A polypeptide targeting b7h3 and preparation method and application thereof

Abstract

The application discloses a polypeptide and derivatives thereof targeting B7H3 (CD276), a drug conjugate, a pharmaceutical composition, a preparation method and a tumor diagnosis and treatment application, and belongs to the technical field of biological medicines. Existing B7H3 monoclonal antibodies and CAR-T have defects such as large molecular weight, poor penetration, high cost, easy immunogenicity and the like, and there are few targeting peptides. The application provides a peptide with a sequence of Gly ‑ Val-Pro-Tyr-Ala-Gln-Pro-Leu, the polypeptide has strong B7H3 binding specificity; derivatives can be obtained by modifying the polypeptide in various ways; the polypeptide can be coupled with a chemotherapeutic drug such as camptothecin to form a polypeptide drug conjugate. The polypeptide and the coupled drug can be prepared into a targeted preparation, are specifically enriched in B7H3 positive tumors such as pancreatic cancer, lung cancer and breast cancer, reduce drug toxic side effects, and have dual application values of diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide targeting immune checkpoint B7H3, its derivatives, polypeptide-drug conjugates, pharmaceutical compositions and their preparation methods, and also relating to their applications in tumor molecular diagnosis and the preparation of targeted anti-tumor drugs. Background Technology

[0002] B7H3 (CD276) is a novel member of the B7 immune protein family. As an important immune checkpoint molecule, its protein and mRNA expression and distribution differ significantly between normal and tumor tissues. It is widely and highly expressed in various malignant tumor tissues and is closely related to tumor progression. B7H3 not only participates in tumor immune regulation but also plays a regulatory role in non-immune functions such as tumor migration, invasion, angiogenesis, glucose metabolism, and drug resistance. It exerts a key biological function in the progression of malignant tumors and is one of the ideal targets for targeted cancer therapy.

[0003] Currently, drug development targeting B7H3 mainly focuses on monoclonal antibodies and chimeric antigen receptors (CARs). Among them, monoclonal antibodies have drawbacks such as large molecular weight, poor tissue penetration, high production cost, and easy to induce immunogenic reactions; CAR-T cell therapy has problems such as complex preparation, limited scope of application, and potential off-target effects, which limit its clinical application.

[0004] Peptide drugs possess advantages such as small molecular weight, strong tissue penetration, low immunogenicity, low production cost, and ease of synthesis and modification, making them uniquely promising for targeted drug development. However, research on peptides targeting B7H3 is currently extremely limited. Therefore, developing a B7H3-targeting peptide with high specificity and binding affinity has significant clinical value and application potential. Summary of the Invention

[0005] Based on the above, this disclosure provides a structurally simple peptide that is easy to synthesize, isolate, and purify, targeting B7H3, which exhibits specific binding to the B7H3 protein in vivo and in vitro. This disclosure also provides a peptide with good ability to target the B7H3 protein in vivo, which is enriched in tumor tissues that highly express B7H3. This disclosure further provides the aforementioned B7H3-targeting peptide with improved affinity for B7H3. The peptides of this disclosure also exhibit improved drug solubility when conjugated with a drug.

[0006] In a first aspect of this disclosure, a peptide having the following formula I that targets B7H3, a derivative thereof, or a pharmaceutically acceptable salt thereof is provided.

[0007] X1-X2-X3-X4-X5-X6-X7-X8 (Formula I)

[0008] Wherein, X1-X8 independently represent any amino acid or its derivative; and the polypeptide, its derivative or its pharmaceutically acceptable salt has a similar or better binding affinity for B7H3 relative to the polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0009] In one embodiment of this disclosure, in Formula I, X1 represents G or A, X2 represents V, I or L, X3 represents P, X4 represents Y, F or W, X5 represents A or G, X6 represents Q or N, X7 represents P, and X8 represents L, I or V.

[0010] In one embodiment of this disclosure, the amino acid sequence of the polypeptide is SEQ ID NO: 1 (Gly-Val-Pro-Tyr-Ala-Gln-Pro-Leu).

[0011] In one embodiment of this disclosure, the derivative comprises, compared to the polypeptide, any one or more modifications selected from: amino acid modification, conserved amino acid substitution, hydrogen substitution of amino acid residues, PEG modification, fatty acid modification, and glycosylation modification.

