Extrahepatic targeting polypeptide mainly based on intestinal tract, polypeptide coupling medicine and application of polypeptide coupling medicine

Abstract

The invention discloses an extrahepatic targeting polypeptide mainly based on intestinal tracts, a polypeptide coupling drug and application thereof. The polypeptide targets extrahepatic tissues, especially intestinal tissues / organs. The extrahepatic targeting polypeptide provided by the invention is coupled with molecules with pharmacological activity, especially oligonucleotide molecules, to form a polypeptide coupled drug, and the molecules with pharmacological activity can be transported outside the liver, especially into intestinal tissues, so that the extrahepatic targeting, especially intestinal disease administration, is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an intestinal-based extrahepatic targeting polypeptide, a polypeptide-coupled drug and applications thereof. Background Art

[0002] With the continuous progress of biomedicine, the development of targeted drug delivery technology is one of the most important biotechnology fields in the era of personalized and precision medicine. Targeted drug delivery can deliver more active drug molecules to the location of the disease and reduce the impact on other non-lesion sites, thereby greatly improving the utilization rate of the drug, reducing the dosage accordingly, and avoiding the toxic side effects caused by drug enrichment in non-target sites. Therefore, the development of targeted drugs is a vital measure to solve major diseases. However, targeted drug delivery technology is still in its early stages of development and is currently most concentrated in the field of liver targeting. Both nanomedicines and conjugated drugs are limited to the liver, and the problems that can be solved are limited. Therefore, the development of more extrahepatic targeted drug delivery technologies is the key to dealing with more major diseases.

[0003] Taking RNA therapy in nucleic acid medicine as an example, although different types of RNA therapy have different mechanisms of action, these RNA-based drug molecules need to be delivered to the correct tissue and cannot stimulate harmful immune responses or other potential toxic side effects. So far, academia and industry have developed a variety of RNA drug delivery systems.

[0004] Vectors that deliver RNA can reach their targeted cells or tissues through two mechanisms, namely passive targeting and active targeting. Passive targeting relies on biomolecules adsorbed on the surface of nanoparticles. Since nanoparticles are very small, their surface area / volume ratio is very high. When nanoparticles are introduced into the biological environment (such as into the blood circulation), many biomolecules in the environment will bind to the surface of the nanoparticles. These chemical and biological molecules bound to the surface of the nanoparticles will change the interaction between the nanoparticles and immune cells and target tissues. Passive targeting uses endogenous transport systems to reach target cells. Studies have shown that the size, composition, charge and hydrophobicity of the delivery vehicle can affect the type of protein that binds to the nanoparticles in the blood, thereby affecting the transport and uptake of nanoparticles.

[0005] Active targeting achieves target delivery through the introduction of a targeting fragment, adding a ligand that can bind to a specific biological molecule to the delivery system. Currently, the most clinically validated example is GalNAc (galactosamine)-coupled siRNA and ASO. FDA-approved therapies Givosiran, Lumasiran, Inclisiran, and Vutrisiran all use this strategy. GalNAc can bind to the asialoglycoprotein receptor (ASGPR), which is an ideal receptor for active targeting. It is highly expressed on the surface of hepatocytes and not expressed in other cell types. After binding to GalNAc, it quickly causes endocytosis and can then be quickly recycled back to the cell surface. The combination of this receptor and the optimized ligand significantly reduces the toxicity of subcutaneously injected siRNA and ASO.

[0006] Liver-targeted drugs, especially oligonucleotide drugs, have been developed relatively well. Several liver-targeted oligonucleotide drugs have been launched, which have achieved the treatment of various diseases caused by abnormal liver gene expression, with significant clinical benefits. However, the development of extrahepatic targeting, including the targeting technology of drugs in the intestine, respiratory tract, brain, etc., is relatively backward, and related problems have not been effectively solved.

[0007] For the treatment of intestinal diseases, oral and rectal administration are simpler, more comfortable, non-invasive and less costly for patients compared to injection or intravenous administration. However, even with oral administration, the compliance of chronic drugs is only about 50%. Therefore, it is very important to choose a more effective gastrointestinal drug delivery method. Although patient compliance is not as good as oral administration, the use of intravenous or subcutaneous injection to treat intestinal diseases still has important application value, especially through the guidance of specific ligands to target the drug to the intestinal site, which can achieve precise treatment of intestinal diseases, improve efficacy while reducing toxic side effects. Summary of the invention

[0008] The purpose of the present invention is to provide a polypeptide capable of achieving extrahepatic targeting, especially intestinal targeting, and a polypeptide-coupled drug constructed based on the polypeptide.

