A polypeptide binding to FGFR2 receptor and its use

Abstract

The present invention belongs to the technical field of biopharmaceutical polypeptides, and specifically relates to a polypeptide binding to an FGFR2 receptor or a pharmaceutically acceptable salt thereof. The present invention also relates to a polypeptide-drug conjugate or a pharmaceutically acceptable salt thereof, and further relates to the use of the polypeptide or a pharmaceutically acceptable salt thereof or the polypeptide-drug conjugate or a pharmaceutically acceptable salt thereof in a drug for a disease associated with FGFR2.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical polypeptides, and specifically relates to a polypeptide binding to FGFR2 receptor and uses thereof. Background Art

[0002] FGFRs belong to the receptor tyrosine kinase (RTK) family in the human genome. They are a member of the tyrosine kinase family, including four receptor subtypes: FGFR1, FGFR2, FGFR3, and FGFR4. Their ligands include 23 subtypes, including FGF1, 4, 7, 8, 9, and 19. FGFRs are a type of single-chain glycoprotein consisting of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region of FGFRs includes three immunoglobulin-like domains (D1-D3), which are connected by a serine-rich region between the D1 and D2 structures. The intracellular region is the tyrosine kinase domain. [1] .

[0003] FGFR2 is widely expressed in normal human tissues, with high expression in tonsils, nasopharynx, bronchi, esophagus, testicles, skin and other tissues; moderate expression in cerebral cortex, hippocampus, lateral ventricle, gallbladder, lung, liver, pancreas and other tissues; low expression in parathyroid glands, lymph nodes, spleen, kidney, colon, kidney and other tissues; almost no expression in salivary glands, prostate, adipose tissue, ovary and other tissues [2] .

[0004] Changes in FGFR, such as overexpression and mutation, can lead to dysregulation of the FGF / FGFR signal transduction axis, promote cell proliferation, angiogenesis, invasion, metastasis, anti-apoptosis, etc., and are related to the pathogenesis of various cancers. There is a lot of evidence to support the correlation between FGFR activation and carcinogenesis. The FGFR signal transduction pathway causes cancer through ligand-dependent mechanisms or independent mechanisms. [3] Fibroblast growth factor receptor (FGFR) plays a vital role in tumor cell growth, survival, metastasis and tumor angiogenesis. Missense mutation and overexpression of FGFR2 gene are closely related to the growth of cancer cells such as endometrial cancer, cervical cancer, breast cancer, lung cancer, bile duct cancer and gastric cancer.

[0005] Juanni Li et al. studied the existing large-scale comprehensive data set, covering more than 10,000 tumor samples of more than 30 cancer types, and analyzed FGFR2 abnormal expression, methylation, changes (mutation / fusion and amplification / deletion) and their clinical relevance. The results showed that the overall mutation rate of FGFR2 in pancreatic cancer was low; bile duct cancer had the highest mutation frequency, and fusion accounted for the majority. In May 2021, the FDA approved a drug, infigratinib, for the treatment of patients with bile duct cancer. It is the second drug targeting bile duct cancer. Further analysis shows that FGFR2 is overexpressed in cancers such as gastric adenocarcinoma, uterine carcinosarcoma, invasive breast cancer, ovarian serous cystadenocarcinoma, pheochromocytoma and paraganglioma, esophageal cancer, and pancreatic cancer. [4] .

[0006] Abnormalities in FGFR are closely related to the occurrence of many tumors, such as bladder cancer, breast cancer, and bile duct cancer. Therefore, FGFR targeted drugs will have direct or indirect anti-cancer effects. FGFR is one of the hot targets currently focused on by the "cancer-free" therapy. Currently, three drugs targeting this target have been approved, namely Johnson & Johnson's Balversa (erdafitinib), Incyte's Pemazyre (pemigatinib), and BridgeBio / Helsinn's Truseltiq (infigratinib). In addition to the above three, many domestic and foreign companies are currently laying out the FGFR inhibitor market. The types of FGFR inhibitors have expanded from small molecules to large molecules, and the indications have also expanded from bile duct cancer and urothelial carcinoma to liver fibrosis, chondrodysplasia, etc. It can be seen that most inhibitors for FGFR are antibodies and small molecule inhibitors. [5] . Peptides have the characteristics of simple spatial structure, small molecular weight, high specificity, strong permeability, easy to reach tissues and organs, not easily recognized and degraded by enzymes, low immunogenicity, less anti-peptide immune response, etc., and peptides can be artificially synthesized and the purification method is simple. Therefore, they are increasingly valued in the development of new drugs. Many peptide drugs have been successfully used in clinical treatment, such as GLP-1 peptides. However, there are no peptides that specifically bind to FGFR2 receptors, so the development of peptides that can specifically bind to FGFR2 and regulate its activity has good application prospects. In addition, for some small molecule compounds with excellent activity and potential clinical application prospects in clinical practice but poor stability or lack of targeting, they are coupled with peptides with good therapeutic effects or targeting FGFR2 to become new peptide-drug conjugates (PDCs), which also have good application prospects.

