NPC1 Monobody and its Monobody Conjugate

Abstract

The present invention relates to Niemann-Pick disease type C1 (NPC1)-binding polypeptides and NPC1-binding peptide conjugates containing these binding polypeptides. Furthermore, the present invention relates to pharmaceutical compositions containing these NPC1-binding polypeptides and binding peptide conjugates, as well as the use of these compositions to treat various conditions, including cancer, infectious diseases, neurodegenerative diseases, inflammatory conditions, and bone conditions. The NPC1-binding conjugates are also useful for enhancing endosomal release of pharmaceutically active moieties. TIFF2023548889000016.tif88165

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 112,031, filed on November 10, 2020. U.S. Provisional Patent Application No. 63 / 112,031 is hereby incorporated by reference in its entirety.

[0002] Field The present invention relates to Niemann-Pick disease type C1 (NPC1) binding polypeptides and NPC1 binding peptide conjugates comprising these binding polypeptides. Further, the present invention relates to pharmaceutical compositions comprising these NPC1 binding polypeptides and binding peptide conjugates, and to the use of these compositions in the treatment of various pathologies.

Background Art

[0003] Background Niemann-Pick disease type C1 (NPC1) is located in the membranes of the endosomal compartment and is essential for transporting cholesterol from the endosome to the plasma membrane. When cholesterol transport to the plasma membrane is disrupted, the integrity of the plasma membrane is destroyed, correct Rac1 localization required for migration and metastasis is blocked, and other cholesterol-dependent proteins such as receptor tyrosine kinases (RTKs) may be disrupted. Further findings are that in certain cancer cells, autophagic flux is blocked when NPC1 is disrupted. Since autophagy is a mechanism of treatment resistance to chemotherapy in cancers such as colorectal cancer and pancreatic cancer, this is clinically interesting.

[0004] Autophagy is a cellular process that aids in the growth and survival of advanced cancers. Although many large pharmaceutical companies have abandoned autophagy-targeting therapeutic strategies for cancer treatment, preclinical evidence is increasing that inhibiting autophagy can enhance the efficacy of currently used cancer therapies, reigniting interest in this field. In fact, hydroxychloroquine (HCQ), an anti-malaria drug approved by the FDA, has been used in combination with chemotherapy for various tumor types, including pancreatic cancer and colorectal cancer, and many clinical trials have been conducted. Due to at least partially resulting from the lysosomal storage disorder phenotype induced by HCQ intracellularly, HCQ has a certain dose-sensitivity effect, for which the amount of drug administered can be limited.

[0005] Similar to HCQ, past research targeting NPC1 has shown promise in cancer treatment. However, small molecule approaches are vulnerable to unwanted off-target effects and toxicity. This disclosure relates to overcoming these and other pharmaceutical constraints in the art. Summary of the Invention

[0006] Summary A first aspect of the disclosure relates to a Niemann-Pick disease type C1 (NPC1) binding polypeptide. This NPC1 binding polypeptide comprises a fibronectin type III (FN3) domain having a modified FG loop amino acid sequence, a modified BC loop amino acid sequence, a modified CD loop amino acid sequence, a modified DE loop amino acid sequence, or a combination thereof, wherein the one or more modified loop sequences enable binding to NPC1.

[0007] Another aspect of this disclosure relates to an NPC1-binding peptide conjugate. This NPC1-binding peptide conjugate comprises a first part and a second part. The first part of the NPC1-binding peptide conjugate comprises an NPC1-binding polypeptide as described herein, and the second part of the conjugate is attached to the first part and is selected from a pharmaceutically active part, a diagnostic part, a half-life extension part, a delivery vehicle, a prodrug, a second binding molecule, a polymer, and an unbinding protein.

[0008] Other aspects of this disclosure relate to isolated polynucleotides encoding NPC1-binding polypeptides as described herein, isolated polynucleotides encoding NPC1-binding peptide conjugates as described herein, and vectors comprising any one of the described polynucleotides. Another aspect of this disclosure relates to host cells containing these polynucleotides or vectors.

[0009] Another aspect of this disclosure relates to a pharmaceutical composition comprising an NPC1-conjugated polypeptide, an NPC1-conjugated peptide conjugate, an isolated polynucleotide, or a vector as described herein, and a pharmaceutical carrier.

[0010] Another aspect of this disclosure relates to combination therapeutic agents. These combination therapeutic agents include the NPC1-binding polypeptide described herein and an oncological agent.

[0011] Another aspect of this disclosure relates to a method for treating cancer in a subject. This method includes administering to a subject having cancer an amount of a pharmaceutical composition described herein that is effective in treating the cancer.

[0012] Another aspect of this disclosure relates to a method for treating an infection in a subject. This method includes administering to a subject having an infection an amount of an NCP1-binding polypeptide or NPC1-binding peptide conjugate described herein that is effective in treating the infection.

[0013] Another aspect of this disclosure relates to a method for enhancing the endosomal release of a pharmaceutically active portion in a subject where such enhancement is needed. This method comprises administering an NPC1-binding peptide conjugate to a subject, wherein the peptide conjugate comprises a first portion and a second portion as described herein, the second portion being the pharmaceutically active portion.

[0014] Another aspect of this disclosure relates to a method for enhancing the endosomal release of a pharmaceutically active moiety in a subject where such enhancement is needed. This method comprises the step of administering a combination therapeutic agent to a subject, the combination therapeutic agent comprising an NPC1-binding polypeptide and a pharmaceutically active moiety as described herein.

