Functional polypeptide carrier and polypeptide drug chimera

Abstract

The invention discloses a functional polypeptide carrier and a polypeptide drug chimera in the technical field of polypeptide drug carriers, and the polypeptide drug chimera is constructed by a recognition segment specifically combined with a target protein and the 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 application belongs to the technical field of polypeptide drug carriers, and particularly relates to a functional polypeptide carrier, a polypeptide drug chimera containing the functional polypeptide carrier and a preparation method of the polypeptide drug chimera. BACKGROUND

[0002] At present, cancer is still one of the most serious health threats to human beings (Mattiuzzi C, Lippi G. Current Cancer Epidemiology. J Epidemiol Glob Health. 2019; 9(4): 217-222.). Traditional anticancer drugs, after intravenous injection or oral administration, only a small part of the effective components can be enriched in the cancer site in the blood circulation, and most of the components are absorbed by normal tissues, resulting in low drug utilization efficiency and large toxic side effects (Randall EC, Emdal KB, Laramy JK, et al. Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma. Nat Commun. 2018; 9(1): 4904.). How to realize the precise delivery of drugs to cancer cell tissues and kill cancer cells targetedly has become a key problem to be solved in the field of drug delivery.

[0003] Polypeptides have been widely used in the construction of targeted drug delivery carriers due to their good biocompatibility, functional diversity, high biological in vivo responsiveness and simple and easy synthesis and modification methods. Based on the functional polypeptides with targeting function and response to stimuli, the drug delivery system constructed can accurately deliver drugs to tumor regions (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. J Am Chem Soc. 2023; 145(34): 19107-19119.). When the drug delivery system reaches the tumor tissue, under the action of special tumor microenvironment or external stimuli, the precise release of drugs is realized. This specific tumor targeting and stimulus responsive functional polypeptide carrier can maximize the anti-tumor effect of drugs and reduce the toxic side effects of drugs.

[0004] Due to the small size of polypeptides, the biochemical properties of the active group will not change significantly after being introduced into the functionalized polypeptide transporter, so when the polypeptide is linked to a specific active group, the targeting is more accurate and controllable. The targeting function of functionalized polypeptides can be divided into two levels of tumor cells and various organelles, and the targeting function of organelles can bypass drug resistance. For example, mitochondria, as the energy center of cells, regulate the synthesis of adenosine triphosphatase in cells. Mitochondria-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, non-specific uptake of drugs by tumor cells and normal cells, etc. Therefore, developing a new type of organelle-targeting transport system is crucial to solve the problems of systemic toxicity and drug resistance.

[0005] Lysosomes, as a targeted organelle for drug transport, have unique advantages. Its main function is to decompose and digest substances inside and outside the cell, and lysosome-targeting functionalized polypeptide targeting transport can help to utilize this natural "garbage processor" to bypass the 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 releasing drugs directly 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 lysosome-targeting strategy provides new ideas and possibilities for developing safer and more effective anticancer drug delivery systems. SUMMARY

[0006] In view of the above, in order to overcome the defects of the prior art, the present application provides a functional polypeptide carrier and a polypeptide drug chimera comprising the same, and the polypeptide drug chimera is constructed by a recognition segment specifically binding to a target protein and a functional polypeptide carrier mediating the transport of the target protein to lysosomes, the antibody is combined with a lysosome signal amino acid sequence, the pathogenic antigen protein is transported into lysosomes by the specific binding of the antibody to different antigens, and is degraded.

[0007] In order to achieve the above object, the technical scheme adopted by the present application is as follows: the present application provides a functional polypeptide carrier, which is a key part of a lysosome targeting structure unit and has the following amino acid sequence representing a peptide: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-S-Y-K-Y-S-K-Xaa12-Xaa13-K-Xaa15; wherein Xaa1=R or is absent; Xaa2=R or is absent; Xaa3=L or is absent; Xaa4=R or is absent; Xaa5=K or is absent; Xaa12=V or L; Xaa13=N or Q; and Xaa15=E or D.

[0008] Preferably, the functional polypeptide carrier has the following amino acid sequence representing a peptide: R-R-L-R-K-S-Y-K-Y-S-K-Xaa12-Xaa13-K-Xaa15; wherein Xaa1=R; Xaa2=R; Xaa3=L; Xaa4=R; Xaa5=K; Xaa12=V or L; Xaa13=N or Q; and Xaa15=E or D.

[0009] Preferably, the functional polypeptide carrier has the following amino acid sequence representing a peptide: S-Y-K-Y-S-K-Xaa12-Xaa13-K-Xaa15; wherein Xaa1= is absent; Xaa2= is absent; Xaa3= is absent; Xaa4= is absent; Xaa5= is absent; Xaa12=V or L; Xaa13=N or Q; and Xaa15=E or D.

[0010] Preferably, the sequence of the functional polypeptide carrier comprises SYKYSKVQKE (SEQ ID NO: 5), SYKYSKVQKD (SEQ ID NO: 6), SYKYSKLQKD (SEQ ID NO: 8), RRLRKSYKYSKVNKE (SEQ ID NO: 10), RRLRKSYKYSKVQKE (SEQ ID NO: 11), and the like.

[0011] The application also provides a polypeptide drug chimera, i.e. a chimera mediating the transport of a target protein to a lysosome, the structure of the polypeptide drug chimera comprising a recognition segment and a lysosome targeting structural unit; the recognition segment recognizes and specifically binds to the target protein; the lysosome targeting structural unit mediates the transport of the target protein to the lysosome; and the lysosome targeting structural unit comprises the one or more functional polypeptide carriers. The recognition segment can specifically bind to the target protein and is a 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, an antibody fragment, an aptamer, a small molecule inhibitor or other molecules capable of binding to a specific protein; its role is to ensure that the polypeptide drug chimera can accurately recognize and bind to a specific target protein, avoiding non-specific binding; after specific binding, a target protein-polypeptide drug chimera complex is formed; 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 for degradation or processing through the endocytosis pathway or other mechanisms.

[0012] Preferably, the functional lysosome targeting structural unit comprises one or more functional polypeptide carriers. Preferably, the recognition segment comprises at least one of a polypeptide, a protein, a nucleic acid, a nanoparticle or a small molecule drug, wherein the small molecule drug is an organic compound with a simple structure, a molecular weight less than 1000 daltons, and is chemically synthesized or extracted from natural products. Preferably, the recognition segment is connected to the functional polypeptide carrier by direct connection or indirect connection. Preferably, the direct connection of the recognition segment and the functional polypeptide carrier can be that the C-terminus of the recognition segment is directly connected to the N-terminus of the functional polypeptide carrier through a peptide bond, or the N-terminus of the recognition segment is directly connected to the C-terminus of the functional polypeptide carrier through a peptide bond. Preferably, the indirect connection of the recognition segment and the functional polypeptide carrier is through a linker peptide, a chemical linker or other biologically active peptide chains, and the amino acid sequence of the linker peptide can be (Leu-Pro-Glu-Thr)x-(Glu-Thr)y1-Asp-Thr-Glu-Thr)x-z, wherein x=0 or 1, y1=3, 4 or 5, y2=0 or 1, and z=1, 2 or 3. - )z, wherein x=0 or 1, y1=3, 4 or 5, y2=0 or 1, and z=1, 2 or 3. Preferably, the recognition segment comprises a polypeptide, and the position of the functional polypeptide carrier connected to the recognition segment comprises 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 recognition segment peptide chain side chain group includes a natural amino acid side chain group or a non-natural amino acid side chain group; the natural amino acid group includes at least one of amino and thiol groups; the non-natural amino acid side chain group includes at least one of nitrogen group, alkynyl group, aldehyde group, ketone group, fluorosulfonate, chlorine group, bromine group, and iodine group. Preferably, the method for linking 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 monospecific antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, humanized antibody, monoclonal antibody, or antigen-binding fragment of monoclonal antibody; Preferably, the antigen-binding fragment of the monoclonal antibody is Fab, Fab', F(ab')2, Fv, dsFv, scFv, Or at least one of VHH.