[0009] In a first aspect of the present invention, an extrahepatic targeting polypeptide is provided, wherein the extrahepatic targeting polypeptide particularly targets intestinal tissues / organs, and the amino acid sequence of the extrahepatic targeting polypeptide is selected from: NYVKSKFRGYNALRVY, SKVGGNYNYLYRLFRK, SKVGANYNYLYRLFRK, EAFNCYFPLQSYGFQPTNGVGYQPY, EGFNCYFPLQSYGFQPTNGVAYQPY, FNCYFPLQSYGFQPTNGVAY, and an amino acid sequence in which one or more amino acids are deleted and / or added and / or substituted in the above amino acid sequence but the targeting function remains unchanged.

[0010] According to one embodiment of the present invention, the amino acids of the above-mentioned polypeptide can be replaced by other amino acids of the same nature without affecting the function of the polypeptide, glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I) can be replaced by each other, glutamic acid (E) and aspartic acid (D) can be replaced by each other, asparagine (N) and glutamine (Q) can be replaced by each other, proline (F), tyrosine (Y) and tryptophan (W) can be replaced by each other, and serine (S) and threonine (T) can be replaced by each other.

[0011] Specifically, the extrahepatic targeting sequence of the present invention is any one or more of the sequences in Table 1 and Table 2 below.

[0012] The second aspect of the present invention provides a polypeptide-drug conjugate comprising the above-mentioned extrahepatic targeting polypeptide, wherein the structure is polypeptide-linker-load, wherein the load is a small molecule drug, an antibody drug, a protein or an oligonucleotide. In particular, by using the polypeptide provided by the present invention to couple active drug molecules through a linker, the active drug molecules can be carried to target locations such as the intestine, and the active drug molecules exert their own effects to achieve effective treatment of intestinal diseases.

[0013] According to a specific embodiment of the present invention, the above-mentioned linker is a cleavable linker or a non-cleavable linker, and the cleavable linker may include a linker containing a disulfide bond, maleimide, a cathepsin cleavable dipeptide, a diphosphate or a linker containing glucuronic acid, and the non-cleavable linker may include N-succinimidyl-4-(N-maleimido) cyclohexane-1-carboxylate. It is used for coupling between the above-mentioned polypeptide molecule and the load, including coupling of endogenous amino acids of the polypeptide, disulfide heavy bridging strategy, polysaccharide coupling, enzymatic site-specific coupling technology, etc.

[0014] According to a specific embodiment of the present invention, the above polypeptide can be coupled to the load via a cleavable linker.

[0015] Specifically, the above polypeptide can be coupled to the payload through a new generation of maleimide (NGM) as a linker. Specifically, the polypeptide is co-incubated with dibromomaleimide (DBM) or dithiophenylmaleimide (DSPh) to bridge the reduced disulfide bonds together to achieve coupling with the payload.

[0016] Specifically, the above polypeptide can be coupled to the load through a cathepsin cleavage dipeptide (-Z1-Z2-), and the coupled drug can be cleaved by a high content of protease B (Cathepsin B) in the lysosomes of mammalian cells. Wherein, Z1 is one of phenylalanine (F), valine (V) or alanine (A), and Z2 is one of arginine (R) or citrulline (Cit). Preferably, the above dipeptide is Val-Cit or Val-Ala dipeptide.

[0017] Specifically, the polypeptide can be coupled to the payload via a linker containing glucuronic acid, and can be decomposed by β-glucuronidase. β-Glucuronidase (GUSB) is an important lysosomal enzyme involved in the degradation of glycosaminoglycans containing glucuronic acid.

[0018] Specifically, the above polypeptide can be coupled to the load through a diphosphate linker. There are acid pyrophosphatase and acid phosphatase in the lysosome, which can hydrolyze the phosphate bond.

[0019] When a cleavable linker is used to couple a polypeptide to a siRNA molecule, the linker is coupled to the sense strand or antisense strand of the siRNA, preferably, the linker is coupled to the sense strand of the siRNA. The linker is connected to the siRNA at the 3'-end or 5'-end of the sense strand or antisense strand, preferably, the connection position is the 3'-end, and further preferably, the connection position is the 3'-end of the sense strand.

[0020] The peptide-siRNA conjugate enters the cytoplasm through endocytosis. After being released into the cell, the peptide is cleaved off in the endosome, and the free siRNA binds to a specific protease to form a gene silencing complex (RISC). The antisense strand guides the RSIC to bind to the mRNA, exerting the expression inhibition effect of nucleic acid interference.

