Targeting egfr cyclizing polypeptide-drug conjugates for tumor immunotherapy and uses thereof

Abstract

This invention discloses a targeted EGFR cyclized peptide-drug conjugate for tumor immunotherapy and its application. The peptide-drug conjugate comprises an EGFR-targeting peptide and a first drug. The EGFR-targeting peptide contains at least one cyclized structure. The first drug, a Pt(IV) prodrug, serves as a bridging unit for at least one cyclized structure. The Pt(IV) prodrug acts as both a chemotherapeutic agent and a bridging structure constraining the peptide conformation. By combining the chemotherapeutic drug oxaliplatin with the IDO enzyme inhibitor NLG919, it exhibits a synergistic effect in inducing immunogenic death (ICD). This conjugate enables sustained release of both oxaliplatin and NLG919 in tumor cells targeting EGFR-high expression, exhibiting a dual synergistic effect of inducing ICD and inhibiting IDO.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a targeted EGFR cyclized polypeptide-drug conjugate for tumor immunotherapy and its application. Background Technology

[0002] Peptide-drug conjugates (PDCs), as an emerging targeted drug delivery method, have shown great potential in tumor immunotherapy, potentially improving efficacy and reducing side effects in cancer treatment. PDCs mainly consist of three basic components: peptides, drugs, and linkers.

[0003] Linkers in PDCs play a crucial role in the cycling time of the conjugate, the activity of the drug at the target site, and drug release. Common linkers in traditional PDCs can be broadly classified into four groups: enzyme-cleavable linkers, acid-cleavable linkers, reducible disulfide linkers, and non-cleavable linkers.

[0004] The peptides used in drug delivery systems (PDS) fall into two categories: cell-penetrating peptides and cell-targeting peptides. Cell-penetrating peptides can interact with the cell membrane and internalize it through various mechanisms; however, their application is limited due to their low cell specificity. Cell-targeting peptides can selectively bind to overexpressed receptors on target cells, delivering the conjugate drug to specific cells. However, cell-targeting peptides are mostly linear peptides, making them easily degraded in vivo and exhibiting poor cell penetration. Cyclic peptides are more stable than linear peptides; therefore, cyclizing linear peptides into cyclic peptides or stapled peptides with a higher helical structure helps constrain peptide conformation and improve their pharmacokinetic properties.

[0005] Currently, peptide cyclization mainly includes main chain cyclization and side chain cyclization. Commonly used main chain cyclization methods include: oxidative coupling of cysteine ​​to form a disulfide bond between two thiol groups after oxidation, and cycloaddition of azide and alkyne to introduce ε-azidolysine at one end of the peptide and propargylglycine at the other end, followed by Cu catalysis to generate a triazole linker. Side chain cyclization mainly involves coupling the side chains of two amino acids with rigid small molecules.

[0006] Commonly used drug molecules in tumor immunotherapy include immunogenic chemotherapeutic drugs such as the ARG inhibitor CB-1158, the adenosine A2A receptor antagonist CPI-444, and the RORγt agonist LYC-55716, as well as antibodies or chemicals that reverse the tumor immunosuppressive microenvironment, such as the IDO enzyme inhibitor NLG919. However, currently, PDCs used in tumor immunotherapy are mainly limited to single-drug research, or the efficacy is not ideal due to the physicochemical properties of the drugs. Summary of the Invention

[0007] This invention provides a targeted EGFR cyclized peptide-drug conjugate for tumor immunotherapy and its application. The targeted peptide is cyclized using the Pt(IV) prodrug Oxal(IV) with an axial ligand. On one hand, the Pt(IV) prodrug acts as a bridging structure to constrain the conformation of the targeted EGFR peptide, improving the peptide's stability and cell penetration. On the other hand, after the PDC drug is cleaved in vivo by endogenous hydrolases, a Pt(IV) complex is released. The Pt(IV) complex is then activated by reduction to become the active product Pt(II). Furthermore, by cleavably linking an IDO enzyme inhibitor to the targeted EGFR peptide, it achieves combined use with an IDO enzyme inhibitor (NLG919), exhibiting a dual synergistic effect of inducing immunogenic death (ICD) and inhibiting IDO.

