Polypeptides binding to the fgfr2 receptor and uses thereof

Abstract

The present application belongs to the technical field of biological medicine polypeptide, and particularly relates to a polypeptide or a pharmaceutically acceptable salt thereof which binds to FGFR2 receptor, and further relates to a polypeptide drug conjugate or a pharmaceutically acceptable salt thereof, and still further relates to application of the polypeptide or the pharmaceutically acceptable salt thereof or the polypeptide drug conjugate or the pharmaceutically acceptable salt thereof in a drug for treating a disease related to FGFR2.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedicine polypeptide, and particularly relates to a polypeptide binding FGFR2 receptor and application thereof. BACKGROUND

[0002] FGFRs belong to the receptor tyrosine kinase (RTK) family in human genome, and are members of the tyrosine kinase family, including FGFR1, FGFR2, FGFR3 and FGFR4 four receptor subtypes, and their ligands include 23 subtypes of FGF1, 4, 7, 8, 9, 19, etc. FGFRs are a kind of single-chain glycoprotein, which is composed 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 D1 and D2 structures, and the intracellular region is a tyrosine kinase domain [1] .

[0003] FGFR2 is widely expressed in normal human tissues, and is highly expressed in tonsil, nasopharynx, bronchus, esophagus, testis, skin and other tissues; is moderately expressed in cerebral cortex, hippocampus, lateral ventricle, gallbladder, lung, liver, pancreas and other tissues; is lowly expressed in parathyroid, lymph node, spleen, kidney, colon, kidney and other tissues; and is almost not expressed in salivary gland, prostate, adipose tissue, ovary and other tissues [2] .

[0004] Changes of FGFR such as overexpression and mutation can cause disorders of FGF / FGFR signal transduction axis, promote cell proliferation, neovascularization, invasion, metastasis, anti-apoptosis and the like, and are related to the pathogenesis of a variety of cancers. There is a large amount of evidence supporting the correlation between FGFR activation and carcinogenesis. FGFR signal transduction pathway causes cancer through ligand-dependent mechanism or independent mechanism [3] : Fibroblast growth factor receptor (FGFR) plays a crucial role in tumor cell growth, survival, metastasis and tumor angiogenesis. Missense mutation and overexpression of FGFR2 gene are closely related to the growth of endometrial cancer, cervical cancer, breast cancer, lung cancer, cholangiocarcinoma and gastric cancer cells.

[0005] Juanni Li et al. studied the existing large comprehensive dataset, covering more than 10,000 tumor samples of 30 kinds of cancer types, analyzed FGFR2 abnormal expression, methylation, changes (mutation / fusion and amplification / deletion) and its clinical relevance, the results showed that the overall mutation rate of FGFR2 in pancreatic cancer was low; the mutation frequency of cholangiocarcinoma was the highest, and the fusion accounted for the majority. In May 2021, FDA approved a drug infigratinib for the treatment of cholangiocarcinoma patients, which is the second drug targeting cholangiocarcinoma. Further analysis can see that there is FGFR2 overexpression in gastric adenocarcinoma, uterine carcinosarcoma, breast invasive carcinoma, ovarian serous cystadenocarcinoma, pheochromocytoma and paraganglioma, esophageal cancer, pancreatic cancer and other cancers [4] .

[0006] The abnormality of FGFR is closely related to the occurrence of many tumors, such as bladder cancer, breast cancer, cholangiocarcinoma, etc. Therefore, FGFR targeted drugs will have a direct or indirect anti-cancer effect. FGFR is one of the popular targets of current "cancer-unlimited" therapy, and three drugs targeting this target have been approved, namely Strong's Balversa (erdafitinib), Incyte's Pemazyre (pemigatinib) and BridgeBio / Helsinn's Truseltiq (infigratinib). In addition to the above 3, many domestic and foreign enterprises have laid out the FGFR inhibitor market, and the types of FGFR inhibitors have expanded from small molecules to macromolecules, and the indications have expanded from cholangiocarcinoma, uroepithelial cancer to liver fibrosis, achondroplasia, etc. It can be seen that FGFR inhibitors are mainly antibodies and small molecule inhibitors [5] . Polypeptides have the characteristics of simple spatial structure, small molecular weight, high specificity, strong penetration, easy to reach tissues and organs, low immunogenicity, and less immune response to polypeptides. In addition, polypeptides can be artificially synthesized, and the purification method is simple, so they are increasingly valued in new drug development. A variety of polypeptide drugs have been successfully applied in clinical treatment, such as GLP-1 polypeptides. However, there is no specific polypeptide that binds to FGFR2 receptor, so the development of polypeptides that can specifically bind to FGFR2 and regulate its activity has good application prospects. In addition, small molecule compounds with excellent activity and potential clinical application prospects but poor stability or lack of targeting can be coupled with polypeptides with good therapeutic effect or targeting FGFR2 to become new peptide-drug conjugate (PDC) also has good application prospects.

