Bicyclic peptide targeting human Nectin-4 protein as well as preparation method and application of bicyclic peptide

Abstract

The invention discloses a bicyclic peptide targeting human Nectin-4 protein as well as a preparation method and application of the bicyclic peptide, and belongs to the field of biological medicines. The amino acid sequence of the bicyclic peptide is shown as SEQ ID NO: 1, the bicyclic peptide can be screened through a phage display technology and prepared through a chemical synthesis method, and a stable bicyclic space structure is formed in a molecule through two pairs of disulfide bonds. A surface plasmon resonance technology proves that the bicyclic peptide can be specifically combined with an extracellular domain of human Nectin-4 protein with micromole affinity KD = 1.79 * 10 <-4 > M. The bicyclic peptide provided by the invention has important application value in the aspects of preparing a Nectin-4 protein detection reagent, serving as a tool compound for researching the biological function of Nectin-4 and serving as a lead molecule for developing a diagnostic or therapeutic product of targeted Nectin-4.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a bicyclic peptide targeting human Nectin-4 protein, its preparation method, and its application. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human health. Treatment strategies have gradually shifted from traditional broad-spectrum radiotherapy and chemotherapy to precision medicine targeting specific molecular targets. In epithelial-derived malignant tumors, specific transmembrane proteins located on the cell membrane surface have become ideal targets for developing targeted drugs due to significant expression differences between cancer cells and normal cells. These "tumor-associated antigens" can be directly recognized by extracellular ligands, thereby achieving specific enrichment of drugs in tumor tissues. This helps to effectively kill cancer cells while significantly reducing off-target toxicity to normal tissues, a key strategy for improving the therapeutic window.

[0003] Nectin-4 (poliovirus receptor-associated protein 4, PVRL4) is a member of the Nectin family and a calcium-independent immunoglobulin-like cell adhesion molecule. It is a type I transmembrane glycoprotein whose structure comprises an extracellular region consisting of three immunoglobulin-like domains (IgV, IgC1, and IgC2), a transmembrane region, and an intracellular region. The intracellular region interacts with the intracellular scaffold protein Afadin via its C-terminal PDZ-binding motif, playing a crucial physiological role in the formation of intercellular adhesion junctions and tight junctions. Nectin-4 expression exhibits distinct developmental stage and tissue specificity, showing widespread high expression during embryonic and placental development, while its expression is significantly restricted in healthy adult tissues, detected at low levels only in a few tissues such as the skin, esophagus, and bladder. However, Nectin-4 exhibits abnormally high expression in various human epithelial malignancies. This stark contrast between "high expression in cancer cells and low expression in normal cells" in multiple cancers, including breast cancer, ovarian cancer, urothelial carcinoma, and lung cancer, makes it a highly attractive tumor-specific target. Its pro-cancer mechanism is complex; it not only directly drives tumor cell proliferation, survival, and invasion by activating intracellular signaling pathways such as PI3K / Akt, but its cleaved extracellular domain can also promote angiogenesis and may participate in immune regulation, thus playing a multifaceted pro-cancer role in the tumor microenvironment.

[0004] Based on this, antibody-drug conjugates targeting Nectin-4 have made breakthrough progress in clinical practice, fully validating the clinical feasibility of this target.

[0005] However, monoclonal antibody-based drugs have inherent limitations, including large molecular size leading to poor penetration into solid tumor tissues, high production costs, potential immunogenicity, and long half-lives that may cause persistent toxicity. To overcome these bottlenecks, the research community is actively developing peptide-conjugated drugs using small molecule peptides as targeting carriers. Compared to antibodies, small molecule peptides have an order-of-magnitude advantage in molecular weight, enabling them to penetrate deeper into solid tumors more effectively and achieve more uniform distribution. Simultaneously, peptides can be produced rapidly, on a large scale, and at low cost using mature solid-phase synthesis techniques, and are easily and precisely chemically modified to optimize their stability and pharmacokinetic properties. Furthermore, short peptide sequences generally have lower immunogenicity and can be flexibly conjugated to various therapeutic payloads or reporter molecules, forming a multifunctional platform.

[0006] Compared to linear peptides, cyclic peptides exhibit greater application value. Their conformational rigidity can enhance binding properties and metabolic stability with target proteins, providing a potential direction for overcoming the limitations of existing targeting vectors. In summary, there is an urgent need in this field for a novel peptide molecule that can bind to Nectin-4 protein with high affinity and specificity to meet the diagnostic and therapeutic needs of targeting Nectin-4. Summary of the Invention

[0007] This invention provides a bicyclic peptide targeting human Nectin-4 protein, its preparation method, and its application, which can solve the technical problem of the limited variety of molecular tools for targeting Nectin-4 protein in the prior art.

