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 refers to a polypeptide drug chimera and a preparation method thereof. Background Art

[0002] At present, cancer remains one of the most serious health threats to humans (Mattiuzzi C, Lippi G. Current Cancer Epidemiology. J Epidemiol Glob Health. 2019;9(4):217-222.). After intravenous injection or oral administration, only a small part of the active ingredients of traditional anticancer drugs can be enriched in the cancer site in the blood circulation, and most of the ingredients are absorbed by normal tissues, resulting in low drug utilization efficiency and large toxic side effects (RandallEC, EmdalKB, LaramyJK, et al. Integrated mapping of pharmacokinetics andpharmacodynamics in a patient-derived xenograft model of glioblastoma. NatCommun. 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 solved 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 function and responsive stimulation, 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.). When the drug transport system reaches the tumor tissue, it can achieve precise release of the drug under the action of the special tumor microenvironment or external stimulation. This specific tumor-targeted and stimulus-responsive functionalized peptide transporter can maximize the anti-tumor effect of the drug and reduce the toxic side effects of the drug.

[0004] Due to the small size of peptides, their biochemical properties usually do not change significantly after the active groups are introduced into functionalized peptide transporters. Therefore, when peptides are bonded with specific active groups, the targeting is more precise and controllable. The targeting function of functionalized peptides can be divided into two levels: 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 in cells. Mitochondrial-based drug delivery systems can affect a series of physiological activities in cells, so they have 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, mitochondria as a targeted anti-tumor strategy still faces some challenges, such as low accumulation of drugs in mitochondria, nonspecific uptake of drugs by tumor cells and normal cells, etc. Therefore, the development of a new subcellular organelle-targeted transport system is crucial to 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 the cell. Targeted transport of functionalized peptides to lysosomes helps to utilize this natural "garbage disposer" and bypass the problems of 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 proteindegradation by lysosome-targeting chimeras. Science. 2023;382(6668):eadf6249.). By releasing drugs directly into lysosomes, it can not only improve the effectiveness of drugs, but also reduce their damage to healthy cells, thereby more effectively inhibiting the growth and spread of cancer cells. This lysosome-targeted strategy provides new ideas and possibilities for the development of safer and more effective anticancer drug delivery systems. Summary of the invention

[0006] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a polypeptide drug chimera and a preparation method thereof. The present invention constructs a polypeptide drug chimera 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 a lysosome, combines an antibody with a lysosomal signal amino acid sequence, and through the specific binding of the antibody with different antigens, transfers the pathogenic antigen protein to the lysosomal subcellular organelle for degradation.

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

[0008] Preferably, the functional polypeptide carrier comprises one or more lysosomal targeting structural units; Preferably, the sequence of the lysosomal 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). 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), 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 comprises at least one of a polypeptide, a protein, a nucleic acid, a nanoparticle or a small molecule compound; Preferably, the recognition segment is a polypeptide, the polypeptide is sugar-modified or not sugar-modified; 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 position where the functional polypeptide carrier is connected to the recognition segment includes 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 groups of the peptide chain of the recognition segment include natural amino acid side chain groups or non-natural amino acid side chain groups; the natural amino acid groups include at least one of amino groups and thiol groups; the side chain groups of the non-natural amino acid groups include at least one of nitrogen groups, alkynyl groups, aldehyde groups, ketone groups, fluorosulfonate groups, chlorine groups, bromine groups, and iodine groups; Preferably, the method for connecting the recognition segment to the functional polypeptide carrier includes at least one of chemical coupling, enzyme catalysis and gene recombination; 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 humanized 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 chimera, which specifically comprises the following steps: synthesizing in sequence according to the above amino acid sequence using a solid phase synthesizer, and obtaining a functional polypeptide carrier after cleavage and purification; and chimerizing the recognition segment with the functional polypeptide carrier using at least one of a chemical coupling method, an enzyme catalysis method and a gene recombination method to obtain a polypeptide drug chimera.