[0015] As disclosed herein, NPC1 inhibition disrupts autophagy in cancer cells. Since autophagy is a mechanism of treatment resistance, NPC1 inhibition can be used to enhance cell resensitization to treatment and improve the efficacy of cancer agents. Current NPC1 inhibitors are not useful for this purpose because they do not selectively target cancer cells. However, the NPC1-binding molecules and NPC1-binding peptide conjugates described herein are specifically translocated into endosomal compartments by macropinocytosis. Macropinocytosis is a process that confers upon cells the ability to internally transport large amounts of extracellular fluid and solute to support metabolic demands, and is a process that is specifically enhanced in cancer caused by mutant Ras, dysregulated growth factor signaling, Src activation, etc. Accordingly, the incorporation of the NCP1-binding molecules and NPC1-binding peptide conjugates described herein, mediated by macropinocytosis, provides both independent cancer therapies, namely, means for selectively delivering cancer agents to cancer cells, and adjuvant therapies for resensitizing cancer cells to treatment with cancer agents. [Invention 1001] A Niemann-Pick disease type C1 (NPC1) conjugating polypeptide containing a fibronectin type III (FN3) domain, The FN3 domain has a modified FG loop amino acid sequence, a modified BC loop amino acid sequence, a modified CD loop amino acid sequence, a modified DE loop amino acid sequence, or a combination thereof. The one or more modified loop sequences enable connection with NPC1. Niemann-Pick disease type C1 (NPC1) binding polypeptide. [Invention 1002] The conjugated polypeptide of the present invention 1001, wherein the modified FG loop amino acid sequence is selected from one of SEQ ID NO:2 to 13. [Invention 1003] A conjugated polypeptide according to Invention 1001 or Invention 1002, wherein the modified BC loop amino acid sequence is selected from one of SEQ ID NO: 15 to 21. [Invention 1004] A conjugated polypeptide according to any of Invention 1001 to 1003, wherein the modified CD loop amino acid sequence is selected from one of SEQ ID NO: 23 to 28. [Invention 1005] A conjugated polypeptide according to any of the present invention 1001 to 1004, wherein the modified DE loop amino acid sequence is selected from one of SEQ ID NO: 30 to 32. [Invention 1006] The FN3 domain is The tenth domain of human fibronectin type III, SEQ ID NO:1, containing at least one modified loop amino acid sequence. 10 Fn3) A conjugated polypeptide according to any of invention 1001 to 1005. [Invention 1007] 10 The conjugated polypeptide of the present invention 1006, wherein the Fn3 domain further comprises amino acid substitutions in one or more of the C, D, E, or Fβ chains. [Invention 1008] The conjugated polypeptide of the present invention 1007, wherein the aforementioned amino acid substitution is located in one or more residues selected from R33, E47, T49, and A74 of SEQ ID NO:1. [Invention 1009] The conjugated polypeptide of the present invention 1008, wherein the amino acid substitution at R33 is selected from the group consisting of R33V, R33D, and R33F. [Invention 1010] The conjugated polypeptide of the present invention 1008, wherein the amino acid substitution at E47 is selected from the group consisting of E47T and E47K. [Invention 1011] The conjugated polypeptide of the present invention 1008, wherein the amino acid substitution at T49 is selected from the group consisting of T49K and T49A. [Invention 1012] The conjugated polypeptide of the present invention 1008, wherein the amino acid substitution at A74 is A74T. [Invention 1013] The conjugated polypeptide of the present invention 1006, further comprising amino acid substitutions in one or more residues selected from D3, R6, and D7 of SEQ ID NO:1. [Invention 1014] The FN3 domain is (i) Modified FG loop amino acid sequence of SEQ ID NO:2, modified BC loop amino acid sequence of SEQ ID NO:15, and modified DE loop amino acid sequence (N8) of SEQ ID NO:30; (ii) Modified FG loop amino acid sequence of SEQ ID NO:3, modified BC loop amino acid sequence of SEQ ID NO:16, and modified DE loop amino acid sequence (N16) of SEQ ID NO:30; (iii) Modified FG loop amino acid sequence of SEQ ID NO:4, modified BC loop amino acid sequence of SEQ ID NO:17, and modified DE loop amino acid sequence (N18) of SEQ ID NO:30; (iv) Modified FG loop amino acid sequence of SEQ ID NO:5, modified BC loop amino acid sequence of SEQ ID NO:18, and modified CD loop amino acid sequence (N22) of SEQ ID NO:23; (v) Modified FG loop amino acid sequence of SEQ ID NO:6, modified BC loop amino acid sequence of SEQ ID NO:19, and modified CD loop amino acid sequence (N23) of SEQ ID NO:23; (vi) Modified FG loop amino acid sequence of SEQ ID NO:7, modified BC loop amino acid sequence of SEQ ID NO:18, and modified CD loop amino acid sequence (N24) of SEQ ID NO:24; (vii) Modified FG loop amino acid sequence of SEQ ID NO:8, modified BC loop amino acid sequence of SEQ ID NO:18, and modified CD loop amino acid sequence (N26) of SEQ ID NO:25; (viii) Modified FG loop amino acid sequence of SEQ ID NO:9, modified BC loop amino acid sequence of SEQ ID NO:18, and modified CD loop amino acid sequence (N31) of SEQ ID NO:26; (ix) Modified FG loop amino acid sequence of SEQ ID NO:10, modified BC loop amino acid sequence of SEQ ID NO:18, and modified CD loop amino acid sequence (N34) of SEQ ID NO:26; (x) Modified FG loop amino acid sequence of SEQ ID NO:11, modified BC loop amino acid sequence of SEQ ID NO:20, and modified CD loop amino acid sequence (N35) of SEQ ID NO:24; (xi) Modified FG loop amino acid sequence of SEQ ID NO:12, modified BC loop amino acid sequence of SEQ ID NO:21, and modified CD loop amino acid sequence (N38) of SEQ ID NO:27; and (xii) Modified FG loop amino acid sequence of SEQ ID NO:13, modified BC loop amino acid sequence of SEQ ID NO:20, and modified CD loop amino acid sequence (C45) of SEQ ID NO:28 A conjugated polypeptide comprising any of the inventions 1001 to 1013. [Invention 1015] A conjugated polypeptide according to any of the present invention 1001 to 1014, wherein the FN3 domain contains an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 32 to 43. [Invention 1016] A conjugated polypeptide according to any of the present invention 1001 to 1014, wherein the FN3 domain contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 32 to 43. [Invention 1017] A conjugated polypeptide according to any of the present invention 1001 to 1014, wherein the FN3 domain contains an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 32 to 43. [Invention 1018] A conjugated polypeptide according to any of the present invention 1001 to 1014, wherein the FN3 domain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 32 to 43. [Invention 1019] A first portion comprising any of the conjugated polypeptides of the present invention 1001 to 1018, A second portion is connected to the first portion and is selected from a pharmaceutically active portion, a diagnostic portion, a half-life extension portion, a delivery vehicle, a prodrug, a second binding molecule, a polymer, and an unbinding protein. A conjugate containing Niemann-Pick disease type C1 (NPC1) conjugated peptide. [Invention 1020] The second portion is the pharmaceutically active portion, the NPC1-binding peptide conjugate of the present invention 1019. [Invention 1021] The NPC1-binding peptide conjugate of the present invention 1020, wherein the pharmaceutically active portion is selected from the group consisting of small molecules, nucleic acid molecules, antibodies or their antigen-binding fragments, antibody derivatives, proteins or their polypeptide fragments, and proteolytic chimeras (PROTACs). [Invention 1022] An NPC1-binding peptide conjugate according to Invention 1020 or Invention 1021, wherein the pharmaceutically active portion is a cancer treatment agent. [Invention 1023] The NPC1-binding peptide conjugate of the present invention 1022, wherein the cancer treatment agent is selected from antimetabolites, alkaloids, alkylating agents, mitotic inhibitors, antitumor antibiotics, DNA binding agents, toxins, antiproliferative agents, DNA antagonists, radionuclides, thermoblisters, proteolytic chimeras (PROTACs), and nucleic acid inhibitors. [Invention 1024] The NPC1-conjugated peptide conjugate of the present invention 1023, wherein the alkaloid is selected from the group consisting of duocalmycin, docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, vindesine, and their analogs and derivatives. [Invention 1025] The alkylating agent is selected from the group consisting of busulfan, improsulfan, pigosulfan, benzodepa, carbocon, metsuredepa, uredepa, altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, chlorambucyl, chloranafadin, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide HCl, melphalan, nobemeviquin, perphosphamide, phenesterine, prednimustine, trophosphamide, uracil mustard, carmustine, chlorozotosine, fotemustine, lomustine, nimustine, semustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitractol, pipobromane, temozolomide, and their analogs and derivatives, wherein the NPC1-conjugated peptide conjugate of the present invention 1023. [Invention 1026] The NPC1-binding peptide conjugate of the present invention 1023, wherein the antitumor antibiotic is selected from the group consisting of acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, calitiamycin, carbicin, cardinophilin, chromomycin, dactinomycin, daunorubicin, 6-diazo-5-oxo-l-norleucine, doxorubicin, epirabicin, idarubicin, menogalil, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, pirarubicin, plicamycin, porphyromycin, puromycin, pyrrolobenzodiazepine, streptonigrin, streptozocin, tubercidine, dinostatin, zolubicin, and their analogs and derivatives. [Invention 1027] The antimetabolite is selected from the group consisting of SN-38, denopterin, edatrexate, mercaptopurine (6-MP), methotrexate, pyritrexime, pteropterin, pentostatin (2'-DCF), tomdex, trimethrexate, cladridine, fludarabine, thiamiprine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, floxuridine, fluorouracil, gemcitabine, tegafur, hydroxyurea, urethane, and their analogs and derivatives, as an NPC1-binding peptide conjugate of the present invention 1023. [Invention 1028] The NPC1-binding peptide conjugate of the present invention 1023, wherein the antiproliferative agent is selected from the group consisting of acegraton, amsacrin, bisanthren, camptothecin, dephosphamide, demecolsin, diaziquan, diflomotecan, eflornithine, eriptinium acetate, etogluside, etopside, fenretinide, gallium nitrate, hydroxyurea, lamelarin D, ronidamin, miltefosine, mitogluzone, mitoxantrone, mopidamol, nitracrin, pentostatin, fenamet, 2-ethyl-hydrazide podophyllate, procarbazine, razoxane, sobuzoxane, spirogermanium, teniposide, tenuazonic acid, triadiquan 2,2',2''-trichlorotriethylamine, and its analogs and derivatives. [Invention 1029] The NPC1-binding peptide conjugate of the present invention 1023, wherein the mitotic inhibitor is selected from the group consisting of auristatin, mytansinoids, drastatin, tubulsin, taxane, eposilone, vinca alkaloids, and their analogs and derivatives. [Invention 1030] An NPC1-binding peptide conjugate according to Invention 1020 or Invention 1021, wherein the pharmaceutically active portion is an immunomodulator. [Invention 1031] The immunomodulator is a macrophage type 1 stimulant, and the NPC1-binding peptide conjugate of the present invention 1030. [Invention 1032] The NPC1-binding peptide conjugate of the present invention 1031, wherein the macrophage type 1 stimulant is selected from the group consisting of paclitaxel, colony-stimulating factor-1 (CSF-1) receptor antagonist, IL-10 receptor antagonist, Toll-like receptor (TLR)-2 agonist, TLR-3 agonist, TLR-4 agonist, TLR-7 agonist, TLR-8 agonist, and TLR-9 agonist. [Invention 1033] The immunomodulator is a macrophage type 2 stimulant, and the NPC1-binding peptide conjugate of the present invention 1030. [Invention 1034] The NPC1-binding peptide conjugate of the present invention 1033, wherein the macrophage type 2 stimulant is selected from the group consisting of IL-33, IL-4 receptor agonists, glucocorticoids, IL-10 receptor agonists, and IL-1 receptor agonists. [Invention 1035] The immunomodulator is a T-cell stimulant, and the NPC1-binding peptide conjugate of the present invention 1030. [Invention 1036] The T-cell stimulant is an interferon gene stimulator (STING) agonist, and is an NPC1-binding peptide conjugate of the present invention 1035. [Invention 1037] The immunomodulator is a dendritic cell stimulant, and the NPC1-binding peptide conjugate of the present invention 1030. [Invention 1038] The NPC1-binding peptide conjugate of the present invention 1037, wherein the dendritic cell stimulant is selected from the group consisting of CpG oligonucleotides, imiquimod, camptothecin, colchicine, podophyllotoxin, and its derivatives. [Invention 1039] The NPC1-binding peptide conjugate of the present invention 1030, wherein the immunomodulator is a neutrophil stimulant. [Invention 1040] The NPC1-binding peptide conjugate of the present invention 1039, wherein the neutrophil stimulator is recombinant granulocyte colony-stimulating factor protein (filgrastim) or pegylated recombinant granulocyte colony-stimulating factor protein. [Invention 1041] The NPC1-binding peptide conjugate of the present invention 1021, wherein the pharmaceutically active portion is a nucleic acid molecule. [Invention 1042] The NPC1-binding peptide conjugate of the present invention 1041, wherein the nucleic acid molecule is selected from the group consisting of siRNA, aptamers, miRNA, immunostimulant oligonucleotides, splice-switching oligonucleotides, and guide RNA. [Invention 1043] An NPC1-binding peptide conjugate according to any of invention 1020 to 1042, wherein the pharmaceutically active portion is connected to a delivery vehicle. [Invention 1044] The NPC1-binding peptide conjugate of the present invention 1019, wherein the second portion of the conjugate is a delivery vehicle. [Invention 1045] An NPC1-conjugated peptide conjugate according to Invention 1043 or 1044, wherein the delivery vehicle is selected from nanoparticles, polymer-based particles, and lipid-based particles. [Invention 1046] The second part is the diagnostic portion, the NPC1-binding peptide conjugate of the present invention 1019. [Invention 1047] The NPC1-binding peptide conjugate of the present invention 1046, wherein the diagnostic portion is selected from the group consisting of a fluorescent dye, a radioisotope, a contrast agent suitable for imaging, a radionuclide having a chelating agent, and a photosensitizer. [Invention 1048] An isolated polynucleotide encoding an NPC1-binding polypeptide of any of Invention 1001 to 1018 or an NPC1-binding peptide conjugate of Invention 1019. [Invention 1049] A vector comprising the isolated polynucleotide of the present invention 1048. [Invention 1050] A host cell containing the vector of the present invention 1049. [Invention 1051] A conjugated polypeptide of any of Invention 1001 to 1018, an NPC1-conjugated peptide conjugate of any of Invention 1019 to 1047, an isolated polynucleotide of Invention 1048, or a vector of Invention 1049, Pharmaceutical carriers and A pharmaceutical composition containing [the specified substance]. [Invention 1052] A conjugated polypeptide according to any of invention 1001 to 1018, The pharmaceutically active part and A combination therapy that includes this. [Invention 1053] The combination therapeutic agent of the present invention 1052, wherein the pharmaceutically active portion is selected from the group consisting of small molecules, nucleic acid molecules, antibodies or their antigen-binding fragments, antibody derivatives, proteins or their polypeptide fragments, and proteolytic chimeras (PROTACs). [Invention 1054] A combination therapeutic agent of the present invention 1052 or 1053, wherein the pharmaceutically active portion is a cancer treatment agent. [Invention 1055] The combination therapeutic agent of the present invention 1054, wherein the cancer treatment agent is a chemotherapy agent. [Invention 1056] A combination therapeutic agent of the present invention 1055, wherein the chemotherapeutic agent is selected from cyclophosphamide, gemcitabine, vorinostat, temozolomide, bortezomib, carmustine, and paclitaxel. [Invention 1057] The combination therapeutic agent of the present invention 1054, wherein the cancer treatment agent is an immune checkpoint inhibitor. [Invention 1058] The combination therapeutic agent of the present invention 1057, wherein the immune checkpoint inhibitor is selected from a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor. [Invention 1059] The combination therapeutic agent of the present invention 1054, wherein the cancer treatment agent is selected from an epidermal growth factor (EGFR) inhibitor and an mTOR inhibitor. [Invention 1060] A method for treating cancer in a subject, comprising the following steps: A step of administering to a subject with cancer an amount of the pharmaceutical composition of the present invention 1051 that is effective in treating the cancer. Methods that include... [Invention 1061] The method of the present invention 1060, characterized in that the cancer cells have increased macropinocytosis compared to the corresponding non-cancerous cells. [Invention 1062] The method of the present invention 1060, wherein the cancer is characterized by cancer cells having oncogenic mutations in H-ras, N-ras, or K-ras. [Invention 1063] Any method of the present invention 1060 to 1062, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, glioma, solid tumor, melanoma, glioblastoma multiforme, leukemia, renal cell carcinoma, hepatocellular carcinoma, prostate cancer, and myeloma. [Invention 1064] The process of administering a cancer treatment agent together with the aforementioned pharmaceutical composition. The method of the present invention 1060, further comprising the above. [Invention 1065] The method of the present invention 1064, wherein the cancer treatment agent is a chemotherapy agent. [Invention 1066] The method of the present invention 1065, wherein the chemotherapeutic agent is selected from cyclophosphamide, gemcitabine, vorinostat, temozolomide, bortezomib, carmustine, paclitaxel, mitoxantrone, and capecitabine. [Invention 1067] The method of the present invention 1064, wherein the cancer treatment agent is an immune checkpoint inhibitor. [Invention 1068] The method of the present invention 1067, wherein the immune checkpoint inhibitor is selected from CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. [Invention 1069] The method of the present invention 1064, wherein the cancer treatment agent is selected from epidermal growth factor (EGFR) inhibitors and mTOR inhibitors. [Invention 1070] The process of administering the pharmaceutical composition together with radiotherapy. The method of the present invention 1060, further comprising the above. [Invention 1071] A method for treating an infectious disease in a subject, comprising the following steps: A step of administering to a subject with an infectious disease an amount effective to treat the infectious disease of any of the conjugated polypeptides of Invention 1001 to 1018 or the NPC1-conjugated peptide conjugate of Invention 1019. Methods that include... [Invention 1072] The method of the present invention 1071, wherein the infectious disease is caused by a filovirus. [Invention 1073] The method of the present invention 1072, wherein the filovirus is the Ebola virus or the Marburg virus. [Invention 1074] The method of the present invention 1071, wherein the infectious disease is caused by a coronavirus. [Invention 1075] The method of the present invention 1074, wherein the coronavirus is severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or Middle East respiratory syndrome coronavirus (MERS-CoV). [Invention 1076] A method for enhancing the endosomal release of a pharmaceutically active portion in a target where this is necessary, comprising the following steps: A step of administering an NPC1-binding peptide conjugate to the subject, wherein the peptide conjugate is A first portion comprising any of the conjugated polypeptides of the present invention 1001 to 1018, A second portion is connected to the first portion and includes a pharmaceutically active portion. Process Methods that include... [Invention 1077] A method for enhancing the endosomal release of a pharmaceutically active portion in a target where this is necessary, comprising the following steps: A step of administering a combination therapeutic agent to the subject, wherein the combination therapeutic agent is An NPC1-binding polypeptide according to any of invention 1001 to 1018, The pharmaceutically active part and Process Methods that include... [Invention 1078] The method of Invention 1076 or Invention 1077, wherein the pharmaceutically active portion is selected from the group consisting of small molecules, nucleic acid molecules, antibodies or their antigen-binding fragments, antibody derivatives, proteins or their polypeptide fragments, and proteolytic chimeras (PROTACs). [Invention 1079] A method according to any one of the present invention 1076 to 1078, wherein the subject has a neurodegenerative disease, and the pharmaceutically active portion is suitable for treating the neurodegenerative disease. [Invention 1080] The method of the present invention 1079, wherein the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease. [Invention 1081] A method according to any one of the present invention 1076 to 1078, wherein the subject has an inflammatory state, and the pharmaceutically active portion is suitable for treating the inflammatory state. [Invention 1082] The method of the present invention 1081, wherein the inflammatory condition is rheumatoid arthritis or atherosclerosis. [Invention 1083] A method according to any of items 1076 to 1078 of the present invention, wherein the subject has a bone pathology condition, and the pharmaceutically active portion is suitable for treating said bone pathology condition. [Invention 1084] The method of the present invention 1083, wherein the bone pathology is osteoporosis or Paget's disease of bone. [Invention 1085] A method according to any one of the present invention 1076 to 1078, wherein the subject has cancer and the pharmaceutically active portion is suitable for treating said cancer. [Invention 1086] The method of the present invention 1085, relating to cancer and RAS pathway activation. [Brief explanation of the drawing]