[0007] Bibliographic references:

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

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

[0010] [3]GFR targeting drugs | multiple cancer species are effective, targeting treatment army a rising star in the new star https: / / bbs.guahao.com / topic / iAbWq135708647153106946.

[0011] [4]Li J, Hu K, Huang J, et al. A Pancancer Analysis of the Expression Landscape and Clinical Relevance of Fibroblast Growth Factor Receptor 2 in Human Cancers[J]. Frontiers in Oncology, 2021, 11: 644854.

[0012] [5]FGFR inhibitor competition is fierce: 3 approved, many in research. https: / / www.cn-healthcare.com / articlewm / 20210713 / content-1242424.html. SUMMARY

[0013] The following is a summary of some aspects of the present application and is not limited thereto. These aspects and other parts are more fully described later. All references in the specification are incorporated by reference in their entirety. When the disclosure of the specification is inconsistent with the cited references, the disclosure of the specification prevails.

[0014] In a first aspect, the present application provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1-14, or a derivative of the amino acid sequence of SEQ ID NO: 1-14.

[0015] In some embodiments, the derivative of the modified amino acid sequence comprises one or more modifications selected from the group consisting of N-terminal and / or C-terminal modification, substitution of one, two or more amino acid residues with one or more natural and / or unnatural amino acid residues.

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

[0017] In a second aspect, the present application also provides a polypeptide drug conjugate or a pharmaceutically acceptable salt thereof, wherein the polypeptide drug conjugate or the pharmaceutically acceptable salt thereof comprises a polypeptide, a linker and a payload, wherein the polypeptide is linked to the payload via the linker, and the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1-14, or a derivative of the amino acid sequence of SEQ ID NO: 1-14.

[0018] In some embodiments, the payload is an anti-cancer agent; preferably, the anti-cancer agent is selected from the group consisting of a small molecule drug or a nucleic acid drug.

[0019] In some embodiments, the anti-cancer agent is selected from the group consisting of maytansine and its derivatives, taxol 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 anti-cancer agent is selected from the group consisting of BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, dacarbazine, triaziquone, 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 application also relates to a pharmaceutical composition comprising any of the polypeptides or pharmaceutically acceptable salts thereof or the polypeptide drug conjugate or pharmaceutically acceptable salts thereof described in the present application.

[0021] In some embodiments, the pharmaceutical composition described in the present application further comprises at least one of pharmaceutically acceptable carriers, excipients, diluents, adjuvants and vehicles.

[0022] In another aspect, the present application relates to the use of the polypeptide or pharmaceutically acceptable salts thereof or the polypeptide drug conjugate or pharmaceutically acceptable salts thereof or the pharmaceutical composition described in the present application in the preparation of a medicament for preventing, managing, treating or alleviating FGFR2-associated disorders.

[0023] In some embodiments, the FGFR2-associated disorder is a cancer, further wherein the cancer is selected from the group consisting of bile duct cancer, intrahepatic bile duct cancer, gastric cancer (e.g., gastric adenocarcinoma), adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, bile duct cancer, bladder cancer, brain cancer (e.g., meningioma, glioma, e.g., astrocytic glioma, oligodendroglioma, medulloblastoma), cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, large intestinal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma), Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), hypereosinophilic syndrome, 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 cancer, oropharyngeal cancer)), hematopoietic cancer (e.g., leukemia, e.g., acute lymphoblastic leukemia (ALL) (e.g., 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, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt 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 lymphoma, e.g., precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (e.g., cutaneous T-cell lymphoma (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, intestinal-type T-cell lymphoma;Multiple myeloma (MM), hemangioblastoma, inflammatory myofibroblastoma, immune-mediated amyloidosis, renal cell carcinoma (such as nephroblastoma, renal cell carcinoma), liver cancer (such as hepatocellular carcinoma, malignant hepatocellular carcinoma), lung cancer (such as bronchial carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, leiomyosarcoma (LMS), mastocytosis (such as generalized mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorders (MPD) (such as polycythemia vera (PV)). Essential thrombocythemia (ET), idiopathic extramedullary metaplasia (AMM), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), eosinophilic syndrome (HES), neuroblastoma, neurofibroma (such as neurofibromatosis type 1 or 2, schwannoma), neuroendocrine carcinoma (such as gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (such as cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, etc. Attached Figure Description