[0008] According to a first aspect of the present invention, a targeted EGFR cyclized polypeptide-drug conjugate for tumor immunotherapy is provided, the polypeptide-drug conjugate comprising an EGFR-targeting polypeptide and a first drug, wherein the EGFR-targeting polypeptide contains at least one cyclized structure, and the first drug is used to form a bridging unit of at least one cyclized structure.

[0009] Epidermal growth factor receptor (EGFR) is a cell-targeting peptide belonging to the ErbB family of receptor tyrosine kinases. Through ligand binding and kinase-dependent activation, it translates various external stimuli into specific cellular responses. In pathological settings, EGFR is a driver of tumorigenesis; it is mutated or overexpressed in various cancers and is a widely used target in current clinical treatments.

[0010] Typically, in PDCs (Proton Transfer Cells), the drug and linker are two different compounds used to achieve cytotoxicity and linkage functions, respectively. In this invention, a specific drug molecule is used, with its structure modified and conjugated to a peptide, enabling it to function both as a chemotherapeutic agent inducing immunogenic death (ICD) and as a bridging structure for cyclization, thereby improving the stability and cell penetration of the EGFR-targeting peptide, enhancing the effective utilization of the drug molecule, and reducing its side effects.

[0011] In another preferred embodiment, the EGFR-targeting polypeptide is selected from any one of NH2-CMYIEALDKYAC-COOH, NH2-CQTPYYMNTC-COOH, NH2-YHWYGYTPQNVI-COOH, and NH2-LARLLT-COOH.

[0012] In another preferred embodiment, the first drug is a platinum-based drug.

[0013] In another preferred embodiment, the platinum-based drug may be selected from cisplatin, carboplatin, and oxaliplatin. Cisplatin is a square planar Pt(II) complex coordinated to two chloride and two amino groups, with the chloride ligands in the cis configuration. It is a non-specific therapeutic agent that can cause systemic toxicity with prolonged use, resulting in severe damage to normal tissues. Carboplatin, compared to cisplatin, exhibits lower hydration and greater biocompatibility, significantly reducing systemic toxicity. However, cross-resistance between carboplatin and cisplatin has been observed in many cancer types. In the platinum-based clinical drug oxaliplatin, the 1,2-diaminocyclohexane (DACH) ligand replaces the amino group of cisplatin. Similar to cisplatin, oxaliplatin forms cross-links primarily on adjacent guanine bases or between guanine and adenine, but to a lesser extent. Due to the larger DACH ligand, oxaliplatin induces different conformational distortions on DNA, thus the Pt-DNA adduct is more effective in inhibiting DNA synthesis. In addition, oxaliplatin can also fight tumors by inducing ICD in tumor cells, thereby triggering a specific anti-tumor immune response in the body and improving the efficacy of chemotherapy.

[0014] In another preferred embodiment, the bridging unit can be selected from... Any one of them.

[0015] In another preferred embodiment, the polypeptide drug conjugate further includes a second drug that is cleavably linked to the EGFR-targeting polypeptide, the second drug being selected from any one of IDO enzyme inhibitors, doxorubicin (DOX), paclitaxel (PTX), camptothecin (CPT), and vascular oxidizing agents (VDA).

[0016] In another preferred embodiment, the IDO enzyme inhibitor is selected from... Any one of them.

[0017] In another preferred embodiment, the breakable chemical bonds are selected from... Any one of them.

[0018] Carbamate bond Ester bonds can be cleaved and broken within cells or lysosomes under enzymatic conditions. and amide bond The hydrazone bond can be cleaved by esterases and amidases within cancer cells and lysosomes. acetal / ketal bonds It is pH sensitive and can break disulfide bonds in the acidic tumor microenvironment or within cells and lysosomes. They are easily broken down by the excessively reducing environment of tumor cells.

[0019] In another preferred embodiment, a spacer molecule is further connected between the second drug and the cyclized EGFR-targeting peptide, the spacer molecule being selected from either 6-Ahx or β-Ala.

[0020] Linking peptides and drugs through spacer molecules not only ensures the stability of their coupling but also increases the flexibility of the coupled molecules, facilitating self-assembly into nanoparticles.