[0016] [Figure 1A] Figures 1A and 1B show NPC1 expression in cancer. Figure 1A shows increased NPC1 expression in pancreatic cancer tissue compared to normal adjacent tissue. Figure 1B is a Kaplan-Meier survival analysis showing that NPC1 is a poor prognostic indicator in pancreatic cancer. The data were derived from the TCGA dataset. [Figure 1B] See the explanation in Figure 1A. [Figure 2] This study demonstrates the effect of NPC1 inhibition on DLD-1 cancer cell proliferation. Proliferation was analyzed by the Syto60 assay 3 days after treatment. n=3. [Figure 3-1]Figure 3A shows endosomal accumulation of free cholesterol upon NPC1 knockdown in DLD-1 and HCT-116 cancer cell lines. The label "Philippines" indicates free cholesterol. Figure 3B shows autophagy flux inhibition upon NPC1 knockdown, as indicated by LC3B accumulation. Figure 3C shows verification of LC3B accumulation by Western blot analysis using tool compounds to inhibit NPC1. [Figure 3-2] See the explanation in Figure 3-1. [Figure 4] This is a schematic diagram showing the NPC1 topology. Monobody libraries were screened for binders for the yellow NTD (cholesterol-binding) domain. [Figure 5] Figures 5A and 5B show NPC1 N-terminal domain (NTD) binding monobodies and C-terminal domain (CTD) binding monobodies. Figure 5A shows the binding affinity (arbitrary unit) of monobody clones to NPC1 NTD, and Figure 5B shows the binding affinity (arbitrary unit) of monobody clones that bind to NPC1 CTD. FC is a non-specific binding control. [Figure 6] This image shows NPC1 NTD binding and NPC1 CTD binding monobodies in the cholesterol-loaded vs. cholesterol-unloaded states. The binding affinity (in arbitrary units) of monobody clones that bind to NPC1 NTD (left) and CTD (right) is shown. FC represents a nonspecific binding control. [Figure 7-1] Figures 7A-7C show the results of screening for NPC1-inhibiting monobodies. Figure 7A is a graph showing the effect of monobody clones on intracellular cholesterol transport. Figure 7B is a representative cell image from Figure 7A. Figure 7C is a heatmap of cholesterol localization from Figure 7B. [Figure 7-2] See the explanation in Figure 7-1. [Figure 8]Figures 8A-8B show the mutant KRas-dependent effects of NPC1 target monobodies. The effects of N23 and N34 clones on macropinocytosis-negative wild-type KRas HeLa cells (Figure 8A) versus macropinocytosis-positive mutant KRas HeLa cells (Figure 8B) were analyzed. FN is a non-target monobody control. Arrows indicate LC3B accumulation. [Figure 9] This study demonstrates the effect of a candidate monobody on HCT-116 cell proliferation. Proliferation was analyzed by the Syto60 assay 3 days after treatment. n=3. [Figure 10A] Figures 10A-10B show monobody selectivity in colorectal DLD-1 and HCT-116 cancer cells (CRCs). Candidate monobody N34 shows selective uptake (Figure 10A) and biological effects (Figure 10B) in mutant KRas CRC cell lines. [Figure 10B] See the explanation in Figure 10A. [Figure 11] This shows in vivo cholesterol changes using NPC1-targeted monobody (N34) versus non-targeted control (FN). [Figure 12] This study demonstrates the in vivo biological effects of an NPC1-targeted monobody. The candidate monobody N34 induces cholesterol and LC3B accumulation in N34-positive tumors versus N34-negative tumors. The monobody (1 μM; 50 μL volume) was injected intratumor 2 hours prior to tumor extraction. [Figure 13A] Figures 13A–13B demonstrate that ERK hyperactivation occurs after NPC1 inhibition in vitro and in vivo. Figure 13A shows that ERK activation increases upon NPC1 knockdown in DLD-1 and HCT-116 cell lines. As shown in Figure 13B, the candidate monobody N34 induces ERK phosphorylation in N34-positive tumors versus N34-negative tumors. The monobody (1 μM; 50 μL volume) was injected intratumor 2 hours prior to tumor extraction. [Figure 13B] See the explanation in Figure 13A. [Figure 14]This study demonstrates that EGFR signaling triggers ERK hyperactivation. ERK hyperactivation after NPC1 knockdown can be reversed by short-term EGFR inhibition with dacomitinib. [Figure 15] This shows EGFR phosphorylation after treatment with an NPC1-targeted monobody. The candidate monobody N34 induces EGFR phosphorylation in N34-positive tumors versus N34-negative tumors. The monobody (1 μM; 50 μl volume) was injected into the tumor 2 hours before tumor extraction. Images of serial sections from Figure 13B were taken. [Figure 16] Using split GFP, we demonstrate that the NCP1 monobody induced endosomal release of GFP11. Mutant Ras PDAC MIA PaCa-2 cells stably expressing cytoplasmic GFP1-10 were treated with 600 mM GFP11 for 24 hours with 1 mM N23 or N34 NCP1 monobody, or without 1 mM N23 or N34 NCP1 monobody. Fluorescence was dependent on GFP11 endosomal release and was observed in cells treated with the NCP1 monobody but not in cells treated with the unbound FN monobody. [Figure 17] This shows endosomal calcein release induced by the NCP1 monobody. Calcein is a membrane-impermeable liquid-phase uptake marker that is semi-quenched when in close proximity to other calcein molecules within the vesicular compartment, but dequenched upon intracellular release and molecular diffusion, resulting in increased cellular fluorescence. We show that calcein fluorescence increases with treatment of N23 and N34 NCP1 monobodies, but not with treatment of unbound FN monobodies. [Figure 18A]Figures 18A–18B show increased endosomal calcein release via NCP1 monobodies, further enhanced in the presence of a nanoparticle delivery vehicle. Figure 18A is a panel of immunocytochemical images of PDAC MIA PaCa3 cells treated with calcein alone (PBS) or in a pegylated nanoparticle delivery vehicle (90 nm nano) (top row of images). Co-treatment of cells with either N23 or N34 NCP1 monobodies enhanced endosomal calcein release under both conditions. Figure 18B is a graph quantifying calcein fluorescence for each treatment condition. The highest levels of calcein fluorescence were observed in cells treated with nanoparticles containing both calcein and NCP1 monobodies. [Figure 18B] See the explanation in Figure 18A. [Modes for carrying out the invention]

[0017] Detailed explanation The present invention relates, in general terms, to Niemann-Pick disease type C1 (NPC1) conjugated polypeptides, NPC1-conjugated peptide conjugates comprising these conjugated polypeptides, and methods for using these NPC1-conjugated polypeptides and NPC1-conjugated peptide conjugates to treat cancer, infections, and other pathological conditions.

[0018] Accordingly, the first aspect of this disclosure relates to a Niemann-Pick disease type C1 (NPC1) binding polypeptide. This NPCI binding polypeptide comprises a fibronectin type III (FN3) domain having a modified FG loop amino acid sequence, a modified BC loop amino acid sequence, a modified CD loop amino acid sequence, a modified DE loop amino acid sequence, or any combination of the above modified loop sequences. One or more modified loop sequences enable binding to NPC1.

