CN118005762A

CN118005762A - Polypeptide ligand of targeted scavenger receptor, non-covalent double-targeted molecule and pharmaceutical application thereof

Info

Publication number: CN118005762A
Application number: CN202311099013.4A
Authority: CN
Inventors: 董甦伟; 王椠; 阳星月; 袁瑞欣
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2023-08-29
Filing date: 2023-08-29
Publication date: 2024-05-10

Abstract

The application relates to the field of biological medicine, in particular to a polypeptide ligand of a targeted scavenger receptor, a non-covalent double-targeted molecule and pharmaceutical application. In particular, the application relates to a linear polypeptide comprising a plurality of glutamic acid alanine units (-EA-), to molecules formed by non-covalent interactions of the above linear polypeptide with a staple peptide, and to pharmaceutical uses of the linear polypeptide or of the molecules. The polypeptide ligand and the stable non-covalent double-targeting molecule formed by the staple peptide can be used for treating diseases such as psoriasis and the like related to IL-17A over-expression. Meanwhile, the polypeptide ligand or non-covalent double-targeting molecule can be targeted to bind with scavenger receptors, has potential capability of cell or tissue targeting and selectivity, and can be used for targeted treatment.

Description

Polypeptide ligand of targeted scavenger receptor, non-covalent double-targeted molecule and pharmaceutical application thereof

Technical Field

The application relates to the field of biological medicine, in particular to a polypeptide ligand of a targeted scavenger receptor, a non-covalent double-targeted molecule and pharmaceutical application.

Background

Currently, development of inhibitors against disease targets remains the dominant drug development strategy. However, the placeholder-driven paradigm employed by target inhibitors suffers from non-negligible drawbacks. For example, it can only inhibit a target having catalytic activity, and is not a strategy for a target that plays a non-catalytic role or skeletal function in the development of disease. This has led to the advent of many non-patentable targets. In addition, when the target is over-expressed or mutated to cope with the inhibitor action or the ligand of the target is over-expressed, drug resistance is generated, so that the drug effect is greatly reduced.

The target protein degradation strategy eliminates the pathogenic activity of the target protein by directly degrading the target protein, and makes up the defect of the inhibitor strategy in drug development. Currently, the mainstream target protein degradation strategies are mainly by the technology of proteolysis targeting chimera (PROTAC) of hijacking ubiquitin-proteasome degradation system, the technology of lysosome targeting chimera (LYTAC) of hijacking lysosome-proteasome degradation system and the technology of autophagy-lysosome targeting chimera (AUTAC). They achieve degradation of the target protein by designing a covalent chimeric molecule (chimera) capable of acting on both the target protein and the degradation inducing molecule (ubiquitin ligase/membrane surface lysosome targeting receptor/autophagy inducing protein) to form a ternary complex of target protein-chimeric molecule-degradation inducing molecule (ternary complex). However, current protein targeted degradation strategies also face challenges. On the one hand, the design of covalent chimeric molecules, especially PROTAC molecules, requires consideration of various factors such as the spatial distance of the two ligands, orientation, linker flexibility, etc., which determines whether the target protein can be successfully ubiquitinated, so matching of the coupling site, length and flexibility of the linker requires extensive effort to optimize the screening. On the other hand, some receptors need to bind to multivalent ligands to function effectively, such as M6PR and ASGPR receptors in LYTAC technology, and the multivalent/multimeric polysaccharide ligand synthesis process such as M6Pn and Tri-GalNAc bound to them is complex, which limits the development of the corresponding target protein degradation technology in practical application to a certain extent.

In addition, lysosomal targeting receptors currently developed for use as a strategy for targeted protein degradation (Targeted Protein Degradation, TPD) are relatively limited, mainly M6PR, ASGPR, CXCR7 and Integrin αvβ3. They are widely expressed on different cell types and are capable of high expression on the surface of hepatocytes or tumor cells. While developing a protein degradation strategy selective for cells or tissues is a development goal of the next stage of TPD. Therefore, the development of more types of lysosomal targeting receptors is important to meet the degradation requirements of different diseases and targets. Recently, it has also been reported that dendritic DNA chimeras are developed for cancer treatment using scavenger receptor SRs as targeting receptors.

Scavenger receptor A (SCAVENGER RECEPTOR A, SR-A) is a pattern recognition receptor that is widely distributed on the surface of immune cells, such as macrophages, monocytes, dendritic cells, microglia, and the like. It can recognize and induce the molecules (DAMPs) related to diseases and molecules (PAMPs) related to injury in organism to be engulfed by immune cells such as macrophages and cleared by lysosome, so as to stabilize the homeostasis of organism. Scavenger receptors prefer to recognize ligands with multiple negative charges, such as Lipopolysaccharide (LPS), oxidized or acetylated low density lipoprotein (Ac/Ox-LDL), lipo-phosphoric acid (LTA), and beta-glucan, etc. And because it is specifically expressed on specific cells, scavenger receptors are expected to be able to effect cell or tissue specific clearance of disease-associated proteins. Furthermore, while scavenger receptors have recently proven useful for targeting cancer therapies, there is still a great potential yet to be developed in the relevant arts of treating immune-related diseases, such as those involving immune diseases initiated by macrophage aggregation. It has been shown that enrichment of macrophages at skin lesions, particularly at the interface of the dermis and epidermis, is an important feature of psoriasis, which plays an important role in psoriasis-like skin inflammation.

Summary of The Invention

The invention develops a scavenger receptor-dependent non-covalent double-targeting protein degradation strategy, which comprises the steps of designing a polypeptide ligand SRAL capable of targeting the scavenger receptor, carrying a polypeptide 17Abp with affinity to IL-17A, and forming a stable non-covalent double-targeting molecule SAncb (SRAL &17Abp non-covalent bispecific molecule) to realize the targeted degradation of IL-17A.

The invention can avoid the complex optimization work faced in the construction of covalent chimeric molecules. The ligand molecules can be designed with structural modules capable of interacting to drive two ligands to form a stable double-targeting non-covalent binding system, and the specific clearance effect of the target protein is exerted through the formation of the target protein and a lysosome targeting receptor.

The polypeptide ligand SRAL and the stable non-covalent double-targeting molecule SAncb formed by the polypeptide 17Abp can be used for treating diseases such as psoriasis and the like related to IL-17A over-expression. Meanwhile, the polypeptide ligand or non-covalent double-targeting molecule can be targeted to bind with scavenger receptor, has potential capability of cell or tissue targeting and selectivity, and can be used for treating targeted immune related diseases.

Thus, the present application provides the following applications:

in a first aspect, the application provides a linear polypeptide comprising n glutamic alanine units (-EA-), n being from 3 to 50; optionally, the polypeptide further comprises a modifying group at the C-or N-terminus, the modifying group comprising a polycyclic group or an alkanoyl group; optionally, the modifying group has a spacer with n glutamic acid alanine units (-EA-), the spacer being an amino acid.

In a second aspect, the present application provides a molecule formed by non-covalent interaction of the linear polypeptide described above with a staple peptide comprising a backbone amino acid sequence selected from the group consisting of:

(a) An amino acid sequence as shown in IHVTIPADLWDWINK (SEQ ID NO: 1);

(b) An amino acid sequence having at least 80%, 86.7% or 93% sequence identity to SEQ ID No. 1; or (b)

(C) An amino acid sequence having an addition or substitution of 1,2 or 3 amino acid residues compared to SEQ ID NO. 1;

Wherein the amino acid residues at positions i and i+3, or at positions i and i+4, or at positions i and i+7 of the backbone of the staple peptide are coupled to form a loop via a linker comprising at least one amino acid;

The i is an integer greater than or equal to 1, preferably an integer less than or equal to 8.

In a third aspect, the application provides a method of preparing the molecule comprising contacting the linear polypeptide and the staple peptide for a time.

In a fourth aspect, the application provides a pharmaceutical composition or formulation comprising said linear polypeptide or said molecule.

In a fifth aspect, the application also provides the use of the linear polypeptide or the molecule in the manufacture of a medicament or formulation.

Detailed Description

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the laboratory procedures referred to herein are all conventional procedures widely used in the respective arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In the present invention, "molecule" refers to an entity composed of atoms bonded together in a certain bonding order and spatial arrangement, and the atoms may be bonded to each other by covalent or non-covalent interactions such as hydrogen bonding, ionic bonding or pi conjugation (pi-pi stacking).

In the present invention, a "receptor" refers to a protein located on the plasma membrane or in the nuclear/cytosol of a target organ, to which a corresponding information molecule called "ligand" is bound. Different ligands can bind to their corresponding receptors, initiating intracellular information delivery systems, resulting in alterations in cellular function.

In the present invention SRAL is a linear polypeptide comprising a plurality (e.g., 3 to 50) of glutamic acid alanine units (-EA-) that can specifically bind to scavenger receptors.

In the present invention, "stapling peptides" refer to polypeptides modified by a stapling strategy, which refers to the crosslinking of the side chains of the anchor residues of polypeptides to side chains or side chains to end groups to form loops, which "staple" the polypeptide backbone in a staple-like fashion to stabilize the secondary structural conformation of the polypeptide. The stapling strategy enables the polypeptide to pre-form a stable helical conformation thereby reducing the "entropy penalty" in the target binding process.

In the present invention, 17Abp is a polypeptide that binds IL-17A. The polypeptide can specifically bind to IL-17A and inhibit the interaction of IL-17A with its receptor IL-17 RA. An exemplary amino acid sequence of 17Abp is shown in SEQ ID NO. 1. In the present invention, the term "17Abp" is intended to encompass variants of 17Abp, which "variant" refers to a polypeptide having an amino acid sequence that differs (e.g., is a conservative amino acid substitution) by one or more (e.g., 1, 2, 3, or more) amino acids from the amino acid sequence of 17Abp or that has at least 60%, 70%, 80%, 86.7%, 93%, 96% or more identity, and which has the same function as 17Abp, which "function" may be one or more of the following functions: i) Binds to IL-17A, ii) inhibits the interaction of IL-17A with its receptor IL-17 RA.

In the present invention, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithm of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which has been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (JMoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available on www.gcg.com) using the Blossum 62 matrix or PAM250 matrix and the GAP weights (GAP WEIGHT) of 16, 14, 12, 10, 8, 6 or 4 and the length weights of 1,2, 3, 4, 5 or 6.

In the present invention, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of a protein/polypeptide comprising an amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, another amino acid residue from the same side chain family may be selected to replace the corresponding amino acid residue. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA94:412-417 (1997), which are incorporated herein by reference).

In the present specification, unless otherwise specified, the numbers indicating the positions of amino acid residues in the amino acid sequence are assigned in order in the C-terminal direction with the N-terminal amino acid residue set to 1.

In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., Remington'sPharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to, fillers, diluents, binders, wetting agents, disintegrants, lubricants, surfactants, preservatives, colorants, flavoring agents, fragrances, effervescent agents, emulsifiers, flocculants, deflocculants, bacteriostats, solubilizing agents).

In the present invention, an "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

The amount of drug administered to a subject depends on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, weight and tolerance to the drug, and also on the type of formulation and mode of administration of the drug, and on factors such as the period or time interval of administration. One skilled in the art will be able to determine the appropriate dosage based on these factors and other factors.