[0013] Preferably, the chimeric sequences include chimeric sequences SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, and SEQ ID 55 targeting the cellular TIGIT protein, and chimeric sequences SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, and SEQ ID 69 targeting the cellular IL6R protein.

[0014] The beneficial effects achieved by this invention are as follows: This invention provides a functional polypeptide carrier and a lysosomal targeting structural unit composed thereof. The invention constructs a polypeptide-drug chimera by using a recognition segment that specifically binds to the target protein and a functional polypeptide carrier that mediates the transport of the target protein to the lysosome. Antibodies bind to the lysosomal signal amino acid sequence. Through the specific binding of the antibody to different antigens, pathogenic antigen proteins are transferred to the lysosomal subcellular organelle for degradation. The polypeptide-drug chimera of this invention utilizes the lysosomal protein sorting mechanism. The lysosomal targeting structural unit forms a complex with clathrin, promoting clathrin-mediated endocytosis, ultimately leading to the enrichment and degradation of the target protein bound to the functional polypeptide carrier within the lysosome. The functional polypeptide carrier of this invention can achieve effective degradation and regulation of target proteins in vitro and / or in vivo, and can be used for the in vivo and / or in vitro knockdown or degradation of target proteins, as well as the prevention and treatment of diseases such as cancer, metabolic diseases, and chronic diseases. Attached Figure Description

[0015] Figure 1 The HPLC (A) and MS (B) characterization results of Ly-a5 in this embodiment are shown in the figure. Figure 2 The HPLC (A) and MS (B) characterization results of Ly-a6 in this embodiment are shown in the figure. Figure 3 The HPLC (A) and MS (B) characterization results of Ly-a8 in this embodiment are shown in the figure. Figure 4 This is a quantitative diagram showing the internalization of Ly-a1~Ly-a14 in SKOV3 cells in Example 2 of the present invention; Figure 5 This is a quantitative diagram showing the internalization of Ly-a15~Ly-a29 in SKOV3 cells in Example 2 of the present invention; Figure 6 This is a quantitative diagram showing the internalization of Ly-a1~Ly-a14 in A549 cells in Example 2 of the present invention; Figure 7 This is a quantitative diagram showing the internalization of Ly-a15~Ly-a29 in A549 cells in Example 2 of the present invention; Figure 8 These are the electrophoretic gel characterization results of LA1~LA10 in this embodiment; Figure 9 This is a quantitative diagram showing the internalization of HER2 in MDA-MB-231 cells by LA1-LA10 in Example 4 of the present invention. Figure 10 This is an immunoblot image showing the degradation of HER2 by LA1-LA10 in MDA-MB-231 cells according to Example 4 of the present invention; Figure 11This is a confocal laser scanning microscope image showing the degradation of HER2 by LA1-LA10 in MDA-MB-231 cells according to Example 4 of the present invention. Figure 12 This is a quantitative diagram of the activity of Ly-a1~Ly-a14 on lysosomal leucine aminopeptidase (PLBD1) in the human neuroblastoma cell line SH-SY5Y of Example 5 of the present invention. Figure 13 This is a confocal laser scanning microscope image showing the degradation of TIGIT by LC1-LC5 cells in human multiple myeloma cells MM1S in Example 6 of the present invention. Figure 14 Immunoblot results of LC1-LC5 degradation of CD226 in MM1S cells in Example 6 of this invention; Figure 15 Quantitative graph of the activation effect of LC1-LC5 on Jurkat T cells in Example 6 of this invention; Figure 16 This is a quantitative graph of the LC1-LC5 inhibition of breast cancer tumor growth curves in Example 7 of the present invention; Figure 17 This is a quantification diagram of tumor weight quantification of breast cancer inhibition by LC1-LC5 in Example 7 of the present invention; Figure 18 This is a diagram showing the tumor tissue results of LC1-LC5 inhibition of breast cancer in Example 7 of the present invention; Figure 19 This is an immunoblot image showing the degradation of IL6R by LD1-LD10 in SaOS-2 cells according to Example 8 of the present invention; Figure 20 This is a diagram showing the results of LD4-LD5 treatment of immune-mediated skin diseases in mice according to Example 9 of the present invention; Figure 21 This is a scanning microscope image of HE staining of the dorsal skin tissue of mice with LD4-LD5-treated immune skin diseases according to Example 9 of the present invention; Figure 22 This is a quantitative diagram of the toxic effects of the introduced cell-penetrating peptide region on cells in Example 10 of the present invention; Figure 23 This is a diagram showing the molecular structure of the chimera with YSKV or YSKL introduced in Example 11 of the present invention. The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art; furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention; the preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the experimental materials used in the following embodiments are all purchased from commercial channels. Example 1: Construction of Functional Peptide Vectors This embodiment provides a series of functional polypeptide carriers as lysosomal targeting structural units for mediating the transport of target proteins to lysosomes in this invention. The sequences of the functional polypeptide carriers of this invention are shown in Table 1. Table 1. Amino acid sequences of the functional polypeptide carriers of the present invention.

[0017] This embodiment also provides a method for preparing a functional polypeptide carrier, specifically including the following steps: S1. Resin swelling: Wang Resin, brand name Shanghai Yuanye Biotechnology Co., Ltd., product number S28287, with a substitution degree of 0.56 mmol / g, 1% DVB, and a particle size of 100-200 mesh, was used. Wang Resin was added to a solid-phase reactor, DCM was added, and the reaction was shaken for 30 min. The mixture was then dried under reduced pressure, and the resin was washed twice. S2. Coupling of Fmoc-1AA-OH: Based on the peptide sequence, the peptide sequence was synthesized sequentially from the C-terminus to the N-terminus using a solid-phase synthesizer. Fmoc-1AA-OH was dissolved in DMF, activated with HOBt, and added to the solid-phase synthesis reactor for coupling condensation reaction with the resin treated in step S1. After reacting at room temperature for 5 hours, the Kaiser method was used for detection. If the detection result was negative, the coupling was complete. The reaction solution was dried under reduced pressure, and the reaction was ended. The resin was washed three times each with DCM and DMF. The resin was deprotected twice with 20% DBLK for 5 min and 8 min respectively. The resin was then washed three times each with DCM and DMF. The fully protected resin was synthesized sequentially 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 added to a solid-phase reactor. It was then coupled with the resin treated in step S2. After a condensation reaction at room temperature for 2 hours, the Kaiser method was used for detection. If the detection result was negative, the coupling was complete. The reaction solution was then dried under reduced pressure. After the reaction was completed, the resin was washed three times each with DCM and DMF. The resin was deprotected twice with 20% DBLK for 5 min and 8 min respectively. Then, the resin was washed three times each with DCM and DMF to remove Fmoc protection. The coupling and deprotection steps were repeated to complete the assembly of the linear peptide resin. The S4 and FITC conjugation involved coupling a fluorescent molecule with a functional polypeptide carrier that mediates the transport of the target protein to the lysosome. After the linear peptide chain was synthesized, the terminal amino group was deprotected with Fmoc to expose the N-terminal amino group. Then, 4 eq of FITC and 8 eq of DIPEA were added and reacted overnight in DMF solution. The Kaiser method was used for detection. If the detection result was negative, the conjugation was considered complete. S5. Cleavage of functional polypeptide carriers mediating target protein transport to lysosomes: The resin was concentrated with methanol, dried under reduced pressure, and weighed. 10-20 times the amount of lysis buffer was added. : : : =90:5:3:2) The pyrolysis reaction was stirred at room temperature for 2-2.5 h, and then filtered. The filtrate was added to ice-cold anhydrous diethyl ether or methyl tert-butyl ether (10 eq), and centrifuged at 2-8℃ for 30 min (3000 rpm). The solid was washed twice with an appropriate amount of precipitate and then vacuum dried to obtain crude peptide. S6. Purification of the functional polypeptide carrier mediating the transport of target proteins to lysosomes: The crude peptide sample prepared in step S5 was dissolved in an appropriate amount of pyrogen-free water to obtain a crude peptide sample solution. The sample solution was filtered through a φ0.45µm filter, and the filtrate was collected and purified by RP-HPLC. The target peptide fraction was collected to obtain the functional polypeptide carrier mediating the transport of target proteins to lysosomes. The synthesized functional polypeptide carrier was characterized by HPLC and MS. Figure 1 The figures show the HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a5 in this embodiment. Figure 2 These are HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a6 in this embodiment. Figure 3 The HPLC (A) and MS (B) characterization results of the functional polypeptide carrier Ly-a8 in this embodiment are shown.