[0024] Figure 1 EC50 values ​​of peptides and FGFR2IIIc as measured by ELISA

[0025] Figure 2 Affinity test pattern of the peptide shown in sequence 1 with FGFR2(IIIC)

[0026] Figure 3 Affinity test pattern of the peptide shown in sequence 2 with FGFR2(IIIC)

[0027] Figure 4 Affinity test pattern of the peptide shown in sequence 3 with FGFR2(IIIC)

[0028] Figure 5 Affinity test pattern of the peptide shown in sequence 4 with FGFR2(IIIC)

[0029] Figure 6 Affinity test pattern of the peptide shown in sequence 5 with FGFR2(IIIC)

[0030] Figure 7 Affinity test pattern of the peptide shown in sequence 6 with FGFR2(IIIC)

[0031] Figure 8 Affinity test pattern of the peptide shown in sequence 7 with FGFR2(IIIC)

[0032] Figure 9 Affinity test pattern of the peptide shown in sequence 8 with FGFR2(IIIC)

[0033] Figure 10 Affinity test pattern of the peptide shown in sequence 9 with FGFR2(IIIC)

[0034] Figure 11 Affinity test pattern of the peptide shown in sequence 10 with FGFR2(IIIC)

[0035] Figure 12 Affinity test pattern of the peptide shown in sequence 11 with FGFR2(IIIC)

[0036] Figure 13 Affinity test pattern of the peptide shown in sequence 12 with FGFR2(IIIC)

[0037] Figure 14 Affinity test pattern of the peptide shown in sequence 13 with FGFR2(IIIC)

[0038] Figure 15 Affinity test pattern of the peptide shown in sequence 14 with FGFR2(IIIC) Detailed Implementation

[0039] The term "thymic stromal lymphopoietin (TSLP)" is a type I tetraalpha-helical cytokine, an epithelial cell-derived cytokine produced in response to pro-inflammatory stimuli. Closely associated with interleukin-7 (IL-7), TSLP initiates allergic responses by stimulating dendritic cells (DCs) and is a crucial factor in regulating the human immune response. The term "TSLP" encompasses variants, isotypes, homologs, orthologs, and paralogs of TSLP.

[0040] The meaning of the terms "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 an adjacent amino acid. Polypeptides described herein may contain naturally occurring or non-naturally occurring amino acids. They can be modified into analogs, derivatives, functional mimics, pseudopeptides, and other compounds containing at least two amino acids. Unless a specific modification is specified at the N-terminus or C-terminus, a polypeptide containing a particular amino acid sequence includes both unmodified and modified amino and / or carboxyl terminals, as is well known to those skilled in the art. A polypeptide with a particular amino acid sequence may include modified amino acids and / or additional amino acids unless the N- and / or C-terminus contain modifications that prevent the 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 application can be modified by engineering to form polypeptide derivatives. As is well known to those skilled in the art, various modifications can be made to polypeptides. Typical modifications include, but are not limited to, N-terminal acetylation, C-terminal amidation, d-form amino acid substitution, non-natural amino acid substitution, fatty acid modification, or combinations of the above. The present application encompasses any modification of a polypeptide that is well known. For example, a polypeptide derivative can include a chemical modification of the polypeptide, such as alkylation, acylation, carbamylation, iodination, or any other modification that results in a polypeptide derivative. Modifications of the polypeptide can include modified amino acids, such as, for example, hydroxyproline or carboxyglutamate, and can include amino acids linked by non-peptide bonds.