[0007] Citation list:

[0008] [1]Maddaluno L, Urwyler C, Werner S. Fibroblast growth factors: keyplayers in regeneration and tissue repair[J]. Development, 2017, 144(22): 4047-4060.

[0009] [2]https: / / v15.proteinatlas.org / ENSG00000066468-FGFR2 / tissue.

[0010] [3] GFR targeted drugs | Effective for multiple cancer types, a rising star in the field of targeted therapy https: / / bbs.guahao.com / topic / iAbWq135708647153106946.

[0011] [4]Li J, Hu K, Huang J, et al. A PancancerAnalysis of the ExpressionLandscape and Clinical Relevance of Fibroblast Growth Factor Receptor2 inHuman Cancers[J]. Frontiers in Oncology, 2021,11:644854.

[0012] [5] Fierce competition among FGFR inhibitors: 3 approved, many under development. https: / / www.cn-healthcare.com / articlewm / 20210713 / content-1242424.html. Summary of the invention

[0013] The following is only an overview of some aspects of the present invention, which is not limited thereto. These aspects and other parts are described in more detail below. All references in this specification are incorporated herein by reference in their entirety. When there is a discrepancy between the disclosure of this specification and the referenced documents, the disclosure of this specification shall prevail.

[0014] The first aspect of the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, characterized in that the amino acid sequence of the polypeptide is any one of the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:14 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1 to SEQ ID NO:14 or a modified derivative of the amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:14.

[0015] In some embodiments, the derivative of the modified amino acid sequence includes one or more modifications selected from the following: N-terminal and / or C-terminal modification; substitution of one, two or more amino acid residues with one or more natural and / or non-natural amino acid residues.

[0016] In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94% or 95% identical to the amino acid sequence of any one of the polypeptides SEQ ID NO:1 to SEQ ID NO:10.

[0017] In a second aspect, the present invention also relates to a polypeptide drug conjugate or a pharmaceutically acceptable salt thereof, wherein the polypeptide drug conjugate or a pharmaceutically acceptable salt thereof comprises a polypeptide, a linker and a payload, wherein the polypeptide is connected to the payload via a linker, and the polypeptide sequence is one of SEQ ID NO:1 to SEQ ID NO:14 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1 to SEQ ID NO:14 or a modified derivative of the amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:14.

[0018] In some embodiments, the payload is an anticancer agent; preferably, the anticancer agent is selected from small molecule drugs or nucleic acid drugs.

[0019] In some embodiments, the anticancer agent is selected from maytansine and its derivatives, paclitaxel and its derivatives, auristatin and its derivatives, epothilone and its derivatives, bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives, and mitomycin C; further, the anticancer agent is selected from BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, dexamethasone, chloramphenicol ... amine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, actinomycin D, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminoglutethimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane, and amifostine.

[0020] In one aspect, the present invention also relates to a pharmaceutical composition comprising any polypeptide or a pharmaceutically acceptable salt thereof according to the present invention or a polypeptide-drug conjugate or a pharmaceutically acceptable salt thereof according to the present invention.

[0021] In some embodiments, the pharmaceutical composition described in the present invention further comprises at least one of a pharmaceutically acceptable carrier, excipient, diluent, adjuvant and vehicle.

[0022] On the other hand, the present invention relates to the use of the polypeptide or its pharmaceutically acceptable salt or the polypeptide drug conjugate or its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of a medicament for preventing, treating, curing or alleviating FGFR2-related disorders.

