Polypeptide drug chimera and preparation method thereof

Abstract

The invention discloses a polypeptide drug chimera and a preparation method thereof, and belongs to the technical field of polypeptide drug carriers. The polypeptide drug chimera is constructed by a recognition segment specifically combined with a target protein and a functional polypeptide carrier mediating the target protein to be transferred to lysosome; an antibody is combined with a lysosome signal amino acid sequence, and pathogenic antigen protein is transferred into a lysosome subcellular organelle for degradation through specific combination of the antibody and different antigens.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide drug carriers, and specifically relates to a polypeptide drug chimera and a preparation method thereof. Background Art

[0002] Cancer remains one of the most serious health threats to humanity (Mattiuzzi C, Lippi G. Current Cancer Epidemiology. J Epidemiol Glob Health. 2019;9(4):217-222.). After intravenous or oral administration, only a small portion of the active ingredients of traditional anticancer drugs can be accumulated in the cancerous site in the blood circulation, while most of the ingredients are absorbed by normal tissues, resulting in low drug utilization efficiency and significant toxic side effects (Randall EC, Emdal KB, Laramy JK, et al. Integrated mapping of pharmacokinetics and pharmacocytic dynamics in a patient-derived xenograft model of glioblastoma. Nat Commun. 2018;9(1):4904.). How to achieve precise drug transport to cancer cell tissues and kill cancer cells in a targeted manner has become a key issue that needs to be addressed in the field of drug delivery.

[0003] Peptides have been widely used in the construction of targeted drug transport carriers due to their good biocompatibility, functional diversity, high in vivo responsiveness, and simple and easy synthetic modification methods. Based on functionalized peptides with targeting functions and responsive stimuli, the constructed drug transport system can accurately deliver drugs to the tumor area (Yu J, Li H, Fang T, et al. Harnessing the Lysosomal Sorting Signals of the Cation-Independent Mannose-6-Phosphate Receptor for Targeted Degradation of Membrane Proteins. JAm Chem Soc. 2023;145(34):19107-19119.). Once the drug transport system reaches the tumor tissue, it can achieve precise release of the drug under the influence of the special tumor microenvironment or external stimulation. This specific tumor-targeting and stimuli-responsive functionalized peptide transporter can maximize the anti-tumor effect of the drug and reduce the drug's toxic side effects.

[0004] Due to the small size of polypeptides, their biochemical properties usually do not change significantly after introducing active groups into functionalized polypeptide transporters. Therefore, when specifically active groups are conjugated to polypeptides, the targeting is more precise and controllable. The targeting function of functionalized polypeptides can be divided into two levels: targeting tumor cells and various subcellular organelles. The targeting function of subcellular organelles can especially bypass drug resistance. For example, mitochondria, as the energy center of cells, regulate the synthesis of adenosine triphosphatase within cells. The drug delivery system based on mitochondria can affect a series of physiological activities within cells and thus has broad application prospects (Lin X, Li L, Li S, et al. Targeting the Opening of Mitochondrial Permeability Transition Pores Potentiates Nanoparticle Drug Delivery and Mitigates Cancer Metastasis. Adv Sci. 2020;8(4):2002834.). However, the anti-tumor strategy targeting mitochondria still faces some challenges, such as low drug accumulation in mitochondria, non-specific uptake of drugs by tumor cells and normal cells, etc. Therefore, developing a new type of targeting subcellular organelle transport system is crucial for solving problems such as systemic toxicity and drug resistance.

[0005] Lysosomes have unique advantages as targeted subcellular organelles for drug transport. Their main function is to decompose and digest substances inside and outside cells. Functionalized polypeptide targeting transport towards lysosomes helps utilize this natural "garbage processor" to bypass problems existing in traditional targeting methods, such as drug resistance and toxic side effects (Ahn G, Riley NM, Kamber RA, et al. Elucidating the cellular determinants of targeted membrane protein degradation by lysosome-targeting chimeras. Science. 2023;382(6668):eadf6249.). By directly releasing drugs into lysosomes, not only can the effectiveness of drugs be improved, but also the damage to healthy cells can be reduced, thereby more effectively inhibiting the growth and spread of cancer cells. This targeting strategy for lysosomes provides new ideas and possibilities for developing safer and more effective anti-cancer drug delivery systems. Summary of the Invention