[0019] The FN3 domain is an evolutionarily conserved protein domain with a β - sandwich structure that is approximately 100 amino acids long. The β - sandwich structure of human FN3 contains seven β - strands called strands A, B, C, D, E, F, and G, and six connecting loops called loops AB, BC, CD, DE, EF, and FG that show structural homology to immunoglobulin - binding domains. Three of the six loops, namely loops DE, BC, and FG, topologically correspond to the complementarity - determining regions of an antibody, i.e., CDR1, CDR2, and CDR3. The remaining three loops are surface - exposed, similar to antibody CDR3. According to the present disclosure, one or more of the loop regions of each FN3 domain of the binding molecule are modified so as to be able to specifically bind to NPC1.

[0020] As used herein, "specifically binds" or "specific binding" means that the FN3 - containing binding molecule of the present disclosure binds to a predetermined antigen, i.e., NPC1, with a dissociation constant (K -6 ) of about 1×10 -7 M or less, for example, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, about 1×10<管理員注:原文のこの部分は、1×10の指数が順に-6から-10まで続いていますが、一部の数字が欠落しています。ここでは、原文通りに表示しています。-17]] -12 M or less, or about 1×10 -13 M or less. Typically, the FN3 domain binds to NPC1 with a K D that is at least 1 / 10 less than the K D for a non - specific antigen (e.g., BSA or casein) when measured by surface plasmon resonance using, for example, a Proteon Instrument (BioRad). D

[0021] ​The modified FN3 domains of the binding molecules of this disclosure may also be FN3 domains derived from any of the diverse extracellular proteins of animals, yeasts, plants, and bacteria that contain these domains. In one embodiment, the FN3 domain is derived from a mammalian FN3 domain. Exemplary FN3 domains include, but are not limited to, any one of the 15 different FN3 domains present in human tenascin C or any one of the 15 different FN3 domains present in human fibronectin (FN), for example, the 10th fibronectin type III domain. Exemplary FN3 domains also include non-natural synthetic FN3 domains, for example, the non-natural synthetic FN3 domain described in U.S. Patent Application Publication 2010 / 0216708 to Jacobs et al., which is incorporated by reference in its entirety herein. Individual FN3 domains are referred to by their domain number and protein name, for example, the 10th FN3 domain of fibronectin (10FN3).

[0022] In some embodiments, the FN3 domain of the binding molecule is derived from the 10th FN domain (10FN3) of fibronectin. In some embodiments, the FN3 domain of the binding molecule is derived from the human 10FN3 domain. The human 10FN3 domain has the amino acid sequence SEQ ID NO:1 as shown below. The locations of the BC (residues 24-30), CD (residues 40-45), DE (residues 51-55), and FG (residues 75-86) loops in the wild-type sequence of SEQ ID NO:1 are underlined. The locations of other amino acid residues mentioned in this disclosure are also identified by their positions within SEQ ID NO:1. TIFF0007841758000001.tif18164

[0023] According to this disclosure, one or more loop regions, or selected residues within one or more of these loop regions, are modified to enable binding specificity and affinity for NPC1. Appropriate modifications include amino acid substitutions, insertions, and / or deletions. In one aspect, amino acid residues in at least one, at least two, at least three, at least four, at least five, or all six loop regions are altered for binding specificity and affinity for NPC1. In one embodiment, one or more amino acid modifications within the loop regions of SEQ ID NO:1, specifically at residues 24-30 (BC loop), 40-45 (CD loop), 51-55 (DE loop), and 75-86 (FG loop), or approximately residues 24-30 (BC loop), 40-45 (CD loop), 51-55 (DE loop), and 75-86 (FG loop), form an NPC1 binding region. In another embodiment, one or more amino acid modifications in any one of these loop regions enable NPC1 binding.

[0024] In some embodiments, the NCP1-binding molecule of the present disclosure includes a modified BC loop. In some embodiments, the modified BC loop is selected from any one of the modified BC loops of SEQ ID NO: 15-21 (see Table 1), or from BC loops having an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the amino acid sequences of SEQ ID NO: 15-21.

[0025] In some embodiments, the NCP1-binding molecule of the present disclosure includes a modified CD loop. In some embodiments, the modified CD loop is selected from any one of the modified CD loops with SEQ ID NO: 23-28 (see Table 1), or from CD loops having an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the amino acid sequences with SEQ ID NO: 23-28.

[0026] In some embodiments, the NCP1-binding molecule of this disclosure includes a modified DE loop. In some embodiments, the modified DE loop has a SEQ ID NO: 14, 22, and 30 amino acid sequences, or SEQ ID NO: (See Table 1) 14, 22, and It includes a DE loop having an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with respect to 30 amino acid sequences.

[0027] In some embodiments, the NCP1-binding molecule of the present disclosure includes a modified FG loop. In some embodiments, the modified FG loop is selected from any one of the modified FG loops of SEQ ID NO: 2 to 13 (see Table 1), or from an FG loop having an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the amino acid sequences of SEQ ID NO: 2 to 13.

[0028] (Table 1) Amino acid sequences of BC, CD, DE, and FG loops of NCP1 binding molecules TIFF0007841758000002.tif131166

[0029] As discussed above, the FN3 domain contains two sets of CDR-like loops on opposite sides of the molecule. The two sets of loops are separated by a β-chain (domain region between the loops) that forms the center of the FN3 structure. Similar to the loops, these β-chains can be modified to enhance the binding specificity and affinity of the target molecule. Preferably, some or all of the surface-exposed residues on the β-chains are randomized without affecting (or minimally affecting) the intrinsic stability of the FN3 domain. In some embodiments, one or more residues on one or more β-chains are modified to enable interaction with NPC1. Appropriate modifications include amino acid substitutions, insertions, and / or deletions. For example, one or more amino acid residues on the Aβ, Bβ, Cβ, Dβ, Eβ, Fβ, or Gβ chains may be modified to enable binding to NPC1 or to enhance the specificity or affinity of NPC1 binding. In one embodiment, one or more amino acid residues of the A, B, C, D, E, and / or Fβ chains are modified in order to bind to NPC1.

[0030] In some embodiments, the NCP1-binding polypeptide described herein includes substitution, addition, or deletion of one or more amino acid residues in the Aβ chain or an upstream region. In some embodiments, the NCP1-binding polypeptide includes amino acid substitutions in one or more residues corresponding to residues D3, R6, and D7 of SEQ ID NO:1. In some embodiments, the amino acid substitutions are aspartic acid to serine (D3S) in the amino acid residue corresponding to aspartic acid at position 3 of SEQ ID NO:1, arginine to threonine (R6T) in the amino acid residue corresponding to arginine at position 6 of SEQ ID NO:1, and / or aspartic acid to lysine (D7K) in the amino acid residue corresponding to aspartic acid at position 7 of SEQ ID NO:1. In some embodiments, the NCP1-binding polypeptide includes amino acid substitutions of aspartic acid to serine, arginine to threonine, and aspartic acid to lysine in the amino acid residues corresponding to D3S, R6T, and D7K of SEQ ID NO:1.

[0031] In some embodiments, the NCP1-binding polypeptide described herein includes substitution, addition, or deletion of one or more amino acid residues in the Cβ chain. In some embodiments, the NCP1-binding polypeptide includes an amino acid substitution in the Cβ chain at the residue corresponding to the tyrosine residue at position 31 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a tyrosine-to-histidine substitution (Y31H) at the amino acid residue corresponding to the tyrosine at position 31 of SEQ ID NO:1. In some embodiments, the NCP1-binding polypeptide includes an amino acid substitution in the Cβ chain at the residue corresponding to the arginine residue at position 33 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine-to-valine substitution (R33V) at the amino acid residue corresponding to the arginine at position 33 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an arginine-to-aspartic acid substitution (R33D) at the amino acid residue corresponding to the arginine at position 33 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a substitution from arginine to phenylalanine (R33F) at the amino acid residue corresponding to arginine at position 33 of SEQ ID NO:1.

[0032] In some embodiments, the NCP1-binding polypeptide described herein includes substitution, addition, or deletion of one or more amino acid residues in the Dβ chain. In some embodiments, the NCP1-binding polypeptide includes an amino acid substitution at the residue corresponding to the glutamate residue at position 47 of SEQ ID NO:1 in the Dβ chain. In some embodiments, the amino acid substitution is a glutamate-to-threonine substitution (E47T) at the amino acid residue corresponding to glutamate at position 47 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a glutamate-to-lysine substitution (E47K) at the amino acid residue corresponding to glutamate at position 47 of SEQ ID NO:1. In some embodiments, the NCP1-binding polypeptide includes an amino acid substitution at the residue corresponding to the threonine residue at position 49 of SEQ ID NO:1 in the Dβ chain. In some embodiments, the amino acid substitution is a threonine-to-lysine substitution (T49K) at the amino acid residue corresponding to threonine at position 49 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a substitution from threonine to alanine (T49A) at the amino acid residue corresponding to threonine at position 49 of SEQ ID NO:1.

[0033] In some embodiments, the NCP1-binding polypeptide described herein comprises substitution, addition, or deletion of one or more amino acid residues in the Fβ chain. In some embodiments, the NCP1-binding polypeptide comprises an amino acid substitution in the Dβ chain at the residue corresponding to the alanine residue at position 74 of SEQ ID NO:1. In some embodiments, the amino acid substitution is an alanine-to-threonine substitution (A74T) at the amino acid residue corresponding to the alanine at position 74 of SEQ ID NO:1.

[0034] In some embodiments, the NCP1-binding polypeptides described herein include substitutions, additions, or deletions of one or more amino acid residues in the A, C, D, E, and Fβ chains. In some embodiments, the NCP1-binding polypeptides described herein include amino acid substitutions at positions corresponding to all of the above amino acid residues, i.e., at residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1.