[0023] In some embodiments, the FGFR2-related disorder is cancer, and further, the cancer is selected from: bile duct cancer, intrahepatic bile duct cancer, gastric cancer (such as gastric adenocarcinoma), adrenal cancer, anal cancer, angiosarcoma (such as lymphangiosarcoma, lymphangioendothelial sarcoma, hemangioma), appendix cancer, bile duct cancer, bladder cancer, brain cancer (such as meningioma, glioma, such as astrocytoma, oligodendroglioma, medulloblastoma), cervical cancer (such as cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (such as colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial cancer, Ependymoma, endothelial sarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), hypereosinophilia, gallbladder cancer, gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma), oral cancer (e.g., oral squamous cell carcinoma, laryngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal carcinoma, oropharyngeal cancer)), hematopoietic cancer (e.g., leukemia, such as acute lymphoblastic leukemia (ALL)) For example, B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CML, T-cell CML), chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, lymph node marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma B-cell lymphoma of the central nervous system (CNS), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma; and T-cell non-Hodgkin's lymphoma, such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (such as cutaneous T-cell lymphoma (e.g., fungal disease, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma;Multiple myeloma (MM), hemangioblastoma, inflammatory myofibroblastic tumor, immune cell amyloidosis, kidney cancer (e.g. Wilms tumor, renal cell carcinoma), liver cancer (e.g. hepatocellular carcinoma, malignant hepatocellular carcinoma), lung cancer (e.g. bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, leiomyosarcoma (LMS), mastocytosis (e.g. systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative diseases (MPD) (e.g. polycythemia vera (PV) ), essential thrombocythemia (ET), idiopathic myeloid metaplasia (AMM), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES), neuroblastoma, neurofibroma (such as type 1 or type 2 neurofibromatosis, neurothecoma), neuroendocrine cancer (such as gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (such as cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, etc. ; BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :ELISA test peptide and FGFR2IIIc EC50 value map

[0025] Figure 2 : Affinity test profile of the polypeptide shown in sequence 1 and FGFR2 (IIIC)

[0026] Figure 3 : Affinity test map of the polypeptide shown in sequence 2 and FGFR2 (IIIC)

[0027] Figure 4 : Affinity test profile of the polypeptide shown in sequence 3 and FGFR2 (IIIC)

[0028] Figure 5 : Affinity test profile of the polypeptide shown in sequence 4 and FGFR2 (IIIC)

[0029] Figure 6 : Affinity test profile of the polypeptide shown in sequence 5 and FGFR2 (IIIC)

[0030] Figure 7 : Affinity test profile of the polypeptide shown in sequence 6 and FGFR2 (IIIC)

[0031] Figure 8 : Affinity test profile of the polypeptide shown in sequence 7 and FGFR2 (IIIC)

[0032] Fig. 9 : Affinity test profile of the polypeptide shown in sequence 8 and FGFR2 (IIIC)

[0033] Fig.10 : Affinity test profile of the polypeptide shown in sequence 9 and FGFR2 (IIIC)

[0034] Fig.11 : Affinity test profile of the polypeptide shown in sequence 10 and FGFR2 (IIIC)

[0035] Fig.12 : Affinity test profile of the polypeptide shown in sequence 11 and FGFR2 (IIIC)

[0036] Fig.13 : Affinity test profile of the polypeptide shown in sequence 12 and FGFR2 (IIIC)

[0037] Fig.14 : Affinity test profile of the polypeptide shown in sequence 13 and FGFR2 (IIIC)

[0038] Fig.15 : Affinity test profile of the polypeptide shown in sequence 14 and FGFR2 (IIIC) DETAILED DESCRIPTION

[0039] The term "Thymic Stromal Lymphopoietin (TSLP)" is a four-α-helical bundle type I cytokine, and is also an epithelial cell-derived cytokine produced in response to pro-inflammatory stimuli. It is closely related to interleukin-7 (IL-7), which initiates allergic reactions by stimulating dendritic cells (DCs) and is an important factor in regulating human immune responses. The term "TSLP" includes variants, isoforms, homologs, orthologs, and paralogs of TSLP.