[0021] In another preferred embodiment, the structure of the EGFR-targeting cyclized polypeptide-drug conjugate is shown in Formula 1:

[0022]

[0023]

[0024] While oxaliplatin has been proven effective against various solid tumors, its systemic toxicity and drug resistance severely limit its clinical application in platinum-based therapy. Pt(IV) prodrugs, obtained by oxidizing Pt(II) drug molecules and introducing an active ligand at its axial position, exhibit improved physicochemical and pharmacological / toxicological properties. Due to the modifiability of the axial ligand, the functions of Pt(IV) complexes are also diverse. When the Pt(II) precursor is oxidized to form a dihydroxy Pt(IV) complex, the axial ligand can retain the oxidized hydroxyl group, or both ligands can be different biologically active molecules. By cyclizing the target peptide with the Pt(IV) prodrug Oxal(IV) containing axial ligands, on the one hand, it serves as a bridging structure that constrains the peptide conformation, improving the peptide's stability and cell penetration ability, while also significantly enhancing the effective utilization rate of platinum-based drugs and reducing their side effects. On the other hand, after being cleaved in vivo by endogenous hydrolases, the Pt(IV) complex is released. The Pt(IV) complex is then activated by reduction to become the active product Pt(II), enabling its combined use with the IDO enzyme inhibitor (NLG919) to achieve a dual synergistic effect of inducing immunogenic death (ICD) and inhibiting IDO.

[0025] NLG919 was selected as an IDO enzyme inhibitor, which can bind to IDO enzymes and block their immunometabolic effects. It was then chemically coupled to an EGFR-targeting peptide with side-chain cyclization modification via a spacer molecule and ester bond. This chemical coupling effectively improved the poor water solubility, short duration of action, and rapid metabolism of NLG919, thus enhancing the efficacy of immunotherapy. The resulting EGFR-targeting bicyclic peptide-drug conjugate exhibited improved cellular uptake and proteolytic stability, ultimately demonstrating stronger apoptosis-inducing and immunogenic cell death-inducing capabilities.

[0026] According to another aspect of the present invention, the use of a targeted EGFR cyclized polypeptide-drug conjugate for tumor immunotherapy in the preparation of a drug for tumor immunotherapy is provided.

[0027] The dosage forms that the drug may be administered include, but are not limited to, injectable dosage forms, tablets, and powders.

[0028] This invention utilizes the Pt(IV) prodrug Oxal(IV) with axial ligands to cyclize EGFR-targeting peptides. On one hand, this serves as a bridging structure to constrain the peptide conformation, improving the peptide's stability and cell penetration ability, while also significantly enhancing the effective utilization rate of platinum-based drugs and reducing their side effects. On the other hand, this PDC drug is cleaved in vivo by endogenous hydrolases to release the Pt(IV) complex. The Pt(IV) complex is then activated by reduction to become the active product Pt(II), thereby achieving a killing effect on tumor cells.

[0029] This invention provides a targeted EGFR-cyclized peptide-drug conjugate for tumor immunotherapy, which combines the chemotherapeutic drug oxaliplatin with the IDO enzyme inhibitor NLG919. The Pt(IV) prodrug not only acts as a chemotherapeutic agent but also serves as a bridging structure constraining the peptide conformation. Combined with the IDO enzyme inhibitor, it exhibits a synergistic effect in inducing immunogenic cell death (ICD). This conjugate enables sustained release of both oxaliplatin and NLG919 in tumor cells targeting EGFR-overexpressing tumors, demonstrating a dual synergistic effect of inducing ICD and inhibiting IDO.

[0030] This invention uses cleavable chemical bonds to link IDO inhibitors to EGFR-targeting peptides. On the one hand, this can significantly improve the shortcomings of IDO inhibitors, such as poor biocompatibility, poor selectivity, and poor permeability. On the other hand, the release mechanism through enzymatic cleavage facilitates the slow release of IDO inhibitors, making it easier to control the dosage.

[0031] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0033] Figure 1 This is the synthetic route for the two-drug linear polypeptide-drug conjugate linear-Oxal(IV)-EBP-NLG.

[0034] Figure 2 This is the synthetic route for the bicyclic polypeptide-drug conjugate cyclic-Oxal(IV)-EBP-NLG.

[0035] Figure 3 This is a chromatogram showing the serum stability analysis of the cyclic-Oxal(IV)-EBP-NLG conjugate.

[0036] Figure 4 CRT immunofluorescence assays of different treatment groups after incubation with CT26 cells.

