Polypeptide drug conjugate, drug delivery system, drug combination system for tumor immunotherapy and applications thereof

Abstract

The present invention discloses a polypeptide drug conjugate, a drug delivery system, a drug combination system and an application for synergistically enhancing tumor immunotherapy. The structural formula of the polypeptide drug conjugate is: P-L-D, wherein P is a polypeptide, D is a drug, and L is a cleavable chemical linker. The polypeptide is a pro-apoptotic polypeptide, and the drug is an indoleamine 2,3-dioxygenase (IDO) inhibitor. The polypeptide drug conjugate is prepared into nanoparticles by simple self-assembly and can be used for multiple tumor immunotherapies in vivo and in vitro. On the one hand, it can achieve the cleavage of tumor cells by the pro-apoptotic polypeptide and induce immunogenic cell death. At the same time, the combined IDO enzyme inhibitor can reverse the IDO enzyme-mediated immune metabolic pathway at the cellular level to relieve the inhibition of the immune microenvironment, and has a synergistic and enhanced tumor immunotherapy effect, and has good application prospects in the field of drug delivery.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a polypeptide drug conjugate, a drug delivery system and an application for synergistically enhancing tumor immunotherapy. Background Art

[0002] At present, the clinical treatment of tumors is not only limited to directly killing tumor cells. Due to the existence of multiple immune escape pathways, tumors still have a very high probability of recurrence and metastasis after treatment. Therefore, further strengthening tumor immunotherapy is an emerging means of tumor treatment.

[0003] Existing tumor immunotherapies mainly enhance the further clearance of residual tumor cells by the body's immune system by using oncolytic viruses, upregulating the body's immune system response, enhancing the induction of immunogenic cell death, or combining with other tumor-killing means.

[0004] An oncolytic virus is an artificial virus that can selectively proliferate and infect specific tumors and kill tumor cells after genetic engineering modification. Due to the mutability of tumors, their cell surfaces often contain variant receptors. The artificially gene-edited oncolytic virus expresses specific viral surface proteins on its surface that can selectively bind to the receptors on the surface of tumor cells to achieve specific receptor-ligand binding, enabling its specific proliferation in tumors, and directly lysing tumor cells to trigger apoptosis or immunogenic death of tumor cells, inducing the release of immunogenic substances such as tumor-associated antigens, inflammatory cytokines, and chemokines to trigger the body's anti-tumor immune response to achieve immunotherapy of tumors.

[0005] Indoleamine 2,3-dioxygenase IDO is a cytoplasmic enzyme that is widely expressed in mammalian tissues and cells, especially lymphoid tissues and placental tissues. It is the only enzyme outside the liver that catalyzes the oxidative cleavage of the indole in the tryptophan molecule. This enzyme can catalyze the conversion of L-tryptophan (Trp) into kynurenine (Kyn). Among them, kynurenine has the effect of inhibiting natural killer cells (NKs) and antigen-presenting cells (APCs) including dendritic cells, monocytes, and macrophages. At the same time, kynurenine also inhibits the proliferation of effector T cells by promoting the proliferation of regulatory T cells (Tregs), inducing apoptosis of T cells. After inhibiting the IDO enzyme, it is expected to upregulate and restore the suppressed immune system and restore some of the immunosuppressive pathways mediated by this pathway.

[0006] Immunogenic cell death (ICD) refers to the process in which cells release danger signal-related molecular patterns after death under the action of radiotherapy, photothermal therapy, photodynamic therapy, or certain chemotherapeutic drugs. Its typical characteristics include the exposure of calreticulin (CRT) on the cell surface, the secretion of high-mobility group protein B1 (HMGB1), and ATP. Inducing immunogenic cell death can not only directly kill tumor cells but also stimulate specific immune responses against tumors. On the one hand, it enhances the immune system response of the body to improve the treatment effect, and on the other hand, it forms long-term immune memory to prevent tumor recurrence and metastasis.

[0007] However, there are certain deficiencies in the above-mentioned various tumor immunotherapy methods, which are specifically reflected in: ① Due to the uniqueness of the virus, oncolytic viruses have high immunogenicity, but the immune effects they trigger tend to be more inclined to the immune pathways that clear the virus. Therefore, the stronger the immunogenicity of oncolytic viruses, the stronger the antiviral immune effects they induce; moreover, the penetration ability of oncolytic viruses is weak, which is very limited for the infiltration treatment effect of deep tumors; ② IDO enzyme inhibitors have problems such as poor water solubility, short action time, and fast metabolism, which severely limit their application in clinical treatment; ③ Relying solely on the immunogenic substances released by inducing immunogenic cell death is far from enough. Due to the existence of multiple immunosuppressive mechanisms, the immune system response is still hindered.