[0040] The meaning of the term "peptide" or "polypeptide" is well known to those skilled in the art. Generally, a peptide or polypeptide is two or more amino acids linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of the adjacent amino acid. The polypeptides described herein may contain naturally occurring amino acids or non-naturally occurring amino acids. They may be modified into analogs, derivatives, functional mimetics, pseudopeptides, and the like containing at least two amino acids. Unless it is specified that the N-terminus or C-terminus has a specific modification, a polypeptide comprising a specific amino acid sequence includes unmodified and modified amino and / or carboxyl termini, which is well known to those skilled in the art. A polypeptide of a specific amino acid sequence may include modified amino acids and / or additional amino acids, unless the N- and / or C-terminus contain modifications that prevent further addition of amino acids. Such modifications include, for example, acetylation of the N-terminus and / or amidation of the C-terminus.

[0041] The polypeptides of the present invention can be modified to form polypeptide derivatives. As is well known to those skilled in the art, various modifications can be made to the polypeptides. Typical modifications include, but are not limited to, N-terminal acetylation, C-terminal amidation, d-amino acid substitution, non-natural amino acid substitution, fatty acid modification, or a combination of the above modifications. The present invention includes any well-known modification of the polypeptide. For example, the polypeptide derivatives may include chemical modifications to the polypeptide, such as alkylation, acylation, carbamylation, iodination, or any other modification that produces a polypeptide derivative. The modification of the polypeptide may include modified amino acids, for example, hydroxyproline or carboxyglutamic acid, and may include amino acids connected by non-peptide bonds.

[0042] For other modifications of the polypeptide of the present invention, non-natural amino acids can be used to replace the natural amino acids in the polypeptide, including but not limited to 2-amino fatty acid (Aad), 3-amino fatty acid (βAad), β-alanine, β-aminopropionic acid (βAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid, piperidine carboxylic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (βAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'- Diaminopimelate (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle) and ornithine (Orn). Of course, all modified α-amino acids can be replaced by the corresponding β-, γ- or ω-aminocarboxylic acids.

[0043] The term "amino acid" refers to a molecule containing an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, the D- and L-isomers of naturally occurring amino acids, and non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, but is not limited to, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

[0044] The term "naturally occurring amino acid" refers to any of the 20 L-amino acids commonly found in peptides synthesized in nature, i.e., the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0045] "Conservative amino acid substitutions" are amino acid substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. 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., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), non-polar side chains (e.g., A, V, L, I, P, F, M, W), β-branched side chains (e.g., T, V, I), and aromatic side chains (e.g., Y, F, W, H). Thus, for example, a predicted non-essential amino acid residue in a polypeptide is preferably replaced by another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g., norleucine replacing methionine) or other properties (e.g., 2-thienylalanine replacing phenylalanine).

[0046] The polypeptides of the present invention can be prepared using methods well known to those skilled in the art, including well-known chemical synthesis methods. Therefore, when a polypeptide or its derivatives contain one or more non-standard amino acids, it is very likely to be prepared by chemical synthesis. In addition to using chemical synthesis methods to prepare polypeptides or their derivatives, they can also be prepared by expressing encoding nucleic acids. This is particularly applicable to the preparation of polypeptides or their derivatives containing only natural amino acids, in which case well-known methods for preparing nucleic acid encoding polypeptide sequences can be used (see Sambrook et al., Molecular Cloning: A La bora tory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The polypeptide can be expressed in an organism and purified by well-known purification techniques.

[0047] The term "analog" refers to a substance that shares one or more specific structural features, elements, components or parts with a reference substance. Typically, an "analog" shows significant structural similarity to a reference substance, such as a shared core or shared structure, and also differs in certain discrete ways. In some embodiments, an analog is a substance that can be produced from a reference substance, for example, by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by the conduction of a synthetic process that is substantially similar to (e.g., shares multiple steps with) the synthetic process that produces the reference substance. In some embodiments, an analog is or can be produced by the conduction of a synthetic process that is different from the synthetic process used to produce the reference substance.