[0008] In a first aspect, the present invention provides a bicyclic peptide targeting human Nectin-4 protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] Furthermore, the nucleotide sequence of the gene encoding the bicyclic peptide is shown in SEQ ID NO:2.

[0010] Furthermore, two pairs of disulfide bonds are formed within the molecule of the bicyclic peptide.

[0011] Secondly, the present invention provides a method for preparing a bicyclic peptide targeting human Nectin-4 protein, comprising the following steps: S1. Synthesis of linear peptides: On the resin, according to the amino acid sequence shown in SEQ ID NO:1, the corresponding Fmoc-protecting amino acids are sequentially linked from the C-terminus to the N-terminus to gradually extend the peptide chain and obtain resin-bound protected linear peptides. S2. Cutting and crude peptide acquisition: Using a cutting reagent, the peptide is cut off from the resin and all side chain protecting groups are removed at the same time. The linear crude peptide is obtained by ether precipitation. S3. Purification of linear crude peptide: The linear crude peptide is purified, the main peak fraction is collected, and the purified linear peptide KP3298 is obtained. S4, Oxidative refolding: The purified linear polypeptide is dissolved in a redox buffer system containing reduced glutathione and oxidized glutathione and incubated under alkaline pH conditions. S5. Purification and identification of the final product: After refolding, the product is purified again using preparative reversed-phase high-performance liquid chromatography to obtain the target bicyclic peptide with correct structure and high purity, and then identified by mass spectrometry.

[0012] Further, in step S2, the cutting reagent is composed of trifluoroacetic acid, water, phenol and triisopropylsilane in a volume ratio of 88:5:5:2.

[0013] Furthermore, in step S3, the instrument used to purify the linear crude peptide is a preparative reversed-phase high-performance liquid chromatography (RP-HPLC).

[0014] Furthermore, in step S4, the pH value of the alkaline pH is 8.5.

[0015] Thirdly, the present invention provides the following applications of a bicyclic peptide targeting human Nectin-4 protein: in the preparation of reagents for the in vitro detection of Nectin-4 protein for non-diagnostic purposes; in the preparation of diagnostic agents targeting Nectin-4; and in the preparation of therapeutic agents targeting Nectin-4.

[0016] The beneficial effects of this invention are: 1. Provides a novel molecular tool: This invention discloses for the first time a novel Nectin-4 binding ligand, a bicyclic peptide (named KP3298). Its novel sequence, obtained through high-throughput screening, ensures its uniqueness and inventiveness. This bicyclic peptide provides a new tool for studying the biological functions of Nectin-4 and developing related targeting strategies.

[0017] 2. A reliable preparation process was established: This invention optimized and verified the entire production process from chemical synthesis to oxidative refolding. This process is stable and reproducible, laying the foundation for the large-scale production and quality control of peptides. In particular, the redox-based refolding method can efficiently guide peptides to form the correct native conformation.

[0018] 3. Clarified binding characteristics and potential advantages: Using SPR technology, not only was the binding ability (micromolar affinity) of the bicyclic peptide to full-length Nectin-4 quantitatively confirmed, but more importantly, it was found that its binding does not depend on a single independent domain. This suggests that the bicyclic peptide may target a conformational epitope, and this binding mode may have higher specificity, providing a possibility for the development of differentiated targeting reagents.

[0019] 4. It opens up broad application prospects: The bicyclic peptide KP3298 provided by this invention can serve as a core binding element for developing in vitro detection reagents for Nectin-4 protein. Simultaneously, as a small molecule lead compound with a unique binding mode, it demonstrates great development potential in constructing peptide-conjugated drugs targeting Nectin-4 or other innovative diagnostic and therapeutic agents. Attached Figure Description

[0020] Figure 1 The high-throughput sequencing results (logo image) are the results of screening using the three-round phage display technology targeting the extracellular domain of Nectin-4 in this invention. Figure 2 Figure 1 shows the chromatographic and mass spectrometric characterization of the bicyclic peptide KP3298 before and after refolding in this invention; Figure 2a shows the chromatographic and mass spectrometric characterization of the bicyclic peptide KP3298 before refolding; Figure 3b shows the chromatographic and mass spectrometric characterization of the bicyclic peptide KP3298 after refolding. Figure 3 This is a size exclusion chromatography and SDS-PAGE electrophoresis image of the extracellular domain of Nectin-4 in this invention; Figure 4 This invention provides a dynamic sensing image of the binding of bicyclic peptide KP3298 to the full-length, IgV, IgC1, and IgC2 of immobilized N4-ECD using surface plasmon resonance technology. Detailed Implementation