[0008] Therefore, it is very necessary to develop a drug carrier that can jointly enhance immune activation and improve the effect of immunotherapy. Summary of the Invention

[0009] To solve the deficiencies of the above-mentioned tumor immunotherapy methods, the present application provides a polypeptide drug conjugate, a drug delivery system, and an application thereof with dual functions of inducing immunogenic cell death and inhibiting IDO. The IDO enzyme inhibitor NLG919 is conjugated with the apoptosis-promoting polypeptide KLA through an ester bond to form a polypeptide drug conjugate. On the one hand, it can not only improve the biocompatibility of the IDO enzyme inhibitor, but more importantly, after the apoptosis-promoting polypeptide KLA directly kills tumors and induces immunogenic cell death to release immunogenic signal molecules to induce immune responses, it can also block the immunosuppression mediated by the IDO enzyme pathway through the NLG919 drug, achieving the effect of synergistic immune enhancement.

[0010] According to the first aspect of the present invention, there is provided a polypeptide drug conjugate for synergistically enhancing tumor immunotherapy. The structural formula of the polypeptide drug conjugate is: P-L-D, where P is a polypeptide, D is a drug, and L is a cleavable chemical linker. The polypeptide is an apoptosis-promoting polypeptide, and the drug is an indoleamine 2,3-dioxygenase (IDO) inhibitor.

[0011] The synergistic enhanced tumor immunotherapy in the present invention is mainly manifested as having a dual synergistic effect of immunogenic death induction and IDO inhibition. Among them, the apoptosis-promoting polypeptide in the polypeptide drug conjugate can not only cause the swelling and permeabilization of mitochondria, leading to the release of apoptotic proteins such as cytochrome C, thereby inducing apoptosis of tumor cells, but more importantly, it has the potential ability to induce immunogenic death. And by side-chain stapling of the apoptosis-promoting peptide, not only can the stability and permeability of the polypeptide be improved, but also the ability to induce immunogenic death can be significantly enhanced. The IDO enzyme inhibitor can up-regulate and restore the inhibited immune system, reverse the IDO enzyme-mediated immune metabolic pathway at the cellular level, achieve dual combination to strengthen immune activation, and improve the immunotherapy effect. In addition, using a cleavable chemical bond to connect the IDO inhibitor to the apoptosis-promoting polypeptide chain can, on the one hand, significantly improve the deficiencies of poor biocompatibility and poor permeability of the IDO inhibitor, and on the other hand, the release mechanism through enzymatic cleavage is conducive to the slow release of the IDO inhibitor, prolong its action time, and achieve a long-term inhibitory effect. Using the chemical bond coupling method instead of the encapsulation method can not only improve the loading rate, but also has an accurate molecular weight, which is convenient for better controlling the dosage of the drug.

[0012] In another preferred example, the apoptosis-promoting polypeptide is KLA, with the sequence of KLAKLAKKLAKLAK; the cationic amphiphilic oncolytic peptide LTX-315, with the sequence of KKWWKKW-Dip-K; the fluorinated modified mitochondrial disrupting polypeptide MDHPs, with the structure of The Ano-3 / 3s oncolytic polypeptide, with the structure of

[0013] The IDO enzyme inhibitor can be selected from any one of

[0014] In another preferred example, the cleavable chemical linking bond is selected from any one of

[0015] The carbamate bond can be cleaved and broken in cells or lysosomes under enzymatic conditions, the ester bond and the amide bond can be cleaved and broken by esterases and amidases in cancer cells and lysosomes, the hydrazone bond and the acetal bond / ketal bond are pH-sensitive and can be broken in the acidic tumor microenvironment or in cells and lysosomes, and the disulfide bond is easily cleaved and broken by the overly reduced environment in tumor cells.

[0016] In another preferred example, the apoptosis-promoting polypeptide is a stapled peptide with a side-chain cyclized structure.

[0017] Due to problems such as poor stability, short half-life, and insufficient uptake of polypeptides, the enzymatic and chemical stability of peptides can be improved through chemical modification, including but not limited to cyclizing the hydrophobic side chains of amino acids at positions i, i+4, i+7, and i+11 in the polypeptide sequence to constrain the conformation of the polypeptide, which is used to enhance the secondary structure of the peptide, including but not limited to α-helix and β-turn.

[0018] In another preferred embodiment, a spacer molecule is further connected between the polypeptide and the drug, and the spacer molecule is selected from any one of 6-Ahx and β-Ala.

[0019] Connecting the polypeptide and the drug through a spacer molecule can not only achieve the stability of their conjugation, but also increase the flexible structure of the conjugate molecule, facilitating self-assembly to form nanoparticles.

[0020] In another preferred embodiment, the structure of the polypeptide drug conjugate is shown in Formula 1:

[0021]

[0022] NLG919 is selected as the IDO enzyme inhibitor, which can bind to the IDO enzyme and block its immunometabolic effect. It is conjugated to the pro-apoptotic polypeptide sKLA with side-chain cyclization modification through a spacer molecule and an ester bond. The chemical conjugation method effectively improves the problems of poor water solubility, short action time, and fast metabolism of NLG919, and enhances the effect of immunotherapy. In addition, the KLA polypeptide is modified with a hydrophobic side chain, and the conjugated polypeptide shows improved cell uptake and proteolytic stability, and finally can show stronger apoptosis induction and immunogenic death induction ability.

[0023] According to another aspect of the present invention, a drug delivery system prepared from the polypeptide drug conjugate is provided, and the drug delivery system includes self-assembling the polypeptide drug conjugate to form oncolytic virus-like nanoparticles.