[0006] In view of the above situation, to overcome the defects of the prior art, the present invention provides a polypeptide drug conjugate and a preparation method thereof. The present invention constructs a polypeptide drug conjugate by using an identification segment that specifically binds to a target protein and a functional polypeptide carrier that mediates the transport of the target protein to lysosomes, binds an antibody to a lysosomal signal amino acid sequence, and transfers a pathogenic antigen protein into a lysosomal sub-organelle through the specific binding of the antibody to different antigens for degradation.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: A polypeptide drug conjugate proposed by the present invention, that is, a conjugate that mediates the transport of a target protein to lysosomes, the conjugate includes an identification segment that binds to the target protein, and the conjugate further includes a functional polypeptide carrier that mediates the transport of the target protein to lysosomes; The identification segment can specifically bind to the target protein, which is a key functional part of the polypeptide drug conjugate and is responsible for recognizing and specifically binding to the target protein; the target protein selective identification segment can be an antibody, an antibody fragment, an aptamer, a small molecule inhibitor, or other molecules that can bind to a specific protein; its function is to ensure that the polypeptide drug conjugate can accurately recognize and bind to a specific target protein, avoid non-specific binding, and form a target protein-polypeptide drug conjugate complex after specific binding; lysosomes can recognize the polypeptide sequence of the functional polypeptide carrier, can guide the complex into lysosomes, and can be recognized by the intracellular transport system, thereby guiding the bound target protein-polypeptide drug conjugate complex to be transported to lysosomes through the endocytosis pathway or other mechanisms for degradation or treatment.

[0008] Preferably, the functional polypeptide carrier includes one or more lysosomal targeting structural units; Preferably, the sequence of the lysosome targeting structural unit includes SKSYKYSKVNKE (SEQ ID NO:1), SYSYKYSKVNKE (SEQ ID NO:2), SYKYSKVNKD (SEQ ID NO:3), SYKYSKLNKE (SEQ ID NO:4), SYKYSKVQKE (SEQ ID NO:5), SYKYSKVQKD (SEQ ID NO:6), SYKYSKLNKD (SEQ ID NO:7), SYKYSKLQKD (SEQ ID NO:8), RRRSYKYSKVNKE (SEQ ID NO:9), RRLRKSYKYSKVNKE (SEQ ID NO:10), RRLRKSYKYSKVQKE (SEQ ID NO:11), RRRKSYLYSKVNKE (SEQ ID NO:12), RRRSYKYSKLQKD (SEQ ID NO:13), EESEERDDHLIPM (SEQ ID NO:14), EESEDRDDHLLPL (SEQ ID NO:15), EESEDRDDHLIPL (SEQ ID NO:16), EESEERDDHLLPL (SEQ ID NO:17), EDSEERDDHLLPL (SEQ IDNO:18), EDSEDRDEHLLPL (SEQ ID NO:19), EDSEDREEHLLPL (SEQ ID NO:20), EDSEDRDDHLIPL (SEQ ID NO:21), DTGDNPIYKSAV (SEQ ID NO:22), DTGEQPIYKSAV (SEQ ID NO:23), DTGDQPIYKSAV (SEQ ID NO:24), ETGENPIYKSAV (SEQ ID NO:25), DTGENPLYKSAV (SEQ ID NO:26), DTGENPIYKSAL (SEQ ID NO:27), DTGENPIYKSAI (SEQ ID NO:28), DTGDNPIYKSAL (SEQ IDNO:29); Preferably, the recognition segment includes at least one of polypeptide, protein, nucleic acid, nanoparticle or small molecule compound; Preferably, the recognition segment is a polypeptide, and the polypeptide is either glycosylated or non-glycosylated; the polypeptide is composed of natural amino acids or non-natural amino acids; Preferably, the recognition segment is connected to the functional polypeptide carrier by direct connection or indirect connection; Preferably, the indirect connection between the recognition segment and the functional polypeptide carrier is through a linker peptide or a chemical linker; Preferably, the positions where the functional polypeptide carrier is connected to the recognition segment include at least one of the C-terminus of the recognition segment peptide chain, the N-terminus of the recognition segment peptide chain, and the side chain group of the recognition segment peptide chain; Preferably, the side chain group of the recognition segment peptide chain includes a natural amino acid side chain group or an unnatural amino acid side chain group; the natural amino acid group includes at least one of an amino group and a thiol group; the side chain group of the unnatural amino acid group includes at least one of a nitrogen group, an alkynyl group, an aldehyde group, a ketone group, a fluorosulfonate group, a chlorine group, a bromine group, and an iodine group; Preferably, the method for connecting the recognition segment and the functional polypeptide carrier includes at least one of a chemical coupling method, an enzyme-catalyzed method, and a gene recombination method; Preferably, the recognition segment is an antibody; the antibody includes at least one of a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a monoclonal antibody, or an antigen-binding fragment of a monoclonal antibody; Preferably, the antigen-binding fragment of the monoclonal antibody is at least one of Fab, Fab’, F(ab’)2, Fv, dsFv, scFv, sc(Fv)2, or VHH.