[0021] According to a specific embodiment of the present invention, the above polypeptide can be coupled to the load via a non-cleavable linker.

[0022] Specifically, the above polypeptide can be coupled to the load by containing N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). The alkylation reaction occurs through nucleophilic Michael addition of the thiol group to form a stable thioether bond, thereby connecting the polypeptide to the load.

[0023] When a non-cleavable linker is used to couple a polypeptide to a siRNA molecule, the linker is coupled to the sense strand of the siRNA, and the linker is connected to the siRNA at the 3'-end or 5'-end of the sense strand. Preferably, the connection position is the 3'-end of the sense strand.

[0024] The peptide-siRNA conjugate enters the cytoplasm through endocytosis. After being released from the endosome, the antisense strand binds to the protease to form RISC, and the sense strand coupled to the peptide dissociates from RISC. The antisense strand guides RSIC to bind to mRNA, exerting the expression inhibition effect of nucleic acid interference.

[0025] According to one embodiment of the present invention, the above-mentioned oligonucleotide can be siRNA, ASO, miRNA, mRNA, gene editing CRISPR-Cas system, gRNA or RNA aptamer.

[0026] Preferably, the oligonucleotide and the polypeptide are connected in an end-to-end form, that is, one end of the polypeptide (or a position close to the end) is connected to one end of the oligonucleotide (or a position close to the end). For polypeptides, the amino acid residues close to the nitrogen end are preferably used as coupling sites, and the first five amino acid residues are further preferably used as coupling sites. Suitable functional groups are selected, including the amino group at the nitrogen end, the functional groups on the side chains of the amino acid residues, such as the side chain hydroxyl groups of serine and threonine, the side chain sulfhydryl groups of cysteine, the amino group of lysine, etc. For oligonucleotide drug molecules, the 3'-end or the 5'-end can be used as coupling sites. Preferably, the polypeptide is coupled to the 3'-end of the oligonucleotide through a linker. Specifically, for siRNA, if the linker is a cleavable linker, the polypeptide can be coupled to the sense strand or antisense strand of the siRNA (mainly the 3'-end), and if the linker is a non-cleavable linker, the polypeptide can be coupled to the sense strand of the siRNA through the linker. For mRNA, it is preferred to couple the polypeptide to the cap structure at the 5'-end.

[0027] A dual-target pharmaceutical composition comprising two RNAs of the same type. Preferably, the two RNAs are small interfering nucleotides (siRNA), antisense nucleotides (ASO), micronucleic acids (miRNA) or messenger RNA (mRNA). Further preferably, a pharmaceutical composition comprising two siRNAs.

[0028] A dual-target pharmaceutical composition comprising two different types of RNA. The two RNAs in the dual-target pharmaceutical composition, one of which is siRNA and the other is ASO; one of the two RNAs is siRNA and the other is miRNA; one of the two RNAs is siRNA and the other is mRNA; one of the two RNAs is ASO and the other is miRNA; one of the two RNAs is ASO and the other is mRNA; one of the two RNAs is miRNA and the other is mRNA. Preferably, a dual-target inhibitory RNA pharmaceutical composition, wherein one RNA is siRNA and the other is ASO or miRNA.

[0029] A dual-target siRNA pharmaceutical composition. One siRNA (siRNA-1) is coupled to a polypeptide at a molar ratio of 1:1 (one siRNA molecule is coupled to one polypeptide molecule), and another siRNA (siRNA-2) is also coupled to a polypeptide at a molar ratio of 1:1. The two polypeptide-coupled siRNAs are mixed in a specific ratio to form a dual-target composition. Preferably, the mixing ratio of the two polypeptide-coupled siRNAs is determined according to the ratio of their respective half-inhibitory concentrations (IC50) for inhibiting the expression of the corresponding genes. In the case where the IC50s of the two siRNAs for inhibiting the target gene are close, the two polypeptide-coupled siRNAs are mixed in a molar ratio of 1:1 to form a dual inhibitory pharmaceutical composition containing two polypeptide-siRNA conjugates (bimolecular structures).

[0030]

[0031] In another embodiment, two siRNA molecules are coupled to a polypeptide at a molar ratio of 1:1:1, i.e., one polypeptide molecule is coupled to two different siRNA molecules to form a dual inhibitory pharmaceutical composition containing a polypeptide-two siRNA conjugates (single molecular structure). The two different siRNA molecules are coupled to amino acid residues at different sites of a polypeptide molecule. Preferably, the siRNA is coupled to two non-continuous non-critical amino acid residues.