[0012] In a second aspect of this disclosure, a drug-peptide conjugate is provided, comprising a polypeptide of the present disclosure, a derivative thereof, or a pharmaceutically acceptable salt thereof, and a drug. In some embodiments, the polypeptide, its derivative, or a pharmaceutically acceptable salt thereof is conjugated to the drug directly (e.g., via a covalent bond) or indirectly (e.g., via a linker). In some preferred embodiments, the drug is selected from at least one cytotoxic agent, such as camptothecin, docetaxel, and paclitaxel.

[0013] In a third aspect of this disclosure, an isolated polynucleotide is provided that encodes a polypeptide or a derivative thereof as described in this disclosure.

[0014] In a fourth aspect of this disclosure, an expression vector is provided that expresses the polynucleotides described in this disclosure.

[0015] In a fifth aspect of this disclosure, a host cell is provided that comprises the polynucleotide or expression vector described in this disclosure.

[0016] In a sixth aspect of this disclosure, a pharmaceutical composition is provided comprising a polypeptide, a derivative thereof or a pharmaceutically acceptable salt thereof, a polynucleotide, an expression vector, a host cell or a drug-peptide conjugate, and a pharmaceutically acceptable carrier or salt thereof, as described in this disclosure.

[0017] In a seventh aspect of this disclosure, the use of the polypeptides, derivatives thereof, or pharmaceutically acceptable salts thereof for increasing the water solubility of a drug is provided. In one embodiment, the polypeptide, derivative thereof, or pharmaceutically acceptable salt thereof is conjugated directly (e.g., via a covalent bond) or indirectly (e.g., via a linker) to a drug. In some preferred embodiments, the drug is selected from at least one of cytotoxic agents, such as camptothecin, docetaxel, and paclitaxel.

[0018] In an eighth aspect of this disclosure, a method for increasing the water solubility of a drug is provided, comprising conjugating a polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof of this disclosure directly (e.g., via a covalent bond) or indirectly (e.g., via a linker). In some preferred embodiments, the drug is selected from at least one of cytotoxic agents, such as camptothecin, docetaxel, and paclitaxel.

[0019] In a ninth aspect of this disclosure, the use of polypeptides, derivatives thereof, or pharmaceutically acceptable salts, polynucleotides, expression vectors, host cells, drug-peptide conjugates, or pharmaceutical compositions according to this disclosure in the manufacture of reagents or medicaments for the detection, prevention, or treatment of diseases is provided. In some preferred embodiments, the diseases include cancer, more preferably, the cancer is a malignant tumor expressing B7H3, such as one or more of pancreatic cancer, lung cancer, breast cancer, liver cancer, gastric cancer, colorectal cancer, ovarian cancer, prostate cancer, melanoma, or glioma.

[0020] In a tenth aspect of this disclosure, a method for detecting, preventing, or treating a disease is provided, the method comprising administering to a subject in need a detectable, preventive, or therapeutically effective amount of a polypeptide, its derivative, or a pharmaceutically acceptable salt thereof, polynucleotide, expression vector, host cell, drug-peptide conjugate, or pharmaceutical composition according to this disclosure. In some preferred embodiments, the disease includes cancer, more preferably, the cancer is a malignant tumor expressing B7H3, such as one or more of pancreatic cancer, lung cancer, breast cancer, liver cancer, gastric cancer, colorectal cancer, ovarian cancer, prostate cancer, melanoma, or glioma. Attached Figure Description

[0021] Figure 1 Structure of peptide B1 targeting B7H3 protein

[0022] Figure 2 Binding diagram of peptide B1 and B7H3

[0023] Figure 3 Selection of pancreatic cancer cell lines and analysis results of cells expressing high B7H3 expression

[0024] Figure 4Structure of B1-CPT (B1-camptothecin), a peptide drug conjugate targeting the B7H3 protein

[0025] Figure 5 PDC molecules' inhibitory activity against tumor cells

[0026] Technical solution

[0027] To achieve the above objectives, the present invention adopts the following technical solution:

[0028] A peptide targeting B7H3

[0029] The amino acid sequence of the polypeptide is: Gly - Val-Pro-Tyr-Ala-GIn-Pro-Leu (Formula 1);

[0030] The polypeptide of this invention is a short peptide composed of 8 amino acids with a small molecular weight (about 1000-1100 Da). It has strong tissue penetration and can quickly penetrate tumor tissue and specifically bind to B7H3, which is highly expressed on the surface of tumor cells.