[0009] The present invention also provides a method for preparing a polypeptide drug conjugate, which specifically includes the following steps: using a solid-phase synthesizer to sequentially synthesize according to the above amino acid sequence, and after cleavage and purification, obtaining a functional polypeptide carrier; using at least one of a chemical coupling method, an enzyme-catalyzed method, and a gene recombination method to chimerize the recognition segment and the functional polypeptide carrier to obtain a polypeptide drug conjugate.

[0010] The beneficial effects achieved by the present invention are as follows: The present invention provides a polypeptide drug conjugate and a method for preparing the same. The present invention constructs a polypeptide drug conjugate by using a recognition segment that specifically binds to a target protein and a functional polypeptide carrier that mediates the transport of the target protein to lysosomes, combines an antibody with a lysosomal signal amino acid sequence, and transfers a pathogenic antigen protein into lysosomal sub-organelles for degradation through the specific binding of the antibody to different antigens; the polypeptide drug conjugate of the present invention utilizes the lysosomal protein sorting mechanism, forms a complex with clathrin through a lysosomal targeting structural unit, triggers clathrin-mediated endocytosis, and ultimately results in the enrichment and degradation of the target protein bound by the functional polypeptide carrier in lysosomes. The functional polypeptide carrier of the present invention can achieve effective degradation and regulation of target proteins in vitro and / or in vivo, and can be used for knockdown or degradation of target proteins in vitro and / or in vivo, as well as the prevention and treatment of diseases such as cancer, metabolic diseases, and chronic diseases. Description of the Drawings

[0011] Figure 1 HPLC (A) and MS (B) characterization result diagrams of Ly-a5 for this example; Figure 2 HPLC (A) and MS (B) characterization result diagrams of Ly-a6 for this example; Figure 3 HPLC (A) and MS (B) characterization result diagrams of Ly-a8 for this example; Figure 4 Quantification diagram of the internalization of Ly-a1 to Ly-a14 in SKOV3 cells in Example 2 of the present invention; Figure 5 Quantification diagram of the internalization of Ly-a15 to Ly-a29 in SKOV3 cells in Example 2 of the present invention; Figure 6 Quantification diagram of the internalization of Ly-a1 to Ly-a14 in A549 cells in Example 2 of the present invention; Figure 7 Quantification diagram of the internalization of Ly-a15 to Ly-a29 in A549 cells in Example 2 of the present invention; Figure 8 Electrophoresis gel characterization result diagrams of LA1 to LA10 for this example; Figure 9 Quantification diagram of the internalization of LA1-LA10 to HER2 in MDA-MB-231 cells in Example 4 of the present invention; Figure 10 Immunoblot result diagram of the degradation of HER2 by LA1-LA10 in MDA-MB-231 cells in Example 4 of the present invention; Figure 11 Confocal laser scanning microscopy result diagram of the degradation of HER2 by LA1-LA10 in MDA-MB-231 cells in Example 4 of the present invention; The accompanying drawings are used to provide a further understanding of the present invention and form a part of the specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation to the present invention; Detailed implementation manners Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention; Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art; in addition, any methods and materials similar or equivalent to the described content can be applied to the present invention; the preferred implementation methods and materials described in the text are for illustrative purposes only and do not limit the content of this application; The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples are all obtained from commercial channels unless otherwise specified; Example 1: This example provides a functional polypeptide carrier, and the functional polypeptide carrier can mediate the transport of a target protein to lysosomes; the functional polypeptide carrier that mediates the transport of the target protein to lysosomes includes one or more lysosome targeting structural units; the lysosome targeting structural unit has the following amino acid sequence representation: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; Xaa1 = R or absent; Xaa2 = R or absent; Xaa3 = R, L, E or absent; Xaa4 = R, S, E, D or absent; Xaa5 = R, S, K, T, Y or absent; Xaa6 = S, G or E; Xaa7 = Y, E or D; Xaa8 = K, L, N, Q or R; Xaa9 = Y, E, P or D; Xaa10 = S, E, L, I or D; Xaa11 = K, Y or H; Xaa12 = V, K or L; Xaa13 = N, Q, L, S or I; Xaa14 = K, A or P; Xaa15 = E, D, M, L, V, or I; Preferably, the lysosome targeting structural unit has the following amino acid sequence excluding the sequences: SYKYSKVNKE, EESEERDDHLLPM and DTGENPIYKSAV; Preferably, the lysosome targeting structural unit has the following amino acid sequence representing a peptide: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; wherein, Xaa1 = R or absent; Xaa2 = R or absent; Xaa3 = R, L or absent; Xaa4 = R, S or absent; Xaa5 = R, K, Y or absent; Xaa6 = S; Xaa7 = Y; Xaa8 = K or L; Xaa9 = Y; Xaa10 = S; Xaa11 = K; Xaa12 = V or L; Xaa13 = N or Q; Xaa14 = K; Xaa15 = E or D; Preferably, the lysosome-targeting structural unit has a peptide represented by the following amino acid sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; Xaa1 = absent; Xaa2 = absent; Xaa3 = E; Xaa4 = E or D; Xaa5 = S; Xaa6 = E; Xaa7 = E or D; Xaa8 = R; Xaa9 = E or D; Xaa10 = E or D; Xaa11 = H; Xaa12 = L; Xaa13 = L or I; Xaa14 = P; Xaa15 = M or L; Preferably, the lysosome-targeting structural unit has a peptide represented by the following amino acid sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; wherein, Xaa1 = absent; Xaa2 = absent; Xaa3 = absent; Xaa4 = E or D; Xaa5 = T; Xaa6 = G; Xaa7 = E or D; Xaa8 = N or Q; Xaa9 = P; Xaa10 = L or I; Xaa11 = Y; Xaa12 = K; Xaa13 = S; Xaa14 = A; Xaa15 = V, I or L.