[0032]

[0033] In another embodiment, one siRNA molecule is coupled to the polypeptide at a molar ratio of 1:1, and another siRNA molecule is coupled to the polypeptide at a molar ratio of 1:1. The two polypeptide-siRNA conjugates are then coupled together in a specific manner to form a double polypeptide-two siRNA conjugates (single molecular structure) dual inhibitory drug composition.

[0034] In such a single molecule containing two different siRNAs and two identical polypeptides, one siRNA is coupled to a polypeptide (polypeptide-siRNA-1), another siRNA is coupled to a polypeptide (polypeptide-siRNA-2), and the two polypeptide-siRNA conjugates are reconnected. The reconnection may be a polypeptide-to-polypeptide connection, or a siRNA-to-another siRNA connection, or a siRNA on one polypeptide-siRNA conjugate is connected to a polypeptide on another polypeptide-siRNA conjugate.

[0035] One siRNA is coupled to another siRNA (siRNA-1 coupled to siRNA-2), two identical polypeptide molecules are coupled (polypeptide coupled to polypeptide), and the two conjugates are reconnected. The reconnection can be one siRNA molecule coupled to one polypeptide molecule (single connection), or two siRNA molecules are coupled to polypeptides (double connection). Preferably, the reconnection is a single connection. When a double connection is used, preferably, the two siRNA molecules are connected to two polypeptide molecules respectively.

[0036]

[0037] A dual-target pharmaceutical composition comprising two different types of RNA, wherein the different types of RNA include siRNA, miRNA, ASO, and mRNA and gRNA, etc. Preferably, the dual-target pharmaceutical composition is a dual inhibitory composition. Further preferably, one of the RNAs is siRNA and the other is ASO. In one embodiment, siRNA is coupled to a polypeptide at a molar ratio of 1:1 (one siRNA molecule is coupled to one polypeptide molecule), and ASO is also coupled to a polypeptide at a molar ratio of 1:1, and the polypeptide-coupled siRNA and the polypeptide-coupled ASO are mixed in a specific ratio to form a dual-target composition. In another embodiment, siRNA, ASO and polypeptide are coupled at a molar ratio of 1:1:1, that is, one polypeptide molecule is coupled to both siRNA molecules and ASO, forming a dual inhibitory pharmaceutical composition containing a polypeptide-siRNA / ASO conjugate (single molecular structure). In another embodiment, siRNA molecules are coupled to polypeptides at a molar ratio of 1:1, and ASO molecules are coupled to polypeptides at a molar ratio of 1:1. The polypeptide-siRNA conjugate and the polypeptide-ASO conjugate are then coupled together in a specific manner to form a double polypeptide-siRNA / ASO conjugate (single molecular structure) dual inhibitory drug composition.

[0038]

[0039] According to a specific embodiment of the present invention, the above-mentioned small molecule drug can be a microtubule protein inhibitor, a DNA damaging compound or an apoptosis inducer.

[0040] Specifically, the above-mentioned microtubule protein inhibitor may include calendula, monomethylcalendula, maytansine derivatives, tubulysin, cryptocolistin or anti-mitotic EG5 inhibitor.

[0041] Specifically, the DNA damaging compound may include pyrrolebenzazepines, indolechlorobenzazepines, dukamycin, camptothecin or calicheamicin.

[0042] Specifically, the apoptosis inducing agent may include a Bcl family inhibitor, tylanstatin and its analogs, amatoxin or amidophosphoribosyltransferase.

[0043] According to one embodiment of the present invention, the molar ratio of the above-mentioned polypeptide to the load may be 10:1-1:10, more preferably, the molar ratio of the polypeptide to the load is between 5:1-1:5, and even more preferably, the molar ratio of the polypeptide to the load is between 2:1-1:2.

[0044] Specifically, multiple polypeptide molecules can be coupled to one load. Preferably, one load is coupled to no more than 5 polypeptide molecules, and further preferably, one load is coupled to no more than 2 polypeptide molecules. Multiple loads can be coupled to one polypeptide molecule. Preferably, no more than 5 loads can be coupled to one polypeptide molecule. Further preferably, no more than 2 loads can be coupled to one polypeptide molecule. Further preferably, one polypeptide molecule is coupled to one load.

[0045] According to one embodiment of the present invention, the above-mentioned conjugated drug may be a polypeptide molecule connected to an oligonucleotide molecule.

[0046] According to one embodiment of the present invention, the above-mentioned conjugated drug may be a polypeptide molecule connected to two or more oligonucleotide molecules.