The inventors have designed a novel class of SR-A targeted multi-negatively charged polypeptides SRAL, which consist of multiple glutamic acid alanine units (-EA-) by taking advantage of the multi-negatively charged ligand preference of SR-A. SRAL can form a non-covalent double targeting molecule SAncb (SRL &17Abp non-covalent bispecific molecule) with a polypeptide 17Abp capable of specifically binding IL-17A through non-covalent action, and the evaluation result of the capability of targeting IL-17A degradation at a cell level towards SAncb shows that compared with Chimera, SAncb can effectively promote IL-17A to be engulfed by cells and degraded through a lysosomal pathway, and the process is dependent on a scavenger receptor SR-A. Thus, the present application provides the following applications:

linear polypeptides

The application provides a linear polypeptide, which comprises n glutamic acid alanine units (-EA-), wherein n is 3-50; optionally, the polypeptide further comprises a modifying group at the C-or N-terminus, the modifying group comprising a polycyclic group or an alkanoyl group; optionally, the modifying group has a spacer with n glutamic acid alanine units (-EA-), the spacer being an amino acid.

In certain embodiments, n is 3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49 or 50.

In certain embodiments, the linear polypeptide has a relative molecular mass of 700 to 12000, e.g., 700～1000、1000～2000、2000～3000、3000～4000、4000～5000、5000～6000、6000～7000、7000～8000、8000～9000、9000～10000、10000～11000 or 11000 to 12000.

In certain embodiments, the linear polypeptide has a modification group attached to the C-terminus or the N-terminus.

In certain embodiments, the modifying group comprises a polycyclic group.

In certain embodiments, the modifying group comprises a 5-6 membered heterocyclo 5-6 membered heterocycle, such as-carbonyl- (C ₁-C₆ alkyl) - (5-6 membered heterocyclo 5-6 membered heterocycle). In certain embodiments, the modifying group comprises a 5-6 membered nitrogen containing heterocyclo 5-6 membered sulfur containing heterocycle, such as-carbonyl- (C ₁-C₆ alkyl) - (5-6 membered nitrogen containing heterocyclo 5-6 membered sulfur containing heterocycle). The 5-6 membered heterocyclo 5-6 membered heterocycle, 5-6 membered nitrogen containing heterocyclo 5-6 membered sulfur containing heterocycle is optionally substituted.

In certain embodiments, the modifying group is biotin, an exemplary chemical structure of which is as follows:

in certain embodiments, the modifying group comprises a dibenzo5-6 membered cycloalkyl, for example, a-carbonyl- (C _1-6 alkoxy) - (dibenzo5-6 membered cycloalkyl). In certain embodiments, the modifying group is fluorenylmethoxycarbonyl (Fmoc), an exemplary chemical structure of which is as follows:

In certain embodiments, the modifying group comprises a dibenzo 5-6 membered heterocyclyl, for example comprising a dibenzo 5-6 membered oxygen containing heterocyclyl. The dibenzo 5-6 membered heterocyclyl, dibenzo 5-6 membered oxygen containing heterocyclyl is optionally substituted.

In certain embodiments, the modifying group is selected from Fluorescein Isothiocyanate (FITC) and Rhodamine B (RB), the exemplary chemical structures of which are respectively as follows:

In certain embodiments, the modifying group may be a fluorescent group including, but not limited to FITC, AMC, FAM, rhodamine B, TAMRA, cy3, cy5, cy7.

In certain embodiments, the modifying group is an alkanoyl group, such as an alkanoyl group containing 1-18 carbon atoms, including, but not limited to, acetyl.

In the present invention, "optionally substituted" means that the group is optionally substituted with one or more groups selected from the group consisting of: deuterium atom, halogen, hydroxy, cyano, amino, nitro, carboxy, carbonyl, C ₁-C₆ alkyl, halo C ₁-C₆ alkyl, C ₁-C₆ alkoxy, C _3-8 cycloalkyl or-N (C ₁-C₆ alkyl) ₂. In certain embodiments, the linear polypeptide has a spacer between the modifying group and n glutamic alanine units (-EA-) to avoid interaction between the modifying group and the-EA-units. The spacer may be an amino acid, for example an amino acid containing 6 to 18 carbon atoms, for example 6-aminocaproic acid.

The linear polypeptides include, but are not limited to, linear polypeptides represented by the following formulas:

In the above formulae, n is preferably 10 to 15 (e.g., 10, 11, 12, 13, 14 or 15).

Non-covalent double targeting molecules

The present application provides a molecule formed by non-covalent interaction of a linear polypeptide as described above with a staple peptide comprising a backbone amino acid sequence selected from the group consisting of:

(a) An amino acid sequence as shown in IHVTIPADLWDWINK (SEQ ID NO: 1);

wherein the amino acid residues at positions i and i+3, or at positions i and i+4, or at positions i and i+7 of the backbone of the staple peptide are coupled to form a loop via a linker comprising at least one amino acid; the i is an integer greater than or equal to 1, preferably an integer less than or equal to 8.

In some embodiments, i is 7 or 10.

In the present invention, the linker may contain an L-amino acid, a D-amino acid, a natural amino acid, or an unnatural amino acid. In some preferred embodiments, the linker comprises an amino acid that is a natural α -amino acid. In some preferred embodiments, the linker comprises an amino acid that is an L-amino acid.

In addition, various types of amino acids may be used to form linkers, such as polar amino acids or nonpolar amino acids, basic amino acids or acidic amino acids, hydrophobic amino acids or hydrophilic amino acids, amino acids with side chains or amino acids without side chains. The linker comprises amino acids including, but not limited to:

Nonpolar amino acids (hydrophobic amino acids): glycine (Gly) alanine (Ala) valine (Val) leucine (Leu) isoleucine (Ile) phenylalanine (Phe) tryptophan (Trp) methionine (Met) proline (Pro);

Polar amino acids (hydrophilic amino acids):

1) Polar uncharged/polar neutral amino acids: threonine (Thr), serine (Ser), cysteine (Cys), asparagine (Asn), glutamine (Gln), tyrosine (Tyr);

2) Basic amino acids (positively charged amino acids) lysine (Lys), arginine (Arg), histidine (His);

3) Acidic amino acids (negatively charged amino acids) aspartic acid (Asp), glutamic acid (Glu).

Further, the side chains of different amino acid linkers are different in size and nature, some of which can play an important role in regulating and controlling the polypeptide, and can be used as secondary modified handles for further functional derivatization. In some preferred embodiments, the linker comprises an amino acid selected from Glu, ala, lys, ser.

In other embodiments, amino acids without side chains may be used to form the linker. In some embodiments, the linker comprises an amino acid that is Gly.

The inventors found that mutation of some sites on the 17Abp sequence is beneficial to eliminating negative influence of the staple peptide side chain loop on the polypeptide helix conformation, and can improve target affinity and enzymolysis resistance.

In some embodiments, the 17Abp backbone amino acid sequence has a substitution (e.g., a conservative substitution or a non-conservative substitution) of 1, 2, or 3 amino acid residues as compared to SEQ ID NO: 1. In some preferred embodiments, the substitution is at position 7, position 10, position 14, or any combination thereof.

In some preferred embodiments, the substitution is selected from: the amino acid residue at position 7 is substituted with Glu, the amino acid residue at position 10 is substituted with Ala, the amino acid residue at position 14 is substituted with Lys, or any combination thereof.

In some more preferred embodiments, the substitution is selected from the group consisting of: substitution of the amino acid residue at position 7 with Glu, the amino acid residue at position 10 with Ala, the amino acid residue at position 14 with Asn with Lys.

Particularly preferred substitutions include, but are not limited to:

(1) Substitution of amino acid residue at position 7 with Glu from Ala;

(2) Replacement of the amino acid residue at position 10 by Trp to Ala;

(3) The amino acid residue at position 14 is replaced by Lys from Asn;

(4) Substitution of the amino acid residue at position 7 with Glu from Ala, and substitution of the amino acid residue at position 10 with Ala from Trp;

(5) Substitution of the amino acid residue at position 7 with Glu from Ala and the amino acid residue at position 14 with Lys from Asn;

(6) Replacement of the amino acid residue at position 10 by Trp to Ala and replacement of the amino acid residue at position 14 by Asn to Lys;

(7) Substitution of the amino acid residue at position 7 with Glu from Ala, the amino acid residue at position 10 with Ala from Trp, and the amino acid residue at position 14 with Lys from Asn;

(8) Substitution of the amino acid residue at position 7 with Glu, the amino acid residue at position 14 with Asn with Lys, and the amino acid residue at position 1, 2, 3, 4, 5, 6, 10, 11 or 12 with Ala.

In some particularly preferred embodiments, the staple peptide comprises a backbone amino acid sequence selected from the group consisting of:

IHVTIPEDLWDWIKK(SEQ ID NO:2)

IHVTIPEDLADWIKK(SEQ ID NO:3)

IHVTIPEDLKDWINK(SEQ ID NO:4)

IHVTIPADLEDWIKK(SEQ ID NO:5)。

further, the N-terminus and/or the C-terminus of the main chain of the staple peptide of the present invention may be modified. In some embodiments, the N-terminus of the staple peptide backbone is modified by acetylation, thereby reducing hydrolysis thereof by the exopolypeptide enzyme and increasing the polypeptide half-life.

The coupling reaction used to form the present staplers may be an amidation reaction. In some embodiments, the linker comprised by the stapled peptide couples the amino acid residue at position i and position i+3, or the amino acid residue at position i and position i+4, or the amino acid residue at position i and position i+7 of the stapled peptide backbone into a loop via a peptide bond.

In some embodiments, the side chain of the amino acid residue at position i of the staple peptide backbone may comprise a free carboxyl group, the side chain of the amino acid residue at position i+3, i+4 or i+7 of the staple peptide backbone may comprise a free amino group, and the linker comprises an amino acid that forms a peptide bond with the free carboxyl group and the free amino group, respectively.

In other embodiments, the side chain of the amino acid residue at position i of the staple peptide backbone may comprise a free amino group, the side chain of the amino acid residue at position i+3, i+4 or i+7 of the staple peptide backbone may comprise a free carboxyl group, and the linker comprises an amino acid forming a peptide bond with the free amino group and the free carboxyl group, respectively.

The peptides include, but are not limited to, peptides represented by the following formulas:

Stapler 17Abp (FITC)

Stapling peptide 17Abp (Fmoc)

Stapled peptide 17Abp (Ac ₂)

Staple peptide 1a

Staple peptide 1b

Staple peptide 2a

Staple peptide 2b

Staple peptide 3a

Staple peptide 3b

Staple peptide 4a

Staple peptide 4b

Staple peptide 5/>

Staple peptide 7a (17 Abp (Ac))

Staple peptide 7b

Staple peptide 8a

Staple peptide 8b

Staple 9a (17 Abp (Glu))

Staple peptide 9b

Staple peptide 11a/>

Staple peptide 11b

Staple peptide 12a

Staple peptide 12b

Staple peptide 13a

Staple peptide 13b

Staple peptide 14

Staple peptide 15

The above-described stapled peptide can be produced by a method comprising the steps of:

(1) Providing a backbone of the linker and the staple peptide;

(2) Coupling the amino acid residues at positions i and i+3, or the amino acid residues at positions i and i+4, or the amino acid residues at positions i and i+7 of the backbone of the staple peptide into a ring by using the linker.