[0031] Preparation method of the B7H3-targeting peptide

[0032] The peptide was prepared using solid-phase peptide synthesis (SPPS), with the Fmoc strategy preferred. The specific steps are as follows:

[0033] 1. Resin pretreatment: Add solid-phase synthesis resin (preferably Rink Amide MBHA resin) into a solid-phase synthesizer and swell the resin with dichloromethane (DCM) for 30-60 minutes to expand the voids between resin particles and facilitate the diffusion of reactants; after swelling, remove the solvent and wash the resin with N,N-dimethylformamide (DMF) 3-5 times to remove impurities.

[0034] 2. Amino acid coupling: The first amino acid at the C-terminus of the peptide (corresponding to Leu at the C-terminus of the peptide sequence) is dissolved in DMF, activated by adding a condensing agent (preferably HBTU), and then added to a solid-phase synthesizer to bind with the pretreated resin. The reaction is carried out for 2-4 hours. After the reaction is completed, the resin is washed with DMF 3-5 times to remove unbound amino acids and condensing agents.

[0035] 3. Deprotection and condensation cycle: The Fmoc protecting group at the N-terminus of the amino acid is removed using piperidine-DMF solution (volume ratio 1:4) for 10-15 minutes. After deprotection, the resin is washed with DMF 3-5 times and the complete deprotection is confirmed by Kaiser reagent. Then, the next activated amino acid is added, and the above deprotection, washing, detection and condensation steps are repeated until all amino acids are linked in sequence.

[0036] 4. Peptide cleavage and crude peptide extraction: The synthesized peptide resin is added to the reaction vessel, and pre-cooled cleavage buffer (TFA:TIS:H2O = 95:2.5:2.5, volume ratio) is added. The mixture is stirred for 2-3 hours to cleave the peptides from the resin. After cleavage, the resin is removed by filtration, and the resin is washed with a small amount of TFA to recover the residual peptides. The cleavage buffer is concentrated to a small volume, and methyl tert-butyl ether is added to precipitate the peptides. After centrifugation and washing, the crude peptides are obtained.

[0037] 5. Purification: The crude peptide was purified by preparative high performance liquid chromatography (HPLC) using a C18 column and an acetonitrile-0.1% trifluoroacetic acid aqueous solution as the mobile phase. Gradient elution was performed (acetonitrile volume fraction increased from 10% to 60%, elution time 30 minutes). The target peak was collected, and the molecular weight of the target peptide was confirmed by mass spectrometry. Qualified components were collected.

[0038] 6. Freeze-drying: After sterile filtration of the purified peptide solution, freeze-dry it in a freeze dryer to obtain a high-purity white powder targeting B7H3 peptide; the freeze-dried peptide is sealed in a package and stored in a low-temperature drying environment at -20℃.

[0039] Furthermore, in steps 2 and 3, the condensing agent can also be DCC, DIC, etc., and the activation time is 30-60 minutes; in step 5, the flow rate of HPLC purification is 1-2 mL / min, and the detection wavelength is 220 nm; the purity of the purified peptide is ≥95%.

[0040] This preparation method employs the Fmoc solid-phase synthesis strategy, which features mild reaction conditions, simple operation, and rapid and efficient synthesis of target peptides. Furthermore, through optimized cleavage and purification processes, high-purity peptide products can be obtained, meeting the requirements of clinical applications while reducing production costs and facilitating large-scale production.

[0041] Application of the B7H3-targeting peptide and composition

[0042] 1. Application in tumor diagnosis: The B7H3-targeting peptide is conjugated with diagnostic reagents (such as fluorescent dyes, radionuclides, enzyme markers, etc.) to prepare a tumor diagnostic agent. This agent can be injected into the body via intravenous injection, subcutaneous injection, etc., and specifically binds to B7H3 on the surface of tumor cells. Through fluorescence imaging, radionuclide imaging and other technologies, early diagnosis, localization, staging and efficacy monitoring of tumors can be achieved.

[0043] The tumors mentioned include, but are not limited to, malignant tumors that express B7H3, such as lung cancer, breast cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, melanoma, and glioma.

