FAP targeting polypeptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an FAP targeting polypeptide and application thereof, and specifically, the FAP targeting polypeptide has an amino acid sequence as shown in formula (I) or (II). Experimental results prove that the FAP targeting polypeptide has excellent binding specificity and affinity, and the FAP targeting polypeptide can be used as a candidate targeting molecule of anti-tumor drugs and is used for treating or diagnosing cancers related to abnormal activation of FAP.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a FAP targeting polypeptide and applications thereof. Background Art

[0002] Cancer, also known as malignant tumors, is a heterogeneous disease that develops within an extremely complex microenvironment. According to cancer statistics in 2020, there were approximately 19.3 million newly diagnosed cancer cases and approximately 10 million cancer deaths worldwide. Malignant tumors are composed not only of cancer cells but also of a large number of endogenous host stromal cells (such as fibroblasts, vascular cells, and immune cells) and extracellular matrix (ECM) components, collectively referred to as the tumor microenvironment (TME).

[0003] Among all cells in the TME matrix, fibroblasts are crucial because their biological functions are strongly correlated with all stages of tumor development. Cancer-associated fibroblasts (CAFs) are a type of continuously activated fibroblast, accounting for 80% of all fibroblasts and possessing strong tumor regulatory effects. They play a key role in tumor development, invasion, metastasis, extracellular matrix remodeling, therapeutic resistance, and immunosuppression.

[0004] CAFs are ubiquitous in a variety of solid tumors, and their exact origin and specific markers remain to be further elucidated. Among them, fibroblast activation protein α (FAP) is an important membrane surface marker of CAFs, highly expressed on CAFs in more than 90% of human epithelial tumors.

[0005] Fibroblast activation protein (FAP) has been identified as a potential prognostic marker. FAP is a type II transmembrane serine protease that belongs to the dipeptidyl peptidase 4 (DPP4) family and possesses both dipeptidyl peptidase (DPP) and proline endopeptidase (PREP) activities, with roles involved in extracellular matrix (ECM) remodeling and fibrosis. Unlike other proteins, FAP is highly expressed in activated mesenchymal fibroblasts of almost all epithelial cancers, but is not expressed in normal fibroblasts or normal tissues. Therefore, FAP has become a marker for activated fibroblasts in tumors, granulation tissue, and fibrotic lesions.

[0006] Because FAP is expressed at low levels in healthy tissues but at elevated levels in various tumors, it is an excellent target for tumor therapy or diagnosis. Clinical approaches targeting FAP include inhibiting FAP enzymatic activity with small molecules or antibodies, prodrugs exploiting FAP enzymatic activity, FAP vaccines, and FAP-targeted CART therapies. Various small molecule inhibitors targeting FAP (FAPIs) have been functionalized for tumor imaging. The boronic acid-based FAP inhibitor MIP-1232 has been shown to bind to FAP-positive SK-MEL-187 (melanoma) xenografts in mice. However, first-generation compounds with boronic acid as the active ingredient lacked selectivity for the relevant enzyme and exhibited poor chemical stability. Talabostat (Val-Boro-pro, PT-100, BXCL-701) was originally designed to inhibit the dipeptidyl peptidase activity shared by DPPIV and FAP. Preclinical studies have demonstrated significant antitumor activity in several tumor types. However, in Phase II clinical trials, no difference in treatment response was observed whether used alone or in combination with other therapies. Inhibitory antibodies targeting FAP have also been reported, but the effects are often poor and research progress is slow.

[0007] So far, there are only a few reports of peptide radiopharmaceuticals targeting FAP, such as FAP-2286 (patent WO2021005125A1) published by 3B-Pharmaceuticals GmbH in 2020. FAP-2286 is a cyclic peptide targeting FAP, which can be labeled after coupling with the chelating agent DOTA. 68 Ga or 177 Lu, and then used for PET imaging or SPECT imaging and targeted radiotherapy. FAP-2286 polypeptide has a high affinity with recombinant human FAP protein and FAP-expressing fibroblasts WI-38. In addition, 177 Imaging results of Lu-FAP-2286 in HEK-FAP tumors demonstrate that the probe has excellent in vivo targeting specificity for FAP-positive tumors and a prolonged retention time in tumors. Detailed preclinical evaluation results of FAP-2286 can be found in the literature [Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy]. Chinese patent CN116410262A discloses a tumor-affinity peptide and its applications.

[0008] FAP can be used as an imaging marker for diseases such as tumors. Although the clinical success rate of FAP targeted therapy is not high, the overexpression of FAP in many diseases indicates that it is a potential molecular diagnostic biomarker. Although FAP inhibitor molecules have poor direct inhibitory effects on tumors, some of them have good specificity and affinity for FAP, and these molecules can be used as structural frameworks for new radiopharmaceuticals. Recently, the FAP-targeted radiotracer developed by the Haberkorn group at Heidelberg University has greatly promoted the research on the clinical application of FAP. The research group optimized a series of FAP inhibitors (FAPI) and coupled them with the radionuclide chelator DOTA. 68 Ga-labeled and used for PET / CT imaging. 68 Ga-FAPI-PET / CT demonstrates significant uptake in various tumor types, providing excellent tumor-to-background contrast and promising clinical applications. This discovery has led to a growing number of studies and clinical trials focusing on the application of FAPI in nuclear medicine diagnosis of tumors.