The stapled peptide can be synthesized by a solid phase synthesis method by using a polypeptide synthesizer. Suitable organic solvents, such as DMF solutions, may be used; using a suitable deprotection reagent (e.g., 20% piperidine in DMF); multiple syntheses are performed using a condensation reaction of an excess of amino acid (e.g., 4 equivalents) with an appropriate condensation reagent (e.g., HATU/HOBt) and an appropriate amount of base (e.g., DIEA) for an appropriate period of time. If desired, after the condensation of highly sterically hindered amino acids such as proline, isoleucine, threonine and valine, the condensation cycle of one amino acid is repeated a plurality of times. Solid phase synthesis can be performed using an amino acid with the α -amino group protected with a 9-fluorenylmethoxycarbonyl protecting group (^α N-Fmoc), e.g Fmoc-Ala-OH,Fmoc-Asn(Trt)-OH,Fmoc-Asp(OtBu)-OH,Fmoc-His(Trt)-OH,Fmoc-Ile-OH,Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH,Fmoc-Pro-OH,Fmoc-Thr(tBu)-OH,Fmoc-Trp(Boc)-OH,Fmoc-Val-OH,Fmoc-Glu(OAll)-OH,Fmoc-Lys(Dde)-OH.

The synthesis of amino acid linkers can be performed using the following route:

An exemplary synthesis method includes:

1) Deprotection of an amino acid in which the α -amino group is protected by a 9-fluorenylmethoxycarbonyl protecting group (^α N-Fmoc);

2) The deprotected amino acid is reacted with an excess of allyl chloroformate to produce an amino acid linker having an alpha amino group protected by an allyloxycarbonyl group.

The deprotection reaction may be carried out at room temperature in the presence of piperidine. Suitable solvents include, but are not limited to, methylene chloride.

The amino acid and allyl chloroformate may be carried out in the presence of a base such as sodium bicarbonate. Suitable solvents include, but are not limited to, tetrahydrofuran/water mixed solvents.

After obtaining the backbone and amino acid linker of the staple peptide, coupling into a ring may be achieved by an amidation reaction. An exemplary method includes:

1) Removing the protecting group of the amino group on the amino acid side chain of one anchoring site on the main chain, for example, removing the Dde protecting group of the amino group by using hydrazine hydrate to obtain the exposed amino group;

2) An amino acid connector with alpha amino protected by allyloxycarbonyl is connected to the amino exposed in the previous step through amidation reaction;

3) Removing allyloxycarbonyl protecting groups on the amino acid linker to obtain a naked amino group, and removing carboxyl protecting groups on amino acid side chains of another anchor site on the backbone to obtain a naked carboxyl group;

For example: treating with tetraphenylphosphine palladium to remove the allyloxycarbonyl protecting group on the amino acid linker while removing the allylic protecting group on the amino acid side chain of the other anchor site on the backbone;

4) The amino group and the carboxyl group exposed in the previous reaction are condensed by amidation reaction to form the staple peptide.

Optionally, the method further comprises: other protecting groups on the backbone are removed, and/or the stapled peptide is purified.

In the non-covalent molecules of the invention, the linear SR-A targeting polypeptide SRL and the IL-17A targeting stapled peptide 17Abp are stably bound together by non-covalent interactions (e.g., ionic bonds and/or pi-pi stacking). The non-covalently acting species are associated with the charged and chemical structures (particularly the terminal modified structures of the polypeptides) of each of SRL and 17 Abp. In certain embodiments, the N-terminus of the linear polypeptide and the N-terminus of the staple peptide bear the same modifying group, such as acetyl, FITC, or Fmoc. When Fmoc is modified at the N-terminal of both the linear polypeptide and the staple peptide, fmoc molecules can increase the stability and compactness of non-covalent interactions through pi-pi stacking, thereby forming more effective SAncb molecules and improving the endocytic efficiency of IL-17A. In certain embodiments, the N-terminus of the linear polypeptide and the N-terminus of the staple peptide may bear different modifying groups.

The affinity of the stapled 17Abp for the linear polypeptide SRL may be tested by, for example, surface Plasmon Resonance (SPR), and in certain embodiments, is not less than 4.25 μm.

In addition, 17Abp and SRL can also produce Fluorescence Resonance Energy Transfer (FRET) effects, demonstrating that two polypeptides are close to each other in solution and very close (e.g., less than 10 nm).

In another aspect, the application also provides a method of preparing the non-covalent bi-targeting molecule comprising contacting the linear polypeptide and the staple peptide for a time (e.g., 8-12 hours). An exemplary method comprises incubating the linear polypeptide and the staple peptide together in a liquid phase (e.g., cell culture medium or buffer solution) for 8-12 hours. In certain embodiments, co-incubation is performed at ambient temperature. In certain embodiments, the linear polypeptide and the staple peptide are each incubated at a concentration of 400 to 800 μm (e.g., 400 μm, 500 μm, 600 μm, 700 μm, or 800 μm).

Pharmaceutical composition and pharmaceutical use

In one aspect, the application provides a pharmaceutical composition comprising a linear polypeptide or a non-covalent dual targeting molecule of the application. Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers useful in the present application include, but are not limited to, fillers, diluents, binders, wetting agents, disintegrants, lubricants, surfactants, preservatives, colorants, flavoring agents, fragrances, effervescent agents, emulsifiers, flocculating agents, deflocculating agents, bacteriostats, solubilizing agents.

The pharmaceutical compositions of the present invention may be formulated into a variety of suitable dosage forms including, but not limited to: oral dosage forms, injectable dosage forms (e.g., dosage forms suitable for subcutaneous, intramuscular, or intravenous injection), inhalable dosage forms, mucosal dosage forms, or topical dosage forms. In certain embodiments, the pharmaceutical composition is formulated into an oral dosage form, such as a tablet, capsule, granule, oral solution, oral suspension, pellet, or microtablet.

In one aspect, the application provides the use of a linear polypeptide or a non-covalent dual targeting molecule of the application in the manufacture of a medicament for the prevention or treatment of a disease associated with IL-17A overexpression and/or with scavenger receptor.

The scavenger receptor (SCAVENGER RECEPTOR A, SR-A) is selectively expressed on the surface of immune cells, such as macrophages, monocytes, dendritic cells, microglia, etc. The linear polypeptide or the non-covalent double-targeting molecule can be targeted to bind with scavenger receptors, thus having potential capability of cell or tissue targeting and selectivity and being used for targeted treatment.

In certain embodiments, the disease involves cells that specifically express scavenger receptors, including but not limited to macrophages, monocytes, dendritic cells, microglia, and the like.

In certain embodiments, the disease may be an inflammatory or immune-related disease, such AS auto-inflammatory disease (AID) and auto-immune disease (AD), such AS Ankylosing Spondylitis (AS), rheumatoid Arthritis (RA), psoriasis (Ps), psoriatic arthritis (PsA), multiple Sclerosis (MS), crohn's Disease (CD).

In one aspect, the application provides a formulation comprising a linear polypeptide or a non-covalent dual targeting molecule of the application. In certain embodiments, the formulation is used to promote uptake, degradation and/or clearance of IL-17A by cells that specifically express a scavenger receptor, including but not limited to macrophages, monocytes, dendritic cells, microglia, and the like.

In one aspect, the application provides the use of a linear polypeptide or non-covalent dual targeting molecule of the application in the preparation of a formulation for promoting uptake, degradation and/or clearance of IL-17A by cells that specifically express a scavenger receptor, including but not limited to macrophages, monocytes, dendritic cells, microglia, and the like.

The formulations of the invention may be administered in vivo or in vitro; for example, the formulation is administered to a subject to promote uptake, degradation, and/or clearance of IL-17A by cells in the subject, or the formulation is administered to cells in vitro (e.g., a cell line) to promote uptake, degradation, and/or clearance of IL-17A by cells in vitro. In certain embodiments, the formulation is used in a method comprising the steps of: the preparation is contacted with the cells for a time (e.g., 0.5 to 48 hours, e.g., 0.5 to 1 hour, 1 to 6 hours, 6 to 12 hours, 12 to 18 hours, 18 to 24 hours, 24 to 36 hours, or 36 to 48 hours).

In one aspect, the application provides a method of treating and/or preventing a disease in a subject comprising administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of a linear polypeptide, a non-covalent bi-targeting molecule or a pharmaceutical composition of the application. The subject may be a mammal, e.g., bovine, equine, porcine, canine, feline, rodent, primate; of these, particularly preferred subjects are humans. The disease may be as described above.

In one aspect, the present application provides the use of a linear polypeptide of the application in combination with a staple peptide as defined above for the manufacture of a medicament for the prevention or treatment of a disease associated with IL-17A overexpression and/or associated with scavenger receptors. In certain embodiments, the treatment is a targeted treatment.

In certain embodiments, the disease involves cells that specifically express scavenger receptors, including but not limited to macrophages, monocytes, dendritic cells, microglia, and the like. The disease may be the disease listed above.

In one aspect, the present application provides a method of treating and/or preventing a disease in a subject, the method comprising co-administering to the subject a linear polypeptide of the application and a stapling peptide as defined above, preferably the linear polypeptide and stapling peptide being present in the same formulation and in contact with each other. In certain embodiments, the treatment is a targeted treatment.

Advantageous effects

The application provides a SAncb non-covalent double-molecule targeting protein degradation system dependent on scavenger receptors, which successfully realizes the targeting degradation of IL-17A in vitro and in vivo. SAncb exerts remarkable anti-inflammatory activity in a psoriasis mouse model by removing IL-17A, improves the psoriasis symptoms of the mice, and shows a certain disease treatment potential. And the SR-A can be developed into a new lysosome targeting degradation receptor successfully, and the application of the targeting degradation strategy in more disease types is expanded. It is worth mentioning that, unlike the traditional strategy of developing inhibitor medicine for IL-17A, the application proves the feasibility of utilizing macrophage to eliminate IL-17A and improve psoriasis symptoms, and provides a new medicine design concept for treating IL-17A related diseases such as psoriasis.

The embodiments of the present application are specifically disclosed below with reference to the drawings as appropriate. However, unnecessary detailed description may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions of the actual same structure may be omitted. This is to avoid that the following description becomes unnecessarily lengthy, facilitating the understanding of those skilled in the art. Furthermore, the drawings and the following description are provided for a full understanding of the present application by those skilled in the art, and are not intended to limit the subject matter recited in the claims.

Drawings

FIG. 1 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRL3 (FITC).

FIG. 2 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (FITC).

FIG. 3 shows the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide chimera (FITC).

FIG. 4 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide SRAL (biotin).

FIG. 5 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRAL (RB).

FIG. 6 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Ac).

FIG. 7 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Glu).

FIG. 8 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide 17Abp (Fmoc).

FIG. 9 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Ac ₂).

FIG. 10 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRAL (Fmoc).

FIG. 11 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRAL (Ac ₂).

FIG. 12 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRL1 (FITC).

FIG. 13 shows the results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRL2 (FITC).

FIG. 14 is a graph showing the results of evaluation of the ability of SRL polypeptide ligands to enter cells at a concentration of 10. Mu.M for polypeptide and cell incubation.

FIG. 15 shows the results of a co-localization experiment of a polypeptide ligand with lysosomes, where the concentration of polypeptide incubated with cells was 10. Mu.M. The red signal is a lysosome tracer; the green signal is a polypeptide; blue signals are nuclei and yellow signals are co-localization signals of lysosomes and polypeptides.

FIG. 16 shows that exogenous IL-17A is taken up by macrophages, the concentration of polypeptide incubated with cells is 50. Mu.M and IL-17A concentration is 0.5. Mu.g/ml.

FIG. 17A shows the effect of SAncb different incubation concentrations on the endocytic efficiency of IL-17A with IL-17A concentration of 0.5. Mu.g/ml; FIG. 17b shows the effect of SAncb different incubation times on IL-17A endocytosis efficiency, SAncb incubated with cells at 50. Mu.M each and IL-17A at 0.5. Mu.g/ml. (n=3, < P <0.05, < P <0.01, < P <0.001, < P < 0.0001).