[0048] About sequence identity. According to methods known in the art, sequence identity is calculated by sequence alignment. In order to determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison. For example, a gap can be introduced in the sequence of the first amino acid sequence so as to optimally align with the second amino acid sequence. The amino acid residues at the corresponding amino acid positions are then compared. When the position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Therefore, % identity = number of identical positions / total number of overlapping positions multiplied by 100. In this comparison, the sequences can be the same length or can be different lengths. The optimal sequence alignment for determining the comparison window can be by the local homology algorithm (J.Theor.Biol., 1981) of Smith and Waterman, by the homology alignment algorithm (J.Mol.Biol, 1972) of Needleman and Wunsch, by the method for Pearson and Lipman, similarity is sought (Proc.Natl.Acad.Sci.USA, 1988) to carry out, by the computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin genetics software package 7.0 versions, Genetic Computer Group, 575, Science Drive, Madison, Wisconsin) or for example use public available computer software such as BLAST.When using this software, preferably use default parameters, such as gap penalty or extension penalty.Select the optimal comparison (i.e. producing the highest identity percentage in the whole comparison window range) produced by the various methods.

[0049] The term "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. Typically, when two sequences are aligned, a comparison is made to give the maximum percentage homology. For example, the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to BLAST algorithms that are often used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL. et al., (1996) Meth. Fnzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other conventional BLAST algorithms such as those provided by NCBI BLAST are also well known to those skilled in the art.

[0050] The term "polypeptide drug conjugate" refers to a polypeptide, a linker and a payload, wherein the linker is a cleavable linker or a non-cleavable linker, and the payload is an anticancer agent. In some embodiments, the "linker" refers to a molecule or part that covalently connects the payload to the polypeptide molecule. In some embodiments, the anticancer agent can be or include one or more alkyl agents, one or more anthracyclines, one or more cytoskeleton disruptors (e.g., microtubule targeting moieties, such as taxanes, maytansine and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors HDAC, one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., having a common related antiproliferative activity).In some specific embodiments, the chemotherapeutic moiety can be or include one or more of the following: Actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, mbucil), Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gem Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and / or their analogs (e.g., DM1), Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Maytansine, The invention relates to vinblastine, vinblastine, vincristine, vindesine, vinorelbine and combinations thereof.

[0051] The term "pharmaceutical composition" refers to a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the present invention and a pharmaceutically acceptable carrier or excipient. As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and physiologically compatible analogs. Examples of pharmaceutically acceptable carriers or excipients include one or more of the following: water, saline, phosphate buffered saline, glucose, glycerol, ethanol, and the like, and combinations thereof. In any case, preferably in the composition it includes an isotonic agent, for example, a sugar, a polyol, such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable substances may also be included, such as a wetting amount or a trace amount of auxiliary substances, such as a wetting or emulsifier, preservative, or buffer that increases the shelf life and effectiveness of the antibody or antibody portion. Optionally, a disintegrant may be included, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate. In addition to excipients, pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin, buffers, binders, sweeteners and other flavoring agents; colorants and polyethylene glycol.

[0052] The composition can be in many forms, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (such as injectable solutions and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form will depend on the established route of administration and therapeutic application. In one embodiment, the composition is in the form of an injectable or infusible liquid, such as those forms similar to those used for passive immunization of humans with antibodies. In one embodiment, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular), in one embodiment, the polypeptide is administered by intravenous injection or infusion. In another embodiment, the polypeptide is administered by intramuscular or subcutaneous injection. Other routes of administration suitable for the pharmaceutical composition include, but are not limited to, rectal, transdermal, transmucosal or enteral administration.