[0009] In recent years, peptide ligands have become a hot topic in research and drug development, primarily due to their advantages: small size, ease of crossing human barriers, strong binding strength and specificity, structural diversity, and programmability. Furthermore, peptide drugs are safe and can avoid unwanted side effects and toxic reactions. In summary, the development of these FAP-targeting peptides has broad application prospects and significant scientific value, and will contribute to the development and advancement of the biomedical field.

[0010] Phage display and panning technology is an effective method for high-throughput screening of peptide ligands targeting proteins. It utilizes proteins or peptides displayed on the surface of phage as recognition molecules to achieve highly selective binding to specific targets. This technology is widely used not only in basic research fields such as biology and pharmacology, but also in the development of targeted drugs, diagnostic reagents, and biosensors. Therefore, using phage display and panning technology to rapidly and efficiently screen bicyclic peptides is a powerful tool for the development of bicyclic peptide ligands. Summary of the Invention

[0011] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a FAP-targeting polypeptide and its application, wherein the FAP-targeting polypeptide is obtained based on phage display technology and the construction of a secondary polypeptide library targeting the target protein FAP.

[0012] To achieve the above objectives, the present invention provides a FAP targeting polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the FAP targeting polypeptide is as shown in the general formula (I).

[0013] GC1FDAC2MHQYX6RNDFC3X 10 DHC4G

[0014] (I)

[0015] in,

[0016] X6 is selected from K, R, H or T;

[0017] X 10 Selected from K, R or H;

[0018] C1, C2, C3, and C4 are all cysteine.

[0019] Another aspect of the present invention provides a FAP targeting polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the FAP targeting polypeptide is as shown in the general formula (II):

[0020] GC1FSX2C2VQVYTHX8FC3X 10 DHC4G

[0021] (II)

[0022] in,

[0023] X2 is selected from R, H or K;

[0024] X8 is selected from N or K;

[0025] X 10 Selected from K, R or H;

[0026] C1, C2, C3, and C4 are all cysteine.

[0027] Preferably, the four cysteines in the amino acid sequence of the FAP targeting polypeptide form intramolecular disulfide bonds between each other through the thiol groups in the residues, and the number of intramolecular disulfide bonds is 1 or 2.

[0028] Preferably, the FAP targeting polypeptide structure contains one intramolecular disulfide bond, an intramolecular disulfide bond is formed between C1-C2, an intramolecular disulfide bond is formed between C1-C3, an intramolecular disulfide bond is formed between C1-C4, an intramolecular disulfide bond is formed between C2-C3, an intramolecular disulfide bond is formed between C2-C4, or an intramolecular disulfide bond is formed between C3-C4.

[0029] Preferably, the FAP targeting polypeptide structure contains two intramolecular disulfide bonds, two intramolecular disulfide bonds are formed between C1-C2 and C3-C4, or two intramolecular disulfide bonds are formed between C1-C3 and C2-C4, or two intramolecular disulfide bonds are formed between C1-C4 and C2-C3.

[0030] Preferably, the amino acid sequence of the FAP targeting polypeptide is as shown in any one of SEQ ID No: 1 to SEQ ID No: 8,

[0031] GCFDACMHQYKRNDFCKDHCG(SEQ ID No:1);

[0032] GCFSKCVQVYTHKFCHDHCG(SEQ ID No:2);

[0033] GCFDACMHQYRRNDFCRDHCG(SEQ ID No:3);

[0034] GCFDACMHQYHRNDFCHDHCG(SEQ ID No:4);

[0035] GCFDACMHQYHRNDFCRDHCG(SEQ ID No:5);

[0036] GCFDACMHQYRRNDFCHDHCG(SEQ ID No:6);

[0037] GCFSRCVQVYTHRFCHDHCG(SEQ ID No:7);

[0038] GCFSHCVQVYTHHFCHDHCG (SEQ ID No: 8).

[0039] Preferably, the amino acid sequence of the FAP targeting polypeptide is shown as SEQ ID No: 1 or SEQ ID No: 2.

[0040] Preferably, the four cysteines in the FAP targeting polypeptide structure form intramolecular disulfide bonds between each other through thiol groups in the residues, and the number of intramolecular disulfide bonds is 1 or 2.

[0041] Preferably, the four cysteines in the amino acid sequence of the FAP targeting polypeptide form intramolecular disulfide bonds between each other through the thiol groups in the residues, the number of intramolecular disulfide bonds is 1, 1 intramolecular disulfide bond is formed between C1-C2, or 1 intramolecular disulfide bond is formed between C1-C3, or 1 intramolecular disulfide bond is formed between C1-C4, or 1 intramolecular disulfide bond is formed between C2-C3, or 1 intramolecular disulfide bond is formed between C2-C4, or 1 intramolecular disulfide bond is formed between C3-C4.

[0042] Preferably, the FAP targeting polypeptide structure contains two intramolecular disulfide bonds, two intramolecular disulfide bonds are formed between C1-C2 and C3-C4, or two intramolecular disulfide bonds are formed between C1-C3 and C2-C4, or two intramolecular disulfide bonds are formed between C1-C4 and C2-C3.

[0043] Preferably, the four cysteines in the FAP targeting polypeptide structure shown in any one of SEQ ID No: 1 to SEQ ID No: 8 form intramolecular disulfide bonds between each other through the thiol groups in the residues, and the number of intramolecular disulfide bonds is 1 or 2.