[0044] 2. Application in tumor treatment: The B7H3-targeting peptide can be used alone as a therapeutic drug, or as a targeted carrier to deliver chemotherapy drugs, immunomodulators, toxins and other therapeutic agents to tumor cells, thereby achieving targeted tumor treatment; through the specific binding of the peptide to B7H3, the therapeutic agent is enriched in the tumor tissue, increasing the local drug concentration, enhancing the therapeutic effect, while reducing damage to normal tissues and reducing toxic side effects.

[0045] Beneficial effects

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. High targeting specificity: The peptides of this invention are designed with specific amino acid sequences to specifically recognize and bind to B7H3, which is highly expressed on the surface of tumor cells.

[0048] 2. Simple preparation and low cost: It is prepared by solid-phase peptide synthesis, which is simple to operate and has mild reaction conditions. It can be mass-produced and the production cost is much lower than that of monoclonal antibodies and CAR-T cells, making it easy to promote and apply in clinical practice.

[0049] 3. Wide range of applications: It can be used for the diagnosis, treatment and prevention of various malignant tumors expressing B7H3, and can work synergistically with chemotherapy drugs, immunomodulators and other agents to further improve the treatment effect, and has broad clinical application prospects. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments, but these descriptions do not limit the scope of protection of the present invention.

[0051] Example 1: Preparation of B7H3-targeting peptide (SEQ ID NO: 1)

[0052] 1. Resin pretreatment: Take 1g of Rink Amide MBHA resin, add it to the solid phase synthesizer, add 10mL of DCM, and swell for 40 minutes; dry the DCM, and wash the resin 4 times with 10mL of DMF, 5 minutes each time.

[0053] 2. Amino acid fixation: Dissolve 0.5 mmol Fmoc-Leu-OH in 5 mL DMF, add 0.5 mmol HBTU as a condensing agent, and activate for 40 minutes; add the activated amino acid solution to the solid phase synthesizer and react with the resin for 3 hours; after the reaction, wash the resin 4 times with 10 mL DMF, 5 minutes each time.

[0054] 3. Deprotection and condensation cycle: Add 10 mL of piperidine-DMF solution (volume ratio 1:4) to remove the Fmoc protecting group and react for 12 minutes; drain the solution and wash the resin 4 times with 10 mL of DMF; add Kaiser reagent to detect that the resin turns blue, confirming complete deprotection; then add activated Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Pro-OH, Fmoc-VaI-OH, and Fmoc-Gly-OH in sequence, and repeat the above deprotection, washing, detection, and condensation steps, with each condensation reaction lasting 3 hours, until all amino acids are linked.

[0055] 4. Peptide lysis and crude peptide extraction: Transfer the peptide resin to a reaction vessel, add 20 mL of pre-cooled lysis buffer (TFA:TIS:H2O = 95:2.5:2.5), and stir for 2.5 hours; remove the resin by filtration, wash the resin twice with 5 mL of TFA, and combine the lysis buffers; concentrate the lysis buffer to 2 mL in a rotary evaporator, add 40 mL of methyl tert-butyl ether, shake well, let stand for 10 minutes, centrifuge (5000 rpm, 10 minutes), and discard the supernatant; wash the precipitate twice with 20 mL of methyl tert-butyl ether, centrifuge, and obtain the crude peptide.

[0056] 5. Fine purification: The crude peptide was purified by preparative high-performance liquid chromatography (HPLC) using a C18 column. Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was acetonitrile. The flow rate was 1.5 mL / min, and the detection wavelength was 220 nm. The gradient elution program was 0-30 minutes, with the volume fraction of mobile phase B increasing from 10% to 60%. The target peak was collected, and the molecular weight was confirmed to be 1274 Da (consistent with the theoretical molecular weight) by mass spectrometry.

[0057] 6. Freeze-drying and storage: After sterile filtration of the purified polypeptide solution, freeze-dry it in a freeze dryer to obtain a white powder polypeptide with a purity of over 95%; after sealing and packaging, store it in a low-temperature drying environment at -20℃.

[0058] Example 2: Detection of binding activity of B7H3-targeting peptides

[0059] Instruments: Fortebio Octet Red96 biolayer interferometer, 96-well black microplate, SA (streptavidin) biosensor, centrifugal ultrafiltration tube.