[0024] The conjugate is formed into nanoparticles by a simple self-assembly method, which is convenient for the nanoparticles to penetrate tumors and induce immunogenic death and up-regulate and restore the inhibited immune system, achieving dual combined enhanced immune activation, thereby realizing intravenous systemic circulation administration.

[0025] In another preferred embodiment, the particle size of the nanoparticles is 150 nm - 180 nm.

[0026] According to another aspect of the present invention, a drug combination system for tumor immunotherapy is provided, and the drug combination system includes the drug delivery system and a PD-L1 antibody.

[0027] Combined therapy in tumor treatment by combining PD-L1 antibody blockade synergistically enhances the efficacy of immune activation. PD-L1 is upregulated in a variety of tumor cells. It binds to PD-1 on T cells, inhibits T cell proliferation and activation, renders T cells in an inactivated state, and ultimately induces immune escape. PD-L1 antibodies can block the binding of PD-1 and PD-L1, upregulate the growth and proliferation of T cells, enhance the recognition of tumor cells by T cells, activate their attack and killing functions, and achieve anti-tumor effects by mobilizing the body's own immune function. That is, both PD-L1 and NLG919 can act on T cells, and the combination of the two can enhance the immune response and improve the curative effect.

[0028] According to another aspect of the present invention, there is provided the use of a polypeptide drug conjugate in the preparation of a drug for tumor immunotherapy.

[0029] The dosage forms that the drug can be formulated into include, but are not limited to, injectable dosage forms, tablets, and powders.

[0030] The polypeptide drug conjugate provided by the present invention is a polypeptide drug conjugate constructed by conjugating the apoptosis-promoting polypeptide KLA and the IDO enzyme inhibitor NLG919 through an ester bond with a peptide chain. On the one hand, it can significantly improve the deficiencies of poor biocompatibility and poor permeability of NLG919. On the other hand, under the action of various lysing enzymes in tumor cells, it can achieve the cleavage of tumor cells by the KLA polypeptide and induce immunogenic death. At the same time, the combined release of the IDO enzyme inhibitor NLG919 reverses the IDO enzyme-mediated immune metabolic pathway at the cellular level, having a synergistic and enhanced tumor immunotherapy effect. Moreover, by performing hydrophobic side chain stapling on the apoptosis-promoting peptide KLA, the stability and permeability of the polypeptide can be improved, and the ability to induce immunogenic death can be enhanced. The obtained stapled polypeptide drug conjugate has the strongest synergistic and enhanced tumor immunotherapy effect.

[0031] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above and other purposes, features, and advantages of the present invention more obvious and understandable, the following specifically illustrates the specific embodiments of the present invention. Brief Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts. In the drawings:

[0033] Figure 1 It is a synthetic route diagram of the polypeptide NLG919 conjugate KLA-NLG.

[0034] Figure 2 It is a synthetic route diagram of the stapled peptide NLG919 conjugate sKLA-NLG.

[0035] Figure 3 It is a circular dichroism spectrum characterization diagram of the polypeptide KLA and the stapled peptide sKLA.

[0036] Figure 4 It is a diagram of the DLS and TEM detection results of the nanoparticles prepared from the sKLA-NLG conjugate.

[0037] Figure 5 It is a diagram of the TEM detection results of the mitochondrial morphology after incubation of different treatment groups with 4T1 cells.

[0038] Figure 6 It is an immunofluorescence detection diagram of CRT and HMGB1 in cells after incubation of different treatment groups with 4T1 cells.

[0039] Figure 7 It is a flow cytometry statistical diagram of the contents of CRT, HMGB1 and ATP in cells after incubation of different treatment groups with 4T1 cells.

[0040] Figure 8 It is a detection diagram of the content of kynurenine in the cell supernatant after incubation of different treatment groups with 4T1 cells.

[0041] Figure 9 It is a diagram of the bilateral tumor sizes 21 days after treating mice with different treatment groups, and the scale bar is 2 cm.

[0042] Figure 10 It is a microscopic diagram of CRT staining of mouse tumor tissue sections after treatment with different treatment groups

[0043] Figure 11 It is a microscopic diagram of HMGB1 staining of mouse tumor tissue sections after treatment with different treatment groups. Detailed implementation manners

[0044] Hereinafter, the exemplary embodiments of the present disclosure will be described in more detail with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present disclosure can be more thoroughly understood and the scope of the present disclosure can be completely conveyed to those skilled in the art.

[0045] Example 1

[0046] This example mainly provides the specific structures and preparation methods of the linear polypeptide NLG919 conjugate KLA-NLG and the stapled peptide NLG919 conjugate sKLA-NLG with dual functions of immunogenic cell death induction and IDO inhibition.