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0022] In a first aspect, the present invention provides a bicyclic peptide targeting human Nectin-4 protein, the amino acid sequence of which is shown in SEQ ID NO:1. This sequence, named KP3298, is a highly enriched sequence obtained by screening from a large-scale peptide library using phage display technology. The bicyclic peptide sequence contains four cysteine ​​residues, enabling it to form intramolecular disulfide bonds under appropriate oxidative conditions.

[0023] In some embodiments, the nucleotide sequence of the gene encoding the bicyclic peptide is shown in SEQ ID NO:2. Knowing the nucleotide sequence of the gene encoding the bicyclic peptide allows for the construction of a recombinant expression system using genetic engineering techniques, enabling large-scale, low-cost production of the bicyclic peptide. This also provides a clear genetic-level operational basis for subsequent molecular modifications (such as site-directed mutagenesis to optimize performance).

[0024] In some embodiments, the bicyclic peptide forms two pairs of disulfide bonds within its molecule. The formation of two pairs of disulfide bonds within the bicyclic peptide molecule enables the construction of a stable, rigid bicyclic conformation, which significantly enhances its metabolic stability during antibody protease degradation and precisely maintains its active conformation for binding to Nectin-4 protein, thereby strengthening binding affinity and specificity.

[0025] Secondly, the present invention provides a method for preparing a bicyclic peptide targeting human Nectin-4 protein, comprising the following steps: S1. Synthesis of linear peptides: Following the amino acid sequence shown in SEQ ID NO:1, Fmoc-protected amino acids were sequentially linked from the C-terminus to the N-terminus on the resin to gradually extend the peptide chain and obtain resin-bound protected linear peptides. The target amino acid sequence was precisely assembled on the resin using the Fmoc solid-phase synthesis strategy, laying the foundation for the correct structure of the subsequent bicyclic peptide. S2. Cutting and crude peptide acquisition: Using a cutting reagent, the peptide is cut off from the resin and all side chain protecting groups are removed at the same time. The linear crude peptide is obtained by ether precipitation. A specific cutting reagent is used to achieve efficient dissociation of the peptide from the resin and complete removal of side chain protecting groups. Combined with ether precipitation, the linear crude peptide is rapidly enriched. S3. Purification of linear crude peptide: The linear crude peptide is purified, and the main peak fraction is collected to obtain the purified linear polypeptide KP3298; this effectively removes synthetic impurities and ensures the efficiency and purity of subsequent oxidative refolding reactions. S4, Oxidative Refolding: The purified linear polypeptide is dissolved in a redox buffer system containing reduced glutathione and oxidized glutathione and incubated under alkaline pH conditions; the linear polypeptide is induced to form correctly paired disulfide bonds and construct a stable bicyclic conformation; S5. Purification and identification of the final product: After refolding, the product is purified again using preparative reversed-phase high-performance liquid chromatography to obtain the target bicyclic peptide with correct structure and high purity, and then identified by mass spectrometry.

[0026] In some embodiments, in step S2, the cleavage reagent is composed of trifluoroacetic acid, water, phenol, and triisopropylsilane in a volume ratio of 88:5:5:2. This method can efficiently achieve the dissociation of peptides from resin and the complete removal of side-chain protecting groups, while reducing peptide oxidative degradation and ensuring the integrity of linear crude peptides.

[0027] In some embodiments, in step S3, the instrument used to purify the linear crude peptide is a preparative reversed-phase high-performance liquid chromatography (RP-HPLC). This can accurately separate synthetic impurities from the target peptide, significantly improving the purity of the crude peptide and laying the foundation for the efficient execution of subsequent oxidative refolding reactions.

[0028] In some embodiments, the pH value of the alkaline pH in step S4 is 8.5. This allows for optimal control of the thermodynamic equilibrium of disulfide bond formation, promoting correct pairing of cysteine ​​residues in linear polypeptides and efficiently constructing a stable bicyclic conformation.