[0060] Core reagents: recombinant human B7H3 extracellular fragment protein (manufacturer: ACRO, catalog number: B73-H5253), B7H3-targeting peptide, PBS-T buffer (pH 7.4, containing 0.05% Tween-20), blocking buffer (PBS-T + 1% BSA), regeneration buffer (10mM Glycine-HCl, pH 2.0), and neutralization buffer (PBS-T, pH 7.4).

[0061] Reagent pretreatment: Recombinant B7H3 protein was desalted by dialyzing with PBS-T buffer, concentrated to 1 mg / mL by centrifugation and ultrafiltration, and aliquoted at -80℃ for storage; diluted to 100 μM with PBS-T, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4℃ for later use.

[0062] Sensor preprocessing

[0063] Take the biosensor and equilibrate it at room temperature for 30 minutes; immerse the sensor in PBS-T buffer and hydrate for 10 minutes to remove the protective solution on the sensor surface and avoid dry film damage to the sensing layer.

[0064] Peptide Immobilization

[0065] 96-well plate setup: Add 200 μL of biotinylated B7H3-targeting peptide solution to each well in columns 1-6; add 200 μL of PBS-T as a blank control to columns 7-8.

[0066] Immobilization procedure: The hydrated sensor was transferred into the peptide solution well and immobilized for 1200 s at room temperature (25℃) and oscillation rate of 1000 rpm. The signal response (nm) was monitored in real time.

[0067] Sealing treatment: After immobilization, the sensor is moved into the sealing liquid pore and sealed for 600s to seal the unbound streptavidin sites on the sensor surface and reduce non-specific adsorption.

[0068] Baseline balance

[0069] After sealing, the sensor was transferred into a well containing PBS-T buffer and equilibrated for 300 seconds to establish a stable baseline.

[0070] Binding-dissociation kinetics detection

[0071] Analyte concentration gradient setup: Recombinant B7H3 protein was serially diluted to different concentrations using PBS-T, 200 μL for each concentration. The calculated affinity Kd value was 396 nM.

[0072] Example 3: Preparation of B7H3-targeting peptide drug conjugates

[0073] Weigh 1g of Rink Amide MBH resin with a degree of substitution of 0.2mmol / g and add it to the peptide solid-phase reactor. Add DCM and continuously bubble with nitrogen to swell for 10 minutes. Dry the mixture under vacuum, then add 0.2ml of methanol and react for 15 minutes. Dry the mixture under vacuum and wash it with DMF 6 times.

[0074] Fmoc was removed twice using a commercially available 20% pyridinium / DMF solution (100ml / 400ml). The reaction was carried out under nitrogen purging at room temperature for 10min and 5min respectively. After the reaction, the resin was washed 6 times with DMF. The resin was then tested with a commercially available ninhydrin:phenol:pyridinium (2:1:1) solution for 30-60s. If the resin turned dark blue, it proved that Fmoc had been successfully removed.

[0075] Then, the following amino acids were added in sequence: Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Val-OH, and Fmoc-Gly-OH.

[0076] CPT (camptothecin, manufacturer: Anaiji Chemical, catalog number: E080008; 348.36 mg, 1.00 mmol) and succinic anhydride (manufacturer: Anaiji Chemical, catalog number: A01079256; 300 mg, 3.00 mmol) were placed in a dry round-bottom flask, and 30 mL of anhydrous dichloromethane was added to dissolve them completely. After cooling the reaction system to 0°C, 1,8-diazabicycloundec-7-ene (manufacturer: Anaiji Chemical, catalog number: D060436; DBU, 0.48 mL, 3.20 mmol) was slowly added dropwise under stirring. After the addition was complete, stirring was continued at 0°C for 15 min, followed by natural warming to room temperature and reacting in the dark for 4 h. After the reaction was complete, 25 mL of ultrapure water was added to quench the reaction, and the pH was adjusted to 2.0 using 1% hydrochloric acid aqueous solution. The brown precipitate was collected by suction filtration and washed three times with 1% hydrochloric acid aqueous solution. The crude product was purified by recrystallization from methanol, and the solvent was evaporated under reduced pressure to obtain a pale yellow modified product—CPT-COOH.

[0077] Subsequently, 300 mg of CPT-COOH, 1.09 g of EDC.Cl, 0.956 g of HBTU and 40 mg of DMAP were weighed and dissolved in 10 ml of DMF. The dissolved mixture was added to the peptide synthesis tube prepared above and reacted for 120 minutes. After the reaction was completed, the K-test was used to check whether the reaction was complete.