[0012] The sequence of the lysosome-targeting structural unit is shown in Table 1; This embodiment also provides a method for preparing a functional polypeptide carrier for mediating the transport of a target protein to lysosomes, which specifically includes the following steps: S1. Resin swelling: Use Wang Resin, brand: Shanghai Yuanye Bio-Technology Co., Ltd., product number: S28287, substitution degree: 0.56 mmol / g, 1% DVB, particle size: 100-200 mesh. Put Wang Resin into a solid-phase reactor, add DCM, shake and react for 30 min, then vacuum-dry under reduced pressure, and repeat the resin washing twice; S2. Coupling of Fmoc-1AA-OH: According to the polypeptide sequence, in the order from the C-terminus to the N-terminus, the polypeptide sequence was synthesized successively using a solid-phase synthesizer. Fmoc-1AA-OH was dissolved in DMF, HOBt was added for activation, and then it was put into the solid-phase synthesis reactor for coupling condensation reaction with the resin treated in step S1. After reacting at room temperature for 5 h, the Kaiser method was used for detection. If the detection result was negative, the coupling was completed. The reaction solution was dried under reduced pressure, and the reaction ended. The resin was washed with DCM and DMF three times each. The resin was subjected to deprotection treatment with 20% DBLK twice, for 5 min and 8 min respectively. Then the resin was washed with DCM and DMF three times each; the fully protected resin was synthesized successively according to the amino acid sequence; S3. Coupling of Fmoc-(2-last)AA-OH: Fmoc-(2-last)AA-OH was dissolved in DMF, activated with HOBt / DIC, and put into the solid-phase reactor for coupling with the resin treated in step S2. Under room temperature conditions, after the condensation reaction for 2 h, the Kaiser method was used for detection. If the detection result was negative, the coupling was completed. The reaction solution was dried under reduced pressure; the reaction ended. The resin was washed with DCM and DMF three times each; the resin was deprotected with 20% DBLK twice, for 5 min and 8 min respectively. Then the resin was washed with DCM and DMF three times each, and Fmoc deprotection was carried out. The coupling and Fmoc deprotection steps were repeated to complete the assembly of the linear peptide resin; S4. Coupling of FITC: A fluorescent molecule was coupled with a functional polypeptide carrier that mediates the transport of the target protein to the lysosome. After the linear peptide chain was synthesized, Fmoc deprotection was carried out on the amino group at the end of the peptide chain to expose the N-terminal amino group. 4 eq of FITC and 8 eq of DIPEA were added and reacted overnight in a DMF solution; the Kaiser method was used for detection. If the detection result was negative, the coupling was completed; S5. Cleavage of the functional polypeptide carrier that mediates the transport of the target protein to the lysosome: The resin was concentrated with methanol and then dried under reduced pressure and weighed. 10 - 20 times the amount of cleavage solution (VTFA:Vbenzyl methyl sulfide:Vanisole:VDODT = 90:5:3:2) was added, and the cleavage reaction was stirred at room temperature for 2 - 2.5 h. Then, the filtrate obtained by suction filtration was added to ice-cold anhydrous ether or methyl tert-butyl ether (10 eq), and left to stand at 2 - 8 °C for 30 min and centrifuged (3000 rpm). The solid was washed and centrifuged twice with an appropriate amount of precipitation solution and then dried in vacuo to obtain the crude peptide; S6. Purification of the functional polypeptide carrier mediating the transport of the target protein to lysosomes: Take the crude peptide sample prepared in step S5, add an appropriate amount of pyrogen-free water to dissolve it to obtain a sample solution of the crude peptide. Filter the sample solution with a φ0.45 µm filter, collect the filtrate, and purify it by RP-HPLC. Collect the target peptide fraction to obtain the functional polypeptide carrier mediating the transport of the target protein to lysosomes, and characterize the synthesized functional polypeptide carrier by HPLC and MS; Figure 1 Figure showing the HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a5 of this example, Figure 2 Figure showing the HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a6 of this example, Figure 3 Figure showing the HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a8 of this example.