[0044] Preferably, the structure of the FAP targeting polypeptide is as follows,

[0045]

[0046]

[0047] Another aspect of the present invention provides a mutant peptide, wherein the amino acid sequence of the mutant peptide is obtained by substituting, deleting, adding and / or replacing 1, 2 or 3 amino acids based on the amino acid sequence of the above-mentioned FAP targeting polypeptide.

[0048] Preferably, the substituted, deleted, added and / or replaced amino acids are natural amino acids and / or unnatural amino acids.

[0049] Preferably, the amino acid sequence of the mutant peptide is obtained by substituting and / or replacing one, two or three amino acids on the basis of the amino acid sequence of the above-mentioned FAP targeting polypeptide.

[0050] Preferably, the amino acid sequence of the mutant peptide is obtained by deleting and / or adding 1, 2 or 3 amino acids based on the amino acid sequence of the above-mentioned FAP targeting polypeptide.

[0051] Preferably, the amino acid sequence of the mutant peptide is obtained by deleting and / or adding 1, 2 or 3 amino acids at the N-terminus and / or C-terminus based on the amino acid sequence of the above-mentioned FAP targeting polypeptide.

[0052] Preferably, the amino acid sequence of the mutant peptide is obtained by deleting and / or adding one amino acid at the N-terminus and / or C-terminus based on the amino acid sequence of the above-mentioned FAP targeting polypeptide.

[0053] Preferably, the amino acid sequence of the mutant peptide is obtained by deleting one amino acid at the N-terminus or C-terminus based on the amino acid sequence of the above-mentioned FAP targeting polypeptide, and the deleted amino acid is G at the first position or G at the 21st position.

[0054] Preferably, the amino acid sequence of the mutant peptide is obtained by deleting one amino acid at the N-terminus and the C-terminus based on the amino acid sequence of the above-mentioned FAP targeting polypeptide, and the deleted amino acids are G at the first position and G at the 21st position.

[0055] Preferably, the amino acid sequence of the mutant peptide is shown in any one of SEQ ID No: 9 to SEQ ID No: 32,

[0056] GCFDACMHQYKRNDFCKDHC(SEQ ID No:9);

[0057] CFDACMHQYKRNDFCKDHCG(SEQ ID No:10);

[0058] CFDACMHQYKRNDFCKDHC(SEQ ID No:11);

[0059] GCFSKCVQVYTHKFCHDHC(SEQ ID No:12);

[0060] CFSKCVQVYTHKFCHDHCG(SEQ ID No:13);

[0061] CFSKCVQVYTHKFCHDHC(SEQ ID No:14);

[0062] GCFDACMHQYRRNDFCRDHC(SEQ ID No:15);

[0063] CFDACMHQYRRNDFCRDHCG(SEQ ID No:16);

[0064] CFDACMHQYRRNDFCRDHC(SEQ ID No:17);

[0065] GCFDACMHQYHRNDFCHDHC(SEQ ID No:18);

[0066] CFDACMHQYHRNDFCHDHCG(SEQ ID No:19);

[0067] CFDACMHQYHRNDFCHDHC(SEQ ID No:20);

[0068] GCFDACMHQYHRNDFCRDHC(SEQ ID No:21);

[0069] CFDACMHQYHRNDFCRDHCG(SEQ ID No:22);

[0070] CFDACMHQYHRNDFCRDHC(SEQ ID No:23);

[0071] GCFDACMHQYRRNDFCHDHC(SEQ ID No:24);

[0072] CFDACMHQYRRNDFCHDHCG(SEQ ID No:25);

[0073] CFDACMHQYRRNDFCHDHC(SEQ ID No:26);

[0074] GCFSRCVQVYTHRFCHDHC(SEQ ID No:27);

[0075] CFSRCVQVYTHRFCHDHCG(SEQ ID No:28);

[0076] CFSRCVQVYTHRFCHDHC(SEQ ID No:29);

[0077] GCFSHCVQVYTHHFCHDHC(SEQ ID No:30);

[0078] CFSHCVQVYTHHFCHDHCG(SEQ ID No:31);

[0079] CFSHCVQVYTHHFCHDHC (SEQ ID No: 32).

[0080] Another aspect of the present invention provides a polynucleotide encoding any of the above-mentioned FAP targeting polypeptides.

[0081] Another aspect of the present invention provides a pharmaceutical preparation, characterized in that the pharmaceutical preparation comprises the above-mentioned FAP targeting polypeptide or a pharmaceutically acceptable salt or polynucleotide thereof as an active ingredient and a drug carrier.

[0082] Another aspect of the present invention provides use of the aforementioned FAP targeting polypeptide or a pharmaceutically acceptable salt, polynucleotide or pharmaceutical preparation thereof in the preparation of a medicament for preventing, diagnosing and / or treating cancer associated with abnormal FAP activation.

[0083] Preferably, the cancer is selected from the group consisting of prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, sarcoma, colorectal cancer, cholangiocarcinoma, chordoma, small intestinal cancer, pheochromocytoma, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, intestinal cancer, and solid tumors such as bone metastasis. Further, the cancer is selected from pancreatic cancer, thyroid cancer, and liver cancer.