[0029] Thirdly, this invention provides the following applications of a bicyclic peptide targeting human Nectin-4 protein: its use in preparing reagents for the in vitro detection of Nectin-4 protein for non-diagnostic purposes; its use in preparing diagnostic agents targeting Nectin-4; and its use in preparing therapeutic agents targeting Nectin-4. This comprehensively covers the needs from basic research to clinical diagnosis and treatment, significantly improving the performance and application value of related products.

[0030] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0031] Example 1

[0032] The bicyclic peptide KP3298 targeting the extracellular domain of Nectin-4 was screened using phage display technology.

[0033] This embodiment is the origin of the discovery of the bicyclic peptide molecule of the present invention, and records in detail the entire process of screening out the specific binding bicyclic peptide from the peptide library.

[0034] 1. Preparation of the target protein - the full-length human Nectin-4 extracellular domain (N4-ECD)

[0035] (1) Plasmid construction: First, based on the coding sequence of human Nectin-4 in the NCBI GeneID database, a cDNA fragment of its extracellular domain (amino acids 1-349, molecular weight 35kDa) was designed and synthesized. This fragment was inserted into the modified mammalian expression vector pcDNA3.1 using molecular cloning technology, and a sequence encoding a 6His tag was introduced at the C-terminus. The resistance was ampicillin (Amp). The successfully constructed plasmid was verified by sequencing.

[0036] (2) Expression in mammalian cells: Human embryonic kidney HEK293F cells were selected as the expression host. Cells were cultured in suspension in 1L shake flasks at 37℃, 5% CO2, and 130rpm using SMM 293-TII medium. When the cell density reached 2.5 × 10⁻⁶ cells / year... 6 At cell / mL, large-scale transient transfection was performed using polyethyleneimine 26k transfection reagent (concentration 1 mg / mL). 15 h after transfection, 10 mM sodium butyrate was added, the temperature was lowered to 30℃, and the cells were cultured at 130 rpm for 48 h before harvesting.

[0037] (3) Protein purification: After 48 hours, cells and culture supernatant were separated by centrifugation (4000 rpm, 20 min), and 500 mL of supernatant was poured into a 750 mL centrifuge jar. 20 μL of the supernatant was retained as a sample.

[0038] b. Prepare 1L binding buffer: 20mM Tris, 200mM NaCl, pH 7.5, and 1L ultrapure water. Both the binding buffer and ultrapure water are filtered through a 0.22μm PES membrane to remove residual particulate matter.

[0039] c. Add 2 ml Ni NTA (for 1 L of supernatant) to the affinity chromatography gravity column, wash with 20 mL ultrapure water and 30 mL binding buffer respectively, leaving 4 mL of binding buffer in the column, blow the Ni column evenly, and divide it into two centrifuge jars containing 500 mL of supernatant. Place the centrifuge jars in a shaker at 4 °C and shake at 100 rpm for 2 h.

[0040] d. Configure wash1, wash2, and Elution buffer

[0041] Wash 1: 30mL binding buffer;

[0042] Wash 2: 30 mL (binding buffer + 20 mM imidazole)

[0043] Elution: 10mL (binding buffer+400mM imidazole)

[0044] After 2 hours, the centrifuge jar was removed, and the supernatant and Ni column were poured into an affinity chromatography gravity column for natural flow-through. The column was then washed with wash1 and wash2, and 20 μL of each of the flow-through, wash1, and wash2 samples were retained.

[0045] f. After washing 2 completely, close the valve, add 10 mL of Elution buffer, let stand for 10 min, then transfer to a 30 kJ protein concentration tube (pre-washed with ultrapure water), and centrifuge at 3200 rpm to less than 1 mL. Measure the protein concentration to determine the subsequent loading volume.

[0046] g. Connect the Superdex 200 Increase 10 / 300GL column to the ÄKTA Pure system and equilibrate with binding buffer pre-cooled to 4°C. Set the flow rate to 0.5 mL / min. -1 Monitor the absorbance and pressure signal at 280nm online; continuously elute with 25mL until the UV baseline and pressure line are stable, which is considered as the equilibration is complete.

[0047] h. After centrifugation, transfer the sample to a 1.5 mL EP tube and centrifuge at 10,000 rpm for 10 min to remove the precipitate. Aspirate the supernatant for AKTA loading. Set the flow rate to 0.5 mL / min. -1 The absorbance at 280 nm and 260 nm and the pressure signal were monitored online; elution was performed continuously for 25 mL. After the procedure, the sample tubes were collected according to the peak shape.