FIG. 18a shows the co-localization of IL-17A with lysosomes, red signal for IL-17A, green signal for lysosomes, white signal for nuclei, and yellow signal for IL-17A with lysosomes. FIG. 18b shows the effect of the lysosomal inhibitor LPT on IL-17A degradation. SAncb incubated with cells at 50 μm and IL-17A at 0.5 μg/ml (n=3, P <0.05, P <0.01, P <0.001, P < 0.0001).

FIG. 19 shows the co-localization of IL-17A, SAncb and lysosomes. IL-17A-647 is interleukin 17A protein labeled with red fluorescent molecules, exhibiting a red signal; both polypeptides in SAncb systems are marked with FITC molecules at the tail end, and the system is green; the lysosomal tracer lysotracker 567 was defined as a blue signal in this experiment.

FIG. 20a shows the energy dependent process of endocytosis of IL-17A by macrophages; FIG. 20b shows that uptake of IL-17A by macrophages is primarily via clathrin-mediated endocytosis and megaloblastic endocytosis. SAncb incubated with cells at 50. Mu.M and IL-17A at 0.5. Mu.g/ml. (n=3, < P <0.05, < P <0.01, < P <0.001, < P < 0.0001).

FIG. 21a shows the effect of SR-A inhibitor on IL-17A uptake by cells; FIG. 21b shows uptake of IL-17A by cells with different SR-A expression levels. SAncb incubated with cells at 50 μm and IL-17A at 0.5 μg/ml (n=3, P <0.05, P <0.01, P <0.001, P < 0.0001).

FIG. 22 shows the effect of SAncb pre-incubation times on IL-17A endocytic efficiency.

FIG. 23a shows the affinity results between MST evaluation 17Abp and SARL; FIG. 23b shows the FRET phenomenon between 17Abp and SARL, with 17Abp (FITC) for the black line, SAncb (FITC/RB) for the red line, and SRAL (RB) for the blue line.

FIG. 24 shows co-localization of polypeptide SRAL and polypeptide 17Abp in cells and FRET phenomena. The red signal is rhodamine B labeled SRAL, the green signal is FITC labeled 17Abp, and the purple signal is FRET signal of both.

Fig. 25 shows the effect of different N-terminal modifications SAncb on IL-17A endocytosis, at a concentration of 50 μm for both polypeptide and cell incubation, and at an IL-17A concentration of 0.5 μg/ml (n=3, P <0.01, P <0.001, P < 0.0001).

Fig. 26 is a flow chart of an animal experiment and a control set up.

Fig. 27a shows the psoriasis skin lesion severity score in mice, fig. 27b shows the body weight change in mice, and fig. 27c shows the spleen index of mice (n=5, P <0.05, P <0.01, P <0.001, P < 0.0001).

Figure 28 shows the skin lesion appearance and skin H & E staining analysis results of mice.

Fig. 29 shows changes in IL-17A protein levels in mouse serum (n=5, P <0.05, P <0.01, P <0.001, P < 0.0001).

Fig. 30 shows changes in IL-17A protein levels at skin lesions in mice (n=5, P <0.05, P <0.01, P <0.001, P < 0.0001).

Fig. 31 shows changes in IL-17A and its downstream inflammatory factor gene levels in mouse skin (n=5, P <0.05, P <0.01, P <0.001, P < 0.0001).

FIG. 32 shows the results of HE staining of heart, liver, spleen, lung and kidney of experimental mice (Scale bar:100 μm).

FIG. 33 illustrates schematically the process by which SRAL and 17Abp achieve SR-A receptor mediated IL-17A targeted degradation by non-covalent interactions to form a SAncb bimolecular system.

Detailed Description

I. Materials and methods

Chemical reagent and consumable: all reagents purchased were used without purification and the sources of the reagents included Sigma-Aldrich, TCI, adamas, J & K, acros, hischi Biotechnology, jil Biochemical, an Naiji, carbofuran, etc. All solvents of the grade of high grade purity (GR), such as ethyl acetate, methylene chloride, acetone, etc., are available from the general company. HPLC grade solvents were purchased from carbofuran, sigma, oceanpak, acros, et al. Purchased with diethyl ether, tetrahydrofuran, toluene, dichloromethane and DMF (N, N-dimethylformamide) and subjected to PURE(Innovative Technology, inc.) the solvent purification system was subjected to a dry purification process. The crude polypeptide solution is filtered by a filter membrane before analysis and purification, and the model of the filter membrane for analysis is Bulk13Mm syringe filter with 0.2 μm GHP membrane, preparation Filter model BulkGHP/>13mm syringe filter with0.45μm GHP membrane。

N.alpha. -Fmoc protected amino acid compound brands for polypeptide solid phase synthesis include Gill Biochemical (GLBiochem), bomichijie, hischi Biotech (CS bio), pichia medicine, available from Novabiochem and An Naiji, among others ：Fmoc-Ala-OH,Fmoc-Asn(Trt)-OH,Fmoc-Asp(OtBu)-OH,FmocCys(Trt)-OH,Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OAll)-OH,Fmoc-Gly-OH,FmocHis(Trt)-OH,Fmoc-Ile-OH,Fmoc-Leu-OH,Fmoc-Lys(Dde)-OH,Fmoc-Lys(Boc)-OH,Fmoc-Phe-OH,Fmoc-Pro-OH,Fmoc-Thr(tBu)-OH,Fmoc-Trp(Boc)-OH,FmocVal-OH.

The resin Fmoc-RINK AMIDE-MBHAResin for solid phase synthesis of polypeptides was purchased from Gill Biochemical Co at a loading rate of 0.355mmol/g.

Sample bottles for polypeptide sample analysis were purchased from the experimental science and technology company of security spectroscopy (Shanghai); analytical column was AGILENT ECLIPSE XDB-C18 column (5 μm, 4.6X150 mm), exsil Pure 300C18 column (5 μm, 20X 150 mm); the preparative chromatography column was Exsil Pure 300C18 column (10 μm, 20X 250 mm).

Biological reagents and consumables: recombinant murine interleukin 17A was purchased from Novoprotein company; macrophage RAW264.7 is purchased from a national biomedical experimental cell resource library; culture medium and PBS buffer were purchased from beijing midkine company; fetal bovine serum was purchased from PAN Biotech company, germany; cell culture consumables were purchased from Thermo and Corning companies; membrane rupture fluid (00-8333-56) was purchased from eBioscience;4% tissue cell fixative was purchased from the feeimers; PE anti-IL-17A streaming antibodies were purchased from Biolegend; IL-17A red fluorescence labeling kit His-tag Labeling Kit RED-tri-NTA2nd Generation was purchased from Nanotemper; lysotracer 567 was purchased from Biyun; trizol (15596026) was purchased from Sieimert (Thermo Fisher); qPCR reagents were purchased from Shanghai next holy Corp, and PCR 8-tube kit was purchased from Fisher; leupeptin Leupeptin, poly I, and Poly C were purchased from Sigma-Aldrich; chlorpromazine, EIPA, methyl- β -cyclodextrin purchased from source foliar organisms; imiquimod cream was purchased from the company Sichuan Ming Xin Lidi; methotrexate was purchased from Sigma; ELISA kits for murine IL-17A were purchased from winter Song Corp.

Instrument: analytical high performance liquid chromatography of polypeptides was acquired by WATERS ALLIANCE E2695 HPLC equipped Waters2489 UV/visual (UV/Vis) Detector or Agilent Technologies 1260Infinity LC equipped 1260VWD Detector; preparation of polypeptide high performance liquid chromatograph was equipped with a hanbang technology NU3010C UV Detector for hanbang technology NP7005CHPLC or a Waters2489 UV/Vis Detector for Waters 1525Binary HPLC. Polypeptide synthesis using solid phase polypeptide autosynthesizer CS Bio peptide synthesizer (CX 136 XT) employs an Fmoc protecting group based synthesis mode. The circular dichromatic signal of the polypeptide is collected from an instrument Jasco J-810circular dichroism spectropolarimeter by a quartz cuvette with an optical path of 1 mm; the flow experiment uses Cytoflex six laser flow cytometry; confocal microscopy using Cai Sichao resolution laser confocal (lsm880+ airyscan); qPCR experiments used an ABI fluorescent quantitative PCR instrument; fluorescence resonance energy transfer experiments and ELISA experiments were measured using a microplate reader BioTek Synergy Neo2 multifunctional microplate detection system. The protein in the skin tissue of the mice is extracted by a Shanghai Jing Xin tissue grinder.

The data processing uses GraphPad software.

General procedure II

2.1Fmoc-SPPS post-treatment-preparation of N-terminal acetyl protected Polypeptides

After the completion of the solid phase synthesis, the resin loaded with the polypeptide was transferred to a polypeptide synthesis tube with DCM, 5mL of a mixed solution of DMF/acetic anhydride/DIEA (8/1, v/v/v) was added, the reaction liquid was discharged after shaking on a shaker for 5 minutes, the resin remaining in the synthesis tube was washed 3 times each in the order DCM-DMF-DCM, and then dried on a vacuum water pump for 15 minutes to perform the next step.

2.2Fmoc-SPPS post-treatment-preparation of polypeptide amide derivatives

After the solid phase synthesis was completed, the resin carrying the polypeptide was transferred to a polypeptide synthesis tube with DCM, after the solvent was drained off on a vacuum water pump, 8mL of a TFA/TIS/H2O (95/2.5, v/v/v) mixed solution was added to cleave the polypeptide from the resin, while protecting groups on the polypeptide side chains were cleaved off, and after shaking on a shaker for 2H, the liquid was transferred to a glass bottle, the solution was blown dry with a nitrogen stream, and the remaining solid was washed 3 times with glacial diethyl ether. And fully dissolving the washed solid by using a water/acetonitrile mixed solution containing 5% acetic acid, and filtering by using a filter membrane to obtain a clear solution, thus analyzing and purifying.

2.3Fmoc-SPPS post-treatment-preparation of N-terminal FITC or rhodamine B tagged Polypeptides

After the solid phase synthesis was completed, the resin loaded with the polypeptide was transferred to a polypeptide synthesis tube with DCM, 5ml of DCM was added to fully swell the resin, then 10eq of DIEA and 1.2eq of FITC or Rhodamine B were added, the polypeptide synthesis tube was covered with tinfoil to avoid light, the reaction liquid was discharged after shaking overnight on a shaker, the remaining resin was washed 3 times each in the order DCM-DMF-DCM, and then dried on a vacuum water pump for 15 minutes to perform the next step.

2.4 Endocytosis of IL-17A by macrophages

Respectively preparing 800 mu M of 17Abp and SRAL aqueous solutions, adding two equal volumes of polypeptide mother solutions into the same EP tube, blowing and mixing uniformly by using a gun head to prepare 400 mu M of mixed liquid SAncb, and placing the mixed liquid in a refrigerator at 4 ℃ for incubation overnight. At the same time, macrophage RAW264.7 was plated in 48-well plates at a volume of 100 tens of thousands of cells per well, 200 μl DMEM medium per well, and cultured overnight until the cells attached. After 12h of cell culture and polypeptide pre-incubation, IL-17A protein was added to SAncb system and both diluted to 50. Mu.M and 0.5. Mu.g/ml with medium. Subsequently, the medium in the 48-well plate was replaced with it, and SAncb and IL-17A proteins were allowed to incubate with the cells for a further 6h. At the end of incubation, cells from each well were washed three times with PBS and gently blown down with a gun and transferred to EP tubes. After undergoing a fixation, membrane rupture, anti-IL-17A fluorescent antibody staining procedure, intracellular IL-17A signal intensity was analyzed by flow cytometry.