[0010] The beneficial effects achieved by the present invention are as follows: The present invention provides a polypeptide drug chimera and a preparation method thereof. The present invention constructs a polypeptide drug chimera 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 a lysosome, combines an antibody with a lysosomal signal amino acid sequence, and transfers the pathogenic antigen protein to a lysosomal subcellular organelle through the specific binding of the antibody to different antigens for degradation; the polypeptide drug chimera of the present invention utilizes a lysosomal protein sorting mechanism, forms a complex with a clathrin through a lysosomal targeting structural unit, and triggers clathrin-mediated endocytosis, ultimately leading to enrichment and degradation of the target protein bound by the functional polypeptide carrier in the lysosome. The functional polypeptide carrier of the present invention can achieve effective degradation and regulation of the target protein in vitro and / or in vivo, and can be used for knockdown or degradation of the target protein in vivo and / or in vitro, as well as the prevention and treatment of diseases such as cancer, metabolic diseases, and chronic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 HPLC (A) and MS (B) characterization results of Ly-a5 of this example; Figure 2 HPLC (A) and MS (B) characterization results of Ly-a6 of this example; Figure 3 HPLC (A) and MS (B) characterization results of Ly-a8 of this example; Figure 4 This is a quantitative diagram of the internalization of Ly-a1 to Ly-a14 in SKOV3 cells according to Example 2 of the present invention; Figure 5 This is a quantitative diagram of the internalization of Ly-a15 to Ly-a29 in SKOV3 cells according to Example 2 of the present invention; Figure 6 This is a quantitative diagram of the internalization of Ly-a1 to Ly-a14 in A549 cells according to Example 2 of the present invention; Figure 7 This is a quantitative diagram of the internalization of Ly-a15 to Ly-a29 in A549 cells according to Example 2 of the present invention; Figure 8 This is the electrophoresis gel image characterization result diagram of LA1~LA10 in this example; Fig. 9 This is a quantitative diagram of the internalization of HER2 by LA1-LA10 in MDA-MB-231 cells according to Example 4 of the present invention; Fig.10 This is a diagram showing the immunoblotting results of LA1-LA10 of Example 4 of the present invention degrading HER2 in MDA-MB-231 cells; Fig.11 This is a confocal laser scanning microscopy result diagram of HER2 degradation in MDA-MB-231 cells by LA1-LA10 of Example 4 of the present invention; The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only 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 ordinary technicians in the field without creative work are within the scope of protection of the present invention; Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art; in addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention; the preferred implementation methods and materials described herein are for demonstration purposes only and cannot limit the content of this application; The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified; Embodiment 1: This embodiment provides a functional polypeptide vector, which can mediate the transport of target proteins to lysosomes; the functional polypeptide vector that mediates the transport of target proteins to lysosomes includes one or more lysosomal targeting structural units; the lysosomal 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 does not exist; Xaa2=R or does not exist; Xaa3=R,L,E or does not exist; Xaa4=R,S,E,D or does not exist; Xaa5=R,S,K ,T,Y or not present;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 lysosomal targeting structural unit has the following amino acid sequence excluding the sequences: SYKYSKVNKE, EESEERDDHLLPM and DTGENPIYKSAV; Preferably, the lysosomal targeting structural unit has the following amino acid sequence to represent the peptide: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; wherein, Xaa1=R or does not exist; Xaa2=R or does not exist; Xaa3=R, L or does not exist; Xaa4=R, S or does not exist; Xaa5=R, K, Y or does not exist; 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 lysosomal targeting structural unit has the following amino acid sequence to represent the peptide: 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 lysosomal 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=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 sequences of the lysosomal targeting structural units are shown in Table 1 ; This embodiment also provides a method for preparing a functional polypeptide carrier that mediates the transport of target proteins to lysosomes, which specifically comprises the following steps: S1, resin swelling: Wang Resin, brand Shanghai Yuanye Biological Co., Ltd., product number S28287, substitution degree of 0.56mmol / g, 1% DVB, particle size of 100-200 mesh, put Wang Resin into a solid phase reactor, add DCM and shake to react for 30 min, then reduce pressure and drain, and repeat washing the resin twice; S2, coupling of Fmoc-1AA-OH: according to the sequence of the polypeptide, the polypeptide sequence is synthesized in sequence from the C-terminus to the N-terminus using a solid phase synthesizer, Fmoc-1AA-OH is dissolved in DMF, HOBt is added for activation, and the mixture is put into a solid phase synthesis reactor for coupling condensation reaction with the resin treated in step S1, and the mixture is reacted at room temperature for 5 hours, and then the Kaiser method is used for detection. If the detection result is negative, the coupling is completed, the reaction solution is decompressed and drained, the reaction is completed, the resin is repeatedly washed with DCM and DMF for 3 times each, the resin is deprotected with 20% DBLK, the deprotection treatment is performed twice, 5 minutes and 8 minutes respectively, and then the resin is repeatedly washed with DCM and DMF for 3 times each; the fully protected resin is synthesized in sequence 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 placed in a solid phase reactor, and coupled with the resin treated in step S2. After the condensation reaction was carried out for 2 hours at room temperature, the Kaiser method was used for detection. If the test result was negative, the coupling was completed, and the reaction solution was decompressed and drained; after the reaction was completed, the resin was repeatedly washed with DCM and DMF for 3 times each; the resin was deprotected twice with 20% DBLK for 5 minutes and 8 minutes respectively, and then the resin was repeatedly washed with DCM and DMF for 3 times each to remove the Fmoc protection, and the coupling and Fmoc protection removal steps were repeated to complete the assembly of the linear peptide resin; S4, FITC coupling, using fluorescent molecules to couple with functional polypeptide carriers that mediate the transport of target proteins to lysosomes. After the linear peptide chain is synthesized, the amino group at the end of the peptide chain is Fmoc deprotected to expose the N-terminal amino group, and 4 eq of FITC and 8 eq of DIPEA are added to react overnight in DMF solution; the Kaiser method is used for detection. If the test result is negative, the coupling is completed; S5. Cleavage of functional polypeptide carriers that mediate target protein transport to lysosomes: The resin is concentrated with methanol, then drained and weighed under reduced pressure, and 10-20 times the amount of cleavage solution (VTFA: Vthioanisole: Vanisole: VDODT = 90:5:3:2) is added to stir the cleavage reaction at room temperature for 2-2.5 hours, and then the filtrate is filtered and added to ice-cold anhydrous ether or methyl tert-butyl ether (10 eq), and the filtrate is allowed to stand at 2-8°C for 30 minutes and centrifuged (3000 rpm). The solid is washed with an appropriate amount of precipitation solution and centrifuged twice, and then vacuum dried to obtain a crude peptide; S6. Purification of the functional polypeptide carrier that mediates the transport of the target protein to the lysosome: Take the crude peptide sample prepared in step S5 and add an appropriate amount of pyrogen-free water to dissolve it to obtain a crude peptide sample solution, filter the sample solution with a φ0.45µm filter, collect the filtrate and purify it by RP-HPLC, collect the target peptide fraction, and obtain the functional polypeptide carrier that mediates the transport of the target protein to the lysosome, and characterize the synthesized functional polypeptide carrier by HPLC and MS; Figure 1 The HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a5 of this example are shown in FIG. Figure 2 The HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a6 of this example are shown in FIG. Figure 3 The HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a8 of this example are shown.