[0047] The specific preparation process is as follows:

[0048] 1. Synthesis method of NLG919 intermediate NLG-SA

[0049] Weigh NLG919, succinic anhydride and DMAP, dissolve them in dichloromethane solvent, and stir for reaction for 24 hours. After the reaction is completed, remove dichloromethane using a rotary evaporator. Then dissolve the product in 5% sodium bicarbonate solution, and extract it 3 times with ethyl acetate to remove the unreacted succinic anhydride, and then collect the sodium bicarbonate aqueous solution. Next, adjust the pH value of the aqueous solution to 2 - 3 using 1mol / L hydrochloric acid solution. Finally, extract and collect the intermediate NLG-SA using dichloromethane solution, and then remove the solvent using a rotary evaporator to obtain the crude product. The crude product is purified using a high-performance liquid chromatograph, the mobile phase is set to change from 10% ACN to 90% within 40 minutes, the detection wavelength is set to 276nm, the purified product is dissolved in DMSO-d6 and detected using nuclear magnetic resonance hydrogen spectrum. At the same time, LC-MS is also used to further confirm the molecular weight of the product.

[0050] 2. Synthesis of linear polypeptide KLA

[0051] First, synthesize the linear KLA polypeptide with the sequence KLAKLAKKLAKLAK. The synthesis method uses solid-phase peptide synthesis technology (SPPS). First, weigh 400mg of Rink Amide-MBHA resin, add DMF to swell it for 30min, then add the deprotection solution (morpholine:DMF = 6:4, v / v) and react for 30min, and repeat the reaction 2 times. After the deprotection reaction is completed, wash the resin 3 times with DCM and DMF in sequence to remove the residual deprotection solution. After washing, weigh the Fmoc-group protected amino acid, fully dissolve it in DMF solvent, add the base-adjusting reagent DIPEA and mix well, then add it to the resin and react for 2 hours, and repeat the reaction 2 times. After the reaction is completed, wash the resin 3 times with DCM and DMF in sequence to remove the residual unreacted liquid. Repeat the above process in sequence until the target sequence is synthesized. Add the cutting solution (trifluoroacetic acid: deionized water: TIS = 95.0%:2.5%:2.5%, v / v / v) to the dried resin and react for 1.5 hours to cut the target product from the resin. Use a nitrogen stream to blow dry the cutting liquid, then add ice ether to precipitate the target product, centrifuge to remove ether and collect the precipitate. Repeat this three times. Finally, dissolve the product in deionized water and filter to remove the resin.

[0052] 3. Synthesis of stapled peptide sKLA

[0053] The sequence of this polypeptide is cyclo - KL[CKLAKKLC]KLAK. The polypeptide synthesis method refers to Step 2. After the synthesis of the polypeptide segment, a side - chain deprotection solution (dichloromethane: trifluoroacetic acid: TIS = 92.0%:1.5%:6.5%, v / v / v) is added to the resin to remove the Trt protecting group of the cysteine side chain. The reaction is stopped when the color of the side - chain deprotection liquid changes from yellow to transparent. The resin is washed 3 times with DCM and DMF to remove the residual side - chain deprotection solution. Weigh 4,4’ - bis(bromomethyl)biphenyl and dissolve it in DMF, add DIPEA and mix well, then react for 5 hours. After the reaction, the Fmoc protecting group is removed, and the stapled peptide is cut and collected from the resin using a cleavage solution.

[0054] 4. Synthesis of linear polypeptide NLG919 conjugate KLA - NLG

[0055] The sequence of this linear polypeptide conjugate is NLG919 - Ahx - KLAKLAKKLAKLAK. After the synthesis of the polypeptide part, the spacer group and the intermediate NLG - SA are sequentially connected to the polypeptide segment according to the polypeptide synthesis method. The specific synthesis route is as Figure 1 shown.

[0056] 5. Synthesis of stapled peptide NLG919 conjugate sKLA - NLG

[0057] The sequence of the stapled peptide conjugate is NLG919 - Ahx - cyclo - KL[CKLAKKLC]KLAK. After the synthesis of the polypeptide segment and grafting the spacer group and the intermediate NLG - SA molecule to the polypeptide segment in turn according to the method in Step 4, the cysteine protecting group is removed using the side - chain deprotection solution, and the polypeptide conjugate is stapled using the method in Step 3. The specific synthesis route is as Figure 2 shown.

[0058] The structures and purities of the polypeptide and the polypeptide - drug conjugate are characterized by proton nuclear magnetic resonance spectroscopy, mass spectrometry and high - performance liquid chromatography to verify that their structures and purities meet the expectations.

[0059] Example 2

[0060] This example mainly provides circular dichroism spectroscopy to detect the polypeptide conformation. Cyclization can effectively constrain the polypeptide conformation, and the polypeptide conformation is considered to have an important impact on its penetration ability. Polypeptides can form various secondary structures such as α - helix and β - sheet. The linear polypeptide KLA and the stapled polypeptide sKLA are mixed at a ratio of 1×10 -5The concentration of M was dissolved in deionized water and detected at room temperature. The detection step resolution of the circular dichroism spectrometer was set to 0.5 nm, the number of accumulations was recorded as 10, the response time was set to 1 s, the bandwidth was set to 1 nm, the path length was set to 10 mm, and the data was recorded and expressed as the mean residue molar ellipticity.

[0061] The obtained circular dichroism spectra are as Figure 3 shown. Analysis shows that the linear polypeptide KLA exhibits a negative peak at 195 nm, indicating its regular conformation; while the stapled polypeptide sKLA detects characteristic negative peaks at 208 nm and 222 nm for the α-helical conformation, indicating that the polypeptide has been successfully constrained to form a secondary α-helical structure by stapling.