[0018] Example 2: Study on the internalization of functional polypeptide carriers in cells In this embodiment, flow cytometry was used to detect the performance of the functional polypeptide carrier mediated by the target protein transport 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 molecules emit signals. After the flow cytometer detects these signals, the content and distribution of the functional polypeptide carrier in the cell are analyzed. The specific testing 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 were both obtained from the American Type Culture Collection Center (ATCC). The SKOV3 cell line was cultured in RPMI 1640 basal medium (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 g / mL added. The A549 cell line was cultured in DMEM basal medium (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 g / mL added. All cells were cultured at 37°C in a humid environment with 5% CO2, and the cells showed normal morphology and good growth. Flow cytometry was used to assess the internalization of functional peptide carriers: SKOV3 and A549 cells were used in this experiment. The vector sequence reported in the patent document (CN118697896A, SEQ ID NO: 4) was selected as the positive control St (amino acid sequence as shown in sequence SEQ ID NO: 30, SYKYSKVNKE). The FITC-labeled functional peptide carrier was diluted to a final concentration of 100 nM in serum-free medium and then co-incubated with SKOV3 or A549 cells for 6 h. Subsequently, the antibody bound to the cell membrane was removed with acid washing buffer, and the residual acid washing buffer was washed with PBS. The cells were then digested from the culture dish and transferred to EP tubes. The fluorescence intensity of the treated cells was observed by flow cytometry. Figure 4 This is a quantitative graph showing the internalization of Ly-a1~Ly-a14 in SKOV3 cells in Example 1. Figure 5 This is a quantitative diagram showing the internalization of Ly-a15~Ly-a29 in SKOV3 cells according to Example 1 of the present invention. Figure 6 This is a quantitative graph showing the internalization of Ly-a1~Ly-a14 in A549 cells according to Example 1. Figure 7 This is a quantitative diagram showing the internalization of Ly-a15~Ly-a29 in A549 cells in Example 1; the control group is the transport peptide St reported in the literature. Figure 1 , Figure 2 ,Figure 3 and Figure 4 All data are expressed as: mean ± SEM (n = 3). P<0.05, and the analysis of the average cell fluorescence value showed that the functional polypeptide carrier of the present invention has an internalization ability that is comparable to or better than that of the positive control sequence. Example 3: Construction of polypeptide drug chimeras This embodiment provides a polypeptide drug chimera: In this embodiment, 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.

[0019] a) Plasmid design: This invention uses the HER2-targeting nanobody 5F7 (SEQ ID NO:31) as the recognition segment, and Ly-a5, Ly-a6, Ly-a8, Ly-a10, Ly-a11, Ly-a16, Ly-a17, Ly-a20, Ly-a27, and Ly-a29 as functional peptide carriers. A linker peptide (amino acid sequence GGGGS) is used to connect the recognition segment and the functional peptide carrier prepared in Example 1. The functional peptide carrier is then linked to the C-terminus of the nanobody 5F7 via homologous recombination to obtain the HER2-targeting peptide-drug chimera LA1-LA10. The amino acid sequences of the peptide-drug chimera LA1-LA10 are shown in Table 2. However, the modified nanobody of this invention is not limited to the amino acid sequences given in this example. Table 2 HER2-targeting peptide drug chimeric sequences

[0020]

[0021] b) Expression, purification, and characterization of polypeptide drug chimeras: In this embodiment, the expression of the polypeptide drug chimera was performed using an E. coli expression system, and the purification method was Ni column affinity purification. The specific method included the following steps: The constructed LA1-LA10 plasmids were respectively transferred into E. coliBL21 competent cells were transformed and plated to obtain glycerol-containing bacteria. 10 mL of glycerol-containing bacteria was transferred to 1 L of fresh LB medium (containing 50 µg / mL kanamycin) and cultured at 37°C with shaking for approximately 8 h. Then, IPTG (final concentration 1 mM) was added, and the culture was continued at 25°C for approximately 16 h. The cultured bacterial solution was centrifuged using a high-speed centrifuge with a horizontal rotor to collect the bacterial cells, discarding the supernatant. An appropriate amount of Ni-NTA Buffer A was added to resuspend the bacterial cells, and they were disrupted using an ultrasonic cell disruptor. The disrupted solution was centrifuged, and the supernatant was collected. LA1-LA10 were purified using a Ni column. The resin was washed with approximately 20 column volumes of Ni-NTA Buffer A, and all Ni-NTA Buffer A flowing through the nickel column was collected for detecting impurity proteins that non-specifically bind to the nickel column. Subsequently, the target proteins on the nickel column resin were eluted using a gradient elution with washing buffers containing different concentrations of imidazole. Each fraction was collected and characterized by SDS-PAGE electrophoresis. Combine the components containing relatively pure target proteins, transfer them to protein concentration tubes, centrifuge with PBS to change the medium, and finally store at -80°C.

[0022] Figure 8 The images show the electrophoretic gel characterization results of LA1~LA10 in this embodiment.