[0047] According to one embodiment of the present invention, the above-mentioned conjugated drug can be a polypeptide molecule connected to an oligonucleotide molecule, a polypeptide molecule connected to another different oligonucleotide molecule, and the two polypeptide molecules connected to different oligonucleotide molecules are then combined together through specific covalent bonds.

[0048] The third aspect of the present invention provides the use of the above-mentioned extrahepatic targeting polypeptide or the above-mentioned polypeptide-coupled drug in the preparation of an intestinal targeting drug.

[0049] A fourth aspect of the present invention provides use of a polypeptide-coupled drug comprising the above-mentioned extrahepatic targeting polypeptide in extrahepatic targeted drug delivery.

[0050] The fifth aspect of the present invention provides an intestinal-targeted multi-component active substance guided by the above-mentioned extrahepatic targeting polypeptide, in particular, a polypeptide-guided intestinal-targeted nanomedicine.

[0051] Specifically, the nanomedicine is a nanomedicine formed by self-assembly of a polyamino acid cationic polypeptide polymer and a nucleic acid molecule, and the polypeptide is coupled to the polyamino acid cationic polypeptide polymer through a covalent bond.

[0052] Specifically, the above polypeptides can be adsorbed onto the nanodrugs through non-covalent bonds such as electrostatic effects, hydrogen bonds, and van der Waals forces, or can be coupled to the surface of nanodrug particles through covalent bonds.

[0053] Compared with the prior art, the present invention has at least the following beneficial effects:

[0054] The present invention designs and screens a polypeptide that can achieve extrahepatic targeting, and couples the polypeptide with a molecule having pharmacological activity, especially an oligonucleotide molecule, to form a polypeptide-coupled drug, which can transport the pharmacologically active molecule to the extrahepatic, especially intestinal tissue, thereby achieving extrahepatic targeting, especially administration of intestinal diseases. Specifically, after the polypeptide / short peptide is coupled with a small interfering nucleotide (siRNA), the siRNA enters the intestinal cells and specifically inhibits the expression of the corresponding gene in the intestine. The polypeptide can also be connected or adsorbed to multi-molecular substances such as nanomedicines, so that the nanomedicines are mainly enriched in the intestinal area. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 , ACE2 expression levels in different organs / tissues, Consensus transcriptomics data.

[0056] Figure 2 , fluorescence results of the specific binding of the polypeptide to the cell surface receptor ACE2.

[0057] Figure 3 , equilibrium constant diagram of specific binding of polypeptide to cell surface receptor ACE2.

[0058] Figure 4 , schematic diagram of the structure of SPR3-siEGFR polypeptide conjugate.

[0059] Figure 5 , HPLC-Mass analysis diagram of SPR3-siEGFR polypeptide conjugate.

[0060] Figure 6 , SPR3-siEGFR gel imaging.

[0061] Figure 7 , The inhibitory effect of SPR3-siEGFR on the target gene.

[0062] Figure 8 , In vivo imaging analysis of mice after intravenous injection of FAM fluorescently labeled SPR3-siEGFR.

[0063] Fig. 9 , Fluorescence distribution of siEGFR in mice after intravenous injection of FAM fluorescently labeled SPR3-siEGFR.

[0064] Fig.10 , The expression levels of TNF-α in different organs of mice after different concentrations of SPR3-siTNF-α peptide conjugates were intravenously injected into mice. DETAILED DESCRIPTION

[0065] The present invention will be further described by the following non-limiting examples. It is well known to those skilled in the art that many modifications may be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention.

[0066] The content of the present invention can be more easily understood by referring to the following examples. These examples are only for further illustrating the present invention and are not intended to limit the scope of the present invention.

[0067] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0068] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Specific embodiments

[0070] Example 1. Targeting peptide design

[0071] In order to screen for extrahepatic targeting, especially intestinal targeting, drug delivery ligands, the present invention analyzes the cell membrane proteins with relatively higher expression levels in tissues such as the intestine and very low expression in the liver, and ultimately determines that angiotensin converting enzyme 2 (ACE2) is most highly expressed in the small intestine, colorectum and other parts, and very low in the liver. As a targeted cell receptor, it is very suitable for screening ligands for intestinal targeted drug delivery (the expression levels of ACE2 in different organs / tissues are as follows Figure 1 (shown, Consensus transcriptomics data).

[0072] Since ACE2 is a membrane protein, many proteins inside and outside the body bind to it as one of the mechanisms for entering cells or transmitting signals. The present invention analyzes the characteristics of proteins that interact with ACE2, clarifies the key amino acids and the arrangement of these amino acids, and designs a series of polypeptide (or short peptide) sequences containing key amino acid residues, as shown in Table 1.