[0084] Beneficial effects:

[0085] 1. This invention has developed a series of novel high-affinity FAP peptides that can be used to target fibroblast activation protein. Taking advantage of the high expression of FAP receptors in tumors, these peptides can be used for the prevention, diagnosis, and / or treatment of FAP-overexpressing tumors.

[0086] 2. These peptides are all low molecular weight peptides with low synthesis cost, and these peptides are relatively stable in plasma.

[0087] As used herein, "amino acid" refers to both natural and unnatural amino acids. The stereo configuration of the amino acid is indicated by a three-letter code prefixed with "L-" or "D-" (except for achiral glycine). For example, the L-amino acids are: alanine ("L-Ala" or "A"), arginine ("L-Arg" or "R"), asparagine ("L-Asn" or "N"), aspartic acid ("L-Asp" or "D"), cysteine ​​("L-Cys" or "C"), glutamine ("L-Gln" or "Q"), glutamic acid ("L-Glu" or "E"), glycine ("Gly" or "G"), histidine ("L- The amino acids denoted as "His" or "H"), "L-Ile" or "I"), "L-Leu" or "L"), "Lysine" or "Lys", "K" or "K"), "L-Met" or "M"), "L-Phe" or "F"), "L-Proline" or "P"), "L-Ser" or "S"), "L-Thr" or "T"), "L-Trp" or "W"), "L-Tyr" or "Y"), and "L-Val" or "V") are tyrosine and valine. L-norleucine and L-norvaline can be represented as (NLeu) and (NVal), respectively. Nineteen naturally occurring chiral amino acids have corresponding D-isomers, which are designated by three-letter codes with the prefix "D-": alanine ("D-Ala" or "a"), arginine ("D-Arg" or "r"), asparagine ("D-Asn" or "a"), aspartic acid ("D-Asp" or "d"), cysteine ​​("D-Cys" or "c"), glutamine ("D-Gln" or "q"), glutamic acid ("D-Glu" or "e"), histidine ("D-His" or "h"), isoleucine ("D-Hydroxysuccinylcholine" or " ... The amino acids D-Ile or D-Ile are listed as “D-Ile” or “i”, D-Leu or “l”, D-Lys or “k”, D-Met or “m”, D-Phe or “f”, D-Proline or “p”, D-Ser or “s”, D-Threonine or “t”, D-Trp or “w”, D-Tyr or “y”, D-Val or “v”, and D-Val or “v”.

[0088] "Non-natural amino acid" refers to any derivative of a natural amino acid, including α- and β-amino acid derivatives. It should be noted that certain amino acids that can be classified as non-natural amino acids in the present invention (e.g., hydroxyproline) can also be present in certain biological tissues or specific proteins in nature. Amino acids with many different protecting groups suitable for direct application in solid-phase peptide synthesis are commercially available. In addition to the twenty most common natural amino acids, the following exemplary non-natural amino acids and amino acid derivatives can be used according to the present invention (common abbreviations are in brackets): 2-aminoadipic acid (Aad), 3-aminoadipic acid (β-Aad), 2-aminobutyric acid (2-Abu), α, β-dehydro-2-aminobutyric acid (8-AU), 1-aminocyclopropane-1-carboxylic acid (ACPC), aminoisobutyric acid (Aib), 3-aminoisobutyric acid (β-Aib), 2-amino-thiazoline-4- Carboxylic acids, 5-aminopentanoic acid (5-Ava), 6-aminohexanoic acid (6-Ahx), 2-aminoheptanoic acid (Ahe), 8-aminooctanoic acid (8-Aoc), 11-aminoundecanoic acid (11-Aun), 12-aminododecanoic acid (12-Ado), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz), 4-aminobenzoic acid (4-Abz), 4-amino-3-hydroxy-6-methylheptanoic acid (statin, Sta), aminooxyacetic acid (Aoa), 2-aminotetralin-2-carboxylic acid (ATC), 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), p-aminophenylalanine (4-NH2-Phe), 2-aminopimelanediol (Apm), biphenylalanine (Bip), p-bromophenylalanine (4-Br-Phe), o-chlorophenylalanine (2-Cl-Phe), m-chlorophenylalanine (3-Cl-Phe), p-chlorophenylalanine (4-Cl-Phe), m-chlorotyrosine (3-C l-Tyr), p-benzoylphenylalanine (Bpa), tert-butylglycine (TLG), cyclohexylalanine (Cha), cyclohexylglycine (Chg), desmosine (Des), 2,2-diaminopimelanediol (Dpm), 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid (Dbu), 3,4-dichlorophenylalanine (3,4-Cl2-Phe), 3,4-difluorophenylalanine (3,4-F2-Phe), 3,5-diiodotyrosine (3,5-I2-Tyr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), o-fluorophenylalanine (2-F-Phe), m-fluorophenylalanine (3-F-Phe), p-fluorophenylalanine (4-F-Phe), m-fluorotyrosine (3-F-Tyr), homoserine (Hse), homophenylalanine (Hfe), homotyrosine (Htyr), hydroxylysine (Hyl) , isohydroxylysine (aHyl), 5-hydroxytryptophan (5-OH-Trp), 3- or 4-hydroxyproline (3- or 4-Hyp), p-iodophenylalanine (4-I-Phe), 3-iodotyrosine (3-I-Tyr), indoline-2-carboxylic acid (Idc), isoiduracil (Ide), isoleucine (α-Ile), isopenecolic acid (Inp), N-methylisoleucine (Melle), N-methyllysine (MeLys), m-methyltyrosine (3-Me-Tyr), N-methylvaline (MeVal), 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), p-nitrophenylalanine (4-NO2-Phe), 3-nitrotyrosine (3-NO2-Tyr), norleucine (Nle), norvaline (Nva), ornithine (Orn), o-phosphotyrosine (H2PO3-Tyr), octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen), pentafluorophenylalanine (F5-Phe), phenylglycine (Phg), pipecolic acid (Pip), propargylglycine (Pra), pyroglutamic acid (PGLU), sarcosine (Sar), tetrahydroisoquinoline-3-carboxylic acid (Tic), thienylalanine, and thiazolidine-4-carboxylic acid (thioproline, Th). ,