[0023] Example 4: Peptide drug chimera LA1-LA10 mediates HER2 degradation in lysosomes In this embodiment, the human breast cancer cell line MDA-MB-231 was used for experiments. 5F7 was used as the recognition segment of the described polypeptide drug chimera, and the functional polypeptide carrier 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 the functional polypeptide carriers for the polypeptide drug chimera. Following the construction method described in Example 3, polypeptide drug chimeras LA1-LA10 were obtained. The ability of LA1-LA10 to endocytose HER2 on the cell membrane surface was evaluated using flow cytometry. Western blotting and laser confocal microscopy were used to detect the degradation of HER2 protein expression on the cell membrane surface by the polypeptide drug chimera, thereby assessing its ability to degrade HER2. The control nanobody used in this embodiment was 5F7 targeting HER2, and the primary antibody used was an 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 Center (ATCC). The MDA-MB-231 cell line was cultured in DMEM basal medium (Gibco) (Procell), supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 g / mL. Cells were cultured at 37°C in a humid environment with 5% CO2; cell morphology was normal and growth was good. Flow cytometry was used to evaluate the internalization of HER2 by a peptide-drug chimera mediated by a functional peptide carrier. In this study, MDA-MB-231 cells were used for the experiment. Cells were plated one day before drug 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. After fixation with 4% paraformaldehyde, the cells were incubated with 1% BSA at room temperature for 1 h. The BSA-treated cells were then incubated with a primary antibody at 37°C for 1 h, followed by incubation with a 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 used in this experiment was Goat anti-rabbit IgG H&L AlexaFluor® 488. Figure 9 This is a flow cytometry diagram showing the HER2 internalization results of LA1-LA10 in MDA-MB-231 cells in Example 4 of the present invention, wherein the control nanobody is TCZ targeting HER2; Figure 9 The results showed that HER2 was significantly internalized after administration of LA1-LA10; this proves that the polypeptide drug chimera with a functional polypeptide carrier has a significant internalization effect on HER2. Western Blot Detection of HER2 Expression: Cells were plated one day before drug 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 with lysis buffer, total cellular protein was extracted, and 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 electroporation; the protein-containing membrane was blocked in 5% skim milk at room temperature for 1 h, then the milk was removed, and the membrane was incubated with primary antibody overnight at 4°C on a shaker; the next day, the membrane was removed and quickly immersed in TBST for three washes, then incubated with secondary antibody at room temperature for 1 h; finally, protein was detected and recorded using chemiluminescence reagents and a Tanon chemiluminescence analyzer, and protein was quantified using ImageJ; Figure 10The image shows the immunoblotting results of LA1-LA10 degrading HER2 in MDA-MB-231 cells in Example 4 of this invention, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. This demonstrates that the polypeptide drug chimera with a functional polypeptide carrier significantly degrades HER2. HER2 expression was observed using a confocal laser scanning microscope: MDA-MB-231 cells were used in this experiment. Cells were plated one day before drug 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. Cell membranes were stained using a cell membrane staining kit (purchased from Beyotime Biotechnology Co., Ltd.), and cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After incubating with 1% BSA at room temperature for 1 h, the cells were first incubated with primary antibody at 37 °C for 1 h, and then with fluorescently labeled secondary antibody at room temperature for 30 min. Cell nuclei were stained with DAPI. HER2 expression after incubation with the peptide-drug carrier was observed using a confocal laser scanning microscope. The results are as follows: Figure 11 As shown; Figure 11 From top to bottom, the signals are: nuclear signal, cell membrane signal, HER2 signal, and the combined signal of the three; Figure 11 It can be seen that, compared with the HER2-targeting nanobody TCZ, the fluorescence intensity of HER2 in cells after incubation with each peptide drug chimera was significantly reduced, indicating that the expression level of HER2 was reduced; thus, it can be proved that the peptide drug chimera has a significant degradation effect on HER2.

[0024] Example 5: Functional polypeptide carrier induces lysosomal leucine aminopeptidase (PLBD1) activity Lysosomal leucine aminopeptidase 1 (PLBD1) is responsible for the specific degradation of proteins within cells, and its activity is closely related to various protein metabolism disorders (such as neurodegenerative diseases and lysosomal storage diseases). This embodiment aims to verify that the functional polypeptide carrier provided by the present invention can specifically induce the activity of PLBD1, thereby achieving the ability to degrade proteins. In this embodiment, the vector sequence reported in patent document (CN118697896A, SEQ ID NO:4) is selected as the positive control St (SEQ ID NO:30: SYKYSKVNKE).

[0025] 1) Cell Culture The human neuroblastoma cell line SH-SY5Y was purchased from ATCC. Cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5%... Incubate in an incubator. Plant cells in each well. Cells were seeded at a density of [number] cells / well in 24-well plates and cultured for 24 hours until the cell density reached 70%-80%. The experimental groups were as follows: Experimental groups: Cells were treated with culture medium containing 10 μM Ly-a1~Ly-a14 functional peptide carriers. Positive control group: Cells were treated with culture medium containing 10 μM St. Blank control group: Cells were treated with only an equal volume of PBS. All groups were treated for 24 hours.

[0026] 2) PLBD1 activity detection Step a: Prepare cell lysis buffer: Add 200 μL of lysis buffer containing 0.1% Triton X-100 to each well and lyse on ice for 30 minutes. Then, centrifuge at 12,000 rpm for 15 minutes at 4°C and collect the supernatant.

[0027] Step b: Protein concentration determination: Protein concentration in each group was determined using the BCA protein quantification kit to standardize subsequent enzyme activity.

[0028] Step c: Enzyme activity assay: Detection was performed using a commercially available leucine aminopeptidase fluorescent substrate. In a 96-well black microplate, add 50 μL of cell lysis buffer (containing an equal volume of 20 μg total protein) and 150 μL of reaction buffer (50 mM sodium citrate buffer, pH 4.5). Then, add 10 μL of substrate (final concentration 200 μM) to initiate the reaction.

[0029] Step d: Fluorescence detection: Immediately place the ELISA plate in a fluorescence microplate reader and monitor the fluorescence intensity at 37°C (excitation wavelength: 360 nm, emission wavelength: 460 nm). Monitor continuously for 60 minutes, and read the data every 5 minutes.

[0030] Figure 12This is a quantitative graph showing the activity of Lysosomal leucine aminopeptidase (PLBD1) in the human neuroblastoma cell line SH-SY5Y using Ly-a1~Ly-a14 from Example 5 of this invention. As shown in the figure, the functional polypeptide carriers Ly-a1 to Ly-a14 provided by this invention can effectively enhance PLBD1 activity, indicating that this series of functional polypeptide carriers exhibits good activity and high degradation capacity for lysosomal leucine aminopeptidase. Particularly noteworthy is that the groups treated with Ly-a5, Ly-a6, Ly-a8, Ly-a10, and Ly-a11 showed significantly better PLBD1 activity than the negative control group and the ST control group, exhibiting an increase of more than tenfold, demonstrating a decisive advantage. This result clearly confirms that the functional peptide carriers represented by Ly-a5 / Ly-a6 / Ly-a8 / Ly-a10 / Ly-a11 exhibit unexpectedly superior performance and unique effectiveness in activating PLBD1. Not all sequences can achieve the same effect, thus highlighting their advanced nature and core value as the core of this invention. Ly-a5 / Ly-a6 / Ly-a8 / Ly-a10 / Ly-a11 strongly demonstrate the high degree of creativity and precision in the peptide carrier structure design of this invention. Their unique sequence composition and spatial structure are key to achieving the superior activation effect of PLBD1, which cannot be achieved by other sequences through conventional substitution or modification.

[0031] Example 6: Chimera-induced TIGIT degradation This embodiment uses the human multiple myeloma cell line MM1S for experiments. The expression level of TIGIT on the cell membrane surface was detected by laser confocal microscopy and Western blotting to evaluate the chimeric ability to degrade TIGIT and its selective / off-target effects. The commercially available naked antibody control used in this embodiment was tiragolumab (LC), and the primary antibody used was an anti-TIGIT antibody.

[0032] 1) Construction of TIGIT-targeting chimeric plasmid The lysosome-targeting functional polypeptide carrier is fused to Ti at at least one of the following locations: the C-terminus of the Ti heavy chain (hereinafter referred to as Ti-HC), the N-terminus of the Ti heavy chain, the C-terminus of the Ti light chain (hereinafter referred to as Ti-LC), and the N-terminus of the Ti light chain. This embodiment modifies Ti at the gene level. For ease of explanation, Ly-a5 is used as an example: Ly-a5 is used as the lysosome-targeting functional polypeptide carrier, and Ly-a5 is fused to the C-terminus of the Ti light chain and / or heavy chain to prepare a chimera. However, the chimera of this invention is not limited to the amino acid sequence given in this embodiment. The lysosome-targeting functional polypeptide carrier of this invention can also be fused to the N-terminus of the heavy chain and / or light chain, and all resulting chimeras are within the protection scope of this invention.

[0033] The construction of the Ti-based chimeric plasmid in this embodiment specifically includes the following steps: First, Ly-a5 was fused to the C-terminus of both the heavy and light chains to construct fragments Ti-LC-Ly-a5 and Ti-HC-Ly-a5. These fragments were then integrated into the vector pcDNA 3.4. The amino acid sequences of Ti-LC, Ti-HC, Ti-LC-Ly-a5, and Ti-HC-Ly-a5 are shown in Table 3.