[0053] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering an antibody or antigen-binding fragment. The carrier can be an antiadhesive, a binder, a coating, a disintegrant, a filler or diluent, a preservative (such as an antioxidant, an antibacterial or antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifier, a buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0054] Methods for synthesizing polypeptides (such as described herein) are known in the art. In some peptide synthesis methods, the amino group of one amino acid (or amino acid derivative) is linked to the carboxyl group of another amino acid (or amino acid derivative), which is activated by reaction with a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group and the activated carboxyl group react chemically, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially reactive groups (such as the α-amino group of the N-terminal amino acid or amino acid derivative and the carboxyl group of the C-terminal amino acid or amino acid derivative) can be blocked ("protected") to prevent them from participating in the chemical reaction. Thus, only specific reactive groups react to form the desired product. Protecting groups that can be used for this purpose include, but are not limited to, tert-butyloxycarbonyl (t-Boc) and benzoyloxy) groups for protecting amine groups; and simple esters (such as methyl and ethyl) and esters for protecting carboxyl groups. The protecting groups can usually be subsequently removed by treatment that leaves the peptide bonds intact (e.g., treatment with dilute acid). The process of protecting reactive groups (which should not react), coupling to form peptide bonds, and deprotecting the reactive groups can be repeated. Peptides can be synthesized by sequentially adding amino acids to a growing peptide chain. According to the present invention, both liquid and solid phase peptide synthesis methods are applicable. In solid phase peptide synthesis methods, the growing peptide chain is usually connected to an insoluble matrix (such as, for example, polystyrene beads) by connecting the C-terminal amino acid to the matrix. At the end of the synthesis, a shearing agent that does not destroy the peptide bonds, such as hydrofluoric acid (HF), can be used to release the peptide from the matrix. At this point, the protecting groups are also usually removed. According to the present invention, automated, high throughput, and / or parallel peptide synthesis methods can also be used. For more information on peptide synthesis methods, see, e.g., Merrifield (1969) "Solid-phase peptide synthesis," Adv Enzymol Relat Areas Mol Biol., 32: 221-96; Fridkin et al. (1974) Annu Rev Biochem., 43(0): 419-43; Merrifield (1997) "Concept and Early Development of Solid Phase Peptide Synthesis," Methods in Enzymology, 289: 3-13; Sabatino et al. (2009) "Advances in automatic, manual and microwave-assisted solid-phase peptide synthesis," Curr Opin Drug Discov Devel., 11(6): 762-70, the entire contents of each of which are incorporated herein by reference.

[0055] In addition, the polypeptides disclosed in the present invention, including their salts, may also exist in the form of their hydrates or in the form of containing their solvents (e.g., ethanol, DMSO, etc.), and may be used for crystallization. The compounds disclosed in the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, the compounds of the present invention include solvated and unsolvated forms.

[0056] In addition, the TSLP-related diseases disclosed in the present invention are not limited as long as they are TSLP-related diseases. For example, the therapeutic response induced by the molecules disclosed in the present invention can be achieved by binding to human TSLP and then inhibiting TSLP from binding to its receptor, or killing cells overexpressing TSLP.

[0057] The details of one or more embodiments of the present disclosure are presented in the above description. Although any method and material similar or identical to that described herein can be used to implement or test the present invention, the preferred method and material are described below. Other features, objects and advantages of the present disclosure will be apparent from the description and claims. In the description and claims, unless otherwise clearly indicated in the context, the singular form includes the case of plural references. Unless otherwise defined, all technical and scientific terms used herein have the common meanings understood by those of ordinary skill in the art to which the present invention belongs. All patents and publications cited in the description are incorporated by reference. The following examples are presented to more fully illustrate the preferred embodiments of the present invention. These examples should not be construed in any way as limiting the scope of the present invention.

[0058] Example

[0059] The polypeptide compounds and derivatives provided by the present disclosure are synthesized by a solid phase synthesis method. The synthetic carrier is Fmoc-Cys (Trt) -2-Chlotrityl Resin resin. During the synthesis process, the Fmoc-Cys (Trt) -2-Chlotrityl Resin resin is first fully swollen in N, N-dimethylformamide (DMF), and then the solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (90: 2.5: 2.5: 5:, v: v: v: v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation by frozen methyl tert-butyl ether. The crude linear precursor after cleavage is subjected to disulfide bond oxidation in an alkaline solution to obtain the crude target polypeptide. The crude polypeptide was purified and separated by C-18 reverse phase preparative chromatography in 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure polypeptide and its derivatives. The obtained amino acid sequence is shown in Table 1.

[0060] Table 1 Amino acid sequences used in the examples

[0061]

[0062]

[0063] Example 1. Screening of phage library to obtain polypeptide sequences with high affinity to FGFR2IIIc

[0064] 1) Selection of strains and vectors

[0065] Escherichia coli strain SS320 (Sidhu, SS, et al. "Methods Enzymol 328 (1999): 333-363.) has a high electroporation efficiency, and the introduced F' fragment is necessary for phage infection and amplification. Escherichia coli strain SS320 is often used as a bacterial host for phage production and expression. M13K07 (New England Biolabs, Beverly, MA) is often used for packaging to obtain single-chain and amplify phage.