[0048] i. Take 20 μL of sample from each sample tube, and add 5 μL of protein loading buffer to each tube, along with the sample retained in the previous step. Mix well and perform SDS-PAGE electrophoresis to determine if the protein is correct. Figure 2 It can be seen that the protein was successfully prepared and has high purity, and can be used for subsequent detection.

[0049] 2. Phage display biological selection

[0050] (1) Peptide library and reagents: The laboratory's existing phage display bicyclic heptadecapeptide library was selected: X2CCX4CX7CGG, with a library capacity of 3×10 9 The peptide is cyclized from four cysteine ​​residues to form a constrained conformation, which helps in screening for high-affinity ligands.

[0051] (2) Selection process: Three rounds of selection are conducted.

[0052] a. Screening of polystyrene ELISA plate media

[0053] Protein incubation: The shaker and 96-well plate were pre-cooled and sterilized with UV light at 4°C and 60 rpm. Proteins were diluted to 200 μL with binding buffer (20 mM Tris pH=7.4, 150 mM NaCl, 0.025% GDN (v / v)), added to single wells of the 96-well plate, and labeled. The first round of selection used 10 μg of protein, the second 5 μg, and the third 2 μg. Proteins were incubated overnight at 4°C and 60 rpm. The control group was not incubated; only an equal volume of binding buffer was added.

[0054] Blocking: Discard the liquid, wash three times with binding buffer (200 μL each time), and add 200 μL of blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.025% GDN (v / v), 1% BSA (w / v)); simultaneously, add the peptide library (200 μL, 1 × 10⁻⁶ ppm) to the buffer. 12 Add 200 μL of blocking buffer to the pfu / mL protein and peptide libraries, and block for 1 h at 4 °C and 60 rpm. Add 200 μL of blocking buffer to the control group.

[0055] Co-incubation: Aspirate the liquid from the 96-well plate, add an equal volume of the sealed peptide library to the experimental or control group, and incubate for 4 hours at 4°C and 60 rpm.

[0056] Washing and elution: Aspirate the liquid and wash 10 times with washing buffer (20 mM Tris pH=7.4, 150 mM NaCl, 0.025% GDN (v / v), 0.1% Tween 20 (v / v)), and 3 times with binding buffer. Invert the 96-well plate onto paper, ensuring no liquid remains. Add 200 μL of 0.2 M Glycine, pH=2.2, to elute, and react on a shaker for 10 min at 37°C and 60 rpm. Add 20 μL of 1 M Tris, pH=8, to neutralize the glycine.

[0057] To determine the titer of the eluted phage: Prepare 2 mL of TG1 monoclonal culture for 5 h in advance. Add 90 μL of bacterial culture to each of 12 EP tubes. Add 10 μL of the collected phage to the first tube, mix well, and then add 10 μL to the second tube. Continuous serial dilutions are performed up to tube number 12. Incubate at 37°C for 45 min. Spread 10 μL from each tube onto a plate and incubate overnight at 37°C for 12 h. Count the colonies at each dilution. The peptide library titer = colony count × dilution factor × 100, which is 8 × 10⁻⁶. 13 The remaining phages were added to 20 mL of TG1 bacterial culture that had been cultured for 5 h, incubated at 37°C for 30 min, and then incubated at 37°C and 220 rpm for 30 min. After centrifugation, the bacterial culture was resuspended with a small amount of culture medium, spread on plates, and incubated at 37°C for 12 h. Colonies were scraped off using a spreader with approximately 7 mL of 2YT medium, and an equal volume of 40% (v / v) glycerol was added. 1 mL of this was amplified according to the peptide library preparation method, and the remaining glycerol was frozen.

[0058] b. Anti-Flag Magnetic Bead Screening

[0059] Protein incubation: Take 100 μL of anti-Flag magnetic beads, wash 3 times with binding buffer, mix well, and aliquot into two tubes of equal volume, labeled as E (experimental group) and C (control group), respectively. Add protein to E and add an equal volume of binding buffer to C. Incubate at 4°C for 1 h on a rotary instrument.

[0060] Blocking: Centrifuge, aspirate the supernatant, wash 3 times with binding buffer, add 200 μL of blocking buffer for blocking, and simultaneously block the peptide library (200 μL, 1×10⁻⁶). 12 pfu / mL, add 200μL Blocking buffer), and incubate E, C and peptide libraries on a gyroscope for 1h.

[0061] Co-incubation: Centrifuge E and C, remove the supernatant, divide the peptide library into two equal volumes and add them to E and C respectively, and incubate for 4 hours.

