(Br);

[0017] A2 is selected from the following group: Chg, Leu, Nle, or Phe(Br);

[0018] A4 is selected from the following groups: Chg, Ile, or Phe(Br);

[0019] A5 is selected from the following groups: Chg, Ile, Nle, or Leu.

[0020] In another preferred embodiment, the polypeptide is selected from the group consisting of:

[0021] (a) Has an amino acid sequence selected from the following group:

[0022] (a1)Phe(Br)-Chg-Chg-Chg-Chg;

[0023] (a2)Nle-Chg-Chg-Chg-Chg;

[0024] (a3)Chg-Chg-Chg-Chg-Chg;

[0025] (a4)Trp-Chg-Chg-Chg-Chg;

[0026] (a5)Leu-Chg-Chg-Chg-Chg;

[0027] (a6)Trp-Chg-Chg-Phe(Br)-Chg;

[0028] (a7)Trp-Chg-Chg-Chg-Ile;

[0029] (a8)Chg-Chg-Chg-Chg-Ile;

[0030] (a9)Trp-Nle-Chg-Chg-Chg;

[0031] (a10)Trp-Chg-Chg-Chg-Nle;

[0032] (a11)Nle-Leu-Chg-Chg-Chg;

[0033] (a12)Trp-Chg-Chg-Chg-Leu;

[0034] (a13)Nle-Phe(Br)-Chg-Chg-Chg;

[0035] (a14)Tic-Chg-Chg-Chg-Chg;

[0036] (a15)Chg-Chg-Chg-Phe(Br)-Chg;

[0037] (a16)Tic-Phe(Br)-Chg-Chg-Chg;

[0038] (a17)Phe(Br)-Chg-Chg-Chg-Leu;

[0039] (a18)Nle-Nle-Chg-Chg-Chg;

[0040] (a19)Phe(Br)-Phe(Br)-Chg-Chg-Chg;

[0041] (a20)Trp-Chg-Chg-Ile-Chg;

[0042] (b) Derivative polypeptides formed by substituting one, two or three amino acids, and / or inserting one, two or three amino acids, and / or deleting one or two amino acids of any of the polypeptides shown in (a1) to (a20).

[0043] In another preferred embodiment, the peptide has a KD ≤ 450 nM for transferrin receptor 1, more preferably ≤ 300 nM, and even more preferably ≤ 200 nM.

[0044] In a second aspect, the present invention provides a peptide ligand specific to transferrin receptor 1, the peptide ligand comprising a polypeptide and a molecular scaffold as described in the first aspect of the present invention, wherein the molecular scaffold forms a covalent bond with a reactive group of the polypeptide.

[0045] In another preferred embodiment, the peptide ligand further includes cysteine ​​residues.

[0046] In another preferred embodiment, the peptide ligand is a linear peptide ligand or a cyclic peptide ligand.

[0047] In a third aspect, the present invention provides a peptide conjugate specific to transferrin receptor 1, the peptide conjugate comprising:

[0048] (a) the polypeptide as described in the first aspect of the invention; and

[0049] (b) A coupling portion conjugated to the polypeptide, the coupling portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0050] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0051] In another preferred embodiment, the structure of the peptide conjugate is shown in formula (II).

[0052] P1-P2-P3(II)

[0053] In the formula, P1 is the polypeptide described in the first aspect of the present invention;

[0054] P2 is AOP; P3 is Lys.

[0055] In another preferred embodiment, P3 is coupled with biotin via side chain modification.

[0056] In a fourth aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide as described in the first aspect of the present invention, or a complementary sequence thereof.

[0057] In a fifth aspect, the present invention provides a carrier containing nucleic acid molecules as described in the fourth aspect of the present invention.

[0058] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0059] In another preferred embodiment, the vector is a retroviral vector.

[0060] In a sixth aspect, the present invention provides a host cell containing a vector as described in the fifth aspect of the present invention or a genome thereof in which an exogenous nucleic acid molecule as described in the fourth aspect of the present invention is integrated.

[0061] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0062] In another preferred embodiment, the cell is a mammalian cell.

[0063] In another preferred embodiment, the cells are NK cells or T cells.