[0035] In some embodiments, the NCP1-binding polypeptide described herein comprises a modified FG loop amino acid sequence of SEQ ID NO:2, a modified BC loop amino acid sequence of SEQ ID NO:15, and SEQ ID NO: 14 The FN3 domain contains a modified DE loop amino acid sequence. In some embodiments, the FN domain further includes amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain includes amino acid substitutions in residues corresponding to residues D3, R6, and D7. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the FN3 domain contains the amino acid sequence of SEQ ID NO:32 (monobody (Mb)NPC1N-N8). TIFF0007841758000003.tif13165

[0036] In some embodiments, the NCP1-binding polypeptide described herein comprises a modified FG loop amino acid sequence of SEQ ID NO:3, a modified BC loop amino acid sequence of SEQ ID NO:16, and SEQ ID NO: 22The FN3 domain contains a modified DE loop amino acid sequence. In some embodiments, the FN domain further includes amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain includes amino acid substitutions in residues corresponding to residues D3, R6, and D7. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FN3 domain contains the amino acid sequence of SEQ ID NO:33 (MbNPC1N-N16). TIFF0007841758000004.tif13160

[0037] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:4, a modified BC loop amino acid sequence of SEQ ID NO:17, and a modified DE loop amino acid sequence of SEQ ID NO:30. In some embodiments, the FN domain further includes amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain includes amino acid substitutions in residues corresponding to residues D3, R6, and D7. In some embodiments, the FN3 domain includes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the FN3 domain includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the FN3 domain includes the amino acid sequence of SEQ ID NO:34 (MbNPC1N-N18). TIFF0007841758000005.tif13164

[0038] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO: 5, a modified BC loop amino acid sequence of SEQ ID NO: 18, and a modified CD loop amino acid sequence of SEQ ID NO: 23. In some embodiments, the FN domain further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO: 1. In some embodiments, the FN domain comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, and E47. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1N-N22) of SEQ ID NO:35. TIFF0007841758000006.tif13165

[0039] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO: 6, a modified BC loop amino acid sequence of SEQ ID NO: 19, and a modified CD loop amino acid sequence of SEQ ID NO: 23. In some embodiments, the FN domain further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO: 1. In some embodiments, the FN domain comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, E47, and A74. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1N-N23) of SEQ ID NO:36. TIFF0007841758000007.tif13164

[0040] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:7, a modified BC loop amino acid sequence of SEQ ID NO:18, and a modified CD loop amino acid sequence of SEQ ID NO:24. In some embodiments, the FN domain further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, E47, and T49. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1N-N24) of SEQ ID NO:37. TIFF0007841758000008.tif13164

[0041] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:8, a modified BC loop amino acid sequence of SEQ ID NO:18, and a modified CD loop amino acid sequence of SEQ ID NO:25. In some embodiments, the FN domain further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, and E47. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1N-N26) of SEQ ID NO:38. TIFF0007841758000009.tif13165

[0042] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:9, a modified BC loop amino acid sequence of SEQ ID NO:18, and a modified CD loop amino acid sequence of SEQ ID NO:26. In some embodiments, the FN domain further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, and E47. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:39. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:39. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1N-N31) of SEQ ID NO:39. TIFF0007841758000010.tif13165

[0043] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:10, a modified BC loop amino acid sequence of SEQ ID NO:18, and a modified CD loop amino acid sequence of SEQ ID NO:26. In some embodiments, the FN domain further includes amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain includes amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, E47, and T49. In some embodiments, the FN3 domain includes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:40. In some embodiments, the FN3 domain includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:40. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1N-N34) of SEQ ID NO:40. TIFF0007841758000011.tif13165

[0044] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:11, a modified BC loop amino acid sequence of SEQ ID NO:20, and a modified CD loop amino acid sequence of SEQ ID NO:24. In some embodiments, the FN domain further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, E47, and T49. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1N-N35) of SEQ ID NO:41. TIFF0007841758000012.tif13164

[0045] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:12, a modified BC loop amino acid sequence of SEQ ID NO:21, and a modified CD loop amino acid sequence of SEQ ID NO:27. In some embodiments, the FN domain further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, Y31, R33, and E47. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:42. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:42. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1N-N38) of SEQ ID NO:42. TIFF0007841758000013.tif13164

[0046] In some embodiments, the NCP1-binding polypeptide described herein includes an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:13, a modified BC loop amino acid sequence of SEQ ID NO:20, and a modified CD loop amino acid sequence of SEQ ID NO:28. In some embodiments, the FN domain further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, Y31, R33, E47, and T49. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the FN3 domain contains the amino acid sequence (MbNPC1C-C45) of SEQ ID NO:43. TIFF0007841758000014.tif13163

[0047] Another aspect of this disclosure relates to an NPC1-binding peptide conjugate comprising a first part and a second part. The first part of the NPC1-binding peptide conjugate comprises an NPCI-binding polypeptide as described above. The second part of the NPC1-binding peptide conjugate is conjugated to the first part of the conjugate and is selected from a pharmaceutically active part, a diagnostic part, a half-life extension part, a prodrug, a second binding molecule, a delivery vehicle, a polymer, an unbound protein, and any combination thereof.

[0048] In this aspect of the present disclosure, the first and second parts of the NPC1-binding peptide conjugate are linked covalently, either directly or via a linker. The first and second parts can be fused and produced directly by standard cloning and expression methods. Alternatively, well-known chemical coupling methods can be used to produce the NPC1-binding peptide conjugate described herein by attaching the parts directly or via a peptide or other linker. For example, covalent conjugation of the first and second parts may be achieved via a lysine side chain using an activated ester or isothiocyanate, or via a cysteine ​​side chain using a maleimide, haloacetyl derivative, or activated disulfide. Site-specific conjugation of the first and second parts can also be achieved by incorporating a non-natural amino acid, a self-labeling tag (e.g., SNAP or DHFR), or a tag that is specifically recognized and modified by another enzyme such as saltase A, lipoic acid ligase, and formylglycine-producing enzyme. In some embodiments, site-specific conjugation of the first and second parts is achieved by introducing a cysteine ​​residue to the C-terminus of the NCP1 binding molecule, or to a specific site as described in Goldberg et al., "Engineering a Targeted Delivery Platform Using Centyrins," Protein Engineering, Design & Selection 29(12):563-572 (2016), which is incorporated herein by reference.

[0049] In some embodiments, the first and second portions of the NPC1-binding peptide conjugate are joined together via a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the amino acid linker is a cleavable linker. In some embodiments, the amino acid linker is an incleavable linker. Suitable linkers include peptides composed of one or more repeating modules of amino acids, such as glycine and serine or alanine and proline. Exemplary linker peptides include, for example, (Gly-Gly) n (Gly-Ser) n (Gly3-Ser) n (Ala-Pro) n It includes, in the formula, n is an integer between 1 and 25. The linker length can be appropriately regulated, as long as it does not affect the function of the unbound protein-drug conjugate. A standard 15-amino acid (Gly4-Ser)3 linker peptide has been well-characterized and shown to adopt structurally indeterminate, flexible conformations. Furthermore, this linker peptide does not interfere with the assembly and activity of the domain to which it connects (Freund et al., "Characterization of the Linker Peptide of the Single-Chain Fv Fragment of an Antibody by NMR Spectroscopy", FEBS 320:97 (1993). This disclosure is incorporated herein by reference in its entirety).

[0050] In some embodiments, the second portion of the NPC1-binding peptide conjugate of this disclosure includes a half-life extension portion. Exemplary half-life extension portions include, but are not limited to, albumin, albumin variants (see, for example, U.S. Patent No. 8,822,417 to Andersen et al., U.S. Patent No. 8,314,156 to Desai et al., and U.S. Patent No. 8,748,380 to Plumridge et al., the whole of which is incorporated herein by reference), albumin-binding proteins and / or domains, transferrin and its fragments and analogs (see, for example, U.S. Patent No. 7,176,278 to Prior et al., the whole of which is incorporated herein by reference), Fc regions and variant Fc regions (see, for example, U.S. Patent No. 8,546,543 to Lazar et al., U.S. Patent Publication No. 20150125444 to Tsui, and U.S. Patent No. 8,722,615 to Seehra et al., the whole of which is incorporated herein by reference).

[0051] The other second half-life extension portion of the NPC1-binding peptide conjugate may, for desirable properties, include, but are not limited to, polyethylene glycol (PEG) molecules, e.g., PEG5000 or PEG20000, fatty acids and fatty acid esters of various chain lengths, e.g., laurate, myristic acid, stearate, arachidate, behenate, oleate, arachidonic acid, octanodioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, polylysine, octane, and carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides). The pegyl portion may be added to the first portion, i.e., the NPC1-binding molecule, for example, by adding a cysteine ​​residue to the C-terminus of the molecule using a method well known in the art, thereby attaching the pegyl group to the cysteine.

[0052] In another embodiment, the second portion of the NPC1-binding peptide conjugate includes a diagnostic portion. A suitable diagnostic portion is a portion that facilitates the detection, quantification, separation, and / or purification of the NPC1-binding peptide conjugate. A suitable diagnostic portion is a purification tag (e.g., polyhistidine (His6-), glutathione-S-transferase (GST-), or maltose-binding protein (MBP-)), a fluorescent dye or tag (e.g., chelate (europium chelate), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lysamine, phycoerythrin, and Texas Red), an enzyme tag, a radioisotope or radiolabel (e.g., 4 C, 11 C, 14 N, 35 S, 3 H, 32 P, 99m Tc, 111 In, 62 / 64 Cu, 125 I, 18 F, 67 / 68 Ga, 90 Y, 177 Lu, and 186 / 188 Re), radionuclides containing chelating agents (e.g., MAG3, DTPA, and DOTA; see also Liu S., "Bifunctional Coupling Agents for Radiolabeling of Biomolecules and Target Specific Delivery of Metallic Radionuclides", Adv. Drug Deli. 60(12):1347-1370 (2008), which are incorporated herein by reference), contrast agents suitable for imaging, or photosensitizers, including but not limited to these.

[0053] Suitable chelating agents used in combination with radionuclides as the diagnostic component include, but are not limited to, NOTA (1,4,7-triaza-cyclononane-N,N',N''-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), DTPA (1,1,4,7,7-diethylenetriaminepentaacetic acid), TETA (p-bromoacetamidobenzyl-tetraethylaminetetraacetic acid), and Df (desferrioxamine B). Each of these can be used with various radiolabels, radionuclides, radioisotopes, metals, and radiometals. DOTA-type chelating agents whose ligands contain hard base chelating functional groups such as carboxylates or amine groups are most effective for chelating hard acid cations. Such metal chelate complexes can be made highly stable by matching the ring size to the metal of interest. Similarly, multiple types of chelating agents can be conjugated with targetable constructs to bind to multiple metal ions, such as diagnostic radionuclides and / or therapeutic radionuclides.

[0054] Chelating agents can be covalently bonded to the NPCI-binding polypeptide (i.e., the FN3 domain) of the conjugate using standard bioconjugation methods. Amine-containing residues in the FN3 domain (e.g., lysine) form amide bonds with chelating agents containing activated esters (e.g., N-hydroxysuccinimidyl ester). Sulfur-containing residues (e.g., cysteine) are conjugated with chelating agents containing activated esters or maleimide moieties. Alternatively, the bioconjugate is formed when activated carboxylate residues in the FN3 domain form amide bonds with amine groups on the chelating agent or thioester bonds with thiol groups. For example, bifunctional linkers such as PEG-maleimide (PEG-Mal), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), or N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) can be used instead.

[0055] Suitable imaging agents for use as the diagnostic portion in NPC1-conjugated peptide conjugates include, but are not limited to, single-photon emission computed tomography (SPECT) agents, positron emission tomography (PET) agents, magnetic resonance imaging (MRI) agents, nuclear magnetic resonance imaging (NMR) agents, X-ray agents, optical agents (e.g., fluorophores, bioluminescent probes, near-infrared dyes, quantum dots), ultrasound agents and neutron capture therapy agents, computed tomography agents, two-photon fluorescence microscopy imaging agents, and multiphoton microscopy imaging agents. Particularly useful diagnostic radiolabels, radionuclides, or radioisotopes that can be bound to the chelating agent are: 110 In, m In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 9 V, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154Gd, 158 Gd, 32 P, n C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 55 Co, 72 As, 75 Br, 76 Br, 82m Rb, 83This includes, but is not limited to, Sr, or other γ, β, or positron emitters, and ultra-small superparamagnetic particles (USPIO) of iron oxide suitable for MRI. Diagnostic radiolabels include decay energies of 25 to 10,000 keV, more preferably 25 to 4,000 keV, even more preferably 20 to 1,000 keV, and even more preferably 70 to 700 keV. The total decay energy of useful positron-emitting radionuclides is preferably <2,000 keV, more preferably less than 1,000 keV, and most preferably <700 keV.