[0042] Other modifications of the polypeptides of the present application can employ the substitution of non-natural amino acids for natural amino acids in the polypeptide, including but not limited to, 2-amino aliphatic acid (Aad), 3-amino aliphatic acid (βAad), β-alanine, β-amino propionic acid (βAla), 2-amino butyric acid (Abu), 4-amino butyric acid, piperidine carboxylic acid (4Abu), 6-amino caproic acid (Acp), 2-amino heptanoic acid (Ahe), 2-amino isobutyric acid (Aib), 3-amino isobutyric acid (βAib), 2-amino pimelic acid (Apm), 2,4-diamino butyric acid (Dbu), desmosine (Des), 2,2'-diamino pimelic acid (Dpm), 2,3-diamino propionic acid (Dpr), N-ethyl glycine (EtGly), N-ethyl asparagine (EtAsn), hydroxylysine (Hyl), iso-hydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), iso-desmosine (Ide), iso-isoleucine (aIle), N-methyl glycine (MeGly), N-methyl isoleucine (MeIle), 6-N-methyl lysine (MeLys), N-methyl valine (MeVal), norvaline (Nva), norleucine (Nle), and ornithine (Orn). Of course, all of the α-amino acids modified can be replaced by the corresponding β-, γ-, or ω-amino carboxylic 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, D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids made 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 one of the 20 L-amino acids commonly found in naturally synthesized peptides, i.e., the L-isomer 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 (lie 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] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having 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), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta- branched side chains (e.g., T, V, I), and aromatic side chains (e.g., Y, F, W, H). Thus, for example, a predicted nonessential amino acid residue in a polypeptide is preferably replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are replacements from analogies based on electron equilibrium considerations (e.g., norleucine for methionine) or other properties (e.g., 2-thienylalanine for phenylalanine).

[0046] The polypeptides of the present application can be prepared using methods well known to those of ordinary skill in the art, including methods of chemical synthesis that are well known. Thus, where the polypeptide or derivative thereof comprises one or more non-standard amino acids, it is likely that the polypeptide or derivative thereof was prepared by chemical synthesis. In addition to using methods of chemical synthesis to prepare the polypeptide or derivative thereof, it can also be prepared by expression from an encoding nucleic acid. This is particularly applicable where the polypeptide or derivative thereof contains only natural amino acids, in which case well known methods of preparing polypeptide sequences encoded by nucleic acids can be used (see Sambrook et al., Molecular Cloning: A Laboratory 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 particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits substantial structural similarity to the reference substance, e.g., shares a core or common structure, and differs in some discrete way. In some embodiments, an analog is a substance that can be produced from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by performance of a synthetic process that is substantially similar to the synthetic process used to produce the reference substance, e.g., shares multiple steps with it. In some embodiments, an analog is produced or can be produced by performance of a synthetic process that is different from the synthetic process used to produce the reference substance.

[0048] Regarding sequence identity. Sequence identity is calculated by sequence alignment according to methods known in the art. To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison. For instance, gaps can be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then 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. Thus, % identity = number of identical positions / total number of positions in the overlap x 100. In this comparison, the sequences can be the same length or can be of different lengths. Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman (J. Theor. Biol., 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol, 1972), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. U.S.A., 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Version 7.0, Genetic Computer Group, 575 Science Drive, Madison, Wisconsin), or by using publicly available computer software such as BLAST, for example. When using such software, default parameters are preferred, such as gap penalties or extension penalties. The optimal alignment is selected from among the various methods (i.e., the alignment that produces the highest percent identity over the entire comparison window).

[0049] The term "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions in the comparison times 100. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous; if 95 of 100 positions in two sequences are matched or homologous, then the two sequences are 95% homologous. Typically, the comparison is performed using the best alignment of two sequences, e.g., the two sequences are optimally aligned to give the greatest percent homology. The comparison can be performed by the BLAST algorithm, for example, where the algorithm is chosen to give the largest match between the sequences over the entire length of the respective reference sequences. The following references are directed to the BLAST algorithm, which is frequently used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410; Gish, W. et al. (1993) Nature Genet. 3:266-272; Madden, T.L. et al. (1996) Meth. Fnzymol. 266:131-141; Altschul, S.F. et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other routine 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" as used herein refers to a polypeptide drug conjugate composed of a polypeptide, a linker, and a payload, wherein the linker is a cleavable linker or a non-cleavable linker, and the payload is an anti-cancer agent. In some embodiments, the "linker" refers to a molecule or moiety that covalently links the payload to the polypeptide molecule. In some embodiments, the anti-cancer agent can be or include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule-targeting moieties such as taxanes, maytansinoids, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), 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 foregoing (i.e., sharing a relevant anti-proliferative activity).In some particular embodiments, the chemotherapeutic moiety can be or include one or more of: Actinomycin, All-trans retinoic acid, Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and / or their analogs (e.g., DM1), Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof.