[0062] Example 3

[0063] This example mainly provides the morphological and particle size characterization of nanoparticles prepared from the linear polypeptide NLG919 conjugate KLA-NLG and the stapled peptide NLG919 conjugate sKLA-NLG (denoted as KLA-NLG NPs and sKLA-NLG NPs, respectively).

[0064] The specific preparation process is as follows: appropriate amounts of KLA-NLG and sKLA-NLG conjugates were respectively dissolved in tetrahydrofuran solvent, and then slowly added dropwise to deionized water and stirred. After the tetrahydrofuran was completely volatilized, nanoparticles could be obtained.

[0065] The hydrodynamic diameters of the two kinds of nanoparticles were detected using a dynamic light scattering particle size analyzer DLS, and the morphologies of the two kinds of particles were observed using a transmission electron microscope TEM. Among them, the DLS and TEM detection results of the nanoparticles prepared from the sKLA-NLG conjugate are as Figure 4 shown. The hydrodynamic diameter of the stapled polypeptide conjugate sKLA-NLG NPs is 145.4 ± 1.9 nm, and the PDI is 0.294 ± 0.036. In addition, the hydrodynamic diameter of the linear polypeptide conjugate KLA-NLG NPs is 189.8 ± 4.8 nm, and the PDI is 0.189 ± 0.031. Both kinds of particles are uniform spherical structures with nanoscale sizes.

[0066] Example 4

[0067] This example mainly provides the study on the mitochondrial targeting and disruption of pro-apoptotic polypeptides.

[0068] After inoculating 4T1 cells in a well plate and incubating for 24 hours, the culture medium was discarded. Four different apoptotic polypeptide treatment groups, namely 15 μM linear apoptotic polypeptide KLA, conjugated polypeptide sKLA, KLA-NLG NPs, and sKLA-NLG NPs, were added respectively, and PBS was used as the control group. After incubating with the cells for another 24 hours, the cells were digested and centrifuged to obtain the cells. Then, 0.5% glutaraldehyde fixative was added to resuspend the cells, and the cells were left standing at 4 °C for 10 minutes. After centrifuging again to collect the cells, 3% glutaraldehyde fixative was added and incubated overnight. Subsequently, after washing with PBS, the cells were stained with 4% osmium tetroxide for 30 minutes, and then dehydrated step by step with 30%, 50%, 70%, and 90% ethanol, and then rinsed 3 times with 100% ethanol. After that, the cells were embedded in epoxy resin and cut into thin slices with a thickness of 50 - 70 nm. After staining with 5% uranyl acetate and 2% lead citrate for 15 minutes, transmission electron microscopy was used for observation. The obtained TEM micrographs are shown in Figure 5 as follows.

[0069] Combined with the morphological observation in Figure 5 , it can be seen that the morphology of mitochondria in the control group cells was normal, specifically manifested as having a clear mitochondrial cristae structure and no cavity structure observed inside the mitochondria; however, different degrees of mitochondrial structure damage were observed in the cells of different apoptotic polypeptide treatment and incubation groups. Among them, the damage of the conjugated polypeptide sKLA to the mitochondrial structure was significantly stronger than that of the linear polypeptide KLA, and the damage was further enhanced after forming particles, which proved that the KLA polypeptide could effectively damage the mitochondrial structure, and both conjugation and self-assembly modification methods could enhance the destructiveness, resulting in more severe mitochondrial morphological damage, thereby inducing apoptosis of tumor cells. This mechanism is different from cytotoxic chemotherapeutic drugs and is not likely to produce tumor drug resistance.

[0070] Example 5

[0071] Currently, the research on apoptotic polypeptides mainly focuses on the mechanism of inducing cell apoptosis. However, the applicant innovatively investigated the ability of apoptotic polypeptides to induce immunogenic cell death and achieved unexpected technical effects. Especially after the polypeptide was conjugated and modified, the ability to induce immunogenic cell death could be further improved, showing potential prospects for tumor immunotherapy.

[0072] The process in which tumor cells, when stimulated by the outside world and undergoing death, transform from non-immunogenic to immunogenic and mediate the body to produce an anti-tumor immune response is called immunogenic cell death. When tumor cells occur, a series of signaling molecules will be produced. Such substances are called damage-associated molecular patterns, mainly including calreticulin (CRT) exposed on the cell surface, high-mobility group protein 1 (HMGB1) secreted by tumor cells to the outside, ATP molecules released by cells, and heat shock proteins (HSP70, HSP90), etc.