[0013] Example 2: In this example, flow cytometry was used to detect the performance of the functional polypeptide carrier mediating the transport of the target protein to lysosomes described in Example 1 during the process of cellular internalization. During the cellular internalization process, after the functional polypeptide carrier enters the cell, the labeled fluorescent molecules emit signals. After the flow cytometer detects these signals, the content and distribution of the functional polypeptide carrier in the cells are analyzed; The specific detection method includes the following steps: Cell culture: The human ovarian cancer cell line SKOV3 and the human non-small cell lung cancer cell line A549 are both from the American Type Culture Collection (ATCC); The SKOV3 cell line uses RPMI 1640 basal medium (Gibco), and 10% fetal bovine serum (Gibco) and 1% double antibody (penicillin 100 U / mL + streptomycin 100 g / mL) are added to the medium; The A549 cell line uses DMEM basal medium (Gibco), and 10% fetal bovine serum (Gibco) and 1% double antibody (penicillin 100 U / mL + streptomycin 100 g / mL) are added to the medium; All cells are cultured in a humid environment containing 5% CO2 at 37 °C, with normal cell morphology and good growth status; Evaluation of the internalization of functional polypeptide carriers by flow cytometry: In this example, SKOV3 cells and A549 cells were used for the experiment. The transport carrier sequence reported in the literature was used as the positive control St (amino acid sequence as shown in SEQ ID NO: 30, SYKYSKVNKE); the functional polypeptide carrier labeled with the fluorescent molecule FITC was diluted to a final concentration of 100 nM with serum-free medium and then co-incubated with SKOV3 cells or A549 cells for 6 h; subsequently, the acid wash solution was used to remove the antibodies bound to the cell membrane, and the residual acid wash solution was washed with PBS; then the cells were digested from the culture dish and transferred to an EP tube, and the fluorescence intensity of the treated cells was observed by flow cytometry; Figure 4 This is the quantification chart of the internalization of Ly-a1 to Ly-a14 in SKOV3 cells in this example, Figure 5 This is the quantification chart of the internalization of Ly-a15 to Ly-a29 in SKOV3 cells in Example 2 of the present invention, Figure 6 This is the quantification chart of the internalization of Ly-a1 to Ly-a14 in A549 cells in this example, Figure 7 This is the quantification chart of the internalization of Ly-a15 to Ly-a29 in A549 cells in this example; among them, the control group is the transport polypeptide St reported in the literature; Figure 1 、 Figure 2 、 Figure 3 and Figure 4 The data are all expressed as: mean ± SEM (n = 3), * P < 0.05. Analysis of the average cell fluorescence value shows that the functional polypeptide carrier of the present invention has an internalization ability equivalent to or better than that of the positive control sequence; Example 3: This example provides a method for constructing a polypeptide-drug conjugate: In this example, a nanobody targeting human epidermal growth factor receptor-2 (HER2) was selected as the recognition segment, and the functional polypeptide carrier prepared in Example 1 was used as the transport carrier.