[0089] As used herein, the terms "peptide" or "polypeptide" have meanings well known to those skilled in the art. Generally, a peptide or polypeptide is composed of two or more amino acids linked by an amide bond, formed by the amino group of one amino acid and the carboxyl group of an adjacent amino acid. The polypeptides described herein may comprise naturally occurring amino acids or non-naturally occurring amino acids. They may be modified to form analogs, derivatives, functional mimetics, pseudopeptides, and the like, comprising at least two amino acids.

[0090] As used herein, "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the compound described in the present invention, or a salt of the drug conjugate, which is safe and effective when used in mammals. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0091] The term "drug carrier" as used in the context of the present disclosure refers to a system that can alter the way a drug enters the human body and its distribution within the body, control the rate of drug release, and deliver the drug to a targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, preferably micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules while having good membrane permeability, making them excellent drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 This is the HPLC test result of compound 2;

[0093] Figure 2 This is the LC / MS test result of compound 2;

[0094] Figure 3 This is the HPLC test result of compound 5;

[0095] Figure 4 This is the LC / MS test result of compound 5;

[0096] Figure 5 Kinetics fitting results of FAP affinity test (SPR single cycle kinetics) of compound 2;

[0097] Figure 6 Kinetics fitting results of compound 5FAP affinity test (SPR single cycle kinetics);

[0098] Figure 7 is the absorbance value of compound 2 at different concentrations in the solubility test;

[0099] Figure 8 is the percentage of compound 2 remaining in mouse plasma at different time points;

[0100] Figure 9This is the prism software fitting result of compound 2's mouse plasma stability;

[0101] Figure 10 This is the logarithmic fitting result of the prism software for the mouse plasma stability of compound 2;

[0102] Figure 11 The residual rate percentage of compound 2 in human plasma at different time points. Specific embodiments

[0103] The present invention will be described in further detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto. Various changes may be made to the form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

[0104] Example 1 Screening of polypeptides

[0105] 1. Phage library selection to obtain peptides with high affinity to FAP target

[0106] The target protein is used as the stationary phase and the phage display library (self-made) is used as the mobile phase. After a period of co-incubation, the unbound free phages are washed away, and then the phages bound to the target molecule are eluted with a competitive receptor or acid. The eluted phages infect the host cells and then multiply and amplify, and then the next round of elution is carried out. After 4 rounds of "adsorption-elution-amplification", a polypeptide with high affinity to the target protein can be obtained.

[0107] 2. Phage ELISA Identification

[0108] 2.1. Coating: Dilute SA to 5 μg / mL in 50 mM sodium bicarbonate, pH 8.5, and coat 50 μL of the solution at 37°C for 2 h.

[0109] 2.2. Blocking: Block with TBST + 20 mg / mL BSA overnight at 4°C;

[0110] Washing: 25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, 0.05% Tween-20, 200 μL washing solution, three times, 3 min each wash;

[0111] 2.4. Target protein binding: 200 ng of FAP target protein was incubated with SA-coated ELISA plates at 37°C for 1 hour.

[0112] Washing: 25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, 0.05% Tween-20, 200 μL washing solution, three times, 3 min each wash;

[0113] 2.6. Phage binding: 10 9 / 10 10 pfu of phage supernatant was bound to the coated FAP ELISA plate at 37°C for 1 h;

[0114] 2.7. Washing: Wash five times with 200 μL of 25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, and 0.05% Tween-20, each wash for 3 min.

[0115] 2.8. Binding Antibody: Dilute M13 phageAntibody to 0.1 μg / mL, take 100 μL and bind to the phage in the ELISA plate at 37°C for 1 hour;

[0116] 2.9. Washing: 25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, 0.05% Tween-20, 200 μL washing solution, wash five times, each wash for 3 min;

[0117] 2.10. Color development: Mix TMB colorimetric solution A and solution B in equal proportions, add 100 μL, and develop at room temperature for 5 min.

[0118] 2.11. Termination: Add 100 μL 2M H2SO4;

[0119] 2.12. Scanning: Microplate reader OD 450 reading.

[0120] 2.13. Select samples with good signals from the above and sequence them to obtain peptide sequences.

[0121] 3. Experimental Results

[0122] As the number of phage library screening rounds increased, the affinity of the eluted peptides for FAP gradually increased. A series of peptides with good affinity for FAP were screened from the phage library. Their binding ability to FAP was verified by further ELISA experiments, in which BSA was used as a blank control. There were significant differences between the experimental groups. The peptides with better verification results were selected for sequencing, and FAP-targeted peptides with high affinity for FAP were obtained.