[0037] Figure 5 The following are flow cytometry plots of CRT after incubation with CT26 cells in different treatment groups: (a) bar chart of semi-quantitative statistical analysis of CRT fluorescence on the surface of CT26 cells; (b) distribution of average fluorescence intensity of CRT on the surface of CT26 cells; (c) bar chart of semi-quantitative statistical analysis of CRT fluorescence on the surface of MCF-7 cells; (d) distribution of average fluorescence intensity of CRT on the surface of MCF-7 cells.

[0038] Figure 6 The images show tumors in mice on day 12 of treatment in different treatment groups. The scale bar in the images is 2 cm.

[0039] Figure 7 Flow cytometry images of mature DC cells and Treg cells in tumor tissues after treatment in different treatment groups, including: (a) bar chart of the percentage of mature DC cells in the tumor area; (b) bar chart of the percentage of Treg cells in the tumor area. Detailed Implementation

[0040] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0041] Appendix Figure 1-2 The synthetic routes for the peptide-drug conjugates linear-Oxal(IV)-EBP-NLG and cyclic-Oxal(IV)-EBP-NLG targeting the EGFR receptor are shown below. The specific preparation process is as follows:

[0042] 1. Synthesis of NLG919-SA intermediate

[0043] Weigh out NLG919 (56.4 mg, 0.2 mmol), succinic anhydride (160 mg, 1.6 mmol), and DMAP (24.4 mg, 0.2 mmol) and dissolve them in DCM (20 mL). Stir for 24 hours. After the reaction is complete, remove the DCM using a rotary evaporator. Dissolve the reaction product in 5% NaHCO3 solution and extract with ethyl acetate (20 mL × 3) 2-3 times to remove unreacted succinic anhydride. Adjust the pH of the NaHCO3 solution to pH 2-3 using 1 M HCl and extract with DCM 5-6 times to ensure complete dissolution of the product in DCM. Remove the DCM using a rotary evaporator to obtain the crude product. Purify the crude product by HPLC. Collect the desired HPLC-grade product and lyophilize to obtain the NLG919-SA intermediate. Dissolve the purified product in DMSO-d6 and determine its structure using 400 M NMR spectroscopy. Further confirm the molecular weight of the product using LC-MS.

[0044] 2. Synthesis of the product EBP-NLG

[0045] The desired peptide EBP (NH2-Ahx-CMYIEALDKYAC-Resin) was successfully synthesized using solid-phase peptide synthesis (SPPS) based on the Fmoc protection strategy. The synthesis method involved weighing 400 mg of Rink MBHA resin and placing it in a reactor. The resin was swollen with DMF under a nitrogen flow for 20-30 minutes. A deprotection solution (30% DMF: 70% morpholine, v / v) was added, and the reaction was repeated twice, each time for 1 hour. After deprotection, the resin was washed three times with DMF and DCM for 30 seconds each time to remove residual deprotection solution. The desired amino acid with the Fmoc protecting group (5 eq.) and the condensing agent HATU (4.9 eq.) were weighed, mixed, and then 6 mL of DMF was added and shaken until fully dissolved. Then, 10 eq. of alkali-adjusting agent DIPEA was added to the reaction solution, mixed thoroughly, and added to the reactor after the resin had been washed in the previous steps. The reaction was repeated twice, each time for 2 hours. After the reaction, the resin was washed twice with DMF and DCM to remove excess reaction solution. The above steps were repeated until the target polypeptide sequence was synthesized. After polypeptide chain synthesis, the spacer group Ahx was coupled to the intermediate NLG919-SA using the same method as for polypeptide synthesis. The polymer was then reacted with the resin in a mixed solution of TFA / deionized water / TIS (9.5 / 0.25 / 0.25, v / v / v) on a shaker for 2 hours to cleave the polypeptide from the resin. Trifluoroacetic acid was dried under a nitrogen stream, and the mixture was centrifuged with cold diethyl ether. The supernatant was discarded, and this process was repeated three times to obtain the product EBP-NLG.