[0034] Table 3. Amino acid sequences of TIGIT-targeting chimeric plasmids

[0035]

[0036]

[0037] The target fragment and vector were amplified separately by PCR, ensuring that the synthesized target fragment and vector had overlapping fragments. Then, the PCR product and plasmid vector were ligated using homologous recombinase, and the ligated product was transformed into DH5α competent cells, plated, and incubated overnight. The next day, a single plaque was picked and cultured, and finally, sequencing was performed to verify successful construction.

[0038] 2) Expression, purification and characterization of chimeras Human embryonic kidney cells (293 Freestyle) were seeded into shake flasks until the cell density reached [value missing]. Transfection can be performed when the DNA concentration is 1 / mL. The transfection reagent is polyethyleneimine (PEI). For transfection, add 10% of the transfection volume of culture medium to two centrifuge tubes. Add 160 μL of PEI to one centrifuge tube and 80 μg of plasmid (heavy chain:light chain = 2:3) to the other. Mix thoroughly and let stand for 5 min. Add DNA dilution buffer to the PEI dilution buffer, mix thoroughly, let stand for 20 min, and transfer to a shake flask. Collect the supernatant 5 days after transfection. Filter the supernatant through a 0.45 μm filter and purify it using a Protein A affinity chromatography column. The purified protein was characterized structurally by SDS-PAGE, HPLC, and MS.

[0039] 3) Laser confocal microscopy detection of the chimera's degradation effect on cellular TIGIT. The human multiple myeloma cell line MM1S was obtained from the American Type Culture Collection Center (ATCC). MM1S cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 basal medium (Procell) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 g / mL. Cell culture was maintained at 37°C with 5% [missing information - likely a specific concentration or concentration]. In a humid environment, the cells have normal morphology and grow well.

[0040] Cells were plated one day before drug administration. The constructed chimeras were diluted with complete culture medium and incubated with MM1S cell lines for 24 h. Cells were then fixed with 4% paraformaldehyde at room temperature and incubated with 1% BSA at room temperature for 1 h. After BSA treatment, cells were first incubated with primary antibody at 37 °C for 1 h, followed by incubation with fluorescently labeled secondary antibody at room temperature for 30 min. Cell nuclei were stained with DAPI, and cell membranes were stained with green fluorescent dye. The degradation of TIGIT by LCs was observed using a confocal laser scanning microscope (CLSM). The fluorescent secondary antibody used in this experiment was Goat anti-rabbit IgG H&L Alexa Fluor® 647.

[0041] Figure 13The figure shows the confocal laser scanning microscopy results of LC1-LC5 degrading TIGIT in human multiple myeloma cells MM1S in Example 6 of this invention. It illustrates the laser confocal microscopy imaging results of various LCs molecules degrading TIGIT protein in MM1S cells in Example 6 of this invention. In the figure, blue represents the cell nucleus, red represents the cell membrane, and green represents the TIGIT protein signal. The experimental results show that, compared with the commercially available naked anti-tirerenzanol (LC) and the ST positive control (LC0), cells treated with the chimeric compounds LC1-LC5 containing functional peptide carriers (Ly-a5 / Ly-a6 / Ly-a8 / Ly-a10 / Ly-a11) showed a significant decrease in green fluorescence signal, indicating that the intracellular TIGIT protein level was significantly degraded. Specifically, the commercially available naked anti-LC itself had no significant effect on TIGIT protein content; while LC0, although reducing TIGIT due to the effect of ST, had a limited effect. In contrast, LC1-LC5 all exhibited strong degradation capabilities against TIGIT protein, with LC4 and LC5 (which are respectively infused with Ly-a10 and Ly-a11) showing the most significant effects. These results fully demonstrate that this invention, by incorporating antibodies with specific functional peptide carriers, successfully achieves highly efficient degradation of the key target TIGIT, overcoming the limitations of traditional antibody drugs that can only block signaling but not directly reduce the target protein. This demonstrates significant innovation and breakthrough in its mode of action.

[0042] 4) Immunoblotting detection of selective / off-target effects of chimeras Figure 14 This is the immunoblotting result of LC1-LC5 in MM1S cells degrading TIGIT in Example 6 of the present invention. TIGIT and its homologous receptor CD226 (DNAM-1) together constitute a key immune regulatory axis. Both are co-expressed on the surface of immune cells and share the same ligand CD155 / PVR, but they play antagonistic biological functions in signal transduction: TIGIT transmits inhibitory signals, while CD226 transmits activating signals. Maintaining the balance between them is crucial for precise regulation of the immune response. This example evaluates the role of a chimera in the degradation of TIGIT protein on CD226 and its effect on immune activation, to examine the target selectivity and functional specificity of the chimera.

[0043] Human multiple myeloma cell line MM1S was plated one day before drug administration. The constructed 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, and total cellular protein was extracted and quantified using a BCA kit (Thermo Fisher). Proteins were then separated using a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane by electroporation. The protein-laden membrane was blocked in 5% skim milk at room temperature for 1 h, then the milk was removed, and the membrane was incubated with primary antibody overnight at 4°C on a shaker. The next day, the membrane was quickly washed three times with TBST and then incubated with secondary antibody at room temperature for 1 h. Finally, protein was detected and recorded using chemiluminescence reagents and a Tanon chemiluminescence analyzer, and quantified using ImageJ.

[0044] Among TIGIT targets, the balance between TIGIT and CD226 is crucial for maintaining normal immune function. An ideal TIGIT degrader must precisely target TIGIT while avoiding off-target effects on structurally similar and functionally antagonistic CD226. Otherwise, it may not only fail to relieve immunosuppression but also produce the opposite and harmful biological consequences. In MM1S cells, the chimeric compounds LC1-LC5, which incorporate functional peptide carriers (Ly-a5 / Ly-a6 / Ly-a8 / Ly-a10 / Ly-a11), efficiently degrade TIGIT while having virtually no effect on CD226 expression levels. However, the st chimeric compound LC0, while achieving similar TIGIT degradation effects, unexpectedly led to a significant downregulation of CD226. This indicates that existing st sequences carry a serious off-target risk and may disrupt immune homeostasis. This result demonstrates that LC1-LC5 unexpectedly endows the entire chimeric molecule with extremely high precision, enabling it to achieve precise differentiation and targeting within a structurally similar protein family (TIGIT / CD226).

[0045] 5) Enzyme-linked immunosorbent assay (ELISA) to investigate the effect of chimeras on T cell activation. Based on the above demonstration that the chimeric LC1-LC5 can specifically degrade TIGIT and maintain the stability of CD226 protein, this embodiment further verifies the precise enhancement effect of the chimeric on T cell immune activity from a functional perspective, thereby confirming its biological superiority and application potential.

[0046] Jurkat cell line was purchased from Procell Biotechnology Co., Ltd. This cell line was cultured in Roswell Park Memorial Institute (RPMI) 1640 basal medium (Procell) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin solution (final penicillin concentration 100 U / mL, final streptomycin concentration 100 µg / mL). Cells were incubated at 37°C with 5% [unclear - likely a specific concentration or product]. The cells were routinely cultured in a humid environment, exhibiting suspension growth, plump morphology, and good growth status. The human chronic myeloid leukemia K562 cell line was purchased from Procell Biotechnology Co., Ltd., and was engineered to stably express the natural ligand CD155 of TIGIT. K562 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Procell) basal medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-dextrose antibody. Cells were incubated at 37°C with 5%... Cultured under specific conditions.

[0047] The constructed chimera was diluted to the target concentration using complete culture medium and incubated with Jurkat cells. The pretreated Jurkat cells were mixed with K562 cells at an effector-target ratio of 10:1 and co-cultured for 48 hours. The culture supernatant was collected to detect the amount of IFN-γ secreted.