[0056] In another embodiment, the second portion of the NPC1-binding peptide conjugate comprises a pharmaceutically active moiety. A suitable pharmaceutically active moiety includes, but is not limited to, a small molecular weight active moiety, a nucleic acid molecule, an antibody or its antigen-binding fragment, an antibody derivative, a protein or its polypeptide fragment, and a proteolytic chimera (PROTAC).

[0057] In some embodiments, the pharmaceutically active portion of the NPC1-binding peptide conjugate is an anticancer agent. Suitable anticancer agents include, but are not limited to, antimetabolites, alkaloids, alkylating agents, mitotic inhibitors, antitumor antibiotics, DNA binding agents, toxins, antiproliferative agents, DNA antagonists, radionuclides, thermoblisters, proteolytic chimeras (PROTACs), nucleic acid inhibitors, and immunomodulators.

[0058] In some embodiments, cancer treatment agents are alkaloids. Suitable alkaloids include, but are not limited to, duocalmycin, docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, vindesine, and their analogues and derivatives.

[0059] In some embodiments, cancer treatment agents are alkylating agents. Suitable alkylating agents include, but are not limited to, busulfan, improsulfan, pigosulfan, benzodepa, carbocon, metsuredepa, uredepa, altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, chlorambucyl, chloranaphazine, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide HCl, melphalan, novemebichin, perphosphamide, fenesterine, prednimustine, trophosphamide, uracil mustard, carmustine, chlorozotosine, fotemustine, lomustine, nimustine, semustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitractol, pipobromane, temozolomide, and their analogues and derivatives.

[0060] In some embodiments, cancer treatment agents are antitumor antibiotics. Suitable antitumor antibiotics include, but are not limited to, acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, kalithiamycin, carbicin, cardinophilin, cromomycin, dactinomycin, daunorubicin, 6-diazo-5-oxo-l-norleucine, doxorubicin, epirabicin, idarubicin, menogalil, mitomycin, mycophenolic acid, nogalamycin, olibomycin, peplomycin, pirarubicin, plicamycin, porphyromycin, puromycin, pyrrolobenzodiazepine, streptonigrin, streptozocin, tubercidine, dinostatin, zolubicin, and their analogs and derivatives.

[0061] In some embodiments, cancer treatment agents are antimetabolites. Suitable antimetabolites include, but are not limited to, SN-38, denopterin, edatrexate, mercaptopurine (6-MP), methotrexate, pyritrexime, pteropterin, pentostatin (2'-DCF), tomdex, trimethrexate, cladridine, fludarabine, thiamiprine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, floxuridine, fluorouracil, gemcitabine, tegafur, hydroxyurea, urethane, and their analogues and derivatives.

[0062] In some embodiments, cancer treatment agents are antiproliferative agents. Suitable antiproliferative agents include, but are not limited to, acegraton, amsacrine, bisanthren, camptothecin, dephosphamide, demecolsin, diaziquan, diflomotecan, eflornithine, eriptinium acetate, etogluside, etopside, fenretinide, gallium nitrate, hydroxyurea, lamelalin D, ronidamin, miltefosine, mitogluzone, mitoxantrone, mopidamol, nitracrine, pentostatin, fenamet, podophillinic acid 2-ethyl-hydrazide, procarbazine, razoxane, sobuzoxane, spirogermanium, teniposide, tenuazonic acid, triaziquan 2,2',2''-trichlorotriethylamine, and their analogs and derivatives.

[0063] In some embodiments, cancer treatment agents are mitotic inhibitors. Suitable mitotic inhibitors include, but are not limited to, auristatins, mytansinoids, drastatins, tubulsin, taxanes, eposilones, vinca alkaloids, and their analogues and derivatives.

[0064] In some embodiments, the pharmaceutically active portion of the NPC1-binding peptide conjugate is an immunomodulator. Suitable immunomodulators include, but are not limited to, macrophage type 1 stimulants, macrophage type 2 stimulants, dendritic cell stimulants, neutrophil stimulants, B cell stimulants, and T cell stimulants.

[0065] In some embodiments, the pharmaceutically active portion of the NPC1-binding peptide conjugate is an immunomodulator that is a macrophage type 1 stimulant. Suitable macrophage type 1 stimulants include, but are not limited to, paclitaxel, colony-stimulating factor-1 (CSF-1) receptor antagonists, IL-10 receptor antagonists, Toll-like receptor (TLR)-2 agonists, TLR-3 agonists, TLR-4 agonists, TLR-7 agonists, TLR-8 agonists, and TLR-9 agonists, as well as their analogues and derivatives.

[0066] In any embodiment, the macrophage type 1 stimulant is a CSF-1 receptor antagonist. Appropriate CSF-1 receptor antagonists include ABT-869 (the whole is incorporated herein by reference, ABT-869 (Guo et al., "Inhibition of Phosphorylation of the Colony-Stimulating Factor-1 Receptor (c-Fms) Tyrosine Kinase in Transfected Cells by ABT-869 and Other Tyrosine Kinase Inhibitors", Mol. Cancer. Ther. 5(4):1007-1012 (2006))) and imatinib (the whole is incorporated herein by reference, Guo et al., "Inhibition of Phosphorylation of the Colony-Stimulating Factor-1 Receptor (c-Fms) Tyrosine Kinase in Transfected Cells by ABT-869 and Other Tyrosine Kinase Inhibitors", Mol. Cancer. Ther. 5(4):1007-1012)) and imatinib (the whole is incorporated herein by reference, Guo et al., "Inhibition of Phosphorylation of the Colony-Stimulating Factor-1 Receptor (c-Fms) Tyrosine Kinase in Transfected Cells by ABT-869 and Other Tyrosine Kinase Inhibitors", Mol. Cancer. Ther. 5(4):1007-1012) (2006)), PLX3397 (the entire text is incorporated herein by reference; Mok et al., "Inhibition of CSF1 Receptor Improves the Antitumor Efficacy of Adoptive Cell Transfer Immunotherapy", Cancer Res. 74(1):153-161 (2014)), PLX5622 (the entire text is incorporated herein by reference; Dagher et al., "Colony-stimulating Factor 1 Receptor Inhibition Prevents Microglial Plaque Association and Improves Cognition in 3xTg-AD Mice", J. Neuroinflamm.This includes, but is not limited to, products such as 12:139 (2015), DCC-3014 (Deciphera Pharmaceuticals), BLZ945 (the entirety of which is incorporated herein by reference, Krauser et al., "Phenotypic and Metabolic Investigation of a CSF-1R Kinase Receptor Inhibitor (BLZ945) and its Pharmacologically Active Metabolite," Xenobiotica 45(2):107-123 (2015)), and GW2580 (the entirety of which is incorporated herein by reference, including, but is not limited to, Olmos-Alonso et al., "Pharmacological Targeting of CSF1R Inhibits Microglial Proliferation and Prevents the Progression of Alzheimer's-like Pathology," Brain 139:891-907 (2016)).

[0067] In any embodiment, the macrophage type 1 stimulant is an IL-10 receptor antagonist. Suitable IL-10 receptor antagonists include, but are not limited to, the peptide antagonists described in Naiyer et al., "Identification and Characterization of a Human IL-10 Receptor Antagonist," Hum. Immunol. 74(1):28-31 (2013), which are incorporated herein by reference in their entirety, and the IL-10 receptor antagonist antibodies described in U.S. Patent No. 7,553,932 to Von Herrath et al., which are also incorporated herein by reference in their entirety.

[0068] In any embodiment, the macrophage type 1 stimulant is a TLR-2 agonist. Suitable TLR-2 agonists for use in the methods described herein include the synthetic triacylated lipoprotein Pam3CSK4 and lipoteichoic acid (LTA) (the whole of which is incorporated herein by reference, Brandt et al., "TLR2 Ligands Induce NF-κB Activation from Endosomal Compartments of Human Monocytes," PLoS One 8(12):e80743). Suitable TLR-3 agonists include, but are not limited to, polyinosine:polycytidylic acid (Poly-I:C) (the whole of which is incorporated herein by reference, Smole et al., "Delivery System for the Enhanced Efficiency of Immunostimulatory Nucleic Acids," Innate Immun. 19(1):53-65 (2013)).Suitable TLR-4 agonists include MPL (the whole text is incorporated herein by reference; Engel et al., "The Pharmacokinetics of Toll-like Receptor Agonists and the Impact on the Immune System," Expert Rev. Clin. Pharmacol. 4(2):275-289 (2011)), glucopyranosyllipid-A (the whole text is incorporated herein by reference; Matzner et al., "Perioperative treatment with the new synthetic TLR-4 agonist GLA-SE reduces cancer metastasis without adverse effects," Int. J. Cancer 138(7):1754-64 (2016)), and Immunomax® (the whole text is incorporated herein by reference; Ghochikyan et al., "Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer," J. Trans. This includes, but is not limited to, Med. 12:322 (2014).

[0069] In any embodiment, the macrophage type 1 stimulant is a TLR-7 agonist. Suitable TLR-7 agonists include uridine / guanidine-rich single-stranded RNA (the whole is incorporated herein by reference, Engel et al., "The Pharmacokinetics of Toll-like Receptor Agonists and the Impact on the Immune System", Expert Rev. Clin. Pharmacol. 4(2):275-289 (2011)), 852A (the whole is incorporated herein by reference, Dudek et al., "First in Human Phase I Trial of 852A, a Novel Systemic Toll-like Receptor 7 Agonist, to Activate Innate Immune Responses in Patients With Advanced Cancer", Clin. Cancer Res. 13(23):7119-7125 (2007)), and resiquimod (the whole is incorporated herein by reference, Chang et al., "Topical resiquimod Promotes Priming of CTL to Parenteral Antigens", Vaccine 27(42):5791-5799 (2009), imidazoquinoline (the whole is incorporated herein by reference, Itoh et al., "The Clathrin-mediated Endocytic Pathway Participates in dsRNA-induced IFN-beta Production", J. Immunol. 181:5522-9 (2008)), ANA975 (the whole is incorporated herein by reference, Fletcher et al., "Masked oral Prodrugs of Toll-like Receptor 7 Agonists: a New Approach for the Treatment of Infectious Disease", Curr. Opin.This includes, but is not limited to, imiquimod (whole imiquimod is incorporated herein by reference in Engel et al., "The Pharmacokinetics of Toll-like Receptor Agonists and the Impact on the Immune System," Expert Rev. Clin. Pharmacol. 4(2):275-289 (2011)), and imiquimod (whole imiquimod is incorporated herein by reference).

[0070] In any embodiment, macrophage type 1 stimulants are TLR-8 agonists. Suitable TLR-8 agonists include, but are not limited to, resiquimod (whole resiquimod is incorporated herein by reference; Chang et al., "Topical resiquimod Promotes Priming of CTL to Parenteral Antigens", Vaccine 27(42):5791-5799 (2009)) and imidazoquinoline (whole resiquimod is incorporated herein by reference; Itoh et al., "The Clathrin-mediated Endocytic Pathway Participates in dsRNA-induced IFN-beta Production", J. Immunol. 181:5522-9 (2008)).