2.5 Co-localization of lysosomes

RAW264.7 cells were plated in a number of 20 ten thousand per dish in a co-Jiao Xiao dish for overnight incubation until they were adherent. Reference to His-tag labeling kit RED-tri-NTA then indicates that IL-17A protein was labeled as IL-17A-647 with a red fluorescent molecule. After SAncb samples were prepared as described in 2.4, they were diluted with IL-17A-647 molecule in medium to concentrations of 50. Mu.M and 0.5. Mu.g/ml, respectively, and incubated with cells for 6h. The nuclear dyes Hoechst 33342 and lysotracker were added to the dishes 20 minutes in advance before the incubation was completed. At the end of incubation, cells from each dish were washed three times with phenol red free medium and observed for co-localization with confocal microscopy.

2.6 Lysosomal degradation experiments

Macrophages RAW264.7 were plated in 48-well plates at 100 tens of thousands of cells per well, 200 μl DMEM medium per well, and cultured overnight until the cells attached. After co-culturing SAncb at 50. Mu.M and 0.5. Mu.g/ml with IL-17A with cells for 6h as described in 2.4, the medium was replaced with fresh medium without protein and polypeptide for further 2h or 4h, allowing intracellular IL-17A to be fully degraded. Wherein, the experimental groups of 2h and 4h are divided into two cross-control groups, one group is added with LPT of 0.1mg/ml, and the other group is not added, so as to study whether the lysosomal inhibitor LPT can inhibit the degradation of IL-17A. Group 0h is a positive control that does not continue culture. After the second incubation, cells from each well were washed three times with PBS, gently blown down with a gun and transferred to EP tubes. After undergoing a fixation, membrane rupture, anti-IL-17A fluorescent antibody staining procedure, intracellular IL-17A signal intensity was analyzed by flow cytometry.

2.7 Verification of endocytic mechanisms

Macrophages RAW264.7 were plated in 48 well plates at 100 ten thousand cells per well and cultured overnight until the cells attached. After half an hour incubation of the endocytosis inhibitor 100mM NaN3, 20. Mu.M EIPA,0.5mM Me-. Beta. -cyclodextrin and 3. Mu.g/ml chlorpromazine with the cells, the SAncb and IL-17A samples prepared as described in 2.4 were added and incubated with the cells for a further 6h. At the end of incubation, cells from each well were washed three times with PBS and gently blown down with a gun and transferred to EP tubes. After undergoing a fixation, membrane rupture, anti-IL-17A fluorescent antibody staining procedure, intracellular IL-17A signal intensity was analyzed by flow cytometry.

2.8 Verification of scavenger receptor mediated TPD

Macrophages RAW264.7 were plated in 48 well plates at 100 ten thousand cells per well and cultured overnight until the cells attached. SAncb and IL-17A samples were prepared as described in 2.4, to which appropriate amounts of Poly I and Poly C were added, diluted with medium to a concentration gradient of 0, 125, 250, 500, 1000. Mu.g/ml, while ensuring that the corresponding SAncb and IL-17A concentrations were 50. Mu.M and 0.5. Mu.g/ml, respectively. Incubate with cells for 6h. At the end of incubation, cells from each well were washed three times with PBS and gently blown down with a gun and transferred to EP tubes. After undergoing a fixation, membrane rupture, anti-IL-17A fluorescent antibody staining procedure, intracellular IL-17A signal intensity was analyzed by flow cytometry.

2.9 Affinity analysis of polypeptide 17Abp and SRAL-Surface Plasmon Resonance (SPR) experiments

Affinity assays for polypeptides 17Abp and SRAL were detected by a Biacore 8K surface plasmon resonance detector. A biotin molecule is coupled to the N-terminus of the polypeptide SARL, and the polypeptide is coupled to the chip by its interaction with the on-chip streptavidin molecule. The sites on the chip that were not coupled to the polypeptide were blocked with free biotin solution. The response signals were then measured by flowing 17Abp solutions of different concentrations through the chip. Calculation of affinity data KD values were calculated by Biacore 8K Evaluation Software evaluation software.

2.10 Fluorescence resonance energy transfer

N-terminal rhodamine B modified SRAL and FITC modified 17Abp were mixed in equal volumes at final concentrations of 320. Mu.M and 160. Mu.M, respectively, pre-incubated overnight to prepare SAncb (RB/FITC), and the presence or absence of FRET was detected with a BioTek SynergyNeo2 microplate reader. Two polypeptides (320. Mu. M SRAL and 160. Mu.M 17 Abp) present alone were first excited with the excitation wavelength of FITC (488 nm) and their emission wavelength was mapped. The same excitation wavelength was used to excite sample SRAncb (RB/FITC), and the emission wavelength spectrum was plotted. The signal increases and decreases of the three emission wavelength spectra are compared.

2.11 Animal experiments

The experiment has been approved by the Beijing university student biomedical ethics committee laboratory animal welfare and Legend committee review (approval number: LA 2022226). Female Balb/c mice were selected for 8-10 weeks, shaved with a 2cm x 3 cm-sized hairless area on their backs, and a psoriasis model was established by applying 5% imiquimod cream (62.5 mg/d) to the backs of the mice after shaving for 7 consecutive days. During this period, mice from the experimental group were injected SAncb daily, designated SAncb (15 mg/kg/d, this concentration calculated with reference to the dose of 17Abp, whereas SRAL concentration was consistent with 17Abp, and both polypeptides were pre-incubated for 12h prior to administration). The negative control group was coated with Vaseline and designated as the Vehicle group (62.5 mg/d). The positive control group was only coated with imiquimod cream and was designated as the Saline group. Both groups of mice were injected with physiological saline in the same volume as SAncb groups. A control group (15 mg/kg/d) injected with 17Abp alone, designated as 17Abp group, was used to investigate the differences in therapeutic effect of IL-17A clearance compared to IL-17A inhibition. In addition, the positive treatment group control group was injected with commercial methotrexate (1 mg/kg/2 d), and was designated as MTX group. Each group had 5 mice. Mice were weighed 8:00 a day in the morning for 7 consecutive days, photographed, recorded on the back skin and evaluated for symptoms, scored according to the degree of erythema, dander, skin thickness, each between 0-4 points. Skin thickness was measured with vernier calipers. The three Index scores are summed as a PASI (Psoriasis AREA AND SEVERITY Index) score. The exposed back is then coated with imiquimod cream or petrolatum. After 6h, i.e. two pm, the sequential administration by intraperitoneal injection was started. Mouse whole blood was obtained by orbital bleeding on day 3 and day 8, respectively. Mice were sacrificed on day 8 and their back skin was cut off for later use. Spleens of mice were removed and weighed, and spleen index was calculated.

2.12 Assessment of cytokine levels in mouse serum

After obtaining whole blood of the mice, the mice were allowed to stand at room temperature for two hours and centrifuged at 6000rpm for 15 minutes. The upper clear yellow serum was aspirated and its IL-17A content was assessed using ELISA kit.

2.13 Extraction and assessment of cytokines in mouse skin

Each mouse was taken with 100mg of skin, minced in an EP tube, then added with 1ml of physiological saline, placed in a tissue grinder, each sample tissue was sufficiently ground under the same conditions, and centrifuged at 1000g for 15min, and the supernatant was evaluated for IL-17A content using ELISA kit.

2.14 Preparation of tissue sections and H & E staining

(1) Drawing and fixing: skin tissue and organs (heart, liver, spleen, lung and kidney) of a mouse are taken and soaked in 4% tissue fixing solution for 12 hours, so that proteins in the tissue are denatured and coagulated, autolysis and decomposition of dead cells are avoided, and the morphology and structure of the cells and the tissues are maintained. (2) tissue dehydration: the tissue is soaked in ethanol with gradient concentration of 30%, 50%, 70%, 80%, 95% and 100% for 3min respectively, so that the water in the tissue is gradually removed. Then standing in 100% ethanol for 1 hr, soaking the tissue in mixed solution of ethanol and xylene (1/1, v/v) for 45min, soaking in xylene, and displacing residual ethanol to make the tissue transparent. Soaking in xylene twice for 45min each time. (3) Paraffin embedding: placing the dehydrated and transparent tissue into a wax dissolving box for heat preservation and temporary storage, then sequentially placing the tissue into a metal embedding box with proper size, injecting paraffin liquid to completely seal the tissue, and taking care to keep the section of the tissue standing at the bottom of the embedding box. And then rapidly placing the tissues soaked with the paraffin liquid into a cooling table to be cooled and solidified into blocks. (4) tissue section: the paraffin block embedded with the tissue is fixed on a slicing machine, cut into slices with the thickness of 5 mu m, put into deionized water with the temperature of 43 ℃ for unfolding, taken out by a hydrophobic glass slide, and put into a baking oven with the temperature of 60 ℃ for baking until the moisture is evaporated to dryness. (5) dewaxing and hydration and H & E staining: dewaxing, hydrating and H & E dyeing with automatic dewaxing and hydrating dyeing instrument, and resin sealing in a sealing machine.

2.15QPCR technique for assessment of mRNA level of target Gene

(1) MRNA extraction: 100mg of mouse skin tissue is weighed and placed in a 5ml EP tube, 1ml of Trizol reagent is added, and after soaking for 5 minutes, the skin tissue is uniformly ground by a grinding rod and kept stand for 10 minutes. mu.L of the supernatant was pipetted into a 1.5ml EP tube, 100. Mu.L of chloroform was added, the mixture was inverted and mixed up and down twice, and the mixture was centrifuged at 12000g for 15 minutes at 4 ℃. The colorless aqueous phase (about 300. Mu.L) at the top was aspirated, transferred to a 1.5ml EP tube, 300. Mu.L of isopropanol was added to precipitate mRNA, mixed up and down again, centrifuged again at 12000g at 4℃for 10 minutes, and the supernatant was discarded, and white mRNA solid adhesion at the bottom was observed. To each tube was added 100. Mu.L of 75% ethanol and centrifuged at 7500g for 5 min at 4℃and the supernatant discarded until the ethanol spontaneously volatilized to a white solid which turned colorless transparent. mu.L of RNAase-free water was added and heated in a water bath for 10 minutes to allow mRNA to be sufficiently dissolved. mRNA concentrations were recorded quantitatively with Nanodrop. (2) cDNA Synthesis: 5X GDNA DIGESTER Mix 3. Mu.L and 2. Mu.g of the extracted mRNA were added to a sterile PCR tube and filled to 15. Mu.L with dd water. Then, 5. Mu.L of 4X was added to the systemIII SuperMixplus, mixing uniformly, and incubating for 2 minutes at 42 ℃ on a PCR instrument to complete cDNA synthesis. The cDNA may be stored in a-20℃refrigerator or may be further processed. (3) qPCR quantitative experiments: mu.L of cDNA was taken, 180. Mu.L of deionized water was added, and vortexed and mixed well. Add 10. Mu.L/> to each well of the eight-strand PCR tubeQPCR SYBR GREEN MASTER Mix (High Rox Plus), 0.4. Mu.L primer and 8. Mu.L cDNA, and finally, the solution was filled to 20. Mu.L with deionized water, and the quantitative experiments were performed on an ABI fluorescent quantitative PCR apparatus.