[0062] The washing and rinsing methods are the same as before.

[0063] The titer of the eluted phages was measured using the method described above. The remaining phages were added to 20 mL of TG1 bacterial culture cultured for 5 h, incubated at 37°C for 30 min, and then incubated at 37°C and 220 rpm for 30 min. After centrifugation, the bacterial culture was resuspended with a small amount of culture medium, plated, and incubated at 37°C for 12 h. Colonies were scraped off using a spreader with approximately 7 mL of 2YT medium, and an equal volume of 40% (v / v) glycerol was added. 1 mL of this was amplified according to the peptide library preparation method, and the remaining glycerol was frozen. Table 1 below compares the phage recovery titers from three rounds of phage display bioscreening. From the recovery titers, it can be seen that with the increase in the number of screening rounds, the recovery titers of the experimental group became significantly higher, indicating obvious enrichment and the possible presence of specific sequences, while the control group only maintained a titer of 10. 7 The level indicates that the control group did not show enrichment. The experimental results show highly specific sequence enrichment, which is in line with theoretical expectations.

[0064] Table 1

[0065] 3. DNA sequencing and sequence alignment

[0066] The bacteriophages were sent to a sequencing company for high-throughput sequencing, with a sequencing volume of approximately 5 × 10⁻⁶. 7 Bioinformatics software was used to translate the obtained nucleic acid sequences into corresponding amino acid sequences. Multiple sequence alignment was performed, and the alignment results are as follows: Figure 1 As shown, from Figure 1 A sequence (amino acid sequence as shown in SEQ ID NO:1) was found to occur at a much higher frequency than other sequences, with an enrichment of approximately 0.8477. We named this common sequence KP3298 and identified it as the bicyclic peptide sequence targeting human Nectin-4 protein of this invention.

[0067] Example 2

[0068] Chemical synthesis, oxidative refolding and quality control of bicyclic peptide KP3298

[0069] This embodiment aims to establish a stable and scalable peptide production process route.

[0070] 1. Solid-phase synthesis of Fmoc

[0071] (1) Resin pretreatment and initiation: Weigh 0.2M Wang resin into a synthesis reaction tube, and swell it at room temperature for 30 min with 5 mL DMF and 5 mL DCM. Drain the solvent using an air pump. Rinse with 10 mL DMF and then drain the solvent.

[0072] (2) Connect the first amino acid

[0073] Peptide formation was achieved by linking amino acids one by one from the C-terminus (rightmost) towards the N-terminus (leftmost) to obtain the peptide structure. 0.8 mmol of amino acids were placed in a 10 mL EP tube, and 6 mL of DMF was added to completely dissolve the amino acids and mix thoroughly. Then, 1.6 mmol of DIEA was added. After mixing, the mixture was transferred to a peptide synthesis tube and incubated overnight at 33°C with shaking. The next day, the peptide synthesis tube was removed, and the resin was rinsed with 10 mL of DMF each time, the solvent being removed after each rinse. This process was repeated three times, followed by three more rinses with DCM, and finally three more rinses with DMF. Then, 10 mL of 20% piperidine solution was added to the synthesis tube until the resin was submerged, and the mixture was incubated at 33°C with shaking for 5 minutes. The above washing steps were then repeated.

[0074] (3) Peptide chain elongation: Weigh 4 equivalents of Fmoc-protected amino acids, HBTU and HOBt, and 8 equivalents of DIEA, dissolve them in 6 mL of DMF, activate for 5 min, and then add them to the reaction tube. Bubble the reaction under nitrogen for 1 h. Wash the resin with DMF. Remove the Fmoc groups with 20% piperidine / DMF solution. Connect the remaining amino acids sequentially according to the above steps.

[0075] (4) Cleavage of crude peptide: After sequence synthesis, the resin was rinsed three times with DMF (10 mL each time), and then the solvent was removed. The resin was rinsed three times with DCM in the same way. In a 50 mL EP tube, the crude peptide was cleaved according to a volume ratio of 88:5:5:2 (TFA: O:Phenol:Tips) Prepare the crude peptide cleavage reagent. Add the prepared liquid to the synthesis tube after the reaction has been completed, and react with shaking at 26°C for 2 hours. The liquid in the tube is the lysis buffer.