[0071] In any embodiment, the macrophage type 1 stimulant is a TLR-9 agonist. Suitable TLR-9 agonists include, but are not limited to, CpG-ODNs (the entirety of which is incorporated herein by reference, Yao et al., "Late Endosome / Lysosome-localized Rab7b Suppresses TLR-9-initiated Proinflammatory Cytokine and Type I IFN Production in Macrophages", J. Immunol. 183:1751-8 (2009)). Specific CpG-ODNs suitable for use are described herein by reference, Engel et al., "The Pharmacokinetics of Toll-like Receptor Agonists and the Impact on the Immune System", Expert Rev. Clin. Pharmacol. 4(2):275-289 (2011).

[0072] Other agents known in the art for reprogramming type 2 macrophages into type 1 macrophages (i.e., macrophage type 1 stimulants) for inclusion in the NPC1-binding peptide conjugate described herein include manganese dioxide nanoparticles (see, for example, Song et al., "Bioconjugated Manganese Dioxide Nanoparticles Enhance Chemotherapy Response by Priming Tumor-Associated Macrophages toward M1-like Phenotype and Attenuating Tumor Hypoxia," ACS Nano. 10:633-647 (2016), which is incorporated herein by reference in whole), and ferumoxytal nanoparticles (see, for example, Zanganeh, et al., "Iron oxide nanoparticles inhibit tumor growth by inducing pro-inflammatory macrophage polarization in tumor tissues," Nat. Nanotechnol. 11:986-994, which is incorporated herein by reference in whole). This includes mannosylated nanoparticle-encapsulated siRNA against IκBα (the entire siRNA is incorporated herein by reference, Ortega et al. "Manipulating the NF-kappaB pathway in macrophages using mannosylated, siRNA-delivering nanoparticles can induce immunostimulatory and tumor cytotoxic functions", Int. J. Nanomed. 2163-2177 (2016)).

[0073] In some embodiments, the pharmaceutically active portion of the NPC1-binding peptide conjugate is a macrophage type II stimulant. Suitable macrophage type II stimulants include, but are not limited to, IL-33, IL-4 receptor agonists, glucocorticoids, IL-10 receptor agonists, IL-1 receptor agonists, and their analogs and derivatives.

[0074] In any embodiment, macrophage type 2 stimulants are IL-4 receptor agonists. Suitable IL-4 receptor agonists include, but are not limited to, mutant IL-4 proteins. Exemplary mutant IL-4 proteins include, but are not limited to, the mutant IL-4 protein described in U.S. Patent No. 5,723,118 to Sebald, which is incorporated in whole by reference herein.

[0075] In any embodiment, the macrophage type 2 stimulant is a glucocorticoid. Glucocorticoids are a type of corticosteroid that is well known in the art and suitable for inducing the macrophage type 2 phenotype. Exemplary glucocorticoids for incorporation into the NPC1-binding peptide conjugate of this disclosure include, but are not limited to, cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone, deoxycorticosterone, and aldosterone.

[0076] In any embodiment, macrophage type 2 stimulants are IL-10 receptor agonists. Suitable IL-10 receptor agonists include, but are not limited to, the mutant IL-10 protein described in U.S. Patent No. 7,749,490 to Sommer, which is incorporated entirely by reference herein.

[0077] In any embodiment, macrophage type 2 stimulants are IL-1 receptor agonists. Suitable IL-1 receptor agonists include, but are not limited to, IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, and IL-36γ (all of which are incorporated herein by reference; Palomo et al., "The Interleukin (IL)-1 Cytokine Family - Balance Between Agonists and Antagonists in Inflammatory Diseases", Cytokine 76(1):25-37 (2015)).

[0078] In any embodiment, the pharmaceutically active moiety of the NPC1-binding peptide conjugate is a T-cell stimulant. In any embodiment, the T-cell stimulant is an interferon gene stimulating (STING) agonist. Suitable STING agonists are cyclic dinucleotides (CDNs), e.g., cyclic dimer guanosine monophosphate (c-di-GMP), cyclic dimer adenosine monophosphate (c-di-AMP), cyclic GMP-AMP (cGAMP), and dithio-(R) P ,R P )-[cyclic[A(2',5')pA(3',5')p(ADU-S100, Aduro Biotech), as well as small molecules, such as 5,6-dimethylxatetenone-4-acetic acid (DMXAA) and linked amidebenzimidazoles, but not limited thereto. Other STING agonists under development that are also suitable immunomodulators according to this disclosure include BMS-986301, E7766, GSK3745417, MK-1454, MK-2118, and SB11285.

[0079] In any embodiment, the pharmaceutically active portion of the NPC1-binding peptide conjugate is a dendritic cell stimulant. Suitable dendritic cell stimulants include, but are not limited to, CpG oligonucleotides, imiquimods, topoisomerase I inhibitors (e.g., camptothecin and its derivatives), microtubule depolymerizers (e.g., colchicine, podophyllotoxin and its derivatives), and their analogues and derivatives.

[0080] In any embodiment, the pharmaceutically active portion of the NPC1-binding peptide conjugate is a neutrophil stimulant. Suitable neutrophil stimulants include recombinant granulocyte colony-stimulating factor protein (filgrastim) and pegylated recombinant granulocyte colony-stimulating factor protein.

[0081] In some embodiments, the pharmaceutically active moiety of the NPC1-binding peptide conjugate is a nucleic acid molecule. Suitable nucleic acid molecule active moieties include, but are not limited to, antisense oligonucleotides, siRNAs, aptamers, miRNAs, immunostimulant oligonucleotides, splice-switching oligonucleotides, and guide RNAs, as well as their analogues and derivatives.

[0082] In any embodiment, the pharmaceutically active portion of the NPC1-conjugated peptide conjugate is attached to or incorporated into the delivery vehicle. Thus, in some embodiments, the NPC1-conjugated peptide conjugate comprises an NPC1-conjugated polypeptide attached to the delivery vehicle. In any embodiment, the delivery vehicle contains the pharmaceutically active portion.

[0083] In this aspect of the present disclosure, any suitable drug delivery vehicle known in the art can be conjugated to an NPC1-binding polypeptide to form the NPC1-binding peptide conjugate described herein. In any embodiment, the drug delivery vehicle may be a nanoparticle delivery vehicle, polymer-based particle, or lipid-based particle delivery vehicle known in the art (see, for example, Xiao et al., "Engineering Nanoparticles for Targeted Delivery of Nucleic Acid Therapeutics in Tumor", Mol. Ther. Meth. Clin. Dev. 12: 1-18 (2019), and Ni et al., "Synthetic Approaches for Nucleic Acid Delivery: Choosing the Right Carriers", Life 9(3): 59 (2019), which are incorporated herein by reference in their entirety, and may be used in the methods described herein.

[0084] Suitable nanoparticle delivery vehicles include, but are not limited to, gold nanoparticles, calcium phosphate nanoparticles, cadinum (quantum dot) nanoparticles, iron oxide nanoparticles, and particles derived from any other solid inorganic material known in the art.

[0085] Suitable polymer-based particles or polyplex carriers include cationic polymers, such as polyethyleneimine (PEI), and / or cationic polymers conjugated with neutral polymers such as polyethylene glycol (PEG) and cyclodextrin. Other suitable PEI conjugates for facilitating the delivery of nucleic acid molecules or expression vectors according to the methods described herein include, but are not limited to, PEI-salicyamide conjugates and PEI-steric acid conjugates. Other synthetic cationic polymers suitable for use as delivery vehicle materials include, but are not limited to, poly-L-lysine (PLL), polyacrylic acid (PAA), polyamidoamine-epichlorohydrin (PAE), and poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA). Natural cationic polymers suitable for use as delivery vehicle materials include, but are not limited to, chitosan, poly(lactic acid-co-glycolic acid) (PLGA), gelatin, dextran, cellulose, and cyclodextrin.

[0086] Suitable lipid-based vehicles include cationic lipid-based lipoplexes (e.g., 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)), neutral lipid-based lipoplexes (e.g., cholesterol and dioleoylphosphatidylethanolamine (DOPE)), anionic lipid-based lipoplexes (e.g., cholesteryl hemisuccinate (CHEMS)), and pH-sensitive lipid lipoplexes (e.g., 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA)). Other suitable lipid-based delivery particles incorporate ionizable DOSPA in lipofectamine and DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate).

[0087] In some embodiments, cancer treatment agents are PROTACs. Suitable PROTACs include, but are not limited to, BET degraders, such as the BET degrader disclosed in Pillow et al., "Antibody Conjugation of a Chimeric BET Degrader Enables In vivo Activity," ChemMedChem 15(1): 17-25 (2020), which is incorporated entirely by reference herein. Suitable PROTACs also include Ras pathway degraders. For example, the following are incorporated herein by reference: Bond et al., "Targeted Degradation of Oncogenic KRAS (G12C) by VHL-recruiting PROTAC", ACS Cent. Sci. 6(8):1367-75 (2020); Nabet et al., "The dTAG system for immediate and target-specific protein degradation", Nat Chem Biol. 14(5):431-41 (2018); Simpson et al., "Inducible degradation of target proteins through a tractable affinity-directed protein missile system", Cell Chem Biol. 27(9):1164-80.e5 (2020); Cheng et al., "Discovery of novel PDEδ degraders for the treatment of KRAS mutant colorectal cancer", J Med Chem. 63(14):7892-905 (2020); Crew et al. al.See also the Ras pathway degraders described in "Identification and Characterization of Von Hippel-Lindau-recruiting proteolysis targeting chimeras (PROTAC) of TANK-binding kinase 1," J Med Chem. 61(2):583-98 (2018); Vollmer et al., "Design, Synthesis, and Biological Evaluation of MEK PROTAC," J Med Chem. 63(1):157-62 (2020); and Yang et al., "Discovery of thalidomide-based PROTAC small molecules as the highly efficient SHP2 degraders," Eur J Med Chem. 218:113341 (2021).

[0088] In another embodiment, the second portion of the NPC1-binding peptide conjugate comprises a second polypeptide. In some embodiments, the second polypeptide is an unbound molecule. In some embodiments, the polypeptide is a second binding molecule. In some embodiments, the second binding molecule is an antibody or its antibody-binding domain. Antibodies used herein include molecules containing any protein or peptide that includes at least a portion of an immunoglobulin molecule, including, but not limited to, at least one, at least two, or at least three complementarity-determining regions (CDRs) of the heavy or light chain, a variable region of the heavy or light chain, a constant region of the heavy or light chain, a framework region, or any portion thereof. Antibodies include, but are not limited to, single-chain antibodies, single-domain antibodies (i.e., antibody fragments containing a single variable domain, which may be VHH, VH, or VL, that independently and specifically binds to an antigen or epitope of another V region or domain), complete antibodies, digested fragments, designated parts thereof, and variants, but are not limited to, parts of antibodies that mimic the structure and / or function of an antibody or a designated fragment or part thereof. Functional fragments include antigen-binding fragments that bind to a specific target. For example, antibody fragments that can bind to a specific target or part thereof include, but are not limited to, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction), and F(ab')2 (e.g., by pepsin digestion), Fd (e.g., by pepsin digestion, partial reduction, and re-aggregation), Fv, or scFv (e.g., by molecular biology techniques) fragments.