[0123] Example 2 Polypeptide Solid Phase Synthesis

[0124] The polypeptide compounds and derivatives disclosed herein utilize a solid-phase synthesis method to synthesize their linear precursors, which are then oxidized with DMSO to form two pairs of disulfide bonds within the molecule. The synthetic support is Fmoc-Gly-Wang Resin. During the synthesis process, the Fmoc-Gly-Wang Resin resin is first fully swollen in N,N-dimethylformamide (DMF). The solid support is then subjected to a repeated condensation step with an activated amino acid derivative, followed by washing, Fmoc deprotection, washing, and subsequent amino acid condensation to achieve the desired polypeptide chain length. Finally, the resin is reacted with a mixture of trifluoroacetic acid, water, triisopropylsilane, and thioanisole (90:2.5:2.5:5, v:v:v:v) to cleave the polypeptide from the solid support. The crude linear precursor solid is then precipitated with chilled methyl tert-butyl ether to yield a crude target polypeptide. The cleaved crude linear precursor is then subjected to disulfide bond oxidation in a weakly alkaline solution to yield the target polypeptide. The crude polypeptide was purified and separated by C-18 reverse phase preparative chromatography in 0.1% trifluoroacetic acid acetonitrile / water system to obtain pure polypeptide and its derivatives.

[0125] Experimental reagents

[0126]

[0127]

[0128] (1) Synthesis of Compound 2

[0129]

[0130] Step 1: Synthesis of linear precursor peptide chain

[0131] The linear precursor peptide chain of compound 2 is GCFDACMHQYKRNDFCKDHCG.

[0132] 306 mg (0.1 mmol) of Fmoc-Gly-Wang Resin was fully swelled in DMF for 1 hour. The linear precursor sequence was then synthesized from the second C at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0133] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0134] The resin was washed 6-8 times with DMF until the pH was neutral.

[0135] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0136] The resin was washed 4-6 times with DMF before coupling the next amino acid.

[0137] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0138] Step 2: Cleavage of the linear precursor peptide chain

[0139] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0140] Step 3: Intramolecular disulfide bond formation

[0141] Dissolve the crude product from step 2 thoroughly in DMSO (20% of the total reaction volume). Add 2 mM GSH to 50 mM ammonium bicarbonate buffer (pH 8.0, containing 50% acetonitrile). Slowly add the dissolved peptide solution dropwise to the buffer to a final peptide concentration of 1 mg / ml. Shake at room temperature for 16 hours. Monitor the reaction by LC-MS. Purify the product immediately after completion.

[0142] Step 4: Peptide purification and preparation

[0143] After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase high-performance liquid chromatography system using buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reverse-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 10-25% acetonitrile over 60 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0144] Step 5: Detection and Characterization Methods

[0145] The purity of the peptide obtained in step 4 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and intramolecular disulfide bond formation of the compound. The test results are shown in Figure 1、 Figure 2 .

[0146] (2) Synthesis of compound 5

[0147]

[0148] Step 1: Synthesis of linear precursor peptide chain

[0149] The linear precursor peptide chain of compound 5 is GCFSKCVQVYTHKFCHDHCG.

[0150] 306 mg (0.1 mmol) of Fmoc-Gly-Wang Resin was fully swelled in DMF for 1 hour. The linear precursor sequence was then synthesized from the second C at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0151] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0152] The resin was washed 6-8 times with DMF until the pH was neutral.

[0153] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0154] The resin was washed 4-6 times with DMF before coupling the next amino acid.

[0155] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0156] Step 2: Cleavage of the linear precursor peptide chain

[0157] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0158] Step 3: Intramolecular disulfide bond formation

[0159] Dissolve the crude product from step 2 thoroughly in DMSO (20% of the total reaction volume). Add 2 mM GSH to 50 mM ammonium bicarbonate buffer (pH 8.0, containing 50% acetonitrile). Slowly add the dissolved peptide solution dropwise to the buffer to a final peptide concentration of 1 mg / ml. Shake at room temperature for 16 hours. Monitor the reaction by LC-MS. Purify the product immediately after completion.

[0160] Step 4: Peptide purification and preparation

[0161] After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase high-performance liquid chromatography system using buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reverse-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 10-30% acetonitrile over 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0162] Step 5: Detection and Characterization Methods

[0163] The purity of the peptide obtained in step 4 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and intramolecular disulfide bond formation of the compound. The test results are shown in Figure 3 、 Figure 4 .

[0164] Example 3 SPR test of affinity between polypeptide sample and FAP protein

[0165] 1. Experimental Materials:

[0166]

[0167] 2. Experimental steps:

[0168] Affinity testing of peptides for FAP was performed using a Biacore T200. Approximately 2000 RU of FAP protein was captured using a Protein A chip at 25°C. Binding experiments were performed using 1× HBS, pH 7.4, as the running buffer at 25°C. The peptide analyte flow rate was 30 μL / min, with an association of 120 s and a dissociation of 600 s. Single-cycle or multi-cycle Kinetics / Affinity analysis was used to detect peptide binding. Chip regeneration was performed using Gly-HCl (pH 1.5) at 30 μL / min for 30 s. Data were fitted using a 1:1 binding model.

[0169] 3. Experimental Results

[0170] The test results are shown in Table 1. Figure 5 、 Figure 6 shown.