[0048] Figure 15 The diagram shows a comparison of the T-cell activation effects of LC1-LC5 in Jurkat cells in Example 6 of this invention. To verify the functional effects of the chimeric compound of this invention, we conducted a T-cell activation experiment. Compared with the blank control (Blank) and negative control (Ctrl), the chimeric compound LC1-LC5, which incorporates functional polypeptide carriers (Ly-a5 / Ly-a6 / Ly-a8 / Ly-a10 / Ly-a11), exhibited strong T-cell activation capabilities. Particularly noteworthy is that the T-cell activation levels induced by LC4 and LC5, i.e., IFN-γ secretion, reached 9301 pg / mL and 9919 pg / mL, respectively, while the IFN-γ secretion level of commercially available naked anti-LC was 2682.5 pg / mL. Although LC0 showed some increase in IFN-γ due to the action of st, the effect was limited. The enhancing effect of LC1-LC5 on T-cell activation far exceeded the reasonable expectations of those skilled in the art, fully demonstrating the unexpected technical effects achieved by this invention through specific sequence optimization.

[0049] Example 7: In vivo tumor-inhibiting effect of chimeric LC1-LC5 To detect the inhibitory activity of LC1-LC5 against breast cancer in vivo, human PBMCs were intravenously injected into NOG mice with severe immunodeficiency. The number of PBMCs injected was 10. 6 Hu-PBMC mice were obtained by subcutaneously inoculating the mice with 200 μL of human breast cancer cells per mouse. A tumor xenograft model was then constructed by subcutaneously inoculating the Hu-PBMC mouse model with MDA-MB-231 human breast cancer cells. The tumors were allowed to grow to 100 mm in size. 3 Chimeric LC1-LC5 were injected starting at approximately 10:00 AM. Similarly, LC0 and LC (the commercially available naked antibody drug tirelinumab) were set up as control drugs. All drugs were administered intravenously twice a week for a total of 6 weeks.

[0050] Figure 16 This is a quantitative graph showing the LC1-LC5 inhibition of breast cancer tumor growth in Example 7 of the present invention. Figure 17 This is a quantification diagram of LC1-LC5 inhibition of breast cancer tumor weight in Example 7 of the present invention. Figure 18 This is a diagram showing the tumor tissue results of LC1-LC5 inhibition of breast cancer in Example 7 of the present invention, as shown. Figure 16 to Figure 18 As shown in Example 7, the antitumor effect of the chimeric molecules (LC1-LC5) constructed in this invention was evaluated through in vivo tumor suppression experiments. Tumor growth curves, endpoint tumor weight, and tumor tissue morphology results consistently showed that all groups treated with LC1-LC5 significantly inhibited breast cancer tumor growth. Among them, LC4 and LC5 (chimeric functional peptide carriers Ly-a10 and Ly-a11, respectively) exhibited the most prominent tumor suppression effects, significantly exceeding existing clinical standards. Compared with naked anti-tirerelimumab (LC), LC1-LC5 showed statistically significant advantages in both tumor volume inhibition and endpoint tumor weight. p The result <0.01 demonstrates that the functional peptide carriers (Ly-a5 / Ly-a6 / Ly-a8 / Ly-a10 / Ly-a11) can fundamentally enhance the therapeutic potential of antibody drugs. More notably, the overall efficacy of the LC1-LC5 groups comprehensively surpasses that of the current first-line treatment, pembrolizumab. This result has significant clinical implications, further highlighting the enormous potential and application prospects of the functional peptide carriers of this invention in enhancing drug activity. The functional peptide carriers of this invention represent an original and innovative therapeutic mechanism. Their superior efficacy data far exceed the reasonable expectations of those skilled in the art regarding peptide carrier technology, providing an effective technical solution to overcome the current bottlenecks in antibody drug efficacy. They possess significant clinical application value and broad market prospects in the field of tumor treatment.

[0051] Example 8: Chimera-induced IL6R degradation In this embodiment, the human osteosarcoma (SaOS-2) cell line was used for experiments. Flow cytometry was used to assess the chimeric endocytosis of IL6R on the cell membrane surface, and Western blotting was used to detect the expression level of IL6R on the cell membrane surface degraded by the chimera, thereby assessing the chimeric IL6R degradation ability. The commercially available naked antibody control used in this embodiment was tocilizumab (hereinafter referred to as To), and all primary antibodies used were anti-IL6R antibodies.

[0052] 1) Construction of IL6R-targeting chimeric plasmid The lysosome-targeting functional polypeptide carrier fused to To at least one of the following positions: the C-terminus of the To heavy chain (hereinafter referred to as To-HC), the N-terminus of the To heavy chain, the C-terminus of the To light chain (hereinafter referred to as To-LC), and the N-terminus of the To light chain. This embodiment modifies To at the gene level. For ease of explanation, Ly-a5 is used as an example: Ly-a5 is used as the lysosome-targeting functional polypeptide carrier, and Ly-a5 is fused to the C-terminus of the To light chain and / or heavy chain to prepare a chimera. However, the chimera of this invention is not limited to the amino acid sequence given in this embodiment. The lysosome-targeting functional polypeptide carrier of this invention can also be fused to the N-terminus of the heavy chain and / or light chain, and all resulting chimeras are within the protection scope of this invention.

[0053] The construction of the To-based chimeric plasmid in this embodiment specifically includes the following steps: First, Ly-a5 was fused to the C-terminus of both the heavy and light chains to construct fragments To-LC-Ly-a5 and To-HC-Ly-a5, which were then integrated into the vector pcDNA 3.4. The amino acid sequences of To-LC, To-HC, To-LC-Ly-a5, and To-HC-Ly-a5 are shown in Table 4.

[0054]

[0055]

[0056]

[0057] The target fragment and vector were amplified separately by PCR, ensuring that the synthesized target fragment and vector had overlapping fragments. Then, the PCR product and plasmid vector were ligated using homologous recombinase, and the ligated product was transformed into DH5α competent cells, plated, and incubated overnight. The next day, a single plaque was picked and cultured, and finally, sequencing was performed to verify successful construction.

[0058] 2) Expression, purification and characterization of chimeric LD1-LD10 Human embryonic kidney cells (293 Freestyle) were seeded into shake flasks until the cell density reached [value missing]. Transfection can be performed when the DNA concentration is 1 / mL. The transfection reagent is polyethyleneimine (PEI). For transfection, add 10% of the transfection volume of culture medium to two centrifuge tubes. Add 160 μL of PEI to one centrifuge tube and 80 μg of plasmid (heavy chain:light chain = 2:3) to the other. Mix thoroughly and let stand for 5 min. Add DNA dilution buffer to the PEI dilution buffer, mix thoroughly, let stand for 20 min, and transfer to a shake flask. Collect the supernatant 5 days after transfection. Filter the supernatant through a 0.45 μm filter and purify it using a Protein A affinity chromatography column. The purified protein was characterized structurally by SDS-PAGE, HPLC, and MS.

[0059] 3) Immunoblotting detection of the degradation effect of chimeric LD1-LD10 on cellular IL6R. The SaOS-2 cell line was obtained from the American Type Culture Collection Center (ATCC). The cell line was cultured in McCoy's 5A basal medium (Procell) supplemented with 15% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 g / mL. Cell culture was maintained at 37°C with 5% [unclear - likely referring to a specific concentration of antibiotics]. In a humid environment, the cells have normal morphology and grow well.

[0060] The constructed 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, and total cellular protein was extracted and quantified using a BCA kit (Thermo Fisher). Proteins were then separated using a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane via electroporation. The protein-laden membrane was blocked in 5% skim milk at room temperature for 1 h, after which the milk was removed, and the membrane was incubated overnight with primary antibody at 4°C on a shaker. The next day, the membrane was quickly washed three times with TBST and then incubated with secondary antibody at room temperature for 1 h. Finally, protein was detected and recorded using chemiluminescence reagents and a Tanon chemiluminescence analyzer, and quantified using ImageJ.