[0013] Embodiment 2: In this example, flow cytometry was used to detect the performance of the functional polypeptide carrier that mediates the transport of target proteins to lysosomes described in Example 1 during the cell internalization process. During the cell internalization process, after the functional polypeptide carrier enters the cell, the labeled fluorescent molecule emits a signal. After the flow cytometer detects these signals, the content and distribution of the functional polypeptide carrier in the cell are analyzed. The specific detection method includes the following steps: Cell culture: Human ovarian cancer cell line SKOV3 and human non-small cell lung cancer cell line A549 were obtained from the American Type Culture Collection (ATCC); SKOV3 cell line used RPMI 1640 basal medium (Gibco), and the medium was supplemented with 10% fetal bovine serum (Gibco) and 1% double antibody (penicillin 100 U / mL + streptomycin 100 g / mL); A549 cell line used DMEM basal medium (Gibco), and the medium was supplemented with 10% fetal bovine serum (Gibco) and 1% double antibody (penicillin 100 U / mL + streptomycin 100 g / mL); All cells were cultured in a humidified environment at 37°C with 5% CO2, and the cell morphology was normal and the growth state was good; Flow cytometry was used to evaluate the internalization of the functional polypeptide vector: In this example, SKOV3 cells and A549 cells were used for the experiment, and the vector sequence reported in the literature was selected as the positive control St (the amino acid sequence is shown in the sequence SEQ ID NO: 30, SYKYSKVNKE); the functional polypeptide vector labeled with the fluorescent molecule FITC was diluted to a final concentration of 100 nM with serum-free culture medium, and then incubated with SKOV3 cells or A549 cells for 6 h; then, an 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 is a quantitative diagram of the internalization of Ly-a1~Ly-a14 in SKOV3 cells in this example, Figure 5 This is a quantitative diagram of the internalization of Ly-a15 to Ly-a29 in Example 2 of the present invention in SKOV3 cells. Figure 6 : is a quantitative diagram of the internalization of Ly-a1 to Ly-a14 in A549 cells of this example, Figure 7 It is a quantitative diagram of the internalization of Ly-a15~Ly-a29 in A549 cells of this example; wherein the control group is the carrier polypeptide St reported in the literature; Figure 1 , Figure 2 , Figure 3 and Figure 4 The data are expressed as mean ± SEM (n = 3), * P < 0.05. The analysis of the mean cell fluorescence value shows that the functional polypeptide vector of the present invention has an internalization ability comparable to or better than that of the positive control sequence; Embodiment 3: This embodiment provides a method for constructing a polypeptide drug chimera: In this example, a nanobody targeting human epidermal growth factor receptor 2 (HER2) is selected as the recognition segment, and the functional polypeptide vector prepared in Example 1 is used as the transport vector.