[0051] The term "pharmaceutical composition," as used herein, relates to a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the present application and a pharmaceutically acceptable carrier or excipient. As used herein, "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 salts and the like. Examples of pharmaceutically acceptable carriers or excipients include one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In any case, it will preferably be in a form suitable for injection, inhalation, or ingestion, including oral ingestion. In any case, it will preferably include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. A pharmaceutically acceptable substance, such as a wetting or minor amount of a supplementary agent, such as a wetting or emulsifying agent, preservative or buffer, can also be included. Optionally, a disintegrating agent, such as a cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate, can be included. In addition to the excipient, the pharmaceutical composition can also include one or more of the following: carrier proteins such as serum albumulin, buffers, binding agents, sweeteners and other flavorings; colorants and polyethylene glycol.

[0052] The composition can be in a number of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form will depend on the intended route of administration and therapeutic application. In one embodiment, the composition is in the form of an injectable or infusible liquid, for example, in a form 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 suitable routes of administration for the pharmaceutical composition include, but are not limited to, rectal, transdermal, transmucosal, or intestinal administration.

[0053] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering an antibody or antigen binding fragment. The carrier can be an anti-adherent, a binder, a coating, a disintegrant, a filler or diluent, a preservative (e.g., an antioxidant, an antibacterial or antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifying agent, a buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyalcohols, 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 attached to the carboxyl group of another amino acid (or amino acid derivative), which is activated by its reaction with a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group and activated carboxyl group chemically react, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially reactive groups (such as the a-amino group of an N-terminal amino acid or amino acid derivative and the carboxyl group of a C-terminal amino acid or amino acid derivative) can be blocked ("protected") so that they are not involved in the chemical reaction. Thus, only the specific reactive groups are allowed to react so that the desired product is formed. Protecting groups that can be used for this purpose include, but are not limited to, t-butyloxycarbonyl (t-Boc) and benzyloxycarbonyl (Cbz) groups to protect amine groups; and simple esters (such as methyl and ethyl) and amides to protect carboxyl groups. The process of protecting reactive groups (which should not react), coupling to form peptide bonds, and deprotecting reactive groups can be repeated, often with subsequent removal of protecting groups by treatment that leaves the peptide bond intact (e.g., treatment with dilute acid). Peptides can be synthesized by adding amino acids sequentially to a growing peptide chain. Both liquid and solid phase peptide synthesis methods are useful according to the present application. In solid phase peptide synthesis methods, the growing peptide chain is typically attached to an insoluble matrix (such as, for example, a polystyrene bead) by attaching the C-terminal amino acid to the matrix. At the end of the synthesis, the peptide can be released from the matrix using a cleavage reagent that does not destroy the peptide bond, such as hydrofluoric acid (HF). At this point, the protecting groups are also typically removed. Automated, high-throughput, and / or parallel peptide synthesis methods can also be used according to the present application. For more information on peptide synthesis methods, see, for example, 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 the foregoing are incorporated herein by reference.

[0055] In addition, the polypeptides disclosed herein, including their salts, can also exist in a hydrate form or a form containing a solvent (e.g., ethanol, DMSO, etc.) and can be used for crystallization. The compounds disclosed herein can inherently or through design form solvates with pharmaceutically acceptable solvents (including water); thus, the compounds of the present disclosure include both solvated and unsolvated forms.

[0056] In addition, the TSLP-related diseases disclosed herein are not limited, as long as it is a disease related to TSLP, for example, the therapeutic response induced by the molecules of the present disclosure can be through binding to human TSLP, then blocking the binding of TSLP to its receptor, or killing cells overexpressing TSLP.

[0057] The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully demonstrate the preferred embodiments of the application. These examples should not be construed as limiting the scope or content of the application.