[0064] In another preferred embodiment, the host cell is an engineered immune cell.

[0065] In a seventh aspect, the present invention provides a delivery system comprising a polypeptide as described in the first aspect of the present invention, a peptide ligand as described in the second aspect of the present invention, a peptide conjugate as described in the third aspect of the present invention, or a combination thereof.

[0066] In another preferred embodiment, the delivery system is suitable for cells expressing transferrin receptor 1, preferably cells that highly express transferrin receptor 1, and more preferably HEK293T cells that highly express transferrin receptor 1.

[0067] In another preferred embodiment, the delivery system is suitable for drug delivery across the blood-brain barrier, capable of mediating therapeutic agents, diagnostic agents, or gene therapy vectors across the blood-brain barrier and into the central nervous system.

[0068] In another preferred embodiment, the delivery system further includes detectable markers, toxins, siRNA, ASO, inhibitors, isotopic elements, viral vectors, nanocarriers, and polymers.

[0069] In another preferred embodiment, the detectable marker includes fluorescent molecules.

[0070] An eighth aspect of the present invention provides a pharmaceutical composition comprising:

[0071] (a) a polypeptide as described in the first aspect of the invention, a peptide ligand as described in the second aspect of the invention, a peptide conjugate as described in the third aspect of the invention, or a combination thereof; and

[0072] (b) Pharmaceutically acceptable carriers, diluents or excipients.

[0073] In another preferred embodiment, the pharmaceutical composition is a formulation, preferably a liquid formulation.

[0074] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0075] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of a polypeptide as described in the first aspect of the invention, a peptide ligand as described in the second aspect of the invention, a peptide conjugate as described in the third aspect of the invention, a nucleic acid molecule as described in the fourth aspect of the invention, a carrier as described in the fifth aspect of the invention, a host cell as described in the sixth aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0076] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of TfR1-related diseases.

[0077] A ninth aspect of the present invention provides the use of the polypeptide as described in the first aspect of the present invention, the peptide ligand as described in the second aspect of the present invention, and the peptide conjugate as described in the third aspect of the present invention for the preparation of:

[0078] (1) The delivery system as described in the seventh aspect of the present invention;

[0079] (2) Medications for the prevention and / or treatment of TfR1-related diseases;

[0080] (3) Immunomodulatory agents; and / or

[0081] (4) TfR1 targeting agents.

[0082] In another preferred embodiment, the TfR1-related disease is a tumor that expresses TfR1.

[0083] In another preferred embodiment, the tumor includes solid tumors, hematologic malignancies, blood vessels, or combinations thereof.

[0084] In another preferred embodiment, the solid tumor includes: liver cancer, lung cancer, breast cancer, non-small cell lung cancer, colon cancer, gastric cancer, ovarian cancer, glioma, pancreatic cancer, lymphoma, leukemia, brain tumor, nasopharyngeal carcinoma, prostate cancer, or combinations thereof.

[0085] In another preferred embodiment, the delivery system targets tissues selected from the group consisting of the brain or muscles.

[0086] In another preferred embodiment, the delivery system targets rapidly proliferating cells, preferably epithelial cells and / or endothelial cells.

[0087] In another preferred embodiment, the TfR1-related diseases include: iron overload diseases, iron deficiency anemia, chronic anemia, rare iron metabolism diseases, and TfR1-related viral infections.

[0088] In another preferred embodiment, the iron overload disease includes hereditary hemochromatosis and secondary iron overload.

[0089] In another preferred embodiment, the chronic disease anemia includes anemia caused by diseases such as chronic inflammation, infection, or tumors.

[0090] In another preferred embodiment, the rare iron metabolism disorders include atransferrinemia and sideroblastic anemia.

[0091] In another preferred embodiment, the immunomodulator includes regulatory effects on T cell activation and proliferation, immune tolerance and inflammation, and macrophage polarization.

[0092] In another preferred embodiment, the TfR1 target is a diagnostic or imaging reagent.