[0014] a) Plasmid design: The present invention uses the HER2-targeting nanoantibody 5F7 (SEQ ID NO: 31) as the recognition segment, Ly-a5, Ly-a6, Ly-a8, Ly-a10, Ly-a11, Ly-a16, Ly-a17, Ly-a20, Ly-a27, and Ly-a29 as functional polypeptide carriers, and uses a linker peptide Linker (amino acid sequence is GGGGS) to connect the recognition segment and the functional polypeptide carrier prepared in Example 1, and connect the functional polypeptide carrier to the C-terminus of the nanoantibody 5F7 by homologous recombination to obtain a polypeptide drug chimera LA1-LA10 that can target HER2 in lysosomes; the amino acid sequence of the polypeptide drug chimera LA1-LA10 is shown in Table 4; however, the modified nanoantibody of the present invention is not limited to the amino acid sequence given in this example; Table 2 HER2 targeting peptide drug chimera sequences b) Expression, purification and characterization of peptide drug chimeras: The expression of the polypeptide drug chimera of this embodiment utilizes the E. coli expression system, and the purification method is Ni column affinity purification. The specific method adopts the following steps: The constructed LA1-LA10 plasmids were respectively transferred into E. coli Transform BL21 competent cells and plate to obtain glycerol bacteria. Take 10mL of glycerol bacteria and transfer to 1L fresh LB medium (containing 50µg / mL kanamycin), place it in a 37℃ shaker for about 8h, then add the inducer IPTG (final concentration is 1mM), and continue to culture at 25℃ for about 16h. Place the cultured bacterial solution in a high-speed centrifuge, use a horizontal rotor to collect the bacteria and discard the supernatant. Add an appropriate amount of Ni-NTA Buffer A to resuspend the bacteria and use an ultrasonic cell disruptor to disrupt the bacteria. Centrifuge the broken solution and collect the supernatant. Use a Ni column to purify LA1-LA10, rinse the resin with about 20 column volumes of Ni-NTA Buffer A, and collect all the Ni-NTA Buffer A that flows through the nickel column to detect impurity proteins that non-specifically bind to the nickel column. Subsequently, the target protein on the nickel column resin is gradient eluted with washing solutions containing different imidazole concentrations, and each component is collected separately for SDS-PAGE electrophoresis analysis and characterization. The fractions containing the purer target protein were combined, transferred to a protein concentration tube, centrifuged and replaced with PBS, and finally stored at -80°C.