[0046] 3. Synthesis of the linear peptide-drug conjugate linear-Oxal(IV)-EBP-NLG

[0047] The linear peptide-drug conjugate linear-Oxal(IV)-EBP-NLG has the following sequence:

[0048] NLG919-SA-Ahx-AMYIEALDKYAC. After synthesizing EBP-NLG according to the above method, EBP-NLG (1 eq.) and OAc-Oxal (IV) (2 eq.) were weighed and dissolved in a mixed solvent (50% DMF: 50% 50mM NH4HCO3 aqueous solution, v / v) to control the final reaction concentration at 0.5mM. The mixture was reacted in a shaker at room temperature for 4 hours. DMF was removed by rotary evaporation, and the crude product was precipitated by adding ice-cold diethyl ether. The crude product was dissolved in a mixed solution (50% acetonitrile: 50% deionized water, v / v). After determining the peak time of the target product by LC-MS, it was purified by C18 preparative column elution with a 15-minute acetonitrile gradient from 10% to 90%. The detection wavelengths were 220 nm and 254 nm. After purification and lyophilization, a linear control product was obtained.

[0049] 4. Synthesis of the cyclic polypeptide-drug conjugate cyclic-Oxal(IV)-EBP-NLG

[0050] Cys has a thiol group in its side chain, so Cys was used to replace Ala in the linear sequence to ensure the uniqueness of the reaction site. The cyclic polypeptide-drug conjugate cyclic-Oxal(IV)-EBP-NLG has the sequence NLG919-SA-Ahx-CMYIEALDKYAC. After synthesizing EBP-NLG according to the above method, EBP-NLG (1 eq.) and Oxal(IV) (2 eq.) were weighed and dissolved in a mixed solvent (50% DMF: 50% 50mM NH4HCO3 aqueous solution, v / v) to control the final reaction concentration at 0.5mM. The mixture was reacted in a shaker at room temperature for 4 h. DMF was removed by rotary evaporation, and after precipitation with ice-cold diethyl ether, the product was dissolved in 50% acetonitrile deionized water and purified by HPLC to obtain the final product.

[0051] Meanwhile, the naked drug Oxal(II), NLG919, and the linear peptide conjugate linear-Oxal(IV)-EBP-NLG were used as controls to conduct the following experiments.

[0052] I. Investigation of Intracellular Platinum Content

[0053] The complex in vivo environment and poor stability of linear peptides lead to their rapid degradation by various proteases, resulting in low uptake. Cycling linear peptides into cyclic peptides or stapled peptides with a high helical structure helps constrain the peptide conformation and improve its permeability, thereby increasing drug uptake. This invention utilizes the Pt(IV) prodrug Oxal(IV) with axial ligands to cyclize EGFR-targeting peptides. Therefore, detecting the effect of intracellular platinum levels after drug administration on the targeting efficacy of the designed conjugate is of great significance.

[0054] CT26 cells were seeded in 6-well plates. After cell attachment and reaching a density of approximately 90%, the culture medium was discarded, and 15 μM of drug-containing culture medium was added again. Cells were cultured for another 24 hours, washed three times with PBS at 4°C, and collected by centrifugation at 2000 rpm for 5 minutes. The cells were then digested at 65°C with 3 mL of aqua regia (25% concentrated nitric acid: 75% concentrated hydrochloric acid, v / v) to remove acidity until the final volume was less than 50 μL. After cooling, the remaining cell solution was diluted to 1 mL with deionized water, filtered, diluted, and the platinum content was determined by inductively coupled plasma mass spectrometry (ICP-MS). The platinum content in CT26 cells was detected by ICP-MS after incubation with different materials, as shown in Table 1. The results showed that after 24 hours of incubation, the intracellular concentration of naked Oxal(II) was 35.04 ng Pt / 10⁻¹⁰. 6 In cells, the cyclic conjugate cyclic-Oxal(IV)-EBP contained 27.85 ng Pt / 10⁻⁶ cells of platinum. 6 The cell uptake was similar to that of the bare drug group, but 2.3 times that of the uncyclized peptide material EBP-NLG-Oxal(IV). Therefore, we hypothesize that Oxal(II) uptake occurs via passive diffusion, while after conjugation with the peptide, it mainly enters cells through receptor-mediated endocytosis. Because the uncyclized peptide is less stable and easily degraded, its uptake is lower. Cyclic peptides have increased stability and improved ability to target tumor cells, resulting in increased drug uptake.