[0073] Table 1

[0074] name Amino acid sequence length Molecular weight SPR-Scr NYVKSKFRGYNALRVY 16 1978.27 RBM1 SKVGGNYNYLYRLFRK 16 1978.27 SRP1 SKVGANYNYLYRLFRK 16 1992.29 SRP2A EAFNCYFPLQSYGFQPTNGVGYQPY 25 2891.14 SRP2B EGFNCYFPLQSYGFQPTNGVAYQPY 25 2891.14 SRP3 FNCYFPLQSYGFQPTNGVAY 20 2316.56

[0075] The amino acids of the above polypeptides can be replaced by other amino acids of the same nature without affecting the function of the polypeptides. Glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I) can be replaced by each other, glutamic acid (E) and aspartic acid (D) can be replaced by each other, asparagine (N) and glutamine (Q) can be replaced by each other, proline (F), tyrosine (Y) and tryptophan (W) can be replaced by each other, and serine (S) and threonine (T) can be replaced by each other. See Table 2 for details.

[0076] Table 2

[0077]

[0078]

[0079]

[0080]

[0081] Example 2. Verification of affinity of targeting peptides at the cellular level

[0082] A549 alveolar type II epithelial cells with relatively high ACE2 expression levels were used as an in vitro model to analyze the ability of the polypeptide designed in the present invention to specifically bind to the cell surface ACE2 receptor.

[0083] The polypeptides designed in Example 1 were all equipped with FAM fluorescent groups. Human lung cancer A549 cells were inoculated into 12-well plates (2-5×10 5 / well), cultured overnight to allow cells to fully adhere to the wall. Different concentrations of peptides were added to the complete cell culture medium to make the final concentrations 10, 20, 30, and 40 μM, respectively. The prepared peptide solutions were added to 12-well plates, incubated in a cell culture incubator for 4-6 hours, the culture medium was discarded, the cells were fully washed with phosphate buffered saline (PBS) (3 times to ensure that non-specifically adsorbed peptides were washed away), the culture medium was added, and the cells were observed and photographed under an inverted fluorescence microscope. The results are shown in Figure 2 As shown in the figure, it can be seen that among the designed and synthesized peptides, SRP2A, SRP2B and SRP3 are significantly enriched on the cell surface and show strong fluorescence. However, peptides with no binding ability or very weak binding ability, such as SRP1-scr, RBM1 and SRP1, have no obvious fluorescence on the cell surface.

[0084] Example 3. Determination of polypeptide targeting and specific binding ability

[0085] After initially screening out peptides that can bind to cell surface receptors, surface plasmon resonance technology (SPR) is used to further analyze the interaction between the peptides and the target receptors. The specific process is as follows:

[0086] First, a new BIAcore CM-5 dextran chip was inserted and the system was equilibrated with a continuous flow system using PBS as the working solution. At the same time, optimal buffer conditions and concentrations were experimentally determined to minimize nonspecific binding of target and ligand proteins to carboxylated dextran. Ligands that nonspecifically absorb to the highly negatively charged dextran may be denatured and may bind nonspecifically to the target protein in solution.

[0087] Next, insert a new chip. Activate the dextran carboxylate by injecting 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). Inject the ligand protein in a low pH sodium acetate buffer so that there is enough protein on the dextran. The ligand density should be optimized to obtain enough molecular surface to present a clear binding reaction, and dilute sufficiently to measure the dissociation rate constant, which should be constant.

[0088] Then, any unreacted activated carboxylates are blocked with ethanolamine treatment. A known concentration of the hypothetical target protein is injected under defined buffer conditions and concentration ranges. Flow rates and analyte concentrations are varied to minimize diffusion or "mass transport" effects.

[0089] After the operation is completed, the BIA evaluation software point-and-click is used to calculate the separation rate and binding rate constant. The equilibrium constant kD is calculated using the average of the binding rate and separation rate obtained at three target protein concentrations. The smaller the kD value, the stronger the affinity. The results are shown in Figure 1. Figure 3 As shown, it can be seen that the peptides SPR-Scr, RBM1 and SPR1 have no kinetic process, indicating that the peptide does not bind to the ligand ACE2, and the kD value is meaningless at this time. However, the peptides SRP3, SRP2A and SRP2B all have kinetic processes and can strongly bind to the ligand ACE2. Among them, SPR3 has the smallest kD value, showing a strong affinity with ACE2. Therefore, SPR3 was selected for the subsequent peptide-coupled drug synthesis.