[0093] A tenth aspect of the present invention provides a delivery method, comprising the steps of:

[0094] (s1) Provide a delivery system as described in the seventh aspect of the present invention and the cells to be delivered;

[0095] (s2) The delivery system is brought into contact with the cell to be delivered, thereby delivering the polypeptide, peptide ligand, peptide conjugate, or combination thereof in the delivery system into or through the cell.

[0096] In another preferred embodiment, the concentration of the delivery system is 1–100 μM, more preferably 1–50 μM, more preferably 1–20 μM, for example, about 10 μM.

[0097] In another preferred embodiment, in step (s2), the delivery system is added to the cell culture medium for contact.

[0098] In another preferred embodiment, the delivery system contacts the cells at 20–40°C, more preferably 25–40°C, more preferably 30–40°C, for example, about 37°C.

[0099] In another preferred embodiment, the delivery system is in contact with cells for 5 to 48 hours, more preferably 10 to 25 hours, and even more preferably 15 to 24 hours.

[0100] In another preferred embodiment, the delivery system further includes a detectable marker.

[0101] In another preferred embodiment, the detectable marker includes fluorescein.

[0102] In another preferred embodiment, the method further includes step (s3) of detecting the delivery effect of the delivery system.

[0103] In another preferred embodiment, the detection includes detection using confocal microscopy imaging or fluorescence signal detection methods.

[0104] In another preferred embodiment, the confocal microscope imaging is a LEICA TCS SP8 confocal microscope.

[0105] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0106] Figure 1 SDS-PAGE analysis of purified TfR1 and biotinylated TfR1 is shown.

[0107] Figure 2 Characterization of TfR1-specific peptides is shown.

[0108] Figure 3 The expression of TfR1 in HEK293T and HEK293T-TfR1 cell lines was detected by Western blot and flow cytometry.

[0109] Figure 4 The results of the cell permeability assay for peptide TR17 are shown. Detailed Implementation

[0110] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a polypeptide specific to transferrin receptor 1 (TfR1). This polypeptide comprises the structure shown below: X1-A1-A2-A3-A4-A5-X2, where A3 is Chg; X1 and X2 are each independently an absent, signal, or transmembrane peptide; A1, A2, A4, and A5 are each independently selected from the group consisting of Chg, Leu, Trp, Ile, Nle, Phe(Br), or Tic; wherein two or more of A1, A2, A4, and A5 are Chg. Experiments show that the polypeptide of this invention has a strong binding affinity to transferrin receptor 1, with a KD ≤ 400 nM. The polypeptide of this invention can penetrate cells overexpressing TfR1 and has the potential to deliver drugs. Based on this, the present invention was completed.

[0111] the term

[0112] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0113] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0114] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0115] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0116] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0117] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0118] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0119] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0120] This invention includes not only complete polypeptides, but also fragments of immunologically active polypeptides or fusion proteins formed by polypeptides and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said polypeptides.

[0121] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the same biological function or activity as the polypeptide of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0122] The variants of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the coding DNA of the polypeptide of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the polypeptide of the present invention.

[0123] The present invention also provides a polynucleotide molecule encoding the aforementioned polypeptide or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0124] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0125] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0126] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0127] The full-length nucleotide sequence or fragments of the polypeptides of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0128] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0129] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0130] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0131] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0132] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0133] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0134] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0135] D-cyclohexylglycine

[0136] D-cyclohexylglycine (Chg) is a derivative of glycine, also known as cyclohexane. Cyclohexylglycine is composed of the amino acid glycine and a cyclohexyl group. It is a white crystalline powder, soluble in water. D-cyclohexylglycine is the enantiomer of L-cyclohexylglycine and is optically active. The unique cyclohexyl structure of cyclohexylglycine endows it with unique physical and chemical properties, making it potentially valuable in drug design, synthetic biology, and materials science. For example, L-cyclohexylglycine is a key intermediate in the synthesis of thiavain. Furthermore, cyclohexylglycine can serve as a model compound for studying amino acid metabolism, protein folding, or regulatory activity.

[0137] Tic

[0138] D-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic) is a quinoline derivative used as a pharmaceutical intermediate, specifically in the synthesis of quinapril. Quinapril, developed by Pfizer, was approved by the FDA in 1991 for the treatment of hypertension, congestive heart failure, and angioedema. Tic can also be used to synthesize bioactive peptide mimics, such as biotinylated derivatives of the opioid receptor antagonist TIPP. Furthermore, Tic can serve as a chiral intermediate in the synthesis of biologically active compounds, for example, as an inhibitor of the coronavirus nonstructural protein 1 (nsp1).