[0076] (5) Obtaining crude peptides: Transfer the lysis buffer to a 50 mL centrifuge tube. At room temperature, dry the buffer as much as possible using nitrogen gas, reducing its volume to less than 5 mL. Add 40 mL of ice-cold diethyl ether to the 50 mL centrifuge tube, mix well, and centrifuge at 4500 rpm for 3 min. After centrifugation, carefully discard the supernatant. Repeat the above ice-cold diethyl ether precipitation and centrifugation operation, discarding the supernatant. The precipitate obtained is the crude peptide. Air dry at room temperature, crush, and obtain the crude peptide.

[0077] 2. Purification of linear crude peptides

[0078] (1) Dissolving: After the crude peptide is lyophilized, weigh 300 mg and dissolve it in 20 mL of 30% acetonitrile / water solution containing 0.1% TFA.

[0079] (2) Preparative HPLC: A Waters XBridge™ BEH C18 OBD Prep Column (130 Å, 5 μm, 19 mm x 250 mm) was used. Mobile phase A: 0.1% TFA in H2O; Mobile phase B: 0.1% TFA in acetonitrile. Gradient: 20% B to 80% Bover for 40 min, detection wavelength 214 nm. The main peak fraction was collected.

[0080] (3) Lyophilization: The collected fractions were combined and lyophilized in a vacuum freeze dryer to obtain approximately 100 mg of pure white flocculent linear polypeptide. A small amount was analyzed by MALDI-TOFMS, showing [M+2H]. + The peak value is 1008.9 (results as follows). Figure 2 As shown in a), it is consistent with the theoretical value of 1009.7 (error < 0.03%).

[0081] 3. Oxidative renaturation forms disulfide bonds.

[0082] (1) Refolding buffer system: 1 mg of polypeptide corresponds to 10 mL of pure water, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, pH 8.5. This pH environment is conducive to disulfide bond exchange, and the appropriate redox ratio promotes correct pairing.

[0083] (2) Renaturation operation: Dissolve 10 mg of linear polypeptide in 2 mL of pure water, weigh 30.7 mg of reduced glutathione and 6.12 mg of oxidized glutathione and dissolve them in 98 mL of pure water. Slowly drop the polypeptide into the pure water with a pipette, shake gently to mix, wrap the mouth of the conical flask with toilet paper and let it stand overnight.

[0084] (3) Monitoring of refolding process: Samples were taken at 0, 2, 4, and 8 hours of reaction and analyzed by analytical HPLC (C18 column, 1-80% acetonitrile gradient, 20 min). It could be observed that the peak of the linear peptide gradually decreased, while the main peak of the peptide gradually increased and tended to stabilize after refolding, indicating that the refolding was completed.

[0085] 4. Final purification and identification of the refolded peptide

[0086] (1) Acidification and concentration: After refolding, add 1M HCl to adjust the pH of the reaction solution to 3.0 to terminate the disulfide bond exchange.

[0087] (2) Final purification: The concentrate was purified again by preparative HPLC, similar to the method for linear peptide purification.

[0088] (3) Mass spectrometry and HPLC identification: The final product was analyzed by MALDI-TOFMS, showing [M+2H] + The peak value is 1007.8 (results are as follows). Figure 2 As shown in b), this is consistent with the theoretical value of 1007.7 (error <0.03%), confirming the successful formation of two pairs of disulfide bonds. Analytical HPLC showed a purity >98% (area normalization method).

[0089] (4) Storage: After aliquoting, store frozen at -80°C for later use. Finally, approximately 20 mg of correctly refolded KP3298 bicyclic peptide can be obtained from 100 mg of linear peptide.

[0090] Example 3

[0091] Surface plasmon resonance analysis of affinity and condition optimization

[0092] This embodiment provides quantitative data on the interaction between the bicyclic peptide and the target protein, further verifying which domain of the Nectin-4 extracellular domain the bicyclic peptide binds to.

[0093] 1. Chip Immobilization

[0094] The experiment used a BIAcore 8K SPR instrument and a Series S CM5 chip. Protein fixation was performed according to the following steps: First, the chip surface was activated using EDC / NHS. Then, the full-length human Nectin-4 extracellular domain protein (N4-ECD) and individual domain proteins (IgV, IgC1, IgC2) were diluted to 10 μg / mL (dilution buffer: 10 mM sodium acetate buffer, pH 4.0) and sequentially immobilized into different test channels of the chip using amine coupling. This ensured that the protein immobilization response value of each channel reached approximately 5000 RU, guaranteeing consistent protein immobilization efficiency. Simultaneously, a reference channel was set up. This channel underwent the same "activation-blocking" process but was not immobilized with any protein. This reference channel was used to subtract buffer bulk effects and non-specific binding signals during subsequent data analysis, ensuring data accuracy.