[0014] a) Plasmid design: In the present invention, the nanobody 5F7 (SEQ ID NO: 31) targeting HER2 is selected as the recognition segment, Ly-a5, Ly-a6, Ly-a8, Ly-a10, Ly-a11, Ly-a16, Ly-a17, Ly-a20, Ly-a27, Ly-a29 are selected as the functional polypeptide carriers, and the linker peptide Linker (amino acid sequence: GGGGS) is used to connect the recognition segment and the functional polypeptide carrier prepared in Example 1. The functional polypeptide carrier is connected to the C-terminus of the nanobody 5F7 by homologous recombination to obtain the polypeptide drug conjugate LA1-LA10 that can target lysosomes; the amino acid sequences of the polypeptide drug conjugate LA1-LA10 are shown in Table 4; however, the modified nanobody of the present invention is not limited to the amino acid sequences given in this example; Table 2 Sequences of HER2-targeting polypeptide drug conjugates b) Expression, purification and characterization of the polypeptide drug conjugate: The expression of the polypeptide drug conjugate in this example uses the Escherichia coli expression system, and the purification method is Ni column affinity purification. The specific method is as follows: The constructed LA1-LA10 plasmid was respectively transferred into E. coli BL21 competent cells for transformation, and glycerol bacteria were obtained by plating on plates. Take 10 mL of glycerol bacteria and transfer them to 1 L of fresh LB medium (containing 50 μg / mL kanamycin), and place them in a shaker at 37 °C for about 8 h of shaking culture. Then add the inducer IPTG (final concentration: 1 mM), and continue to culture at 25 °C for about 16 h. The cultured bacterial solution was placed in a high-speed centrifuge, and the cells were collected by centrifugation using a horizontal rotor, and the supernatant was discarded. Add an appropriate amount of Ni-NTA Buffer A to resuspend the cells and break the cells using an ultrasonic cell disruptor. The lysed solution was centrifuged using a centrifuge, and the supernatant was collected. LA1-LA10 was purified using a Ni column, and the resin was rinsed with about 20 column volumes of Ni-NTA Buffer A, and all the Ni-NTA Buffer A flowing through the nickel column was collected for detecting impurity proteins non-specifically bound to the nickel column. Subsequently, the target protein on the nickel column resin was eluted stepwise with washing solutions containing different imidazole concentrations, and each fraction was collected for SDS-PAGE electrophoresis analysis and characterization. The fractions containing relatively pure target protein were combined and transferred to a protein concentration tube, and the buffer was exchanged by centrifugation with PBS, and finally stored at -80 °C.

[0015] Figure 8 This is the electrophoretic gel characterization result diagram of LA1~LA10 in this example.