[0139] D-Leucine

[0140] D-Leucine (Nle), also known as 2-aminohexanoic acid, is a white crystalline powder, slightly soluble in water and well soluble in anhydrous alcohol. Nleucine (Nle) is an aliphatic amino acid, a non-natural amino acid not found in proteins. It is an isomer of leucine and isoleucine and can influence protein synthesis in skeletal muscle. As an amino acid derivative, Nleucine (Nle) can be used in the synthesis of pharmaceutical intermediates.

[0141] The polypeptide of the present invention

[0142] The polypeptide of the present invention is a polypeptide specific to transferrin receptor 1, said polypeptide comprising the structure shown in formula (I):

[0143] X1-A1-A2-A3-A4-A5-X2(I)

[0144] In the formula, A3 represents Chg;

[0145] X1 and X2 are independently either non-signal peptides, signal peptides, or membrane-penetrating peptides;

[0146] A1, A2, A4, and A5 are each independently selected from the following groups: Chg, Leu, Trp, Ile, Nle, Phe(Br), or Tic;

[0147] Among them, A1, A2, A4 and A5 contain two or more Chg;

[0148] Chg represents cyclohexylglycine.

[0149] In a preferred embodiment, at least two cyclohexylglycine residues are adjacent in the polypeptide.

[0150] In a preferred embodiment, the cyclohexylglycine is of type D.

[0151] In a preferred embodiment, all amino acids in the polypeptide are D-type amino acids.

[0152] In a preferred embodiment, A1 is selected from the group consisting of: Chg, Leu, Trp, Nle, or Phe(Br);

[0153] A2 is selected from the following group: Chg, Leu, Nle, or Phe(Br);

[0154] A4 is selected from the following groups: Chg, Ile, or Phe(Br);

[0155] A5 is selected from the following groups: Chg, Ile, Nle, or Leu.

[0156] In a preferred embodiment, the polypeptide is selected from the group consisting of:

[0157] (a) Has an amino acid sequence selected from the following group:

[0158] (a1)Phe(Br)-Chg-Chg-Chg-Chg;

[0159] (a2)Nle-Chg-Chg-Chg-Chg;

[0160] (a3)Chg-Chg-Chg-Chg-Chg;

[0161] (a4)Trp-Chg-Chg-Chg-Chg;

[0162] (a5)Leu-Chg-Chg-Chg-Chg;

[0163] (a6)Trp-Chg-Chg-Phe(Br)-Chg;

[0164] (a7)Trp-Chg-Chg-Chg-Ile;

[0165] (a8)Chg-Chg-Chg-Chg-Ile;

[0166] (a9)Trp-Nle-Chg-Chg-Chg;

[0167] (a10)Trp-Chg-Chg-Chg-Nle;

[0168] (a11)Nle-Leu-Chg-Chg-Chg;

[0169] (a12)Trp-Chg-Chg-Chg-Leu;

[0170] (a13)Nle-Phe(Br)-Chg-Chg-Chg;

[0171] (a14)Tic-Chg-Chg-Chg-Chg;

[0172] (a15)Chg-Chg-Chg-Phe(Br)-Chg;

[0173] (a16)Tic-Phe(Br)-Chg-Chg-Chg;

[0174] (a17)Phe(Br)-Chg-Chg-Chg-Leu;

[0175] (a18)Nle-Nle-Chg-Chg-Chg;

[0176] (a19)Phe(Br)-Phe(Br)-Chg-Chg-Chg;

[0177] (a20)Trp-Chg-Chg-Ile-Chg;

[0178] (b) Derivative polypeptides formed by substituting one, two or three amino acids, and / or inserting one, two or three amino acids, and / or deleting one or two amino acids of any of the polypeptides shown in (a1) to (a20).

[0179] In a preferred embodiment, the peptide has a KD ≤ 450 nM for transferrin receptor 1, more preferably ≤ 300 nM, and even more preferably ≤ 200 nM.