[0058] Examples

[0059] The polypeptide compounds and derivatives thereof provided by the present disclosure are synthesized by solid phase synthesis. The synthesis support is Fmoc-Cys(Trt)-2-Chlotrityl Resin resin. During the synthesis, the Fmoc-Cys(Trt)-2-Chlotrityl Resin resin is first fully swelled in N,N-dimethylformamide (DMF), then the solid support is repeatedly condensed with activated amino acid derivatives, washed, deprotected Fmoc, washed, and subjected to the next round of amino acid condensation to achieve the desired length of the polypeptide chain, and finally the polypeptide is cleaved from the solid support by reacting the resin with a mixture of trifluoroacetic acid: water: triisopropylsilane: benzyl thioether (90:2.5:2.5:5:, v:v:v:v), and then the solid crude product of the linear precursor is obtained after being precipitated in chilled methyl tert-butyl ether. The cleaved linear precursor crude product is subjected to disulfide bond oxidation in an alkaline solution to obtain the target polypeptide crude product. The polypeptide crude product is purified and separated by C-18 reverse phase preparative chromatography column in a 0.1% trifluoroacetic acid acetonitrile / water system to obtain the 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. Obtaining polypeptide sequences with high affinity to FGFR2IIIc by screening phage library

[0064] 1) Selection of strains and vectors

[0065] Escherichia coli strain SS320 (Sidhu, S. S., et al. "Methods Enzymol 328 (1999): 333-363.) has 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-stranded and amplified phage.

[0066] Vector selection and phagemid construction methods were described in Tonikian, R., et al. Nature Protocol 2.6 (2007): 1368-1386, pGEX4T-2 vector was used for display phage library, the vector contains a replicon for single-stranded (fl ori) and double-stranded (dsDNA ori) DNA replication, in addition to a selectable marker, such as the beta-lactamase gene (Ampr), while making it resistant to ampicillin and carbenicillin. After polypeptide sequence insertion, a fusion protein was formed with mature coat protein (P3) of phage, and N-terminal display was performed.

[0067] 2) Preparation of phage display library

[0068] The present application 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 method of Liu, B., S. Long, and J. Liu. "New Biotechnology 56 (2019)" was used for random library generation. Subsequently, the random CCC-dsDNA library was electroporated into E. coli host SS320 (M13KO7) containing F' fragments, and the M13KO7 pre-infected SS320 strain was used to prepare electroporation competent, and finally the construction of phage library was completed according to (Tonikian, R., et al. Nature Protocol 2.6 (2007): 1368-1386). The phage titer produced by PEG / NaCl precipitation was about 10 13 pfu / ml.

[0069] 3) Obtain polypeptide sequences with high affinity to FGFR2 target (IIIC) by phage library panning

[0070] Fibroblast growth factor receptor-2: FGF R2 (IIIc) was used as the target protein for phage library panning (FGFR2 IIIc (His / Avi): Kaybio FGR-HM4CDB).

[0071] Bacteriophage selection method, known as biopanning, is an affinity selection process to isolate target binding molecules. With target protein as stationary phase and bacteriophage display library as mobile phase, after a period of incubation, the unbound free phage is washed away, then the phage adsorbed with target molecule is eluted with competitive receptor or acid, the eluted phage infects host cells after propagation and expansion, the next round of elution is carried out, after 3-5 rounds of "adsorption-elution-expansion", the antibody with high affinity to target protein can be obtained. Padmanaban, G., et al. Journal of Biotechnology 187 (2014): 43-50 discloses the method of biopanning of bacteriophage library. The present application makes MyOne Streptavidin T1 Dynabeads (10 mg / mL, Invitrogen, cat# 65602) magnetic beads and FGFR2 IIIc (His / Avi) target protein refer to the method to carry out 4 rounds of panning from the biopanning of bacteriophage library to obtain high affinity polypeptide sequence to FGFR2 target (IIIc). After resuspending and washing the Dynabeads according to the instruction method, 60 μg of FGFR2 IIIc (His / Avi) is fixed to 105 μL of magnetic beads, and 60 μL of PBS is added, and finally the total volume is 180 μL. Place on a rotating mixer, mix at 4°C for 16-24 h. After incubation, the magnetic beads combined with the target protein are precipitated with a magnetic stand, the supernatant is removed, and the magnetic beads are washed with 1 ml of 0.1% PBST for 4 times. Then, the magnetic beads are blocked, and the magnetic beads are blocked with 0.5% BSA-PBS at room temperature for 30 min. In the first round, 5 μL of 1×10 11 pfu of M13 phage library (dissolved in PBS) is added to the blocked magnetic beads, mixed gently at 4°C for 1 h. After incubation, the unbound phage is repeatedly washed with PBS containing 0.1% Tween (PBST), and in the next round of panning, the concentration of Tween is gradually increased from 0.1% to 0.4%. The elution method uses 0.1 M citrate (pH 3.1) incubation for 2 min to elute the bound phage, and immediately neutralized with 1 M Tris-HCl (pH 9.1). Take 20 μL for phage titer determination, the remaining phage is amplified using SS320 strain, the purified phage is used for the next round of screening, and the amount of phage input is 1×10 11 pfu each time. The polypeptides selected are obtained by chemical synthesis.