[0016] Example 4: Degradation of HER2 in Lysosomes Mediated by Polypeptide Drug Conjugate In this example, the human breast cancer cell line MDA-MB-231 was selected for the experiment. 5F7 was used as the recognition segment of the polypeptide drug conjugate, and the lysosome-targeting structural unit sequences Ly-a5, Ly-a6, Ly-a8, Ly-a10, Ly-a11, Ly-a16, Ly-a17, Ly-a20, Ly-a27, Ly-a29 were used as the functional polypeptide carriers of the polypeptide drug conjugate. Through the construction method of the polypeptide drug conjugate described in Example 3, the polypeptide drug conjugates LA1-LA10 were obtained. The ability of the polypeptide drug conjugate to endocytose HER2 on the cell membrane surface was evaluated by flow cytometry experiments. Immunoblot (Western Blot) and laser confocal experiments were used to detect the expression level of HER2 protein degraded by the polypeptide drug conjugate on the cell membrane surface, so as to evaluate the ability of the polypeptide drug conjugate to degrade HER2; the control nanobody used in this example was 5F7 targeting HER2, and the primary antibody used was anti-HER2 antibody; The specific experimental steps include: Cell culture: The human breast cancer cell line MDA-MB-231 was obtained from the American Type Culture Collection (ATCC). The MDA-MB-231 cell line was cultured using DMEM basal medium (Gibco) basal medium (Procell), and 10% fetal bovine serum (Gibco) and 1% double antibody (penicillin 100 U / mL + streptomycin 100 μg / mL) were added to the medium. The cells were cultured in a humid environment containing 5% CO2 at 37°C, and the cell morphology was normal and the growth state was good; Flow cytometry was used to evaluate the internalization of HER2 by the polypeptide drug conjugate mediated by the functional polypeptide carrier: In this example, MDA-MB-231 cells were used for the experiment, and the cells were plated 1 day before drug administration; the polypeptide drug conjugate was diluted with complete medium and then incubated with MDA-MB-231 cells in the dark for 24 h; after the cells were fixed with 4% paraformaldehyde, they were incubated with 1% BSA at room temperature for 1 h; the cells treated with BSA were first incubated with the primary antibody at 37°C for 1 h and then incubated with the fluorescently labeled secondary antibody at room temperature for 30 min; the internalization of HER2 by the polypeptide drug conjugate was observed by flow cytometry; the fluorescent secondary antibody used in this experiment was Goat anti-rabbit IgG H&L AlexaFluor® 488; Figure 9 This is the flow cytometry internalization result graph of HER2 of LA1-LA10 in MDA-MB-231 cells in Example 4 of the present invention, where the control nanobody is TCZ targeting HER2; Figure 9The results showed that HER2 was significantly internalized after administration of LA1-LA10; thus, it can be demonstrated that the polypeptide drug conjugate fused with the functional polypeptide carrier has a significant internalization effect on HER2; Detection of HER2 expression by Western Blot: Plate the cells 1 day before drug administration; dilute the constructed polypeptide drug conjugate to the desired concentration with complete medium and incubate with the cells for 48 h; then lyse the cells with lysis buffer, extract the total cellular protein and quantify the protein using a BCA kit (Thermo Fisher); then separate the protein using a 10% SDS-PAGE gel and transfer the protein to a polyvinylidene difluoride (PVDF) membrane by electrotransfer; block the membrane with 5% non-fat milk at room temperature for 1 h, then remove the milk and incubate the membrane with the primary antibody on a shaker at 4 °C overnight; the next day, take out the membrane and quickly immerse it in TBST and wash it 3 times, then incubate the membrane with the secondary antibody at room temperature for 1 h; finally, detect and record the protein using a chemiluminescent reagent and a Tanon chemiluminescence imager, and perform quantitative analysis of the protein using Image J; Figure 10 This is the immunoblot result diagram of LA1-LA10 in MDA-MB-231 cells for degrading HER2 in Example 4 of the present invention, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as an internal reference; thus, it can be demonstrated that the polypeptide drug conjugate fused with the functional polypeptide carrier has a significant degradation effect on HER2; Observation of HER2 expression by confocal laser scanning microscopy: In this example, MDA-MB-231 cells were used for the experiment, and the cells were plated 1 day before drug administration; dilute the constructed polypeptide drug conjugate with complete medium, and then incubate it with MDA-MB-23l cells in the dark for 48 h; stain the cell membrane using a cell membrane staining kit (purchased from Beyotime Biotechnology Co., Ltd.), and fix the cells with 4% paraformaldehyde at room temperature for 15 min; incubate the cells with 1% BSA at room temperature for 1 h, then the cells were first incubated with the primary antibody at 37 °C for 1 h, and secondly incubated with the fluorescently labeled secondary antibody at room temperature for 30 min, and the cell nuclei were stained with DAPI; observe the expression of HER2 after incubation of the polypeptide drug carrier with the cells by a confocal laser scanning microscope, and the results are as Figure 11 shown; Figure 11 From top to bottom are the cell nucleus signal, cell membrane signal, HER2 signal, and the merged signal of the three; as Figure 11 can be seen, compared with the anti-HER2 nanobody TCZ, the fluorescence intensity of HER2 in the cells incubated with each polypeptide drug conjugate was significantly reduced, indicating a decrease in the expression level of HER2; thus, it can be demonstrated that the polypeptide drug conjugate has a significant degradation effect on HER2.

[0017] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention; The present invention and its embodiments have been described above. Such description is not restrictive. What is shown in the drawings is only one of the embodiments of the present invention, and the actual application is not limited thereto. In general, if those of ordinary skill in the art are inspired by it and, without departing from the purpose of the present invention, design similar methods and embodiments to this technical solution without creative efforts, they shall fall within the protection scope of the present invention.

Claims

1. A polypeptide drug conjugate, characterized in that: The structure of the polypeptide drug conjugate includes a recognition segment and a functional polypeptide carrier; the recognition segment recognizes and specifically binds to a target protein; the functional polypeptide carrier mediates the transport of the target protein to lysosomes.

2. The polypeptide drug conjugate according to claim 1, wherein: The functional polypeptide carrier that mediates the transport of the target protein to lysosomes includes one or more lysosome-targeting structural units; the amino acid sequence of the lysosome-targeting structural unit includes at least 10-15 amino acids; the lysosome-targeting structural unit has the following peptide represented by an amino acid sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; Xaa1 = R or absent; Xaa2 = R or absent; Xaa3 = R, L, E or absent; Xaa4 = R, S, E, D or absent; Xaa5 = R, S, K, T, Y or absent; Xaa6 = S, G or E; Xaa7 = Y, E or D; Xaa8 = K, L, N, Q or R; Xaa9 = Y, E, P or D; Xaa10 = S, E, L, I or D; Xaa11 = K, Y or H; Xaa12 = V, K or L; Xaa13 = N, Q, L, S or I; Xaa14 = K, A or P; Xaa15 = E, D, M, L, V, or I; wherein, the sequences SYKYSKVNKE, EESEERDDHLLPM and DTGENPIYKSAV are not included.