[0066] The vector selection and phagemid construction method were described by Tonikian, R., et al. Nature Protocol 2.6 (2007): 1368-1386. The pGEX4T-2 vector was used to display the phage library. The vector contained replicons for single-stranded (f1 ori) and double-stranded (dsDNA ori) DNA replication, and a selective marker, such as the β-lactamase gene (Ampr), which also made it resistant to ampicillin and carbenicillin. After the polypeptide sequence was inserted, it formed a fusion protein with the mature major capsid protein (P3) of the phage for N-terminal display.

[0067] 2) Preparation of phage display library

[0068] The present invention uses oligonucleotide-directed mutagenesis and efficient bacterial transformation to generate highly diverse libraries. The oligonucleotide-directed mutagenesis technique first described by Kunkel is a reliable method for constructing libraries by introducing random mutations into ssDNA templates. The random library was generated using the method of Liu, B., S. Long, and J. Liu. "New Biotechnology 56 (2019). Subsequently, the random CCC-dsDNA library was electroporated into the Escherichia coli host SS320 (M13KO7) containing the F' fragment, and the electroporation competent state was prepared using the SS320 strain pre-infected with M13KO7. Finally, the phage library was packaged according to (Tonikian, R., et al. Nature Protocol 2.6 (2007): 1368-1386) to complete the construction of the phage library. The phage titer produced by PEG / NaCl precipitation is approximately 10 13 pfu / ml.

[0069] 3) Phage library selection to obtain peptide sequences with high affinity to FGFR2 target (IIIC)

[0070] Fibroblast growth factor receptor-2: FGF R2 (IIIc) was used as the target protein for phage library panning (FGFR2IIIc (His / Avi): Kaixia Bio FGR-HM4CDB).

[0071] The phage selection method, called bio-panning, is an affinity selection process for isolating target binding molecules. The target protein is used as the stationary phase and the phage display library is used as the mobile phase. After a period of co-incubation, the unbound free phage is washed away, and then the phage bound to the target molecule is eluted with a competitive receptor or acid. The eluted phage infects the host cell and then multiplies and amplifies, and then the next round of elution is carried out. After 3 to 5 rounds of "adsorption-elution-amplification", antibodies with high affinity to the target protein can be obtained. Padmanaban, G., et al. Journal of Biotechnology 187 (2014): 43-50 discloses a method for biological screening of phage libraries. The present invention uses MyOne Streptavidin T1Dynabeads (10 mg / mL, Invitrogen, cat#65602) magnetic beads and FGFR2 IIIC (His / Avi) target protein to perform 4 rounds of panning according to the method. A polypeptide sequence with high affinity to the FGFR2 target (IIIC) is obtained from the biological screening of the phage library. After the Dynabeads are resuspended and washed according to the instructions, 60 μg of FGFR2 IIIC (His / Avi) is fixed to 105 μL of magnetic beads, and 60 μL of PBS is added, and the final total volume is 180 μL. Place on a rotating mixer and mix at 4°C for 16-24 hours. After the incubation is completed, the magnetic beads bound to the target protein are precipitated with a magnetic stand, the supernatant is removed, and the magnetic beads are washed 4 times with 1 ml of 0.1% PBST. Subsequently, the magnetic beads are blocked and the magnetic beads are blocked with 0.5% BSA-PBS at room temperature for 30 minutes. In the first round, 5 μL of 1×10 11 pfu of M13 phage library (dissolved in PBS), gently mix at 4°C, and incubate for 1 hour. After incubation, unbound phages were repeatedly washed with PBS containing 0.1% Tween (PBST). In the next round of panning, the concentration of Tween was gradually increased from 0.1% to 0.4%. The elution method used 0.1M citrate (pH 3.1) to incubate for 2 minutes to elute the bound phages, and immediately neutralized with 1M TrisHCl (pH 9.1). Take 20μL for phage titer determination, and the remaining phages are amplified using SS320 strains. The phages are purified and used for the next round of screening. The amount of phage input in each round is 1×10 11 pfu. The selected peptides were obtained by chemical synthesis.