[0180] Preparation of peptides

[0181] The DNA sequences of the polypeptides or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form a single-chain polypeptide.

[0182] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0183] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0184] Currently, the DNA sequence encoding the polypeptide (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0185] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0186] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0187] Typically, host cells transformed with the present invention are cultured under conditions suitable for the expression of the polypeptides of the present invention. The polypeptides of the present invention are then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, and other conventional separation and purification methods well known to those skilled in the art.

[0188] The obtained monoclonal peptides can be identified using conventional methods. For example, the binding specificity of monoclonal peptides can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal peptides can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0189] The polypeptides of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0190] The delivery system of the present invention

[0191] The delivery system of the present invention comprises polypeptides as described in the first aspect of the present invention, peptide ligands as described in the second aspect of the present invention, peptide conjugates as described in the third aspect of the present invention, or combinations thereof.

[0192] In a preferred embodiment, the delivery system is suitable for cells expressing transferrin receptor 1, preferably cells highly expressing transferrin receptor 1, and more preferably HEK293T cells highly expressing transferrin receptor 1. The delivery system is suitable for drug delivery across the blood-brain barrier, capable of mediating therapeutic agents, diagnostic agents, or gene therapy vectors across the blood-brain barrier and into the central nervous system.

[0193] In a preferred embodiment, the delivery system further includes detectable markers, toxins, siRNA, ASO, inhibitors, isotopic elements, viral vectors, nanocarriers, and polymers.

[0194] Pharmaceutical Composition

[0195] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned polypeptide or its active fragment, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0196] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0197] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases related to TfR1.

[0198] The pharmaceutical compositions of the present invention can be directly used to bind TfR1, and therefore can be used for the prevention and treatment of diseases such as tumors.

[0199] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal polypeptide (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the polypeptides of the present invention can also be used with other therapeutic agents.

[0200] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0201] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0202] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0203] The present invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of diseases related to TfR1.

[0204] The main advantages of this invention include:

[0205] 1. The polypeptide of the present invention is specific to transferrin receptor 1, exhibiting high specificity.

[0206] 2. The polypeptide of the present invention has a strong binding affinity to transferrin receptor 1, with a KD ≤ 400 nM.

[0207] 3. The polypeptide of the present invention can penetrate cells overexpressing TfR1 and has the potential to deliver drugs.

[0208] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0209] Example 1: Screening for peptides specific to transferrin receptor 1

[0210] Based on the DEL library prepared according to application number CN 202411051631.6, peptides specific to transferrin receptor 1 were screened.

[0211] 40 μL of Dynabeads MyOne T1 Streptavidin magnetic beads were washed three times with 300 μL of wash buffer (PBS pH 7.4, 0.1% Tween-20). Then, 25 μg of biotinylated TfR1 dissolved in 500 μL of wash buffer was added to the magnetic beads, and the mixture was incubated at room temperature for 1 hour to immobilize the target protein. After discarding the supernatant, the magnetic beads were washed three more times with wash buffer, followed by blocking with blocking buffer (PBS pH 7.4, 0.1% Tween-20, 1% BSA) for 1 hour. After washing twice with blocking buffer, the magnetic beads were divided into two equal aliquots for screening the DEL library.

[0212] After blocking the DEL library with selection buffer for one hour, it was incubated with magnetic beads in 100 μL of selection buffer (PBS pH 7.4, 0.1% Tween-20, 0.1 mg / mL cleaved salmon sperm DNA) at room temperature for one hour. Then, the magnetic beads were washed 10 times with washing buffer, followed by pyrolysis of the bound DEL peptide library by heating at 95°C for 10 minutes in 100 μL of washing buffer. The recovered DNA was used as the output for the first round of selection and the input for the second round. The selection procedure was repeated as in the first round, using the pyrolyzed bound library as the output for the second round of selection. The output DNA was then quantified using qPCR, recovering 0.218 pmol and 0.02665 pmol of DNA in the first and second rounds of selection, respectively.