[0061] Figure 19 This is an immunoblotting result of LD1-LD10 degrading IL6R in SaOS-2 cells according to Example 8 of the present invention. Figure 19This paper presents the immunoblotting results of various LDs molecules in Example 6 of this invention degrading IL6R protein in SaOS-2 cells. β-actin was used as an internal control, and LD was a commercially available naked antibody control. The results showed that, compared with commercially available tocilizumab (LD) and the st positive control (LD0), cells treated with chimeric compounds LD1-LD5 containing functional peptide carriers (Ly-a5 / Ly-a6 / Ly-a8 / Ly-a10 / Ly-a11) showed lighter blot bands, indicating a significant degradation of intracellular IL6R protein levels. Specifically, the commercially available naked antibody LD itself has a certain inhibitory effect on IL6R protein levels; while LD0, although reducing IL6R due to the effect of st, has an effect basically similar to that of the commercially available naked antibody LD. In contrast, LD1-LD5 all exhibited a strong ability to degrade IL6R protein. This result fully demonstrates that the present invention successfully achieved efficient degradation of the key target IL6R by embedding the antibody with a specific functional polypeptide carrier.

[0062] Example 9: Chimeric LD4-LD5 improves skin inflammation in mice This study used imiquimod to induce a mouse model of psoriasis and examined the therapeutic effects of the chimeric LD4 and LD5 on psoriatic skin inflammation in mice. After hair removal, the skin in the dorsal modeling area of ​​mice was treated with 62.5 mg of 5% imiquimod cream. On day 5 of modeling, obvious erythema and scaling were observed, indicating successful modeling. Control group mice received an equal amount of petrolatum on their dorsal skin, while treatment group mice were injected with the chimeric LD4 and LD5 once a week for a total of two administrations. LD0 and LD5 (the commercially available naked anti-psoriasis drug itocizumab) were used as control drugs.

[0063] Figure 20 This is a diagram showing the results of LD4-LD5 treatment of immune-mediated skin diseases in mice according to Example 9 of the present invention. Figure 21 This is a scanning microscope image of HE staining of the dorsal skin tissue of mice with LD4-LD5 used to treat immune skin diseases, as described in Example 9 of this invention. Figure 20 to Figure 21As shown in Example 9, the chimeric molecules (LD4 and LD5) constructed in this invention were evaluated to improve psoriasis-like lesions using an imiquimod-induced psoriasis model. LD4 and LD5 (chimeric functional peptide carriers Ly-a10 and Ly-a11, respectively) showed the most significant effects in improving erythema, scaling, and epidermal thickening. Compared with commercially available naked anti-tirerelimumab (LD) and LDO (chimeric st), LD4 and LD5 showed significant advantages across the board, demonstrating that the functional peptide carriers can effectively enhance the therapeutic efficacy of antibody drugs against psoriasis-like lesions. At the histopathological level, the LD4 and LD5 treatment groups showed a significant reversal of psoriatic characteristics. Compared with the model control group, the treatment groups showed a significant reduction in epidermal hyperkeratosis and parakeratosis, and a decrease in Munro microabscess formation; inflammatory cell infiltration in the dermis significantly subsided, especially the effective inhibition of neutrophil and lymphocyte aggregation. Furthermore, the epidermal acanthosis (acanthosis) condition was significantly improved, and the extension of epidermal ridges was reduced, approaching the structure of normal skin tissue. Compared with commercially available naked anti-tirerelinumab (LD) and LD0 (chimeric st), LD4 and LD5 showed significant advantages in various inflammatory and pathological indicators, demonstrating that the functional peptide carrier can enhance the therapeutic efficacy of traditional antibody drugs against psoriatic lesions through a novel degradation mechanism, revealing the important application potential of the functional peptide carrier of this invention in the treatment of immune dermatitis.

[0064] Example 10: Toxicity Study of Membrane-Penetrating Peptides Introduced by Functional Peptide Carriers This embodiment aims to investigate the effects of the functional polypeptide carriers of the present invention, incorporating different membrane-penetrating peptides, on cell viability on human peripheral blood mononuclear cells (PBMCs). By comparing polypeptide carriers modified with highly active membrane-penetrating peptides and those without such modifications, the differences in toxicity were clarified, providing crucial experimental evidence for screening preferred polypeptide carriers that combine efficient internalization, degradation, and good biocompatibility. For ease of explanation, Ly-a4 (SYKYSKLNKE) was used as an example: Ly-a4 was used as the functional polypeptide carrier, and membrane-penetrating peptides of different lengths were introduced onto Ly-a4.

[0065] (1) Cell viability detection Human PBMCs were cultured in RPMI-1640 complete medium (Procell) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 μg / mL. Cells were cultured at 37°C in a humidified environment with 5% CO2, resulting in well-rounded cells and good growth. The test solutions were diluted to the target concentration using complete medium and divided into 2 × 10⁶ cells per well. 5Cells were incubated at a specific density in 96-well plates for 48 h. Afterward, the old culture medium was removed, and 110 μL of fresh complete culture medium containing 10% CCK-8 reagent was added to each well. The plates were then incubated at 37°C in the dark for 2–4 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader, and the relative cell viability was calculated using the formula: "Cell viability (%) = (OD experimental group - OD blank group) / (OD negative control group - OD blank group) × 100%".

[0066] Figure 22 This is a quantitative diagram of the cytotoxic effect of the introduced cell-penetrating peptide region on cells in Example 10 of the present invention. The results are as follows: Figure 22 The results showed that introducing the structural region of the cell-penetrating peptide into Ly-a4 has potential cytotoxicity, and the cell death rate gradually increases with the increase of the cell-penetrating peptide arginine. Figure 22 As shown in the right figure, the cytotoxicity is increasing, which is a major drawback for the clinical application of chimeras. However, the Ly-a1~Ly-a13 of this patent do not involve the membrane-penetrating peptide region, do not affect cell survival rate, and have no potential toxic side effects, demonstrating the significant advancement of our strategy of not involving the cell membrane-penetrating peptide structural region.

[0067] Example 11: Study on the role of key sites in functional polypeptide carriers This embodiment aims to illustrate the significant unpredictability inherent in designing functional peptide carriers based on the known YXXØ signaling theory and successfully applying them to chimeric drug development. Through systematic screening, we discovered that although the theory allows various hydrophobic amino acids (V, F, I, L, M) to substitute for the Ø site while retaining the peptide's internalization function, only when Ø = V or L can a functional peptide carrier possessing both high-efficiency internalization, degradation, and antibody development potential be achieved. This crucial discovery surpasses the predictive capabilities of existing theories and is the core inventive aspect of this invention.

[0068] (1) Screening of key site substitution and internalization function of functional peptide vectors Based on the core sequence RRLRKSYKYSKØQKE, the Ø site was replaced with valine (V), phenylalanine (F), isoleucine (I), leucine (L), methionine (M), asparagine (N), glutamine (Q), serine (S) and threonine (T), respectively. All peptides were synthesized using the standard Fmoc solid-phase synthesis method and purified by high performance liquid chromatography with a purity >95%.