Primer sequence:

IL-6-Forward Primer:5’-TAGTCCTTCCTACCCCAATTTCC-3’

IL-6-Reverse Primer:5’-TTGGTCCTTAGCCACTCCTTC-3’

CCL20-Forward Primer:5’-GCCTCTCGTACATACAGACGC-3’

CCL20-Reverse Primer:5’-CCAGTTCTGCTTTGGATCAGC-3’

IL-22-Forward Primer:5’-GGTGACGACCAGAACATCCA-3’

IL-22-Reverse Primer:5’-CAGCAGGTCCAGTTCCCCAAT-3’

IL-17A-Reverse Primer:5’-TTTAACTCCCTTGGCGCAAAA-3’

IL-17A-Reverse Primer:5’-CTTTCCCTCCGCATTGACAC-3’

Example one preparation and characterization of the polypeptide

1. Polypeptide SRL3 (FITC) or SRAL (FITC) (n=15)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B25-40%, using Exsil Pure C18 column). The retention time of the product was 9.50-11.30 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide SRL3 (FITC) (12.6 mg, overall yield 7.2%) as a fluffy yellow solid. The result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide SRL3 (FITC) is shown in FIG. 1. Left: UV trace (linear concentration gradient of mobile phase B over 30 min 25-40% using Agilent C18 column), t _R =10.68 min; right: mass spectrum data, relative molecular mass of C ₁₄₇H₂₀₅N₃₃O₆₆ S was calculated: 3520.34Da (isotope average), [ m+2h ] ²⁺m/z＝1761.17,[M+3H]³⁺ M/z= 1174.45 assay: 1761.75, 1174.77.

2. Polypeptide 17Abp (FITC)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-55%, using Exsil Pure C18 column). The retention time of the product was 25.00-26.00 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide 17Abp (FITC) (12.6 mg, overall yield 6.5%) as a fluffy yellow solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide 17Abp (FITC) are shown in FIG. 2. Left: UV trace (linear concentration gradient of mobile phase B over 30 min 30-55% using Agilent C18 column), t _R =25.43 min; right: mass spectrum data, relative molecular mass of C ₁₁₆H₁₆₅N₂₅O₂₈ S was calculated: 2388.20Da (isotope average), [ m+2h ] ²⁺m/z＝1195.10,[M+3H]³⁺ M/z= 797.07 assay: 1195.31, 797.07.

3. Polypeptide chimera (FITC) (for control)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-60% using Exsil Pure C18 column). The retention time of the product was 16.50-18.30 minutes. The product fractions were collected and lyophilized under reduced pressure to give the polypeptide chimer (FITC) (5.3 mg, overall yield 1.9%) as a fluffy yellow solid. The result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide chimera (FITC) is shown in FIG. 3. Left: UV trace (linear concentration gradient of mobile phase B over 30min 30-60% using Agilent C18 column), t _R =17.95 min; right: mass spectrum data, relative molecular mass of C ₂₄₂H₃₅₆N₅₆O₈₉ S was calculated: 5502.48Da (isotope average), [ m+3h ] ²⁺m/z＝1835.16,[M+4H]³⁺ M/z= 1376.62 assay: 1836.15, 1377.40.

4. Polypeptide SRAL (biotin)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B25-40%, using Exsil Pure C18 column). The retention time of the product was 15.00-17.30 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide SRAL (biotin) (13 mg, overall yield 8.0%) as a fluffy solid. The result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide SRAL (biotin) is shown in FIG. 4. Left: UV trace (linear concentration gradient of mobile phase B over 30 min 25-40% using Agilent C18 column), t _R =16.03 min; right: mass spectrum data, relative molecular mass of C ₁₃₀H₁₉₇N₃₃O₆₂ S was calculated: 3244.30Da (isotope average), [ m+2h ] ²⁺m/z＝1623.15,[M+3H]³⁺ M/z= 1077.43 assay: 1623.72, 1077.22

5. Polypeptide SRAL (RB)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-60% using Exsil Pure C18 column). The retention time of the product was 11.00-12.00 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide SRAL (RB) (mg, total yield 6.2%) as a fluffy solid. The results of the high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide SRAL (RB) are shown in FIG. 5. Left: UVtrace (linear concentration gradient of mobile phase B over 30 min 30-60% using Agilent C18 column), t _R =11.33 min; right: mass spectrum data, relative molecular mass of C ₁₅₄H₂₂₄N₃₄O₆₃ was calculated: 3557.54Da (isotope average), [ m+2h ] ²⁺m/z＝1779.77,[M+3H]³⁺ M/z= 1186.85, 890.39 assay: 1779.98, 1186.98, 890.63.

6. Polypeptide 17Abp (Ac)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-55%, using Exsil Pure C18 column). The retention time of the product was 15.00-16.25 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide 17Abp (Ac) (12 mg, 12.4% overall yield) as a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Ac) are shown in FIG. 6. Left: UV trace (linear concentration gradient of mobile phase B over 30 min 30-55% using Agilent C18 column), t _R =15.85 min; right: mass spectrum data, relative molecular mass of C ₉₁H₁₄₅N₂₃O₂₃ was calculated: 1929.30Da (isotope average) [ m+2h ] ²⁺m/z＝965.65,[M+3H]²⁺ M/z= 644.10; detection result: 965.38, 643.86.

7. Polypeptide 17Abp (Glu)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-55%, using Exsil Pure C18 column). The retention time of the product was 16.25-17.75 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide 17Abp (Glu) (16 mg, total yield 16.0%) as a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Glu) are shown in FIG. 7. Left: UV trace (linear concentration gradient of mobile phase B over 30 min 30-55% using Agilent C18 column), t _R =17.43 min; right: mass spectrum data, relative molecular mass of C ₉₀H₁₄₀N₂₂O₂₅ was calculated: 1930.24Da (isotope average) [ m+2h ] ²⁺ M/z= 966.12; detection result: 965.45.

8. Polypeptide 17Abp (Fmoc)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B40-55%, using Exsil Pure C18 column). The retention time of the product was 25.00-27.25 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide 17Abp (Fmoc) (15 mg, 6.8% overall yield) as a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide 17Abp (Fmoc) are shown in FIG. 8. Left: UV trace (linear concentration gradient of mobile phase B over 30 min 40-55% using Agilent C18 column), t _R =25.45 min; right: mass spectrum data, relative molecular mass of C ₁₁₀H₁₆₄N₂₄O₂₅ was calculated: 2221.23Da (isotope average) [ m+2h ] ²⁺m/z＝1111.62,[M+3H]³⁺ M/z= 741.41; detection results 1112.52, 741.98.

9. Polypeptide 17Abp (Ac ₂)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-60% using Exsil Pure C18 column). The retention time of the product was 15.00-17.00 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide 17Abp (Ac ₂) (11 mg, overall yield 5.4%) as a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Ac ₂) are shown in FIG. 9. Left: UV trace (linear concentration gradient of mobile phase B over 30min 30-60% using Agilent C18 column), t _R =16.20 min; right: mass spectrum data, relative molecular mass of C ₉₇H₁₅₆N₂₄O₂₄ was calculated: 2041.17Da (isotope average) [ m+2h ] ²⁺m/z＝1021.59,[M+3H]³⁺ M/z= 681.39; detection results 1021.87, 681.72.

10. Polypeptide SRAL (Fmoc)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-60% using Exsil Pure C18 column). The retention time of the product was 16.00-18.00 minutes. The product fractions were collected and lyophilized under reduced pressure to give the polypeptide SRAL (Fmoc) (30 mg, overall yield 8.9%) as a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide SRAL (Fmoc) are shown in FIG. 10. Left: UV trace (linear concentration gradient of mobile phase B over 30 min 30-60% using Agilent C18 column), t _R =17.20 min; right: mass spectrum data, relative molecular mass of C ₁₄₁H₂₀₄N₃₂O₆₃ was calculated: 3353.37Da (isotope average) [ m+2h ] ²⁺m/z＝1677.69,[M+3H]³⁺ M/z= 1118.79; detection results 1678.60, 1118.79.

11. Polypeptide SRAL (Ac ₂)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B20-40%, using Exsil Pure C18 column). The retention time of the product was 16.00-18.00 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide SRAL (Ac ₂) (21 mg, 6.6% overall yield) as a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRAL (Ac ₂) are shown in FIG. 11. Left: UV trace (linear concentration gradient of mobile phase B over 30 min 20-40% using Agilent C18 column), t _R =18.63 min; right: mass spectrum data, relative molecular mass of C ₁₂₈H₁₉₆N₃₂O₆₀ was calculated: 3173.32Da (isotope average) [ m+2h ] ²⁺ M/z= 1587.66; and (3) detecting a result 1588.86.

12. Polypeptide SRL1 (FITC) (for control)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-60% using Exsil Pure C18 column). The retention time of the product was 20.00-22.00 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide SRL1 (FITC) (22.5 mg, 20.6% overall yield) as a fluffy yellow solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRL1 (FITC) are shown in FIG. 12. Left: UV trace (linear concentration gradient of mobile phase B over 30min 30-60% using Agilent C18 column), t _R =20.91 min; right: mass spectrum data, relative molecular mass of C ₁₁₀H₁₂₄N₂₀O₂₅ S was calculated: 2156.88Da (isotope average), [ m+2h ] ²⁺ M/z= 1079.44, detection results: 1079.54.

13. Polypeptide SRL2 (FITC) (for control)

After removal of the protecting groups, the crude polypeptide was dissolved in 16 ml of acetonitrile/water (50/50, v/v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (linear concentration gradient of mobile phase B30-60% using Exsil Pure C18 column). The retention time of the product was 26.90-28.30 minutes. The product fractions were collected and lyophilized under reduced pressure to give polypeptide SRL2 (FITC) (23.5 mg, 17.0% overall yield) as a fluffy yellow solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRL2 (FITC) are shown in FIG. 13. Left: UV trace (linear concentration gradient of mobile phase B over 30min 30-60% using Agilent C18 column), t _R =27.03 min; right: mass spectrum data, relative molecular mass of C ₁₃₉H₁₇₆N₂₆O₃₃ S was calculated: 2769.26Da (isotope average), [ m+2h ] ²⁺m/z＝1385.63,[M+3H]³⁺ M/z= 924.09 assay: 1385.92, 924.37. Signal 1191.43 is the starting material prior to coupling FITC.

Example two assessment of Linear Polypeptides ability to enter macrophages and lysosomal Co-localization

Flow experiments the ability of the following three polypeptides to enter macrophages was assessed.

As a result, as shown in FIG. 14, SRL3, which is a SR-A receptor-targeting polypeptide of the present invention, exhibited absolute intracellular advantages over the polypeptides reported in the other two documents. Whereas the SR-B targeting polypeptide SRL2 exhibits the lowest cell entry efficiency.

To determine whether three polypeptides, after uptake by macrophages, escape from endosomes and eventually diffuse into the cytoplasm or can successfully enter lysosomes, confocal microscopy was used to observe whether polypeptide ligands could co-localize with lysosomes after entering cells. The N-terminal ends of the three polypeptides were first labeled with FITC (6-aminocaproic acid as spacer), then they were added to the medium and incubated with the cells for a further period of time, after which the fluorescently labeled polypeptides were washed off and observed under confocal microscopy (FIG. 15). The dotted green polypeptide signal indicates that it is not spread in the cytoplasm, but is present in the organelle. And, the green signals of the three polypeptides are highly overlapped with the red lysosome signals, and the three polypeptides show yellow co-localization signals, which indicate that the three polypeptides can be transported into lysosomes after entering cells. The superior performance of the SR-A receptor-targeting polypeptide SRL3 (SRAL) of the invention can be seen by combining the results of two experiments, namely a cell-entering experiment and co-localization experiment.