[0213] Example 2: Synthesis of peptides and polypeptide conjugates specific to transferrin receptor 1

[0214] The TFR1-binding peptide was synthesized using the standard Fmoc solid-phase peptide synthesis method on Rink amide MHBC PS resin (0.458 mmol / g loading; GL Biochem Ltd., 49001). Fmoc-Lys(biotin)-OH was pre-activated by adding Fmoc-Lys(biotin)-OH (1 eq), HATU (1 eq), and DIEA (2 eq) to DMF (720 μL). The pre-activated solution was then added to Rink amide MHBC PS resin (25 mg) to couple the Fmoc-Lys(biotin)-OH onto the resin. After shaking for 30 min, the solution was dried off the resin, and the coupling reaction was repeated. The Fmoc group was then removed using 1000 μL of a 20% (v / v) solution of piperidine in N,N-dimethylformamide. Then, the subsequent coupling of Aop and amino acids was achieved under the conditions of Fmoc-Aop-OH / Fmoc-D-AA-OH (4 eq), HATU (4 eq), and DIEA (8 eq), with two coupling reactions performed in each round. After the peptide synthesis was completed in the solid phase, the resin supporting the peptide was placed in a 20% (v / v) DMF solution of piperidine. Finally, after TFA removal and side chain deprotection, the peptide was purified by high performance liquid chromatography (HPLC) and dried under vacuum to constant weight.

[0215] A fluorescent cell membrane-penetrating peptide (DPhe(Br)-DChg-DChg-DChg-DLue-AOP-Lys(fluorescein)) was synthesized on Rink amide MHBC PS resin (0.458 mmol / g loading; GL Biochem Ltd., 49001) using the standard Fmoc solid-phase peptide synthesis method. First, Fmoc-Lys(dde)-OH was coupled to Rink Amide resin (25 mg), and Fmoc-Lys(dde)-OH (4 eq), HATU (4 eq), and DIEA (8 eq) were added to dry DMF (720 μL). After shaking for 30 min, the solution was dried off the resin. Subsequently, Aop and amino acids were coupled under Fmoc-Aop-OH / Fmoc-D-AA-OH (4 eq), HATU (4 eq), and DIEA (8 eq) conditions. After solid-phase peptide synthesis, the resin supporting the peptide was placed in 2% (v / v) hydrazine hydrate DMF (4 mL) to remove the DDE protecting group. The resin was then washed five times with DMF (4 mL), and carboxyfluorescein (2 eq), DIC (2 eq), and HOBt (2 eq) were added to DMF (300 μL) and shaken at room temperature for 2 hours. Following TFA removal and side-chain deprotection, the fluorescent peptide was purified by high-performance liquid chromatography (HPLC).

[0216] Figure 1 SDS-PAGE analysis of purified TfR1 and biotinylated TfR1 is shown.

[0217] The peptides obtained through screening are shown in Table 1.

[0218] Table 1

[0219]

[0220] The selected peptides and peptide conjugates were synthesized and analyzed by LCMS. The results are shown in Table 2.

[0221] Table 2

[0222]

[0223] Note: AOP stands for 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionic acid, used to improve peptide solubility.

[0224] The structural formulas of the selected and synthesized peptides (partial) are shown below:

[0225]

[0226] Example 3: Enzyme-linked immunosorbent assay (ELISA) of peptides

[0227] The binding activity of the candidate peptides was first verified by enzyme-linked immunosorbent assay (ELISA). TfR1 protein was diluted with PBS and coated onto ELISA plates (Sangon Biotech, catalog number: F605031) at a rate of 1 μg per well, then incubated overnight at 4°C. The coating buffer was discarded, and each well was washed four times with 200 μL of PBS containing 0.1% (v / v) Tween 20. Next, 200 μL of blocking buffer (1% (w / v) bovine serum albumin in PBS, containing 0.1% (v / v) Triton X-100) was added to each well, and the plates were incubated at 37°C for 1 hour. After blocking, the blocking buffer was discarded, and each well was washed four times with washing buffer. The peptide was pre-diluted to a final concentration of 5 μM in blocking buffer, and 100 μL of the peptide solution was added to each well. Wells containing only BSA and wells without TfR1 protein coating served as negative controls. After incubating at 37°C for 1 hour, discard the liquid and wash each well four times with washing buffer. Streptavidin-horseradish peroxidase (Smart Lifesciences, catalog number: SLP001H) was diluted 1:5000 in blocking buffer, and 100 μL was added to each well. Incubation was continued at 37°C for another hour. After incubation, discard the liquid and wash each well four times with washing buffer. Then, 100 μL of TMB substrate solution was added to each well, and incubation was continued at 37°C until a clear color change was observed. The reaction was terminated by adding 50 μL of 2M sulfuric acid to each well, and absorbance was measured at 450 nm.