[0072] Biological evaluation

[0073] Example 2. ELISA binding method to test the binding of polypeptide and FGFR2IIIc

[0074] 1) Main experimental materials

[0075]

[0076] 2) Experimental procedures

[0077] Dilute His TagAntibody to 0.5μg / ml with coating solution (0.05mol / L carbonate buffer pH 9.6), add 25μl / well to 384-well plate (Thermo or Greiner), and place at 4℃ for overnight coating. The next day, wash 3-5 times with washing solution (0.05% Tween-20in TBS, pH7.4). Add blocking solution (2% (w / v) BSAin TBS, pH 7.4.) and place at 37℃ for blocking. After washing 3-5 times, add gradient peptide to be tested (such as 100nM, 33nM, 11nM, 3.7nM, 1.24nM, 0.412nM, 0.137nM, 0.046nM) and incubate at 37℃ for 1h. After washing away the unbound peptide, add 0.4μg / ml FGFR2IIIc and incubate at 37℃ for 1h to wash away the unbound FGFR2IIIc protein. Then add anti-Human Fc-HRP and incubate at 37℃ for 1 hour to wash away the unbound HRP protein. Add TMB for color development, and finally terminate the reaction and detect OD450. Graphpadprism is used to draw a graph and calculate the EC50 value of peptide binding to FGFR2IIIc.

[0078] 3) Experimental results

[0079] The ELISA method was used to test the binding of the peptides screened from the phage library to the FGFR2IIIc protein. The measured results were shown in Table 2 through the obtained EC50 values, and the peptides screened had low nM binding to the FGFR2IIIc protein; the Graphpad plot analysis of the ELISA test results is shown in Figure 1 .

[0080] Table 2: ELISA test peptide EC50 values

[0081] serial number EC50(nM) serial number EC50(nM) 1 1.12 8 1.22 2 0.40 9 0.52 3 0.79 10 34.73 4 0.44 11 0.87 5 1.15 12 0.68 6 1.25 13 2.15 7 0.39 14 3.70

[0082] The ELISA method was used to test the binding of the polypeptide to FGFR2 (IIIC) and the results showed that the polypeptide found in the present invention has a high binding ability to the FGFR2 (IIIC) receptor.

[0083] Example 3. BLI (biomembrane interference technique) test of affinity (KD) between polypeptide and FGFR2IIIc

[0084] 1) Main experimental materials

[0085]

[0086] BLI is Bio-Layer Interferometry (BLI for short), which is a label-free, real-time monitoring optical detection technology, mainly used for all-round quantitative analysis of biomolecular interactions and protein concentration determination. BLI can monitor the entire molecular binding process in real time and calculate important data such as molecular affinity (KD), binding rate (ka), and dissociation rate (kd).

[0087] 2) Experimental procedures

[0088] The peptides screened by phage were labeled with His tag during synthesis, and the peptide to be tested was diluted to 10 μg / ml with Kbuffer (PBS pH7.4 + 0.02% Tween), and the FGFR2IIIc protein was diluted to the concentrations of 1000 nM, 333 nM, 111 nM, 37 nM, 12.4 nM, and 4.12 nM with Kbuffer (PBS pH7.4 + 0.02% Tween). The protein and peptide were placed in a 96-well plate and shaken for 100-120 seconds at 25°C. 10 μg / ml peptide was fixed on the His probe of the biosensor. The sensor was then immersed in KBuffer and shaken for 120 seconds for equilibrium. The sensor was then immersed in diluted FGFR2IIIc solutions of different concentrations and shaken for 120 seconds for the binding reaction of the FGFR2IIIc protein and peptide. The sensor was then immersed in KBuffer and shaken for 120 seconds for the dissociation reaction of the FGFR2IIIc protein and peptide. Kon and Koff were calculated and the affinity KD value was calculated using a 1:1 fitting method.

[0089] 3) Experimental results

[0090] The present invention firstly uses BLI technology to screen polypeptides having binding signals with 100 nM FGFR2IIIc, and then performs affinity tests at multiple concentrations. The results are shown in Table 3 below.

[0091] Table 3: Affinity test of peptide binding to FGFR2IIIc

[0092] serial number KD(nM) serial number KD(nM) 1 4.46 8 7.37 2 7.89 9 6.79 3 10.5 10 12.5 4 10.5 11 9.8 5 12.1 12 10.9 6 12.1 13 24.7 7 4.02 14 13.8

[0093] As can be seen from the above table, the polypeptides described in the present invention have high affinity for binding to FGFR2IIIc, and the KD values ​​are all at the nM level.

[0094] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all within the scope defined by the claims of this application.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

8.

2. A pharmaceutical composition, characterized in that The composition comprises the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 2, further comprising a pharmaceutically acceptable carrier or excipient.

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