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

[0016] Example 4: Peptide drug chimera mediates HER2 degradation in lysosomes In this example, the human breast cancer cell line MDA-MB-231 was used for the experiment, 5F7 was used as the recognition segment of the polypeptide drug chimera, 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, and Ly-a29 were used as functional polypeptide carriers of the polypeptide drug chimera. The polypeptide drug chimera LA1-LA10 was obtained by the construction method of the polypeptide drug chimera described in Example 3. The ability of the polypeptide drug chimera to internalize HER2 on the cell membrane surface was evaluated by flow cytometry experiments, and the expression amount of HER2 protein on the cell membrane surface was detected by immunoblotting (Western Blot) and laser confocal experiments, thereby evaluating the ability of the polypeptide drug chimera to degrade HER2. The control nanoantibody used in this example is 5F7 targeting HER2, and the primary antibody used is an anti-HER2 antibody. The specific experimental steps include: Cell culture: The human breast cancer cell MDA-MB-231 cell line was obtained from the American Type Culture Collection (ATCC). The MDA-MB-231 cell line used DMEM basal medium (Gibco) basal medium (Procell), and 10% fetal bovine serum (Gibco) and 1% double antibody (penicillin 100 U / mL + streptomycin 100 g / mL) were added to the medium. The cells were cultured in a humidified environment at 37°C with 5% CO2, and the cell morphology was normal and the growth state was good; Flow cytometry was used to evaluate the internalization of HER2 by the peptide drug chimera under the mediation of the functional peptide carrier: In this example, MDA-MB-231 cells were used for the experiment, and the cells were plated one day before administration; the peptide drug chimera was diluted with complete culture medium, and then incubated with MDA-MB-231 cells in the dark for 24 h; the cells were fixed with 4% paraformaldehyde, and then incubated with the cells with 1% BSA at room temperature for 1 h; the BSA-treated cells 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 peptide drug chimera was observed by flow cytometry; the fluorescent secondary antibody in this experiment was Goat anti-rabbit IgG H&L AlexaFluor® 488; Fig. 9 This is a flow cytometry result diagram of HER2 internalization of LA1-LA10 in MDA-MB-231 cells according to Example 4 of the present invention, wherein the control nanobody is TCZ targeting HER2; Fig. 9The results showed that HER2 was significantly internalized after administration of LA1-LA10, which proved that the peptide drug chimera fused with a functional peptide carrier had a significant internalization effect on HER2. Western Blot detection of HER2 expression: cells were plated one day before administration; the constructed peptide drug chimera was diluted to the target concentration using complete culture medium and incubated with cells for 48 h; cells were then lysed using lysis buffer, total cell protein was extracted, and the protein was quantified using a BCA kit (Thermo Fisher); proteins were then separated using 10% SDS-PAGE gel, and transferred to a polyvinylidene fluoride (PVDF) membrane by electrotransfer; the membrane with protein was blocked in 5% skim milk at room temperature for 1 h, the milk was then removed, and the membrane was incubated with the primary antibody at 4°C overnight on a shaker; the membrane was removed the next day and quickly immersed in TBST for washing 3 times, and then incubated with the secondary antibody at room temperature for 1 h; finally, the protein was detected and recorded using a chemiluminescent reagent and a Tanon chemiluminometer, and the protein was quantitatively analyzed using Image J; Fig.10 This is the immunoblotting result of LA1-LA10 in Example 4 of the present invention degrading HER2 in MDA-MB-231 cells, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an internal reference; thus, it can be proved that the polypeptide drug chimera fused with a functional polypeptide carrier has a significant degradation effect on HER2; Laser confocal experiment to observe the expression of HER2: In this example, MDA-MB-231 cells were used for the experiment, and cells were plated 1 day before administration; the constructed peptide drug chimera was diluted with complete culture medium, and then incubated with MDA-MB-231 cells in the dark for 48 h; the cell membrane was stained with a cell membrane staining kit (purchased from Bio-Technology Co., Ltd.), and the cells were fixed with 4% paraformaldehyde at room temperature for 15 min; after incubating the cells with 1% BSA at room temperature for 1 h, the cells 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, and the cell nuclei were stained with DAPI; the expression of HER2 after incubation of the peptide drug carrier with the cells was observed by confocal laser scanning microscopy, and the results are shown as follows: Fig.11 As shown; Fig.11 From top to bottom, they are the nuclear signal, cell membrane signal, HER2 signal, and the combined signal of the three; Fig.11 It can be seen that compared with the HER2-targeting nanoantibody TCZ, the fluorescence intensity of HER2 in cells incubated with each peptide drug chimera was significantly reduced, indicating that the expression level of HER2 was reduced; this proves that the peptide drug chimera has a significant degradation effect on HER2.