[0090] Example 4. Coupling and characterization of polypeptide and siRNA

[0091] The siRNA used is a siRNA molecule that targets and inhibits EGFR (hereinafter referred to as siEGFR), and its molecular sequence is:

[0092] Sense strand: 5'-CAGAUUUUGGGCUGGCCAAACUGCU-3'

[0093] Antisense strand: 5'-AGCAGUUUGGCCAGCCCAAAAUCUG-3'

[0094] Based on the maleimide linker, the siEGFR molecule is connected to the third amino acid (cysteine ​​thiol) of the SPR3 polypeptide. The maleimide and thiol addition reaction is performed to obtain the coupled SPR3-siEGFR, the structure of which is as follows: Figure 4 shown.

[0095] The purity of the conjugate SPR3-siEGFR (molecular weight 19208.3) was determined by HPLC-Mass. Figure 5 shown.

[0096] In order to determine the stability of peptide-coupled siRNA, 200 ng of SPR3-siEGFR was mixed with 10 ng of RNA enzyme and incubated at 37°C for 0.5, 1, 3, and 6 h. 1 μL of proteinase K was added 10 min before the end of the time. 15% page gel non-denaturing gel electrophoresis was performed, 10 ul of sample was loaded into each well, and after GelRed staining for 5-10 min, the gel imager was used for analysis. The results are shown in Figure 2. Figure 6 shown.

[0097] Example 5. Inhibitory effect of SPR3-siEGFR conjugate drug on target gene (in vitro analysis)

[0098] In order to analyze whether siEGFR coupled with SPR3 peptide still has the effect of inhibiting the expression of target gene EGFR, SPR3-siEGFR was co-incubated into mice by in vitro cell co-incubation. RNA was extracted after a certain period of time. After reverse transcription, the mRNA level corresponding to the target gene was analyzed by real-time fluorescence quantitative nucleic acid amplification (real-time PCR) technology.

[0099] Vero cells were seeded in 12-well plates with DMEM complete medium containing 10% peptide bovine serum at a density of 2-5×10 5 cells / well, 1 mL of culture medium per well, culture overnight at 37°C. Aspirate the cell culture medium in the 12-well plate and add 0.5 mL of serum-free culture medium to each well.

[0100] Vero cells were treated with different concentrations of SPR3-siEGFR conjugate:

[0101] Sample a: 400ul SRP3-siEGFR+400ul opti-MEM, prepared into SPR3-siEGFR peptide conjugates of different concentrations (1nM, 10nM)

[0102] Sample b: 400ul scr-siEGFR+400ul opti-MEM

[0103] The prepared samples were co-cultured with Vero cells for 72 hours, and the cells were collected at 72 hours to extract RNA. The expression of EGFR in the cells was detected by real-time fluorescence quantitative PCR (Quantitative Real-Time PCR). The results are as follows: Figure 7 As shown, it can be seen that the SPR3-siEGFR drug conjugate can still effectively inhibit the expression of the target gene EGFR.

[0104] Example 6. Distribution of SPR3-siEGFR drug conjugate after systemic administration (in vivo analysis)

[0105] The siEGFR was conjugated with a FAM fluorescent group and then coupled to the targeting peptide SPR3. The distribution of fluorescently labeled siEGFR in BALB / c mice was analyzed by intravenous injection. In vivo imaging analysis of small animals showed that the fluorescent siRNA was mainly distributed in the small intestine 1 hour after intravenous injection. Figure 8 shown.

[0106] After further sampling, the tissues and organs of the mice were embedded in OCT, frozen sections (10 μm), and DAPI staining was used to detect the distribution of SPR3-siEGFR-FAM in the organs using a laser confocal fluorescence microscope. It was found that siEGFR was mainly concentrated in the intestine and spleen. The results are as follows Fig. 9 shown.

[0107] Example 7. Inhibitory effect of SPR3-siTNF-α conjugate drug on target gene (in vivo analysis)

[0108] Referring to Example 4, the polypeptide SPR3 was conjugated with siTNF-α, and 100 ul of different concentrations of SPR3-siTNF-α polypeptide conjugates (0.2 mg / ml, 0.4 mg / ml, 0.8 mg / ml) were injected into BALB / c mice by intravenous injection. After 24 hours, the expression of the target gene TNF-α in the mouse tissues was detected by real-time fluorescence quantitative PCR. The results are as follows: Fig.10 shown.

[0109] Although the present invention describes certain embodiments of the above-mentioned compositions and methods, and many details have been set forth for the purpose of illustration, it is obvious to those skilled in the art that these compositions and methods are susceptible to other embodiments, and certain details can be changed from the embodiments described herein without departing from the basic principles of the invention.