[0055] Table 1. Platinum content in CT26 cells after 24 hours of incubation with different materials (unit: μM)

[0056]

[0057] II. Serum stability test

[0058] Compared to small molecule drugs, peptide drugs have many advantages, but their poor penetration and serum stability, as well as their susceptibility to degradation by a series of proteases in vivo, are major obstacles to peptide drug research.

[0059] To verify the serum stability of the materials, the peptides linear-Oxal(IV)-EBP-NLG and cyclic-Oxal(IV)-EBP-NLG were dissolved in PBS solution containing 20% ​​serum (FBS) at a final concentration of 100 μM and incubated at 37°C on a shaker. At time points of 0 h, 1 h, 2 h, 4 h, and 8 h, 20 μL of the sample was added to 180 μL of protein precipitation buffer. The protein precipitation buffer was prepared as a mixture of 12% trichloroacetic acid (75% ACN: 25% H2O, v / v). After vortexing for 1 minute, the diluted solution was refrigerated at 4°C for 30 minutes to precipitate serum proteins. After centrifugation at 12000 rpm for 15 minutes at 4°C, the supernatant was collected and the content of undegraded peptides was measured by HPLC. The half-life of the linear-Oxal(IV)-EBP-NLG and cyclic-Oxal(IV)-EBP-NLG materials was obtained by dividing the undegraded peptide content (expressed as peak area) at 1h, 2h, 4h, and 8h by the undegraded peptide content (expressed as peak area) at 0h and then fitting the data. The results are as follows: Figure 3 As shown, the remaining material decreases with longer incubation time with serum. Fitting the half-life curve reveals that the half-life of the cyclic peptide-drug conjugate cyclic-Oxal(IV)-EBP-NLG is 14.4 hours, which is three times longer than that of the linear peptide conjugate linear-Oxal(IV)-EBP-NLG. Therefore, it can be concluded that the cyclic peptide-drug conjugate is more stable and has higher resistance to protease hydrolysis compared to the linear conjugate.

[0060] III. Cytotoxicity Test

[0061] Five cell lines—HCT116, SW480, CT26, MCF-7, and LO2—were seeded at a density of 5000 cells / well in 96-well plates. 200 μL of medium containing 10% FBS was added, and the plates were incubated for 24 hours. Then, the medium was aspirated, and medium containing 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, and 0 μM of Oxal(II), NLG919, linear-Oxal(IV)-EBP-NLG, and cyclic-Oxal(IV)-EBP-NLG, respectively, was added. The plates were incubated for 48 hours, and the medium was aspirated. The cells were washed three times with PBS, and 10 μL of CCK-8 was added. After incubation for 1.5 hours, the OD value was measured at 450 nm using a microplate reader. Cell viability was calculated as follows:

[0062]

[0063] As shown in Table 2, after 48 hours of incubation, in all EGFR-overexpressing cell lines (HCT116, SW480, and CT26), the cytotoxicity of cyclic-Oxal(IV)-EBP-NLG was significantly stronger after cyclization and restriction of the peptide compared to the uncyclized linear group. Conversely, in MCF-7 cells with low EGFR expression, the IC50 of the Oxal(II) naked group was significantly lower. 50 At a concentration of 12.79 μM, both cyclic and linear conjugates exhibited limited cytotoxicity in MCF-7 cells. For normal hepatocytes (LO2), the bare drug group still showed strong killing activity, while the cyclic peptide-drug conjugate cyclic-Oxal(IV)-EBP-NLG achieved a cell viability of approximately 73.0% at a concentration of 12 μM. We also found that NLG919 had no killing effect on any of the involved cell types. These experimental results demonstrate that the cyclic peptide-drug conjugate cyclic-Oxal(IV)-EBP-NLG exhibits good selectivity for tumor cells with high EGFR receptor expression and minimal damage to normal cells.

[0064] Table 2 IC50 values ​​after 48 hours of incubation with different materials 50 Value (unit: μM)

[0065]

[0066] IV. Ability to induce immunogenic cell death in vitro

[0067] When cells are under stress, increased pressure in the endoplasmic reticulum causes calreticulin (CRT) to migrate from the endoplasmic reticulum to the cell membrane surface, releasing an "eat me" signal that activates the body's immune response. To verify whether the material has the ability to induce immunogenic cell death, we used fluorescence microscopy to qualitatively examine the CRT exposure on the cell surface after incubation with the material, and used flow cytometry to quantitatively examine the fluorescence intensity of CRT on the cell surface.