[0078] Using 7.6 ml of commercially available TFA, 0.2 ml of H2O, and 0.2 ml of commercially available Tis as lysis buffer, the B1-CPT protective peptide resin and lysis buffer were placed together in a centrifuge tube and reacted for 1.5-2 h. After precipitation with ice-cold ether and centrifugation three times, a yellow solid was obtained. The solid was dried in a vacuum drying oven to obtain the crude product. The crude product was purified by HPLC and lyophilized to obtain a pale yellow powder peptide B1-CPT.

[0079] Example 4: Selection of Cell Lines

[0080] This study first found through pan-cancer expression analysis that B7H3 was significantly highly expressed in pancreatic ductal adenocarcinoma (p = 2 × 10⁻⁶). -1 4) The difference was significantly higher than that of other tumor types such as glioblastoma and renal cell carcinoma. Survival analysis showed that pancreatic cancer patients with high B7H3 expression had significantly shorter overall survival, suggesting that it could serve as a potential prognostic marker for pancreatic cancer. The Panc 02.13 cell line showed the highest expression level, while the MIA PaCa-2 cell line showed a lower expression level, and the RNA and protein expression trends of most cell lines were consistent.

[0081] Example 5: Activity assay of B7H3-targeting peptide drug conjugate

[0082] Cells in the logarithmic growth phase were digested, counted, and prepared into 1×10⁻⁶ cells. 5 Cell suspension of 100 μL / well was seeded into 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, different concentrations of camptothecin or peptide conjugate (B1-CPT) were added, with negative control and blank groups established. Each group had 5 replicates. The plates were incubated for 72 hours. After incubation, 10 μL of CCK8 solution was added to each well, and the cells were incubated for another hour. The absorbance was measured at 450 nm, and the proliferation inhibition rate was calculated. The results showed that the B7H3-targeting peptide drug conjugate significantly inhibited tumor cell activity and exhibited target selectivity.

[0083] To further verify its efficacy, the activity of the B7H3-targeting peptide drug conjugate was tested in different types of tumor cells. The results showed that it had a significant inhibitory effect on tumor cell proliferation in human gastric cancer Fu97 and SNU484 cells, human breast cancer MDA-MB-436 cells, human lung cancer H1819 cells, and human prostate cancer LNCap cells.

[0084] Table 1: Control of PDC activity against Fu97 cells

[0085] 10μM 93.94±1.84 93.03±1.17 5μM 85.75±2.33 90.18±1.66 2.5μM 81.25±1.134 89.94±2.78 1.25μM 76.57±3.22 80.33±2.46 0.625μM 66.99±3.13 70.77±3.43 013125μM 59.43±3.22 62.49±2.69 0.156μM 52.68±1.95 49.39±5.35 0.078μM 37.27±2.32 31.89±1.49 0.039μM 22.29±5.74 13.33±4.26 0.019μM 1.17±1.21 3.25±2.45 IC50(μM) 0.211μM 0.207μM

[0086] Table 2: Control of PDC activity against SNU484 cells

[0087] 10μM 94.379±1.013 94.129±1.877 5μM 91.724±0.751 90.6±2.716 2.5μM 86.196±1.372 85.696±2.064 1.25μM 83.854±1.237 82.886±1.988 0.625μM 76.796±3.394 76.64±2.417 0.3125μM 67.114±2.209 69.8±3.081 0.156μM 60.119±2.182 56.34±3.233 0.078μM 44.941±3.092 50.75±1.557 0.039μM 32.386±2.762 30.606±3.358 0.019μM 16.021±5.8 21.549±5.746 IC50 0.1143μM 0.1054μM

[0088] Table 3: Control table of PDC cell viability against MDA-MB-436 cells

[0089]

[0090]

[0091] Table 4: Control Table of PDC Cell Viability in H1819 Cells

[0092] 10μM 91.927±1.012 90.063±1.34 5μM 88.836±1.498 80.948±0.843 2.5μM 81.783±1.624 74.202±1.629 1.25μM 66.206±6.557 58.342±3.492 0.625μM 56.032±2.803 50.378±2.631 0.3125μM 49.64±4.009 41.066±2.617 0.156μM 16.387±4.502 14.676±4.135 0.078μM 6.963±2.393 4.607±4.991 0.039μM 1.591±2.401 2.994±1.733 0.019μM .1.591±2.624 -2.896±1.699 lC50 0.529μM 0.75μM