Example three demonstration SAncb of IL-17A degradation via SR-A mediated degradation

1. Verification SAncb promotes endocytosis of IL-17A by macrophages

To determine whether two polypeptide ligands promote endocytosis of IL-17A by macrophages, the inventors designed five experimental groups: includes chimeric polypeptide chimera with which 17Abp is covalently linked to SARL, control mix with equal volumes of SARL and 17Abp, control SARL with two polypeptides acting alone, 17Abp, and control without polypeptide added. The polypeptides are all N-terminal FITC modified forms. Five groups were added with equal concentrations of commercial IL-17A and after 6h incubation with macrophages, the amount of IL-17A entering the cells was assessed using a flow cytometer. Flow results indicate that neither 17Abp nor SARL alone have a way to promote massive endocytosis of IL-17A, demonstrating the importance of simultaneous targeting of SR-a receptor and target protein. Surprisingly, however, the migratory is highly effective in promoting uptake of IL-17A by macrophages. Whereas the chimera group IL-17A intracellular signal was close to the control group, indicating that it hardly assisted in transporting IL-17A into cells (FIG. 16). This suggests that two polypeptide ligands in the mix may form a dual targeting system (designated SAncb (SRL &17Abp non-covalent bispecificmolecule)) through non-covalent interactions, which captures IL-17A and binds to the SR-A receptor, thereby transporting IL-17A into cells.

To further confirm that SAncb is capable of promoting endocytosis of IL-17A by macrophages, the inventors studied the effect of incubation concentration and incubation time of SAncb in the medium. The results show that uptake of IL-17A by macrophages is SAncb dependent on concentration, i.e., the higher the SAncb concentration, the stronger the IL-17A signal that is transported into the macrophages. And the uptake efficiency reached plateau when SAncb concentration reached 50 μm (fig. 17 a). Whereas with increasing SAncb incubation time, the intracellular signal of IL-17A peaked at 6 h. Then with increasing time, the uptake efficiency gradually decreased to be comparable to the blank signal (fig. 17 b). The inventors speculate that the uptake is greater than the intracellular degradation amount due to the higher concentration of IL-17A in the medium during the rise of intracellular IL-17A signal. Thereafter, as the incubation time is extended, the concentration of IL-17A in the medium is reduced, at which time the uptake is less than the intracellular degradation, resulting in a decrease in intracellular IL-17A signal. This result also shows, to some extent, that uptake of IL-17A into the cell by macrophages will degrade it, providing evidence for the effectiveness of the targeted protein degradation strategy.

2. Verification of macrophage degradation of IL-17A

To determine whether IL-17A undergoes degradation via the lysosomal pathway after uptake by macrophages, the inventors first studied the co-localization of IL-17A by transport into cells and lysosomes. Confocal microscopy results (FIG. 18 a) showed that the experimental group with SAncb molecules added was able to observe co-localization of IL-17A-647 with lysosomes, whereas the control group did not observe red IL-17A signal intracellularly, indicating that exogenous IL-17A could not be taken up directly by macrophages without SAncb involvement (FIG. 19). Lysosomal inhibitor LPT was used to investigate whether the middle hydrolase activity of lysosomes would affect the degradation of IL-17A.

In another experiment, the inventors first cultured macrophages with a medium containing SAncb and IL-17A for 6 hours, allowing them to take up the IL-17A in the medium. The medium was then replaced with fresh medium with or without LPT and the cells were further cultured for 0h,2h and 4h (with 0h cells as positive control), and finally the residual amount of IL-17A in the cells was assessed. The results showed that the intracellular IL-17A content decreased with time after the medium was changed and the culture continued. Whereas degradation of IL-17A by LPT treated cells was significantly less than that of cells not treated by LPT at the same time, indicating that low lysosomal enzyme activity delayed degradation of IL-17A (FIG. 18 b). In summary, SAncb molecules promote the endocytosis of IL-17A by macrophages and then transport to lysosomes for degradation, thereby achieving clearance of IL-17A.

3. Verification SAncb of endocytic mechanism inducing uptake of IL-17A by cells

Next, to verify what endocytic degradation of IL-17A underwent endocytic pattern, the inventors first selected low temperature conditions (4 ℃) and ATP inhibitors (NaN ₃) to explore whether this process is an energy-consuming active uptake process. As shown in fig. 20a, both low temperature and ATP inhibitors significantly reduced the endocytosis of IL-17A by macrophages (< P < 0.0001), with a more pronounced inhibition effect of NaN ₃ (< P < 0.01). Indicating that endocytosis of IL-17A by macrophages is an energy dependent process under SAncb. Next, in a specific model study of endocytic behavior, the inventors selected three endocytic inhibitors to conduct experiments including the lipid valve pathway inhibitor methyl- β -cyclodextrin (Me- β -CD), megaloblastic inhibition 5- (N-ethyl-N-isopropyl) amiloride (EIPA), and clathrin pathway inhibitor Chlorpromazine (CPM). The results (FIG. 20 b) show that all three inhibitors reduced IL-17A uptake by cells to varying degrees, with the inhibitory effects of EIPA and CPZ being more pronounced (P)

< 0.0001), Indicating that clathrin-mediated endocytosis and megaloblastic drinks are the main processes of uptake of IL-17A by macrophages. The above results indicate that SAncb molecules induce the process of IL-17A uptake by macrophages, an energy dependent, clathrin mediated and macropolytic endocytic pathway. This is similar to SR-A mediated endocytic behavior.

4. Verification of scavenger receptor SR-A mediated uptake of IL-17A by cells

Although the above results have demonstrated that the endocytic pattern of IL-17A under SAncb is similar to the SR-A mediated pathway, more direct evidence is needed to demonstrate that the SR-A receptor is involved in this SAncb-induced protein degradation. Thus, the inventors selected a specific inhibitor of SR-A, poly I, to investigate whether inhibition of SR-A would reduce endocytosis of IL-17A by cells, wherein Poly C is the negative control group. Flow-through results showed that the endocytosis efficiency of IL-17A decreased with increasing Poly I concentration, but was unaffected by Poly C (21 a). Furthermore, the results of studies on the endocytosis efficiency of IL-17A in four cell lines having different SR-A expression levels revealed that the endocytosis level of IL-17A and the SR-A expression level on the cell surface were positively correlated (Table 21 b). This suggests that SAncb molecules could take advantage of this difference to achieve cell or tissue specific targeted protein degradation. From the above two experimental results, SAncb can be considered to promote endocytosis and degradation of IL-17A via the SR-A receptor mediated lysosomal targeting degradation process.

Example IV verifies SAncb molecular mechanisms of degradation of IL-17A

Cell experiments showed that the longer the pre-incubation time for SAncb molecule preparation, the more efficient the IL-17A endocytosis (FIG. 22), and it is speculated that a certain amount of time is required to complete the formation of stable non-covalent interactions between the two polypeptides.

To verify the interaction between two polypeptides, the inventors first tested the affinity magnitude between 17Abp and SARL using Surface Plasmon Resonance (SPR). The results indicate that 17Abp has an affinity for SARL polypeptide of up to 4.25 μm (fig. 23 a). In addition, 17Abp and SARL also produced FRET effects (fig. 23 b), demonstrating that the two polypeptides are close to each other in solution and very close (less than 10 nm). This also demonstrates the possibility of forming non-covalent conjugates of the two polypeptides at another angle. At the same time, confocal microscopy also observed that two molecules could be co-localized in lysosomes after entering the cell, and also that FRET phenomenon was observed in the cell (fig. 24). This result suggests that the two polypeptides are endocytosed together into the cell at very close distances and eventually localize to the lysosome. Taken together with the experimental conclusion above, the inventors believe that 17Abp and SARL can form a compact dual-targeting SAncb system through non-covalent interactions, which is capable of mediating uptake and degradation of IL-17A by macrophages.

EXAMPLE five evaluation of the ability of different group-modified polypeptide ligands to promote IL-17A endocytosis

Using 6-aminocaproic acid as spacer, this example designed three polypeptide ligand pairs for N-terminal acetyl Ac modification, FITC modification and Fmoc modification, SAncb (Ac ₂), SAncb (FITC) and SAncb (Fmoc), which were evaluated for their ability to promote IL-17A endocytosis. As shown in fig. 25, all three SAncb molecules significantly promoted endocytosis of IL-17A by macrophages compared to the control, with Fmoc modified polypeptides being most effective. The inventors speculate that Fmoc molecules at the N-terminus of both polypeptides can increase the stability and compactness of this non-covalent interaction by pi-pi stacking, thereby forming more potent SAncb molecules, increasing the endocytic efficiency of IL-17A.

EXAMPLE six SAncb treatment of psoriasis in mice by IL-17A clearance

1 Design and flow of animal experiments

After verifying the feasibility of SAncb to clear IL-17A at the cellular level, this example further explored the ability of co-assembled targeted degradation strategies to clear IL-17A in vivo and whether mice can be treated for psoriasis by clearing IL-17A in a mouse psoriasis model. Here, the SAncb non-covalent molecular system that gave the best performance of the N-terminal Fmoc modification in the cell experiments was chosen for the study. Psoriasis models were established by applying imiquimod cream to the backs of mice after shaving every day for 7 consecutive days (fig. 26). During this period, mice from the experimental group were injected with SAncb (SAncb) daily, and mice from the Vaseline negative control group (Vehicle group) and the IMQ positive control group (Saline group) were injected with equal volumes of physiological Saline. A control group injected with 17Abp alone (designated 17Abp group) was used to explore the differences in therapeutic effect of IL-17A clearance compared to IL-17A inhibition. In addition, the positive treatment group was set up to inject commercial methotrexate (noted MTX group).

Evaluation of therapeutic Effect of SAncb on mouse psoriasis

We scored the degree of back skin damage in mice during 7 days of modeling and dosing (figure 27 a), including the degree of skin thickening, the severity of erythema and dander. And calculate their cumulative score PASI (Psoriasis AREA AND SEVERITY Index). The results show that SAncb and MTX groups have equivalent efficacy in improving three skin lesions of psoriasis, while 17Abp only plays a role in alleviating skin thickening. IMQ cream treated mice all had varying degrees of weight loss, which was one of the manifestations of the disease. SAncb was observed in the experiment to have a better effect in alleviating weight loss in mice than in the MTX treated group (fig. 27 b). Swelling of the spleen is also one of the inflammatory manifestations caused by psoriasis. After mice were sacrificed on day eight, spleen index (spleen weight/mouse weight, mg/g) was assessed for each group of mice. The results show that SAncb treatment significantly improved the extent of spleen swelling in mice compared to the Saline group (< P < 0.0001) and even better than the improvement effect of MTX (< P < 0.05). Whereas 17Abp alone did not alleviate spleen swelling in mice (fig. 27 c). Taken together, SAncb exhibited better effects than MTX in improving weight loss and spleen swelling in mice. This demonstrates to some extent that SAncb has lower toxicity than MTX, representing an advantage of polypeptide molecules in terms of biocompatibility.

From pictures of the back skin of mice and their corresponding H & E staining results (fig. 28), significant dander, erythema and thickened skin of the back skin of mice of the Saline group could be visually observed. The tissue staining results show that the stratum corneum has obvious thickening and incomplete keratinization, the acantha is obviously thickened, the epidermis network ridge is prolonged, the nipple top of the dermis layer is vasodilated, and Munro corpuscles exist. The Saline group showed significant psoriasis symptoms, both macroscopically and microscopically, and the 17Abp group showed similar behavior. In contrast, SAncb and MTX treated groups were significantly relieved of the above symptoms.