3. The polypeptide drug conjugate according to claim 2, wherein: The lysosome-targeting structural unit has the following peptide represented by an amino acid sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; wherein, Xaa1 = R or absent; Xaa2 = R or absent; Xaa3 = R, L or absent; Xaa4 = R, S or absent; Xaa5 = R, K, Y or absent; Xaa6 = S; Xaa7 = Y; Xaa8 = K, L; Xaa9 = Y; Xaa10 = S; Xaa11 = K; Xaa12 = V or L; Xaa13 = N or Q; Xaa14 = K; Xaa15 = E or D.

4. The polypeptide drug conjugate according to claim 2, wherein: The lysosome-targeting structural unit has the following peptide represented by an amino acid sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; Xaa1 = absent; Xaa2 = absent; Xaa3 = E; Xaa4 = E or D; Xaa5 = S; Xaa6 = E; Xaa7 = E or D; Xaa8 = R; Xaa9 = E or D; Xaa10 = E or D; Xaa11 = H; Xaa12 = L; Xaa13 = L or I; Xaa14 = P; Xaa15 = M or L.

5. The polypeptide drug conjugate according to claim 2, wherein: The lysosome-targeting structural unit has the following peptide represented by an amino acid sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; wherein, Xaa1 = absent; Xaa2 = absent; Xaa3 = absent; Xaa4 = E or D; Xaa5 = T; Xaa6 = G; Xaa7 = E or D; Xaa8 = N or Q; Xaa9 = P; Xaa10 = L or I; Xaa11 = Y; Xaa12 = K; Xaa13 = S; Xaa14 = A; Xaa15 = V, I or L.

6. The polypeptide drug conjugate according to claim 2, wherein: The sequences of the lysosome-targeting structural unit include EESEERDDHLIPM (SEQ ID NO: 14), EESEDRDDHLLPL (SEQ ID NO: 15), EESEDRDDHLIPL (SEQ ID NO: 16), EESEERDDHLLPL (SEQ ID NO: 17), EDSEERDDHLLPL (SEQ ID NO: 18), EDSEDRDEHLLPL (SEQ ID NO: 19), EDSEDREEHLLPL (SEQ ID NO: 20), EDSEDRDDHLIPL (SEQ ID NO: 21).

7. A polypeptide drug conjugate according to claim 1, wherein: The recognition segment includes at least one of a polypeptide, a protein, a nucleic acid, a nanoparticle or a small molecule compound; the recognition segment is a polypeptide, and the polypeptide is either glycosylated or non-glycosylated; the polypeptide is composed of natural amino acids or non-natural amino acids.

8. A polypeptide drug conjugate according to claim 1, wherein: The recognition segment is connected to the functional polypeptide carrier by direct connection or indirect connection; the indirect connection between the recognition segment and the functional polypeptide carrier is through a linker peptide or a chemical linker.

9. A polypeptide drug conjugate according to claim 1, wherein: The positions where the functional polypeptide carrier is connected to the recognition segment include at least one of the C-terminus of the binding segment peptide chain, the N-terminus of the recognition segment peptide chain, and the side chain group of the recognition segment peptide chain; the side chain group of the recognition segment peptide chain includes a natural amino acid side chain group or a non-natural amino acid side chain group.

10. A polypeptide drug conjugate according to claim 1, wherein: The natural amino acid group includes at least one of an amino group and a thiol group; the side chain group of the non-natural amino acid group includes at least one of a nitrogen group, an alkyne group, an aldehyde group, a ketone group, a fluorosulfonate group, a chlorine group, a bromine group, and an iodine group; the connection method between the recognition segment and the functional polypeptide carrier includes at least one of a chemical coupling method, an enzyme-catalyzed method, and a gene recombination method.

11. A polypeptide drug conjugate according to claim 1, characterized in that: The recognition segment is an antibody; the antibody includes at least one of a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a monoclonal antibody, or an antigen-binding fragment of a monoclonal antibody; the antigen-binding fragment of the monoclonal antibody is at least one of Fab, Fab’, F(ab’)2, Fv, dsFv, scFv, sc(Fv)2, or VHH.

12. A method for preparing a polypeptide drug conjugate according to any one of claims 1-11, characterized in that, Specifically, it includes the following steps: Using a solid-phase synthesizer, the functional polypeptide carrier is synthesized sequentially according to the above amino acid sequence, and after cleavage and purification, the functional polypeptide carrier is obtained; at least one of a chemical coupling method, an enzyme-catalyzed method, and a gene recombination method is used to chimerize the recognition segment with the functional polypeptide carrier to obtain a polypeptide drug chimera.

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