[0171] Table 1 Affinity results of SPR test peptide samples and FAP protein

[0172]

[0173] Example 4 Polypeptide Solubility Detection

[0174] 1. Main reagents and consumables

[0175] name factory Item No. Normal saline Kefu none dimethyl sulfoxide Aladdin D103272-500mL 96-well ELISA plate Corning / Constant temperature water bath ZHUANZHAN DZKW-S-8 Vortex mixer Hangzhou Miou MIX-25P Constant temperature electric shaker Taicang Huamei Biochemical Instrument Factory THZ-C microplate reader BioTek SynergyNeo2

[0176] 2. Experimental Procedure

[0177] (1) Preparation of test peptides

[0178] The test peptides were diluted in DMSO to a 10 mM stock solution.

[0179] (2) Preparation of PBS buffer (0.01M phosphate buffered saline)

[0180] Take a 1L beaker, wash it with tap water first, and then rinse it with ultrapure water.

[0181] Add about 850 mL of ultrapure water to a beaker. Measure the following reagents according to the order in Table 2, add them to the beaker containing ultrapure water, and stir thoroughly to dissolve.

[0182] Adjust the pH of the solution to 7.4 ± 0.05 with HCl

[0183] Add ultrapure water to make up to 1 L and store at 4°C.

[0184] The compounds were diluted to different solubility working solutions using DMSO, and then diluted to incubation solution using normal saline. The total DMSO content was 0.5%, as shown in Table 2.

[0185] Table 2 Peptide dilution concentration table

[0186]

[0187] (3) Absorbance detection

[0188] The culture was shaken in a constant temperature shaking incubator at 37°C and 200 rpm for 30 min. The incubation solution was mixed and the absorbance was detected at 620 nm using a microplate reader.

[0189] (4) Data Analysis

[0190] The graph is plotted with concentration as the horizontal axis and absorbance as the vertical axis. The first point where the absorbance value begins to change significantly is the solubility. The "significant change" means that the subsequent point increases by more than 1.5 times the previous point, and the subsequent points continue to rise. This point is considered to be the first point with the largest change, that is, the maximum solubility. The experimental results of compound 2 are shown in Table 3. Figure 7 .

[0191] Table 3 Solubility absorbance results of compound 2

[0192] Final concentration (uM) Parallel sample 1 Parallel sample 2 Parallel sample 3 average value SD CV 0 0.038 0.038 0.037 0.038 0.0006 1.5 0.78125 0.038 0.038 0.037 0.038 0.0006 1.5 1.5625 0.042 0.037 0.038 0.039 0.0026 6.8 3.125 0.037 0.039 0.044 0.040 0.0036 9.0 6.25 0.043 0.038 0.038 0.040 0.0029 7.3 12.5 0.037 0.037 0.037 0.037 0.0000 0.0 25 0.04 0.038 0.037 0.038 0.0015 4.0 50 0.038 0.038 0.041 0.039 0.0017 4.4

[0193] The experimental results show that the polypeptide of the present invention has high solubility. Within the experimental range, the absorbance values ​​of compound 2 at each gradient did not change much, with no significant increase, that is, the maximum solubility of compound 2 was greater than 50 μM.

[0194] Example 5 Mouse plasma stability

[0195] 1. Main reagents and consumables

[0196] name factory Item No. name factory model Formic acid Aladdin F301957-50ml Constant temperature water bath ZHUANZHAN DZKW-S-8 Acetonitrile Sigma 34851-4L Vortex mixer Hangzhou Miou MIX-25P Methanol Sigma 34860-4L-R High-speed refrigerated centrifuge Xiangyi H1750R dichloromethane Sigma 650463-4L

[0197] Mouse plasma (heparin sodium) was obtained by Slack.

[0198] 2. Preparation of Test Compounds

[0199] The samples were diluted to 0.1 mM using DMSO.

[0200] 3. Pretreatment solution and preparation method

[0201] Compound name Pre-treatment method Tat-NR2B9c 0.1% formic acid in 75% acetonitrile in water Teriparatide acetate 0.1% formic acid in 75% acetonitrile in water Compound 2 1% formic acid in methanol

[0202] 4. Experimental steps:

[0203] (1) Prepare the mixed solution: Take (50 μL × (6 time points) + 1) 350 μL plasma (heparin sodium) and add it to a 1.5 ml EP tube. Prepare at least 3 replicates for each time point. Prepare one tube of mixed solution and incubate on ice for 5 min. Add 3.5 μL of the sample to be tested to each tube to a final concentration of 1 μM. Vortex the mixture and repeatedly aliquot 50 μL into EP tubes according to the time gradient and incubate.

[0204] (2) Incubation: Incubate in a 37°C water bath at six time points: 0 min, 30 min, 60 min, 240 min, 480 min, and 1440 min.

[0205] (3) Termination of reaction: After incubation, different pretreatment methods are performed according to the pretreatment methods required for biological analysis.

[0206] (4) Mixing: Mix by vortexing on a vortex shaker.

[0207] (5) Centrifugation: Centrifuge in a low-temperature high-speed centrifuge at 4°C, 13,000 rpm for 10 min. Transfer 70 μL of the supernatant to a sample injection vial for analysis by LC-MS / MS.