[0073] Calreticulin is a highly conserved calcium-binding protein that is located in the endoplasmic reticulum organelle of normal cells. When cells undergo immunogenic cell death, this protein will transfer from the endoplasmic reticulum organelle to the cell membrane surface and release signals to induce the recruitment of dendritic cells, thereby enabling the recognition and presentation of tumor antigens and promoting the phagocytosis of dead tumor cells by dendritic cells. After that, dendritic cells will provide certain co-stimulatory signals, and then bind the tumor antigen peptide complex with immunological recognition to the MHC molecules on the surface of dendritic cells and present it to T cells for the recognition and killing of tumor cells by T cells. High-mobility group protein B1 exists in the cell nucleus and cytoplasm of normal cells and is a cytokine related to the inflammatory response. When tumor cells undergo immunogenic cell death, high-mobility group protein B1, as an inflammatory signal molecule, will be released from the cell nucleus to the outside of the cell. This signal molecule can bind to and interact with the TLR4 receptor on the surface of dendritic cells, promoting the maturation of dendritic cells and the presentation of tumor-associated antigens to T lymphocytes. Adenosine triphosphate (ATP), as a chemoattractant, can be sensed by the P2Y(2) receptor on the surface of phagocytes after being released from cells, thereby recruiting antigen-presenting cells (APCs) and ultimately inducing the infiltration of cytotoxic T cells into tumors.

[0074] Therefore, in this experiment, the induced expression of three important danger signal-related molecules, calreticulin, high-mobility group protein B1, and ATP, in immunogenic cell death was measured, mainly including:

[0075] 1): Immunofluorescence detection of calreticulin (CRT) and high-mobility group protein B1

[0076] The specific process is as follows:

[0077] 4T1 cells were seeded in a well plate and incubated for 24 hours, then the culture medium was discarded. Different materials at a concentration of 15 μM were added respectively and incubated for another 24 hours, and then the culture medium was discarded. The cells were washed 3 times with PBS buffer. Then 2.5% glutaraldehyde was added to fix the cells. After 2 hours, the glutaraldehyde was discarded and the cells were washed 3 times with PBS buffer. Then the diluted CRT monoclonal antibody or HMGB1 monoclonal antibody was added to the well plate and incubated on a side-sway shaker for 1 hour. After discarding the CRT antibody solution or HMGB1 monoclonal antibody, the cells were washed 3 times with PBS buffer, and then Alexa Fluor 488 fluorescently labeled secondary antibody was added for incubation. After 1 hour, the secondary antibody was discarded and the cells were washed 3 times with PBS buffer. Finally, Hoechst 33342 was added and incubated for 15 minutes, and then the well plate was placed under a fluorescence microscope for observation and recording. The obtained fluorescence detection results are as Figure 6 described.

[0078] 2): Detection of calreticulin (CRT) expression by flow cytometry and detection of high-mobility group protein B1 and ATP secretion by kits

[0079] The specific procedures are as follows:

[0080] After inoculating cells and co-incubating with materials in each group for 24 hours, use cell digestive solution to digest and centrifuge to collect cells. After washing 3 times with PBS buffer, incubate with CRT primary antibody and Alexa Fluor 488 fluorescently labeled secondary antibody in sequence. Finally, after filtering the cells, use flow cytometry to detect the content of calreticulin (CRT).

[0081] After inoculating cells and co-incubating with materials in each group for 24 hours, take 20 μL of culture supernatant and add 100 μL of prepared ATP detection working solution. After an interval of 2 seconds, place it in an enzyme-labeled instrument and measure and calculate the content of ATP in chemiluminescence mode (Luminometer).

[0082] After inoculating cells and co-incubating with materials in each group for 24 hours, detect the content of high-mobility group protein B1 in the cell culture supernatant according to the operation guide provided by the kit manufacturer.

[0083] The obtained test results are as Figure 7 shown.

[0084] Combined with Figure 6 Immunofluorescence imaging analysis of CRT shows that after cells undergo immunogenic death, CRT will transfer to the cell membrane surface to induce DC cells to capture it. CRT is labeled with green fluorescence, and the cell nucleus is labeled with blue fluorescence. It can be observed that green fluorescence signals are detected on the cell surfaces of all treatment groups, indicating that CRT has transferred to the cell membrane surface. And by quantitatively comparing the green fluorescence intensity on the cell surface after incubation by flow cytometry, it can be known that the relative fluorescence intensity induced by the sKLA polypeptide conjugated and modified with the linear polypeptide KLA is increased by 3.1 times. Due to stronger cytotoxicity, the CRT signal intensity detected on the cell surface after incubation of sKLA-NLG NPs is 2.2 times that of the KLA linear polypeptide. Immunofluorescence detection analysis of HMGB1 shows that different from the CRT fluorescence signal, since the HMGB1 protein is usually expressed in the cell nucleus, it can be observed that there is a green fluorescence signal in the cell nucleus region under the incubation of PBS in the control group. When cells undergo immunogenic death, its HMGB1 protein will leak from the cell nucleus to the extracellular space, resulting in the absence of fluorescence signal; it can be seen from Figure 6 that the green fluorescence signals in the cell nuclei of the treatment groups are all attenuated, indicating that after co-incubation with these materials, HMGB1 in the cell nucleus has leaked out of the cell. Figure 6

[0085] Combined with Figure 7 ​From the quantitative detection results, it can be found that the detected amount of HMGB1 corresponds to the lost signals in the immunofluorescence signal map. The detected contents in the conjugated polypeptide sKLA and the conjugated polypeptide conjugate sKLA-NLG NPs groups are 681.9 pg / mL and 854.8 pg / mL respectively, which are significantly higher than that in the linear KLA polypeptide group (137.1 pg / mL). At the same time, it can be found that after the linear peptide KLA self-assembled into nanoparticles KLA-NLG NPs through drug conjugation, its detected amount increased by 3.3 times. Finally, the results of detecting the ATP content in the cell supernatant after co-incubating the materials with an enhanced ATP detection kit show that the induced release of ATP is consistent with the detection results of CRT and HMGB1. The ATP content induced by the optimal group KLA-NLG NPs reached 0.086 nM.