[0228] Although not all peptides are soluble at 5 μM, a significant number (11 out of 20) exhibit strong binding to TfR1, which was confirmed by chromogenic substrate detection. Figure 2 (a) In contrast, the control peptide (GGGGG-AOP-Lys(Biotin)) did not show binding to TfR1, which confirms the specificity of the interaction between the selected peptide and TfR1.

[0229] Example 4: Combination Affinity Measurement

[0230] The procedure was performed at 30 °C using an Octet Red 96 system (ForteBio, California, USA). Biotinylated peptides were diluted in assay buffer (PBS containing 0.02% Tween-20) and incubated with a streptavidin-coated sensor (ForteBio, catalog number 18-5019) to achieve stable immobilization until the immobilization level reached approximately 0.6 nm. Recombinant 6His-TfR1 protein was serially diluted in assay buffer to generate a concentration gradient covering the expected dissociation constant (KD) range. Subsequently, the peptide-immobilized sensor was exposed to a protein solution for a binding phase (300 s) followed by a dissociation phase (200 s) in assay buffer to monitor real-time binding kinetics. For each interaction, a dual reference was used: a peptide-loaded streptavidin-coated biosensor incubated in assay buffer (without analyte) and an unloaded streptavidin-coated biosensor incubated with the analyte to ensure that the measured RU values ​​reflected only the specific interaction between the target molecules. Binding kinetics were analyzed using a 1:1 Langmuir binding model, which assumes a simple two-state reaction mechanism (binding and dissociation). Experimental data were fitted using a global fitting algorithm in ForteBio data analysis software (version 12.2.2.4) to obtain the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD).

[0231] like Figure 2 As shown in Figure b, the selected peptides exhibit strong binding affinity to TfR1 in the low nanomolar range.

[0232] Example 5 Cell permeabilization experiment

[0233] HEK293T-TfR1 cells were seeded into 12-well plates and cultured for 24 hours. The medium was then replaced with fresh serum-free medium, and peptides were added at a final concentration of 10 μM. Serum was replaced after 4 hours, followed by culturing at 37°C for another 20 hours. After culturing, cells were collected, washed three times with PBS, and evenly seeded onto coverslips pre-treated with poly-L-lysine. After air-drying, cells were fixed with 4% paraformaldehyde and washed three times with PBS. Cell nuclei were counterstained with Hoechst 33258 dye (Beyotime, catalog number: C0003) according to the manufacturer's instructions. Images were acquired using a LEICA TCS SP8 confocal microscope (100× objective, 1.0 magnification), and data were analyzed using LAS X software.

[0234] Confocal microscopy imaging revealed that the linked peptides significantly enhanced the internalization of fluorescein, confirming that they promote peptide-based drug delivery and cell penetration by binding to TfR1. Figure 2 (c)

[0235] Figure 3 The expression of TfR1 in HEK293T and HEK293T-TfR1 cell lines was detected by Western blot and flow cytometry.

[0236] Figure 4 The results of a cell permeability assay for the peptide TR17 are shown. After microscopic observation confirmed that TR17 could penetrate the stable HEK293T (TfR1) cell line overexpressing TfR1, its ability to cross the K562 cell membrane and enter cells using fluorescently labeled TR17-luciferin was further evaluated. K562 is a human cell line derived from patients with chronic myeloid leukemia (CML) that exhibits high levels of TfR1 protein expression, making it a widely used model for studying TfR1 function. Fluorescence imaging of K562 cells incubated with the peptide probe showed membrane permeability comparable to that observed in stable HEK293T (TfR1) cells.