[0093] Table 5: Control Table of PDC on LNCap Cell Viability

[0094] 10μM 92.903±2.987 85.375±1.959 5μM 85.666±3.264 75.754±2.79 2.5μM 66.199±3.606 63.391±2.981 1.25μM 59.158±5.904 55.233±2.635 0.625μM 21.234±4.113 39.625±2.675 0.3125μM 13.324±4.026 29.41±2.787 0.156μM 7.349±2.579 16.408±2.663 0.078μM 1.851±2.048 7.77±1.608 0.039μM 0.898±5.087 4.296±1.617 0.019μM -2.496±1.857 -0.686±4.429 IC50 1.271μM 1.104μM

[0095] Example 6: In vivo efficacy study

[0096] In Panc 02.13 tumor cell subcutaneous xenograft mice (nude mice purchased from Changzhou Cavens Laboratory Animal Co., Ltd.), 5 x 10 6 Panc 02.13 cells were introduced subcutaneously into nude mice, and the tumor volume was increased to 100 mm². 3 Around 10:00 AM, the drug was administered via the tail vein at a dose of 3 mg / kg (calculated based on the camptothecin dose), once every two days. The results showed that the peptide drug conjugate significantly inhibited the growth rate of the tumor and was more effective than camptothecin.

[0097] Table 6: Comparison of in vivo efficacy of PDC and camptothecin

[0098]

[0099]

Claims

1. A polypeptide and its derivatives or a pharmaceutically acceptable salt thereof, having an amino acid sequence as shown in Formula I: X1-X2-X3-X4-X5-X6-X7-X8 Equation I in, X1-X8 can independently represent any amino acid or its derivative. X1 represents G or A. X2 represents V, I, or L. X3 represents P. X4 represents Y, F, or W. X5 represents A or G. X6 represents Q or N. X7 represents P. X8 represents L, I, or V.

2. The polypeptide and its derivatives or a pharmaceutically acceptable salt thereof according to claim 1, wherein the amino acid sequence of the polypeptide is SEQ ID NO:

1.

3. The polypeptide, its derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-2, wherein the derivative comprises, compared with the polypeptide, any one or more modifications selected from: amino acid modification, conserved amino acid substitution, hydrogen substitution of an amino acid residue, PEG modification, fatty acid modification, glycosylation modification.

4. The polypeptide and its derivatives or pharmaceutically acceptable salts according to claims 1-3, wherein the polypeptide has good binding ability with B7H3.

5. A drug-peptide conjugate comprising a polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, and a drug, wherein the polypeptide, its derivative, or a pharmaceutically acceptable salt thereof is conjugated to the drug directly or via a linker; the drug is selected from at least one of cytotoxic agents, chemotherapeutic agents, immunomodulators, or toxins, such as camptothecin, docetaxel, and paclitaxel.

6. A pharmaceutical composition comprising a polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical-peptide conjugate according to any one of claims 1-4, and a pharmaceutically acceptable carrier or diluent.

7. The pharmaceutical composition according to claim 6, wherein the dosage form of the pharmaceutical composition is an injection, a lyophilized powder for injection, an oral preparation, a topical preparation, or a sustained-release preparation; and the pharmaceutically acceptable carrier or diluent is selected from one or more of physiological saline, phosphate buffer, glucose solution, mannitol, lactose, starch, and magnesium stearate.

8. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition further comprises one or more of a chemotherapeutic agent, an immunomodulator, or a toxin.

9. The use of the polypeptide, its derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 in the preparation of a tumor diagnostic agent, wherein the diagnostic agent is prepared by coupling the polypeptide, its derivative or a pharmaceutically acceptable salt thereof with a detectable marker selected from fluorescent dyes, radionuclides or enzyme markers; the diagnostic agent is used for early diagnosis, localization, staging or efficacy monitoring of tumors.

10. The use of the polypeptide, its derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, the drug-peptide conjugate according to claim 5, or the pharmaceutical composition according to any one of claims 6-8 in the preparation of a medicament for detecting, preventing or treating tumors, said tumors including pancreatic cancer, lung cancer, breast cancer, liver cancer, gastric cancer, and colorectal cancer.

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