[0095] 2. Combined with experiments

[0096] KP3298 was serially diluted (0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μM) using PBS + 0.05% (v / v) Tween-20 (pH 7.4) buffer as the run buffer. Binding time was 120 s, dissociation time was 300 s, and flow rate was 30 μL / min. The instrument monitored and recorded the binding kinetics in real time. Figure 4 This figure directly reflects the change of the response signal over time during the binding and dissociation processes, and serves as the original data basis for subsequent quantitative analysis.

[0097] 3. Data Analysis and KD Value Determination

[0098] The collected original kinetic sensorgrams were analyzed using the BIAevaluation software supplied with the BIAcore 8K system. First, the signal of the reference channel was subtracted, and then global fitting was performed using a 1:1 Langmuir binding model. The fitting curve was in good agreement with the experimental data. From this, accurate association rate constants and dissociation rate constants were calculated, and further the equilibrium dissociation constant was obtained. The data of the dissociation constant are shown in Table 2.

[0099] Table 2

[0100] This data indicates that KP3298 has a micromolar-level affinity for the full-length N4-ECD, and the affinity for the three individual domains is significantly reduced. This result shows that although the IgC2 domain may be involved in the interaction, the high-affinity binding of KP3298 depends on the full-length conformation of the Nectin-4 extracellular domain or the conformational epitope formed by multiple domains together, rather than a simple binding to any isolated domain, and also provides a benchmark and starting point for subsequent optimization.

[0101] The above are only several specific embodiments of the present invention disclosed. However, the embodiments of the present invention are not limited thereto, and any changes that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A bicyclic peptide targeting human Nectin-4 protein, characterized in that, The amino acid sequence is shown as SEQ ID NO:

1.

2. The bicyclic peptide targeted to human Nectin-4 protein of claim 1, wherein, The nucleotide sequence of the gene encoding the bicyclic peptide is shown as SEQ ID NO:

2.

3. The bicyclic peptide targeting human Nectin-4 protein of claim 1, wherein, The bicyclic peptide forms two pairs of disulfide bonds intramolecularly.

4. A method for preparing a bicyclic peptide targeting human Nectin-4 protein according to claims 1-3, characterized in that, The method comprises the following steps: S1, synthesis of a linear peptide: according to the amino acid sequence shown in SEQ ID NO: 1, corresponding Fmoc-protected amino acids are sequentially connected from the C-terminal to the N-terminal on Wang resin, the peptide chain is gradually extended, and a resin-bound protected linear peptide is obtained; S2, cleavage and crude peptide acquisition: using a cleavage reagent, the polypeptide is cut off from the resin and all side chain protection groups are removed at the same time, and a linear crude peptide is obtained by an ether precipitation method; S3, purification of the linear crude peptide: the linear crude peptide is purified, and a main peak fraction is collected to obtain a purified linear polypeptide KP3298; S4, oxidative refolding: the purified linear polypeptide is dissolved in a redox buffer system, and incubation is performed under alkaline pH conditions; S5, purification and identification of the final product: after refolding is completed, the product is further purified by using a preparative reverse-phase high-performance liquid chromatography to obtain the target bicyclic peptide, and the target bicyclic peptide is identified by mass spectrometry.

5. The method for preparing the bicyclic peptide targeting human Nectin-4 protein as described in claim 4, characterized in that, In step S2, the cleavage reagent is composed of trifluoroacetic acid, water, phenol and triisopropylsilane in a volume ratio of 88:5:5:

2.

6. The method for preparing the bicyclic peptide targeting human Nectin-4 protein as described in claim 4, characterized in that, In step S3, the instrument for purifying the linear crude peptide is a preparative reverse-phase high-performance liquid chromatography.

7. The method for preparing the bicyclic peptide targeting human Nectin-4 protein as described in claim 4, characterized in that, In step S4, the pH value of the alkaline pH is 8.

5.

8. Use of the bicyclic peptide targeting human Nectin-4 protein according to claims 1-3 in the preparation of a reagent for detecting Nectin-4 protein in vitro for non-diagnostic purposes.

9. Use of the bicyclic peptide targeting human Nectin-4 protein according to claims 1-3 in the preparation of a diagnostic agent targeting Nectin-4.

10. Use of the bicyclic peptide targeting human Nectin-4 protein according to claims 1-3 in the preparation of a therapeutic agent targeting Nectin-4.

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