[0237] sequence list

[0238]

[0239]

[0240] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A polypeptide specific to transferrin receptor 1, characterized in that, The polypeptide comprises a structure as shown in formula (I): X1-A1-A2-A3-A4-A5-X2(I) In the formula, A3 represents Chg; X1 and X2 are independently either non-signal peptides, signal peptides, or membrane-penetrating peptides; A1, A2, A4, and A5 are each independently selected from the following groups: Chg, Leu, Trp, Ile, Nle, Phe(Br), or Tic; Among them, A1, A2, A4 and A5 contain two or more Chg; Chg represents cyclohexylglycine.

2. The polypeptide according to claim 1, characterized in that, The polypeptide contains at least two adjacent cyclohexylglycine residues.

3. The polypeptide according to claim 1, characterized in that, The cyclohexylglycine is of type D.

4. The polypeptide according to claim 1, characterized in that, A1 is selected from the following groups: Chg, Leu, Trp, Nle, Tic, or Phe(Br); A2 is selected from the following group: Chg, Leu, Nle, or Phe(Br); A4 is selected from the following groups: Chg, Ile, or Phe(Br); A5 is selected from the following groups: Chg, Ile, Nle, or Leu.

5. The polypeptide according to claim 1, characterized in that, The polypeptide is selected from the following group: (a) Has an amino acid sequence selected from the following group: (a1)Phe(Br)-Chg-Chg-Chg-Chg; (a2)Nle-Chg-Chg-Chg-Chg; (a3)Chg-Chg-Chg-Chg-Chg; (a4)Trp-Chg-Chg-Chg-Chg; (a5)Leu-Chg-Chg-Chg-Chg; (a6)Trp-Chg-Chg-Phe(Br)-Chg; (a7)Trp-Chg-Chg-Chg-Ile; (a8)Chg-Chg-Chg-Chg-Ile; (a9)Trp-Nle-Chg-Chg-Chg; (a10)Trp-Chg-Chg-Chg-Nle; (a11)Nle-Leu-Chg-Chg-Chg; (a12)Trp-Chg-Chg-Chg-Leu; (a13)Nle-Phe(Br)-Chg-Chg-Chg; (a14)Tic-Chg-Chg-Chg-Chg; (a15)Chg-Chg-Chg-Phe(Br)-Chg; (a16)Tic-Phe(Br)-Chg-Chg-Chg; (a17)Phe(Br)-Chg-Chg-Chg-Leu; (a18)Nle-Nle-Chg-Chg-Chg; (a19)Phe(Br)-Phe(Br)-Chg-Chg-Chg; (a20)Trp-Chg-Chg-Ile-Chg; (b) Derivative polypeptides formed by substituting one, two or three amino acids, and / or inserting one, two or three amino acids, and / or deleting one or two amino acids of any of the polypeptides shown in (a1) to (a20).

6. A peptide ligand specific to transferrin receptor 1, characterized in that, The peptide ligand comprises the polypeptide and molecular scaffold as described in claim 1, wherein the molecular scaffold forms a covalent bond with the reactive group of the polypeptide.

7. A peptide conjugate specific to transferrin receptor 1, characterized in that, The peptide conjugate comprises: (a) the polypeptide as claimed in claim 1; and (b) A coupling portion conjugated to the polypeptide, the coupling portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

8. A delivery system, characterized in that, It comprises the polypeptide as described in claim 1, the peptide ligand as described in claim 6, the peptide conjugate as described in claim 7, or a combination thereof.

9. The use of the polypeptide as described in claim 1, the peptide ligand as described in claim 6, and the peptide conjugate as described in claim 7, characterized in that, Used for preparation: (1) The delivery system as claimed in claim 8; (2) Medications for the prevention and / or treatment of TfR1-related diseases; (3) Immunomodulators; (4) TfR1 targeting agents.

10. A delivery method, characterized in that, Including the following steps: (s1) Provides the delivery system as described in claim 8 and the cells to be delivered; (s2) The delivery system is brought into contact with the cell to be delivered, thereby delivering the polypeptide, peptide ligand, peptide conjugate, or combination thereof in the delivery system into or through the cell.

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