[0068] The specific operation process is as follows:

[0069] Calreticulin (CRT) expression: CT26 cells were expressed at a rate of 2 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / well in 6-well plates and incubated for 24 hours. The culture medium was discarded, and 15 μM of different materials were added to the medium for re-incubation. After 24 hours, the cells were washed three times with PBS at 4°C, and fixed with 4% paraformaldehyde. After 30 minutes, the paraformaldehyde was discarded, and the cells were washed three times with PBS at 4°C. Diluted CRT monoclonal antibody (1:400) was added to the wells and incubated for 1 hour. After recovering the CRT antibody solution, the cells were washed three times with PBS at 4°C and incubated with Alexa Fluor 488 fluorescently labeled secondary antibody (1:500). After 1 hour, the cells were washed three times with PBS at 4°C, and then incubated with Hoechst 33342 for 15 minutes to label the nuclei. Finally, the distribution of calreticulin on the tumor cell membrane was observed under a fluorescence microscope.

[0070] Flow cytometry detection of CRT expression on cell surface: CT26 cells were seeded at 2 × 10⁵ cells per well in 6-well plates and cultured for 24 hours. After incubation, the cells were incubated again for 24 hours with 15 μM of different materials. The culture medium was discarded, and the cells were digested from the bottom of the wells with trypsin to collect tumor cells. After washing three times with PBS at 4°C, CRT monoclonal antibody (1:100) was added and incubated for 1 hour. The cells were then centrifuged at 2000 rpm for 5 minutes at 4°C, washed three times with PBS at 4°C, and then incubated with Alexa Fluor488 fluorescently labeled secondary antibody (1:100) for 1 hour. After incubation, the cells were resuspended in PBS containing 1% FBS, filtered through a 70 μm cell filter, and analyzed by flow cytometry.

[0071] CRT immunofluorescence assay results are as follows Figure 4 As shown, incubation with Oxal(II) and the cyclized cyclic-Oxal(IV)-EBP-NLG conjugate resulted in strong green fluorescence signals on the cell surface. However, due to the poor stability of the uncyclized peptide, the fluorescence signal in the linear treatment group was weaker. No signal was detected in the NLG919 drug group, demonstrating that it does not have the ability to induce immunogenic cell death.

[0072] After qualitative observation using fluorescence microscopy, we quantitatively examined the fluorescence intensity of CRT on the cell surface using flow cytometry, such as... Figure 5 As shown, for CT26 cells, the relative fluorescence intensity of the cyclized cyclic-Oxal(IV)-EBP-NLG conjugate was 3.8 times higher than that of the linear treatment group. However, since the material did not have the ability to target MCF-7 cells, neither the linear nor the cyclized peptide-drug conjugate treatment groups showed almost any fluorescence signal.

[0073] V. Ability to induce immunogenic cell death in vivo

[0074] This study primarily investigates the ability of the prepared peptide drug conjugate to induce immunogenic death in vivo and its ability to inhibit the IDO enzyme immune metabolic pathway in the tumor microenvironment, and comprehensively evaluates its anti-tumor immunotherapy efficacy.

[0075] The establishment of the in vivo tumor model is as follows:

[0076] CT26 cells were administered at a rate of 2 × 10⁻⁶. 7 The cells were resuspended at a density of 100 cells / ml in serum-free RPMI 1640 medium, and 100 μl was subcutaneously injected into the right back of Balb / c mice.

[0077] The specific treatment plan is as follows:

[0078] After the model was established, the experimental mice were randomly divided into 5 groups and treated with different materials: (1) saline, (2) Oxal(II), (3) linear-Oxal(IV)-EBP-NLG, (4) cyclic-Oxal(IV)-EBP-NLG, and (5) combined administration of cyclic-Oxal(IV)-EBP-NLG and PD-L1 antibody. The administration method was tail vein injection of mice, the drug dose was 3 mg / kg platinum content, the administration volume was 100 μL per dose, and the administration frequency was once every 2 days. The treatment group with combined antibody was injected with 200 μg of PD-L1 antibody via tail vein injection on the second day after each administration.