Claims

1. An extrahepatic targeting polypeptide, characterized in that: The extrahepatic targeting polypeptide particularly targets intestinal tissues / organs, and the amino acid sequence of the extrahepatic targeting polypeptide is selected from: NYVKSKFRGYNALRVY, SKVGGNYNYLYRLFRK, SKVGANYNYLYRLFRK, EAFNCYFPLQSYGFQPTNGVGYQPY, EGFNCYFPLQSYGFQPTNGVAYQPY, FNCYFPLQSYGFQPTNGVAY, and an amino acid sequence in which one or several amino acids are deleted and / or added and / or substituted in the above amino acid sequence but the targeting function remains unchanged.

2. The extrahepatic targeting polypeptide according to claim 1, characterized in that The amino acids of the extrahepatic targeting polypeptide can be replaced by other amino acids of the same nature without affecting the function of the polypeptide, G, A, V, L and I can be replaced by each other, E and D can be replaced by each other, N and Q can be replaced by each other, F, Y and W can be replaced by each other, and S and T can be replaced by each other; And / or, the amino acid sequence of the extrahepatic targeting polypeptide is any one or more of Table 1 and / or Table 2, Table 1 Table 2 3. A polypeptide-conjugated drug comprising the extrahepatic targeting polypeptide according to any one of claims 1 or 2, characterized in that: The structure of the polypeptide-coupled drug is: polypeptide-linker-load, and the load is a small molecule drug, an antibody drug, a protein or an oligonucleotide.

4. The polypeptide-conjugated drug according to claim 3, characterized in that: The linker is a cleavable linker or a non-cleavable linker, wherein the cleavable linker includes a linker containing a disulfide bond, maleimide, a cathepsin-cleavable dipeptide, a diphosphate or a linker containing glucuronic acid, and the non-cleavable linker includes N-succinimidyl-4-(N-maleimido)cyclohexane-1-carboxylate; and / or, the oligonucleotide is siRNA, ASO, miRNA, mRNA, gene editing CRISPR-Cas system, gRNA or RNA aptamer; And / or, the small molecule drug is a tubulin inhibitor, a DNA damaging compound or an apoptosis inducer, the tubulin inhibitor preferably includes calendula, monomethyl calendula, maytansine derivatives, tubulysin, cryptocolistin or an anti-mitotic EG5 inhibitor; the tubulin inhibitor preferably includes calendula, monomethyl calendula, maytansine derivatives, tubulysin, cryptocolistin or an anti-mitotic EG5 inhibitor; the DNA damaging compound preferably includes pyrrolobenzazepines, indolechlorobenzazepines, dukamycin, camptothecin or calicheamicin; the apoptosis inducer preferably includes a Bcl family inhibitor, talanstatin and its analogs, amatoxin or amidophosphoribosyltransferase.

5. The polypeptide-conjugated drug according to claim 3, characterized in that: The molar ratio of the polypeptide to the load is 10:1-1:

10.

6. The polypeptide-conjugated drug according to claim 3, characterized in that: The conjugated drug is a polypeptide molecule connected to an oligonucleotide molecule; or the conjugated drug is a polypeptide molecule connected to two or more oligonucleotide molecules; or the conjugated drug is a polypeptide molecule connected to an oligonucleotide molecule, a polypeptide molecule connected to another different oligonucleotide molecule, and the two polypeptide molecules connected to different oligonucleotide molecules are then combined together through specific covalent bonds.

7. Use of the extrahepatic targeting polypeptide according to claim 1 or 2 or the polypeptide-coupled drug according to any one of claims 3 to 6 in the preparation of an intestinal targeted drug.

8. An intestinal targeted nanomedicine comprising the extrahepatic targeting polypeptide according to any one of claims 1 or 2.

9. The intestinal targeted nanomedicine according to claim 8, characterized in that: The intestinal targeted nanomedicine is a nanomedicine formed by self-assembly of a polyamino acid cationic polypeptide polymer and a nucleic acid molecule, and the extrahepatic targeting polypeptide is coupled to the polyamino acid cationic polypeptide polymer through a covalent bond.

10. The intestinal targeted nanomedicine according to claim 9, characterized in that: The extrahepatic targeting polypeptide is adsorbed onto the nano drug via non-covalent bonds, wherein the non-covalent bonds include electrostatic effects, hydrogen bonds, and van der Waals forces, or is coupled to the surface of the nano drug particles via covalent bonds.

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