To assess whether SAncb can clear IL-17A in psoriatic mice, we performed a determination of the amount of IL-17A protein in serum and skin (FIG. 29). The results showed that the serum IL-17A protein concentration was significantly lower in mice from the group SAncb on day four and day seven than in mice from the group Saline (< 0.05P), whereas 17Abp alone did not decrease IL-17A. This suggests that the SAncb strategy can also successfully achieve clearance of the target protein IL-17A in vivo.

The skin IL-17A content assessment showed that both MTX and SAncb were able to significantly reduce IL-17A content compared to the Saline group (< 0.001, <0.01, < P), whereas the 17Abp alone effect was not significantly altered (fig. 30). Taken together, SAncb successfully reduced the levels of IL-17A in mouse serum and skin, demonstrating the feasibility of the protein degradation strategy of SAncb for in vivo application.

Subsequently, the present study assessed SAncb system anti-inflammatory activity in vivo by changes in the expression levels of IL-17A and its downstream related inflammatory factors (IL-6, CCL20, IL-22) genes in mouse skin. The results show that SAncb and MTX both significantly reduced IL-in-a-room compared to the Saline group

MRNA levels of 17A (P < 0.0001), IL-6 (P < 0.001), CCL-20 (P < 0.0001), and IL-22 (P < 0.0001). Whereas 17Abp alone was able to down-regulate mRNA levels of CCL20 (< 0.001) and IL-22 (< 0.05) only and was significantly weaker than the SAncb effect (fig. 31). Therefore, it is reasonable to believe that SAncb can achieve the effect of down-regulating the expression level of IL-17A and its downstream cytokine genes by clearing the IL-17A protein. And its anti-inflammatory effect is significantly better than that of the 17Abp inhibitor alone.

After verifying SAncb's feasibility of treating psoriasis by in vivo clearance of IL-17A, this example continued to evaluate its in vivo toxicity profile. The H & E staining of the individual important organs (heart, liver, spleen, lung and kidney) of the mice showed (fig. 32) that the individual organs of mice in group SAncb, MTX and 17Abp were not abnormally altered compared to the control group injected with physiological saline alone. This result shows that SAncb has good biocompatibility and does not cause systemic toxicity to mice.

In summary, the present invention devised a scavenger receptor SR-A dependent two-component non-covalent polypeptide targeting protein degradation strategy and used to scavenge IL-17A. First, utilizing the multi-negatively charged ligand preference of SR-A, the present invention contemplates SRAL polypeptide ligands targeting SR-A. SRAL and 17Abp were then allowed to interact by pre-incubation to form SAncb non-covalent bimolecular system. The results of the ability assessment of cell-side facing SAncb to target IL-17A degradation show that compared to Chimera, SAncb is capable of efficiently promoting IL-17A endocytosis and degradation via the lysosomal pathway. At the same time, the present invention also verifies that this process is scavenger receptor SR-A dependent.

Subsequent validation of SAncb non-covalent bimolecular system formation mechanism, SPR and FRET results demonstrated that there was an interaction between 17Abp and SRAL.

Furthermore, the invention verifies SAncb molecules in a psoriasis mouse model to reduce the content of IL-17A protein in the serum and skin lesion parts of the mice and realize the in vivo targeted degradation of IL-17A. Meanwhile, SAncb molecules showed improvement of psoriasis symptoms (dandruff, erythema, epidermis thickening and histopathological manifestations) in mice, and more superior ability to slow down weight loss in mice and reduce the extent of spleen swelling in mice than the treatment control group MTX, indicating the advantage of SAncb polypeptide system in terms of biocompatibility. SAncb also down-regulates the mRNA expression level of IL-17A and its downstream inflammatory factors, whose anti-inflammatory activity is significantly higher than that of the IL-17A inhibitor polypeptide 17Abp acting alone. In addition, SAncb molecules have good biocompatibility, and cannot cause toxic damage to organs (heart, liver, spleen, lung, kidney and the like) of mice.

The present application is not limited to the above embodiment. The above embodiments are merely examples, and embodiments having substantially the same configuration and the same effects as those of the technical idea within the scope of the present application are included in the technical scope of the present application. Further, various modifications that can be made to the embodiments and other modes of combining some of the constituent elements in the embodiments, which are conceivable to those skilled in the art, are also included in the scope of the present application within the scope not departing from the gist of the present application.

Claims

1. A linear polypeptide comprising n glutamic acid alanine units (-EA-), n being 3 to 50; optionally, the polypeptide further comprises a modifying group at the C-or N-terminus, the modifying group comprising a polycyclic group or an alkanoyl group; optionally, the modifying group has a spacer with n glutamic acid alanine units (-EA-), the spacer being an amino acid.

2. The linear polypeptide of claim 1, wherein n is 5 to 20.

3. The linear polypeptide of claim 1 or 2, wherein the polycyclic group is a 5-6 membered heterocyclo 5-6 membered heterocycle, dibenzo 5-6 membered cycloalkyl or dibenzo 5-6 membered heterocyclyl; the alkanoyl group is an alkanoyl group having 1-18 carbon atoms, such as acetyl.

4. A linear polypeptide according to any one of claims 1 to 3, wherein the spacer is an amino alkanoic acid having 6 to 18 carbon atoms, such as 6-aminocaproic acid.

5. The linear polypeptide of any one of claims 1-4, selected from the group consisting of linear polypeptides represented by the formulae:

SRAL(FITC)

SRAL(biotin)

SRAL(RB)

SRAL(Fmoc)

SRAL(Ac₂)

preferably, n in the above formulae is 10 to 15.

6. A molecule formed by non-covalent interaction of the linear polypeptide of any one of claims 1-5 with a staple peptide comprising a backbone amino acid sequence selected from the group consisting of:

(a) An amino acid sequence as shown in IHVTIPADLWDWINK (SEQ ID NO: 1);

the i is an integer greater than or equal to 1, preferably an integer less than or equal to 8;

Preferably i is 7 or 10;

Preferably, the linker comprises an amino acid which is an L-amino acid.

7. The molecule of claim 6, wherein the linker in the staple peptide comprises an amino acid selected from the group consisting of: glu, ala, gly, lys, ser.

8. The molecule of claim 6 or 7, wherein the staple peptide backbone amino acid sequence has a substitution (e.g., a conservative substitution or a non-conservative substitution) of 1,2, or 3 amino acid residues as compared to SEQ ID NO: 1;

preferably, the substitution is at position 7, position 10, position 14, or any combination thereof;

preferably, the substitution is selected from: substitution of Glu for the amino acid residue at position 7, ala for the amino acid residue at position 10, lys for the amino acid residue at position 14, or any combination thereof;

more preferably, the substitution is selected from: substitution of the amino acid residue at position 7 with Glu from Ala, the amino acid residue at position 10 with Ala from Trp, and the amino acid residue at position 14 with Lys from Asn;

preferably, the substitution is selected from:

(1) Substitution of amino acid residue at position 7 with Glu from Ala;

(2) Replacement of the amino acid residue at position 10 by Trp to Ala;

(3) The amino acid residue at position 14 is replaced by Lys from Asn;

9. The molecule of any one of claims 6-8, wherein the staple peptide comprises a backbone amino acid sequence selected from the group consisting of seq id nos:

IHVTIPEDLWDWIKK(SEQ ID NO:2)

IHVTIPEDLADWIKK(SEQ ID NO:3)

IHVTIPEDLKDWINK(SEQ ID NO:4)

IHVTIPADLEDWIKK(SEQ ID NO:5)。

10. The molecule of any one of claims 6-9, wherein the N-terminus and/or the C-terminus of the staple peptide backbone is modified;

preferably, the N-terminus of the staple peptide backbone is modified by acetylation.

11. The molecule of any one of claims 6-10, wherein the linker of the staple peptide couples the amino acid residues at positions i and i+3, or at positions i and i+4, or at positions i and i+7 of the staple peptide backbone into a loop via a peptide bond;

Preferably, the side chain of the amino acid residue at position i of the staple peptide backbone comprises a free carboxyl group, the side chain of the amino acid residue at position i+3, i+4 or i+7 of the staple peptide backbone comprises a free amino group, and the linker comprises an amino acid which forms a peptide bond with the free carboxyl group and the free amino group, respectively;

preferably, the side chain of the amino acid residue at position i of the staple peptide backbone comprises a free amino group, the side chain of the amino acid residue at position i+3, i+4 or i+7 of the staple peptide backbone comprises a free carboxyl group, and the linker comprises an amino acid which forms a peptide bond with the free amino group and the free carboxyl group, respectively.

12. The molecule of any one of claims 6-11, wherein the staple peptide is selected from any one of the following peptides represented by the formulas:

Stapler 17Abp (FITC)

Stapling peptide 17Abp (Fmoc)

Stapled peptide 17Abp (Ac ₂)

Staple peptide 1a

Staple peptide 1b

Staple peptide 2a

Staple peptide 2b

Staple peptide 3a

Staple peptide 3b

Staple peptide 4a

Staple peptide 4b

Staple peptide 5

Staple peptide 7a (17 Abp (Ac))

Staple peptide 7b

Staple peptide 8a

Staple peptide 8b

Staple 9a (17 Abp (Glu))

Staple peptide 9b

Staple peptide 11a

Staple peptide 11b

Staple peptide 12a

Staple peptide 12b

Staple peptide 13a

Staple peptide 13b

Staple peptide 14

Staple peptide 15

13. The molecule of any one of claims 6-12, wherein the N-terminus of the linear polypeptide and the N-terminus of the staple peptide bear the same or different modifying groups.

14. A method of preparing the molecule of any one of claims 6-13, comprising contacting the linear polypeptide and the staple peptide for a time.

15. A pharmaceutical composition comprising the linear polypeptide of any one of claims 1-5 or the molecule of any one of claims 6-13;

Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

16. Use of the linear polypeptide of any one of claims 1-5 or the molecule of any one of claims 6-13 in the manufacture of a medicament for the prevention or treatment of a disease associated with IL-17A overexpression and/or associated with scavenger receptor; preferably, the treatment is a targeted treatment;

Preferably, the disease is an inflammatory or immune related disease, such AS auto-inflammatory disease (AID) and Autoimmune Disease (AD), such AS Ankylosing Spondylitis (AS), rheumatoid Arthritis (RA), psoriasis (Ps), psoriatic arthritis (PsA), multiple Sclerosis (MS), crohn's Disease (CD).

17. A formulation comprising the linear polypeptide of any one of claims 1-5 or the molecule of any one of claims 6-13; preferably, the formulation is for promoting uptake, degradation and/or clearance of IL-17A by cells that specifically express a scavenger receptor; preferably, the cells are selected from macrophages, monocytes, dendritic cells, microglia.

18. Use of the linear polypeptide of any one of claims 1-5 or the molecule of any one of claims 6-13 in the preparation of a formulation for promoting uptake, degradation and/or clearance of IL-17A by a cell that specifically expresses a scavenger receptor; preferably, the cells are selected from macrophages, monocytes, dendritic cells, microglia.

19. Use of a linear polypeptide as defined in any one of claims 1 to 5 in combination with a stapling peptide as defined in any one of claims 6 to 12 for the manufacture of a medicament for the prevention or treatment of a disease associated with IL-17A overexpression and/or associated with scavenger receptors; preferably, the treatment is a targeted treatment;