[0079] In cancer treatment, combining PD-L1 antibody blockade with other therapies can synergistically enhance immune activation. PD-L1 is upregulated in various tumor cells; it binds to PD-1 on T cells, inhibiting T cell proliferation and activation, rendering T cells inactive, and ultimately inducing immune escape. PD-L1 antibodies can block the binding of PD-1 and PD-L1, upregulate T cell growth and proliferation, enhance T cell recognition of tumor cells, and activate their attack and killing functions, thereby achieving anti-tumor effects by mobilizing the body's own immune function.

[0080] like Figure 6 As shown in the in vitro tumor images on day 12, the average tumor volume in the saline group increased rapidly due to the faster proliferation rate of CT26 cells, reaching approximately 1452 mm on day 12. 3 During the first 8 days of treatment, the Oxal(II) bare-metal group showed some tumor-suppressing ability, but after day 8, it could not further inhibit tumor growth, and by day 12, the tumor volume rapidly reached approximately 929 mmHg. 3Furthermore, due to its systemic toxicity, two out of five mice died (indicated by the red dashed line in the figure). The tumor volume was smallest in the group treated with the combination of cyclic-Oxal(IV)-EBP-NLG and PD-L1 antibody, demonstrating the best anti-tumor effect. In addition, the cyclic-Oxal(IV)-EBP-NLG material group showed better therapeutic effects than the bare drug Oxal(II) due to its superior targeting ability, allowing the drug to remain at the tumor site for a long time during in vivo treatment, achieving a long-term drug release effect.

[0081] Regulatory T cells (Tregs) in the tumor microenvironment suppress the immune response by expressing related proteins or releasing cytokines, such as interleukin-10 (IL-10), leading to tumor cell escape. Therefore, it is important to detect the number of Treg cells (CD4+FOXP3+CD25+) in tumor tissues of treated mice. Figure 7 As shown, compared with the control group (26.3%) and the Oxal(IV) bare drug group (21.6%), the proportion of Treg cells in tumor tissues treated with linear-Oxal(IV)-EBP-NLG and cyclic-Oxal(IV)-EBP-NLG was significantly reduced to 16.6% and 11.7%, respectively. The average proportion of Treg cells in mice treated with cyclic-Oxal(IV)-EBP-NLG combined with aPD-L1 was 10.1%. This indicates that cyclic-Oxal(IV)-EBP-NLG can effectively alleviate immunosuppression in the tumor microenvironment.

[0082] This invention synthesizes the targeted peptide EBP using solid-phase peptide synthesis technology and couples the IDO enzyme inhibitor NLG919 to the peptide via an ester bond. Finally, the peptide is cyclized with the tetravalent oxaliplatin prodrug Oxal(IV) to prepare a dual-drug cyclized peptide-drug conjugate targeting EGFR. This dual-drug cyclized peptide conjugate, by linking to a GSH-sensitive linker, can cleave upon entering tumor cells, releasing Oxaliplatin and NLG919 for tumor treatment. The peptide is an EGFR-targeting peptide that can target and deliver drugs to tumor cells, improving the off-target toxicity of traditional chemotherapy drugs, thereby achieving precise drug delivery and reducing the systemic toxic side effects of chemotherapy drugs. The Pt(IV) prodrug not only functions as a chemotherapy drug but also serves as a bridging structure to constrain the peptide conformation. Cyclization constrains the peptide conformation, enhancing its stability and improving therapeutic efficacy. Simultaneously, the combination with the IDO enzyme inhibitor reverses the IDO enzyme-mediated immune metabolic pathway at the cellular level, relieving the inhibition of the immune microenvironment, resulting in a synergistic tumor immunotherapy effect.

[0083] Those skilled in the art will understand that, throughout this specification, unless otherwise specified, the terminology used is to be understood as it is commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0084] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0085] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A targeted EGFR-cyclized polypeptide-drug conjugate for tumor immunotherapy, characterized in that, The structure of the EGFR-targeting cyclized polypeptide-drug conjugate is shown in Formula 1: Formula 1.

2. The use of the targeted EGFR cyclized peptide-drug conjugate for tumor immunotherapy as described in claim 1 in the preparation of drugs for tumor immunotherapy, characterized in that, The tumor was selected from colon cancer or breast cancer.

3. The application according to claim 2, characterized in that, The drug is an injectable dosage form, tablet, or powder.

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