[0086] Through the above qualitative and quantitative characterizations, it can be effectively proved that the KLA polypeptide has the ability to induce immunogenic cell death, and it is found that after the KLA polypeptide is conjugated and modified with drugs, sKLA-NLG NPs have the most potent ability to induce immunogenic cell death.

[0087] Example 6

[0088] This example examines the ability of nanoparticles prepared from polypeptide drug conjugates to block the activity of IDO enzyme in vitro and reverse the IDO enzyme-mediated immunometabolic pathway at the cellular level.

[0089] The specific process is as follows: IFN-γ was added to the cell culture medium containing tryptophan to stimulate the expression of IDO enzyme in 4T1 cells, and the inhibitory effects of different treatment groups on IDO enzyme were compared by HPLC tracing and quantifying the kynurenine, the catalytic product of IDO enzyme. The detection results of kynurenine are as Figure 8 shown. The average inhibition rate of the NLG919 free drug group is 75.8%, which is the highest among all material groups. The average inhibition rate of the sKLA-NLG NPs group is 65.9%, which is also higher than that of the KLA-NLG NPs treatment group (41.5%), second only to the free drug group. Thus, it can be seen that the conjugated modification of the polypeptide improves the cell uptake, making the drug release of sKLA-NLG NPs more sufficient and obtaining a stronger IDO enzyme inhibitory effect. Since the IDO enzyme inhibitor is not conjugated, the KLA polypeptide and the sKLA polypeptide do not show IDO enzyme inhibitory effects.

[0090] Example 7

[0091] Immune checkpoint inhibitor monotherapy is insensitive or unable to achieve long-term anti-tumor effects in some patients. The main reason is the poor immunogenicity of the tumor microenvironment, namely the so-called "cold tumor", due to 1) defective antigen presentation to T cells, 2) lack of T cell activation, 3) lack or less infiltration of activated T cells in tumor tissues, and 4) the presence of immunosuppressive cells, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Therefore, in order to overcome the limitations of immune checkpoint inhibitor monotherapy, PD-L1 antibody is combined with materials.

[0092] This example mainly examines the ability of nanoparticles prepared from polypeptide drug conjugates to induce immunogenic cell death in vivo and their inhibitory effect on the IDO enzyme immunometabolic pathway in the tumor microenvironment, and comprehensively evaluates their anti-tumor immunotherapy effect.

[0093] The establishment of the in vivo tumor model is as follows:

[0094] Resuspend 4T1 cells in DMEM medium without serum, and inject 100 μL of 4T1 cell suspension into the left mammary fat pad of experimental mice to establish the main side tumor. Six days later, inject 100 μL of the suspension of 4T1 cells into the right mammary fat pad of experimental mice to establish the distal tumor, thereby establishing a bilateral breast cancer model.

[0095] The specific treatment plan is as follows:

[0096] After the model is established, randomly divide the experimental mice into 6 groups and start treatment from the seventh day. The treatment is divided into (1) PBS buffer, (2) linear polypeptide KLA, (3) stapled polypeptide sKLA, (4) nanoparticle KLA-NLG NPs, (5) nanoparticle sKLA-NLG NPs, and (6) combined treatment group of nanoparticle sKLA-NLG NPs and PD-L1 antibody. The administration method is intratumoral injection on the main side, and the administration dose is calculated based on the optimal group of 15 mg / kg, and the single-dose volume is 100 μL. A total of 3 doses are administered on the 1st, 3rd, and 5th days at the start of treatment. The combined treatment group injects PD-L1 antibody with a dose of 200 μg on the second day after injecting the drug, and a total of 3 doses are administered.

[0097] In the remaining optional examples, each treatment group can also be made into tablets or powdered drugs for easy storage, and is injected after reconstitution with solution during administration.

[0098] Obtain in vitro tumor photos 21 days after administration treatment as Figure 9As shown, it can be observed that in the experimental mice treated with the combination of sKLA-NLG NPs and the PD-L1 antibody, the volumes of the main and distal tumors were the smallest. The tumor inhibition rate of the main tumor was 88.0%, and that of the distal tumor was 97.3%, showing the best therapeutic effect and distal tumor inhibition effect. At the same time, the distal tumors of two mice in this treatment group had been ablated. Meanwhile, the therapeutic effect of the conjugated polypeptide sKLA was also stronger than that of the linear polypeptide KLA, and the combination with IDO enzyme inhibition could further inhibit tumor growth.