[0208] (6) Data Analysis

[0209] Result evaluation: The peak area of ​​the peptide at different time points was detected by LC-MS / MS method, and the results were expressed as the percentage of the remaining rate of the original drug. The results are shown in Table 4, Table 5, Figures 8 to 10 As shown in Table 5, Figure 9 、 Figure 10 The results of the Prism software fitting are shown. It can be seen that the half-life of compound 2 is greater than 24 hours, and the half-life of compound 2 obtained using Prism software is approximately 46.58 hours.

[0210] Table 4 Percentage of peptide remaining rate at different time points

[0211]

[0212] Table 5 Prism software fitting results

[0213]

[0214] Example 6 Human Plasma Stability

[0215] 1. Main reagents and consumables

[0216]

[0217]

[0218] 2. Preparation of Test Compounds

[0219] The samples were diluted to 0.1 mM using DMSO.

[0220] 3. Pretreatment solution and preparation method

[0221] Compound name Pre-treatment method Tat-NR2B9c 0.1% formic acid in 75% acetonitrile in water Teriparatide acetate 0.1% formic acid in 75% acetonitrile in water Compound 2 1% formic acid in methanol

[0222] 4. Experimental steps:

[0223] (1) Prepare the mixed solution: Take (50 μL × (6 time points) + 1) 350 μL plasma (heparin sodium) and add it to a 1.5 ml EP tube. Prepare at least 3 replicates for each time point. Prepare one tube of mixed solution and incubate on ice for 5 min. Add 3.5 μL of the sample to be tested to each tube to a final concentration of 1 μM. Vortex the mixture and repeatedly aliquot 50 μL into EP tubes according to the time gradient and incubate.

[0224] (2) Incubation: Incubate in a 37°C water bath at six time points: 0 min, 30 min, 60 min, 240 min, 480 min, and 1440 min.

[0225] (3) Termination of reaction: After incubation, different pretreatment methods are performed according to the pretreatment methods required for biological analysis.

[0226] (4) Mixing: Mix by vortexing on a vortex shaker.

[0227] (5) Centrifugation: Centrifuge in a low-temperature high-speed centrifuge at 4°C, 13,000 rpm for 10 min. Transfer 70 μL of the supernatant to a sample injection vial for analysis by LC-MS / MS.

[0228] (6) Data Analysis

[0229] Result evaluation: The peak area of ​​the peptide at different time points was detected by LC-MS / MS method, and the results were expressed as the percentage of the remaining rate of the original drug. The results are shown in Table 6. Figure 11 As shown in the results, it can be seen that the half-life of compound 2 is greater than 24 hours.

[0230] Table 6 Percentage of peptide remaining rate at different time points

[0231]

[0232] The foregoing description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be readily conceived by a person skilled in the art within the technical scope disclosed herein are intended to be encompassed within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A FAP targeting polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the FAP targeting polypeptide is shown in general formula (I), GC1FDAC2MHQYX6RNDFC3X 10 DHC4G (I) in, X6 is selected from K, R, H or T; X 10 Selected from K, R or H.

2. A FAP targeting polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the FAP targeting polypeptide is shown in general formula (II), GC1FSX2C2VQVYTHX8FC3X 10 DHC4G (II) in, X2 is selected from R, H or K; X8 is selected from N or K; X 10 Selected from K, R or H.

3. The FAP targeting polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The amino acid sequence of the FAP targeting polypeptide is shown in any one of SEQ ID No: 1 to SEQ ID No:

8.

4. The FAP targeting polypeptide or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that The four cysteines in the amino acid sequence of the FAP targeting polypeptide form intramolecular disulfide bonds between each other through the thiol groups in the residues, and the number of intramolecular disulfide bonds is 1 or 2.

5. The FAP targeting polypeptide or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The FAP targeting polypeptide structure contains two intramolecular disulfide bonds, two intramolecular disulfide bonds are formed between C1-C2 and C3-C4, or two intramolecular disulfide bonds are formed between C1-C3 and C2-C4, or two intramolecular disulfide bonds are formed between C1-C4 and C2-C3; preferably, the structure of the FAP targeting polypeptide is shown in any one of Compounds 1 to 6.

6. A mutant peptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the mutant peptide is obtained by substituting, deleting, adding and / or replacing 1, 2 or 3 amino acids on the basis of the amino acid sequence of the FAP targeting polypeptide according to any one of claims 1 to 5; preferably, the amino acid sequence of the mutant peptide is shown in any one of SEQ ID No: 9 to SEQ ID No:

32.

7. A polynucleotide, characterized in that The polynucleotide encodes any one of the FAP targeting polypeptides according to any one of claims 1 to 5 or the mutant peptide according to claim 6.

8. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises as active ingredients the FAP targeting polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, the mutant peptide or a pharmaceutically acceptable salt thereof according to claim 6, or the polynucleotide according to claim 7, and a drug carrier.

9. Use of the FAP targeting polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, the mutant peptide or a pharmaceutically acceptable salt thereof according to claim 6, the polynucleotide according to claim 7, or the pharmaceutical preparation according to claim 8 in the preparation of a medicament for preventing, diagnosing and / or treating cancer associated with abnormal FAP activation.

10. The method of claim 9, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, sarcoma, colorectal cancer, cholangiocarcinoma, chordoma, small intestine cancer, pheochromocytoma, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, intestinal cancer, and solid tumors such as bone metastasis.

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