[0072] Biological evaluation

[0073] Example 2. ELISA binding method to test the binding of polypeptide and FGFR2 IIIc

[0074] 1) Main experimental materials

[0075]

[0076] 2) Experimental procedure

[0077] His Tag Antibody was diluted to 0.5 μg / ml in coating buffer (0.05 M carbonate buffer pH 9.6) and 25 μl / well was added to a 384 well plate (Thermo or Greiner) and coated at 4°C overnight. The next day, the plate was washed 3-5 times with wash buffer (0.05% Tween-20 in TBS, pH 7.4). Blocking buffer (2% (w / v) BSA in TBS, pH 7.4) was added and the plate was blocked at 37°C. After washing 3-5 times, a gradient of test polypeptide (e.g. 100 nM, 33 nM, 11 nM, 3.7 nM, 1.24 nM, 0.412 nM, 0.137 nM, 0.046 nM) was added and incubated at 37°C for 1 hour. Unbound polypeptide was washed away and 0.4 μg / ml FGFR2 IIIc was added and incubated at 37°C for 1 hour. Unbound FGFR2 IIIc protein was washed away and anti-Human Fc-HRP was added and incubated at 37°C for 1 hour. Unbound HRP protein was washed away and TMB was added to develop the color. The reaction was finally stopped and the OD450 was measured. The data was plotted using Graphpad Prism and the EC50 value for polypeptide binding to FGFR2 IIIc was calculated.

[0078] 3) Experimental results

[0079] The polypeptides selected from the phage library were tested for binding to FGFR2 IIIc protein using the ELISA method. The results are shown in Table 2 as EC50 values. Polypeptides with low nM binding to FGFR2 IIIc protein were selected. The ELISA test results were analyzed using Graphpad Prism as shown in Figure 2. Figure 1 .

[0080] Table 2: ELISA test polypeptide EC50 values

[0081] No. EC50 (nM) No. 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 polypeptides were tested for binding to FGFR2 (IIIC) using the ELISA method. The results show that the polypeptides of the present application have high binding affinity to FGFR2 (IIIC) receptor.

[0083] Example 3. BLI (Biolayer Interferometry) test of polypeptide affinity (KD) to FGFR2 IIIc

[0084] 1) Main experimental materials

[0085]

[0086] BLI is a bio-layer interferometry (Bio-Layer Interferometry, BLI for short) which is a label-free and real-time monitoring optical detection technology, mainly used for full quantitative analysis of interaction between biological molecules and protein concentration determination. BLI can monitor the whole molecular binding process in real time, and calculate important data such as affinity (KD), binding rate (ka), dissociation rate (kd) and the like between molecules.

[0087] 2) Experimental steps

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

[0089] 3) Experimental results

[0090] Firstly, the polypeptide having a binding signal with 100 nM FGFR2IIIc is screened by the BLI technology in the application, and then the affinity test of multiple concentrations is carried out, and the results are shown in the following table 3.

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

[0092] No. KD (nM) No. 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] From the above table, it can be seen that the affinity of the polypeptide to FGFR2IIIc is high, and the KD value is in the nM level.

[0094] The preferred embodiments of the present application have been disclosed with the above particularity, but the application is not limited to the embodiments disclosed, and variations and modifications can be made by those skilled in the art without deviating from the spirit of the application, and such variations and modifications are intended to be included within the scope of the application as defined in the following claims.

Claims

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

2.

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

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

Images