[0099] In addition, the tumor inhibition rate was calculated based on the weight of the excised tumors. After statistics, on the main treatment side, the tumor inhibition rate of the linear polypeptide KLA was only 23.3%, which could not effectively inhibit tumors. However, after conjugation, the stability of the polypeptide and its cellular uptake were enhanced, and its therapeutic effect was significantly improved to 52.3%. After the combination of KLA and IDO enzyme blockade, the linear group and the conjugated group were further increased by 2.4 times and 1.5 times respectively. After combination with the PD-L1 antibody, the tumor inhibition rate was further increased to 88.0%, indicating that after restoring the IDO enzyme-mediated immunosuppression, the immune system further ablated the tumors, demonstrating the synergistic effect of IDO enzyme inhibition and PD-L1 blockade.

[0100] On the day after the third administration, the mice were sacrificed, the tumor tissues were removed, and tumor tissue sections were prepared. Immunofluorescence staining was performed on CRT and HMGB1 expressed in the tumor area to investigate. As Figure 10 - 11 shown, among them, the treatment group with the linear polypeptide KLA had the ability to induce immunogenic cell death. It was found that after conjugation and drug conjugation modification of the linear polypeptide KLA, the ability to induce immunogenic cell death could be improved, and the sKLA-NLG NPs had the strongest ability to induce immunogenic cell death. The positive rates of CRT and HMGB1 in different treatment groups were consistent with the in vitro induction of immunogenic cell death, and could effectively achieve the ability to induce immunogenic cell death in vivo.

[0101] On the day after the third administration, three experimental mice were randomly selected from each experimental group, sacrificed, and the tumor tissues were obtained. Flow cytometry was used to detect and analyze the immune-related cells infiltrating the tumor tissues to investigate the immunotherapeutic efficacy, as Figure 11 shown.

[0102] Mature DC cells can promote immune responses. Therefore, the detection of mature DC cells showed that the average proportion of mature DC cells in the experimental mice of the linear polypeptide administration group was 10.2%. Benefiting from the stronger ability to induce immunogenic cell death and release more danger signal-related molecules and tumor-associated antigens, the number of infiltrating mature DC cells in the experimental mice administered with the conjugated polypeptide sKLA increased to 15.3%. The average proportions of KLA-NLG NPs and sKLA-NLG NPs in the polypeptide conjugate treatment group were 12.7% and 26.5% respectively, which were higher than those in the polypeptide treatment group. The DC cell maturation proportion in the sKLA-NLG NPs and aPD-L1 combination treatment group was the highest, reaching 37.6%.

[0103] Since Treg cells can downregulate immune responses and cause tumor cell escape, the Treg cells infiltrating the tumor region were detected. The NLG919 drug can inhibit the IDO enzyme and reverse the immunosuppression mediated by its immunometabolic pathway. Therefore, compared with KLA and sKLA in the polypeptide treatment group, the proportions of Treg cells in the KLA-NLG NPs and sKLA-NLG NPs administration groups of the polypeptide conjugate NLG919 nanoparticle treatment group were significantly downregulated by 32.0% and 32.1% respectively.

[0104] CD8 + T cells are immune cells that directly kill tumors in immune responses. The infiltrating CD8 + T cells in the conjugated polypeptide sKLA group were higher than those in the linear polypeptide group. After blocking the immunosuppression mediated by the IDO enzyme, 32.4% of CD8 + T cells were detected in the tumor region of the sKLA-NLG NPs administration group. After combination treatment with aPD-L1, the effector T cells infiltrating the tumor region of the experimental mice reached the highest level, and an average of 44.8% of CD8 + T cells were detected respectively, indicating that the inhibition of the IDO enzyme and the blockade of the PD-1 / PD-L1 pathway have a synergistic effect.

[0105] In the present invention, an apoptosis-promoting polypeptide KLA and an IDO enzyme inhibitor are conjugated through an ester bond to a peptide chain to construct a polypeptide drug conjugate. On the one hand, it can achieve the cleavage of tumor cells by the KLA polypeptide and induce immunogenic cell death. At the same time, in combination with the IDO enzyme inhibitor, it can reverse the IDO enzyme-mediated immunometabolic pathway at the cellular level to relieve the inhibition of the immune microenvironment, and has a synergistic and enhanced tumor immunotherapy effect.

[0106] Those skilled in the art should understand that throughout the specification, unless otherwise specifically stated, the terms used herein should be construed as having the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of any contradiction, the present specification shall prevail.

[0107] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0108] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A polypeptide drug conjugate for immunotherapy of breast tumors, characterized in that, the structure of the polypeptide drug conjugate is shown in Formula 1: Formula 1.

2. A drug delivery system, characterized in that, the drug delivery system comprises the polypeptide drug conjugate according to claim 1 self-assembled to form oncolytic virus-like nanoparticles.

3. The drug delivery system according to claim 2, characterized in that, the particle size of the nanoparticles is 150 nm - 180 nm.

4. A drug combination system for immunotherapy of breast tumors, characterized in that, it comprises the drug delivery system according to any one of claims 2 - 3 and a PD-L1 antibody.

5. Use of the polypeptide drug conjugate according to claim 1 in the preparation of a drug for immunotherapy of breast tumors.

6. The use according to claim 5, characterized in that, the drug is an injectable dosage form, tablet or powder.

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