[0017] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention; The present invention and its embodiments are described above, which is not restrictive. What is shown in the accompanying drawings is only one of the embodiments of the present invention, and the actual application is not limited to this. In short, if ordinary technicians in the field are inspired by it and do not deviate from the purpose of the invention, they can creatively design methods and embodiments similar to the technical solution, which should all fall within the scope of protection of the present invention.

Claims

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

2. A polypeptide drug chimera according to claim 1, characterized in that: The functional polypeptide carrier that mediates the transport of target proteins to lysosomes comprises one or more lysosomal targeting structural units; the amino acid sequence of the lysosomal targeting structural unit comprises at least 10-15 amino acids; the lysosomal targeting structural unit has the following amino acid sequence to represent the peptide: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; Xaa1=R or does not exist; Xaa2=R or does not exist; Xaa3=R, L, E or does not exist; 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; among which, the sequences: SYKYSKVNKE, EESEERDDHLLPM and DTGENPIYKSAV are not included.

3. A polypeptide drug chimera according to claim 2, characterized in that: The lysosomal targeting structural unit has the following amino acid sequence to represent the peptide: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; wherein, Xaa1=R or does not exist; Xaa2=R or does not exist; Xaa3=R, L or does not exist; Xaa4=R, S or does not exist; Xaa5=R, K, Y or does not exist; 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. A polypeptide drug chimera according to claim 2, characterized in that: The lysosomal targeting structural unit has the following amino acid sequence to represent a peptide: 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. A polypeptide drug chimera according to claim 2, characterized in that: The lysosomal targeting structural unit has the following amino acid sequence to represent a peptide: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15; wherein, Xaa1=not present; Xaa2=not present; Xaa3=not present; 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. A polypeptide drug chimera according to claim 2, characterized in that: The sequences of the lysosomal targeting structural unit include 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), and EESEDRDDHLIPL (SEQ ID NO: 17). 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), 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: 27) NO:28), DTGDNPIYKSAL (SEQ ID NO:29).

7. A polypeptide drug chimera according to claim 1, characterized in that: 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 sugar-modified or not sugar-modified; the polypeptide is composed of natural amino acids or non-natural amino acids.

8. A polypeptide drug chimera according to claim 1, characterized in that: 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 chimera according to claim 1, characterized in that: The position where the functional polypeptide carrier is connected to the recognition segment includes 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 chimera according to claim 1, characterized in that: 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 alkynyl group, an aldehyde group, a ketone group, a fluorosulfonate, a chlorine group, a bromine group, and an iodine group; and the method for connecting the recognition segment to the functional polypeptide carrier includes at least one of a chemical coupling method, an enzyme catalysis method, and a gene recombination method.

11. A polypeptide drug chimera 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 chimera according to any one of claims 1 to 11, characterized in that: The specific steps include: A solid phase synthesizer is used to synthesize in sequence according to the above amino acid sequence, and after cleavage and purification, a functional polypeptide carrier is obtained; the recognition segment is chimerized with the functional polypeptide carrier by at least one of a chemical coupling method, an enzyme catalysis method and a gene recombination method to obtain a polypeptide drug chimera.

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