[0069] The specific testing method includes the following steps: Cell culture: The human ovarian cancer cell line SKOV3 was obtained from the American Type Culture Collection Center (ATCC). The SKOV3 cell line was cultured in RPMI 1640 basal medium (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 g / mL added to the medium. The cells were cultured at 37°C in a humid environment with 5% CO2. The cells had normal morphology and good growth. Flow cytometry was used to assess the internalization of the functional polypeptide carrier RRLRKSYKYSKØQKE: In this embodiment, SKOV3 cells were used for the experiment. The vector sequence reported in the patent document (CN118697896A, SEQ ID NO: 4) was selected as the positive control St (amino acid sequence as shown in sequence SEQ ID NO: 30, SYKYSKVNKE). The FITC-labeled functional polypeptide carrier was diluted to a final concentration of 100 nM in serum-free medium and then co-incubated with SKOV3 cells for 6 h. Subsequently, the antibody bound to the cell membrane was removed with acid washing buffer, and the residual acid washing buffer was washed with PBS. Then, the cells were digested from the culture dish and transferred to EP tubes. The fluorescence intensity of the treated cells was observed by flow cytometry. The results showed (as shown in Table 5) that when Ø was replaced with hydrophobic amino acids V / F / I / L / M, the internalization function of these functional peptide carriers could be retained, with Ø=V or L showing the best effect and outstanding advantages; when Ø was replaced with hydrophilic amino acids, these peptides lost their internalization function, indicating that the hydrophobicity of Ø is very important for the internalization function of peptides.

[0070] (2) Screening and production evaluation of chimeric degradation function The coding sequences of selected functional peptide vectors with high internalization efficiency (Ø = V, F, I, L, M) were fused to the C-terminus of the heavy chain of atezolizumab (hereinafter referred to as Az). Simultaneously, the core domains YSKV and YSKL were also fused separately as controls of the functional peptide vectors. Transient transfection expression was performed using the CHO expression system. After 7 days of culture following transfection, the supernatant was collected. The chimeras were purified using Protein A affinity chromatography, and protein concentration was determined by the BCA method. Chimeras with a yield below 1 mg / L were considered expression failures.

[0071] The specific degradation detection method includes the following steps: One day before drug administration, SKOV3 cells were plated; the constructed peptide-drug chimera was diluted to the target concentration using complete culture medium and incubated with cells for 48 h; then, cells were lysed using lysis buffer, total cellular protein was extracted, and protein was quantified using a BCA kit (Thermo Fisher); then, proteins were separated using 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane by electroporation; the protein-containing membrane was blocked in 5% skim milk at room temperature for 1 h, then the milk was removed, and the membrane was incubated with primary antibody overnight at 4°C on a shaker; the next day, the membrane was removed and quickly immersed in TBST for 3 washes, then incubated with secondary antibody at room temperature for 1 h; finally, protein was detected and recorded using chemiluminescence reagents and a Tanon chemiluminescence analyzer, and protein was quantified using ImageJ; The results (as shown in Table 5) revealed that, in terms of degradation efficiency, both Ø=L and V exhibited unique advantages, with Ø=V showing the best performance, achieving a PD-L1 degradation rate of 80%, superior to the 60% achieved with Ø=L. Regarding antibody production efficiency, once these functional peptide carriers were fused into full-length antibodies to form chimeric molecules, Ø could only be V / L. If Ø was any of the amino acids F / I / M, the antibody yield would be significantly reduced, rendering it unsuitable for practical antibody production. This conclusion required extensive structural design and experimental validation. A similar phenomenon existed when only the core domains YSKV or YSKL were retained. Individual peptides YSKV or YSKL could be internalized, but once linked to the full-length antibody Az to form a chimeric compound, the small size of the groups resulted in them being encapsulated by the antibody. Figure 21 The diagram shows the molecular structure of the chimeric YSKV or YSKL introduced in Example 11 of this invention. The functional peptide cannot be exposed and therefore cannot be recognized by cells. Ultimately, not only is the lysosomal targeted degradation effect poor (Table 5), but significant internalization is also impossible. In other words, the core domains YSKV or YSKL cannot be applied to the actual production of antibodies and have no transformative significance. The above results cannot be obtained based on the YXXØ signal structure theory and require extensive practical work to understand. This indicates that even if the site of this type of functional peptide is replaced with hydrophobic amino acids as described in the literature, the peptide's activity can be preserved. However, once the functional peptide carrier is applied, i.e., fused to an antibody, even hydrophobic amino acids pose a risk of poor antibody expression. This suggests that the replacement of this site requires extensive experimental verification and involves a great deal of uncertainty and unpredictability.

[0072] Table 5 Comparison of sequence and activity data

[0073] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the invention; The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In short, if a person skilled in the art is inspired by this description and designs a similar method and embodiment without departing from the spirit of the present invention, such design should fall within the protection scope of the present invention.

Claims

1. A functional polypeptide carrier, characterized in that: A peptide has the following amino acid sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-SYKYSK-Xaa12-Xaa13-K-Xaa15; Among them, Xaa1=R or does not exist; Xaa2=R or does not exist; Xaa3=L or does not exist; Xaa4=R or does not exist; Xaa5=K or does not exist; Xaa12=V or L; Xaa13=N or Q; Xaa15=E or D.

2. The functional polypeptide carrier according to claim 1, characterized in that, The peptide has the following amino acid sequence: RRLRKSYKYSK-Xaa12-Xaa13-K-Xaa15; where Xaa12=V or L; Xaa13=N or Q; and Xaa15=E or D.

3. The functional polypeptide carrier according to claim 1, having the following amino acid sequence representing the peptide: SYKYSK-Xaa12-Xaa13-K-Xaa15; in, Xaa12 = V or L; Xaa13 = N or Q; Xaa15 = E or D.

4. The functional polypeptide carrier according to claim 1, characterized in that, Including the sequence shown in SEQ ID NO: 5: SYKYSKVQKE, the sequence shown in SEQ ID NO: 6: SYKYSKVQKD, the sequence shown in SEQ ID NO: 8: SYKYSKLQKD, the sequence shown in SEQ ID NO: 10: RRLRKSYKYSKVNKE, and the sequence shown in SEQ ID NO: 11: RRLRKSYKYSKVQKE.

5. A polypeptide drug chimera, characterized in that: The structure of the polypeptide drug chimera includes a recognition segment and a lysosome-targeting structural unit; the recognition segment recognizes and specifically binds to the target protein; the lysosome-targeting structural unit mediates the transport of the target protein to the lysosome; the lysosome-targeting structural unit includes one or more functional polypeptide carriers as described in claims 1-4.

6. A polypeptide drug chimera according to claim 5, characterized in that: The recognition segment includes at least one of peptides, proteins, nucleic acids, nanoparticles, or small molecule drugs.

7. A polypeptide drug chimera according to claim 5, characterized in that: The recognition segment is connected to the functional polypeptide carrier through direct or indirect connection.

8. A polypeptide drug chimera according to claim 5, characterized in that: The recognition segment includes a polypeptide, and the functional polypeptide carrier is attached to the recognition segment at one or more of the following positions: 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. When the identification segment includes a side chain group, the side chain group includes a natural amino acid side chain group or a non-natural amino acid side chain group; the natural amino acid group includes at least one of amino and thiol groups; the non-natural amino acid side chain group includes at least one of nitrogen group, alkynyl group, aldehyde group, ketone group, fluorosulfonate, chlorine group, bromine group, and iodine group.

9. A polypeptide drug chimera according to claim 5, characterized in that: The recognition segment is an antibody; the antibody includes at least one of monospecific antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, humanized antibody, monoclonal antibody, and antigen-binding fragment of monoclonal antibody; when the recognition segment is a monoclonal antibody, the antigen-binding fragment of the monoclonal antibody includes at least one of Fab, Fab', F(ab')2, Fv, dsFv, scFv, sc(Fv)2, or VHH.

10. A polypeptide drug chimera according to claim 5, characterized in that: The chimeric sequences include chimeric sequences SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, and SEQ ID 55 targeting the cellular TIGIT protein, and chimeric sequences SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, and SEQ ID 69 targeting the cellular IL6R protein.

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