[0089] Another aspect of this disclosure relates to polynucleotides encoding NPC1-binding molecules or NPC1-binding peptide conjugates as described herein. The nucleic acid molecules of this disclosure include isolated polynucleotides, portions of expression vectors, or portions of linear DNA sequences containing linear DNA sequences used for in vitro transcription / translation, or vectors adapted for prokaryotic, eukaryotic, or filamentous phage expression, secretion, and / or display of the composition or its directed mutagen.

[0090] In one embodiment, the isolated polynucleotides of the present disclosure include isolated polynucleotides encoding the aforementioned binding molecules. Exemplary isolated polynucleotide molecules include a modified FG loop amino acid sequence of SEQ ID NO:2, a modified BC loop amino acid sequence of SEQ ID NO:15, and SEQ ID NO: 14 The present invention comprises an isolated polynucleotide molecule encoding an FN3 domain containing a modified DE loop amino acid sequence. In some embodiments, the FN domain encoded by the polynucleotide further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotide of the present invention comprises amino acid substitutions in residues corresponding to residues D3, R6, and D7. In some embodiments, the FN3 domain encoded by the polynucleotide of the present invention contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the FN3 domain encoded by the polynucleotide of the present invention contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the polynucleotide of the present invention encodes an FN3 domain containing the amino acid sequence (MbNPC1N-N8) of SEQ ID NO:32.

[0091] In some embodiments, the isolated polynucleotides of this disclosure include a modified FG loop amino acid sequence of SEQ ID NO:3, a modified BC loop amino acid sequence of SEQ ID NO:16, and SEQ ID NO: 22The polynucleotide encodes an NCP1-binding polypeptide having an FN3 domain containing a modified DE loop amino acid sequence. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further includes amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure includes amino acid substitutions in residues corresponding to residues D3, R6, and D7. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the polynucleotide of the Disclosure encodes an FN3 domain containing the amino acid sequence (MbNPC1N-N16) of SEQ ID NO:33.

[0092] In some embodiments, the isolated polynucleotide of the Disclosure encodes an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:4, a modified BC loop amino acid sequence of SEQ ID NO:17, and a modified DE loop amino acid sequence of SEQ ID NO:30. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, and D7. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the polynucleotides of this disclosure encode an FN3 domain containing the amino acid sequence (MbNPC1N-N18) of SEQ ID NO:34.

[0093] In some embodiments, the isolated polynucleotide of the Disclosure encodes an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO: 5, a modified BC loop amino acid sequence of SEQ ID NO: 18, and a modified CD loop amino acid sequence of SEQ ID NO: 23. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO: 1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, and E47. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the polynucleotides of this disclosure encode an FN3 domain containing the amino acid sequence (MbNPC1N-N22) of SEQ ID NO:35.

[0094] In some embodiments, the isolated polynucleotide of the Disclosure encodes an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO: 6, a modified BC loop amino acid sequence of SEQ ID NO: 19, and a modified CD loop amino acid sequence of SEQ ID NO: 23. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO: 1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, E47, and A74. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the polynucleotides of this disclosure encode an FN3 domain comprising the amino acid sequence (MbNPC1N-N23) of SEQ ID NO:36.

[0095] In some embodiments, the isolated polynucleotide of the Disclosure encodes an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:7, a modified BC loop amino acid sequence of SEQ ID NO:18, and a modified CD loop amino acid sequence of SEQ ID NO:24. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, E47, and T49. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the polynucleotide of this disclosure encodes an FN3 domain containing the amino acid sequence (MbNPC1N-N24) of SEQ ID NO:37.

[0096] In some embodiments, the isolated polynucleotide of the Disclosure encodes an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO: 8, a modified BC loop amino acid sequence of SEQ ID NO: 18, and a modified CD loop amino acid sequence of SEQ ID NO: 25. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO: 1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, and E47. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the polynucleotides of this disclosure encode an FN3 domain comprising the amino acid sequence (MbNPC1N-N26) of SEQ ID NO:38.

[0097] In some embodiments, the isolated polynucleotide of the Disclosure encodes an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:9, a modified BC loop amino acid sequence of SEQ ID NO:18, and a modified CD loop amino acid sequence of SEQ ID NO:26. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, and E47. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:39. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:39. In some embodiments, the polynucleotides of this disclosure encode an FN3 domain containing the amino acid sequence (MbNPC1N-N31) of SEQ ID NO:39.

[0098] In some embodiments, the isolated polynucleotides of the Disclosure encode an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:10, a modified BC loop amino acid sequence of SEQ ID NO:18, and a modified CD loop amino acid sequence of SEQ ID NO:26. In some embodiments, the FN domain encoded by the polynucleotides of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotides of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, E47, and T49. In some embodiments, the FN3 domain encoded by the polynucleotides of the Disclosure comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:40. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:40. In some embodiments, the polynucleotide of this disclosure encodes an FN3 domain containing the amino acid sequence (MbNPC1N-N34) of SEQ ID NO:40.

[0099] In some embodiments, the isolated polynucleotides of the Disclosure encode an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:11, a modified BC loop amino acid sequence of SEQ ID NO:20, and a modified CD loop amino acid sequence of SEQ ID NO:24. In some embodiments, the FN domain encoded by the polynucleotides of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotides of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, R33, E47, and T49. In some embodiments, the FN3 domain encoded by the polynucleotides of the Disclosure comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the polynucleotide of this disclosure encodes an FN3 domain containing the amino acid sequence (MbNPC1N-N35) of SEQ ID NO:41.

[0100] In some embodiments, the isolated polynucleotide of the Disclosure encodes an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:12, a modified BC loop amino acid sequence of SEQ ID NO:21, and a modified CD loop amino acid sequence of SEQ ID NO:27. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, Y31, R33, and E47. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:42. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:42. In some embodiments, the polynucleotide of this disclosure encodes an FN3 domain containing the amino acid sequence (MbNPC1N-N38) of SEQ ID NO:42.

[0101] In some embodiments, the isolated polynucleotide of the Disclosure encodes an NCP1-binding polypeptide having an FN3 domain comprising a modified FG loop amino acid sequence of SEQ ID NO:13, a modified BC loop amino acid sequence of SEQ ID NO:20, and a modified CD loop amino acid sequence of SEQ ID NO:28. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure further comprises amino acid substitutions in one or more residues corresponding to residues D3, R6, D7, Y31, R33, E47, T49, and A74 of SEQ ID NO:1. In some embodiments, the FN domain encoded by the polynucleotide of the Disclosure comprises amino acid substitutions in residues corresponding to residues D3, R6, D7, Y31, R33, E47, and T49. In some embodiments, the FN3 domain encoded by the polynucleotide of the Disclosure comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the FN3 domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the polynucleotide of this disclosure encodes an FN3 domain containing the amino acid sequence (MbNPC1C-C45) of SEQ ID NO:43.

[0102] The polynucleotides of this disclosure may be produced by chemical synthesis, such as solid-phase polynucleotide synthesis using an automated polynucleotide synthesizer, and assembled into complete single-stranded or double-stranded molecules. Alternatively, the polynucleotides of this disclosure may be produced by other techniques, such as PCR, followed by routine cloning. Techniques for producing or obtaining polynucleotides of a particular known sequence are well known in the art.

[0103] The polynucleotides described herein may include at least one non-coding sequence, such as a promoter or enhancer sequence, an intron, a polyadenylation signal, a cis sequence facilitating RepA binding, etc. The polynucleotide sequence may also include, for example, a marker or tag sequence, such as a histidine tag or HA tag facilitating protein purification or detection, a signal sequence, a fusion protein partner, such as RepA, Fc or a bacteriophage coat protein, such as pIX or pIII, or additional sequences encoding additional amino acids.

[0104] Another aspect of the disclosure is a vector comprising at least one or more of the polynucleotides described herein. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vector suitable for introducing the polynucleotides of the invention into a particular organism or genetic background by any means. Such vectors may also be expression vectors comprising nucleic acid sequence elements capable of controlling, regulating, inducing, or enabling the expression of the polypeptide encoded by such vectors. Such elements may include transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites facilitating the expression of the encoded polypeptide in a particular expression system. Such expression systems may be cell-based or cell-free systems well-known in the art.

[0105] Another aspect of this disclosure is a host cell containing the vector described above. The binding molecules and / or NPC1-binding peptide conjugates disclosed herein may optionally be produced by cell lines, mixed cell lines, immortalized cells, or clonal populations of immortalized cells, as is well known in the art (for example, the whole of which is incorporated herein by reference: Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001)).

[0106] The host cell selected for expression may be derived from a mammal and may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HeG2, SP2 / 0, HeLa, myeloma cells, lymphoma cells, yeast cells, insect cells, or plant cells, or any derivative, immortalized, or transformed cells thereof. Alternatively, the host cell may be a species or organism that cannot glycosylate polypeptides, such as a prokaryotic cell or prokaryote, such as BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any strain of the species of natural or engineered Escherichia coli (E. coli), the genus Klebsiella, or the genus Pseudomonas.

[0107] Another aspect of the present disclosure relates to methods for producing and isolating the binding molecules and NPC1-binding peptide conjugates described herein. The method includes culturing the isolated host cells of the present disclosure under conditions such that the binding molecule or NPC1-binding peptide conjugate is expressed, and purifying the expressed binding molecule or NPC1-binding peptide conjugate from the host cell culture.

[0108] The binding molecules and NPC1-binding peptide conjugates described herein can be purified from recombinant cell cultures by well-known methods, such as, for example, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography, or high performance liquid chromatography (HPLC).

[0109] The purified or isolated binding molecules and NPC1-binding peptide conjugates described herein may be linked to a variety of non-proteinoid polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. The binding molecules and / or NPC1-binding peptide conjugates may be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions prepared, for example, by coacervation or interfacial polymerization. Such techniques are disclosed in REMINGTON'S PHARMACEUTICAL SCIENCES, 16th edition, Oslo, A., Ed., (1980), which is incorporated in its entirety by reference herein.

[0110] For therapeutic use, the binding molecules and NPC1-binding peptide conjugates described herein may be prepared as pharmaceutical compositions containing an effective amount of the binding molecule or NPC1-binding peptide conjugate as the active ingredient in a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle administered together with the active compound. Such vehicles may be liquids, such as water, and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, or sesame oil. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. These solutions can be sterilized by conventional, well-known sterilization methods (e.g., filtration). The compositions may, if necessary, contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants, to approximate physiological conditions. The concentration of the binding molecule or NPC1-binding peptide conjugate described herein in such pharmaceutical formulations can vary considerably, ranging from less than about 0.5% by weight to typically about 1% by weight or at least 1% by weight to 15% or 20% by weight, and is primarily selected based on the required dose, fluid volume, viscosity, etc., depending on the specific administration method chosen. Suitable vehicles and formulations containing other human proteins, such as human serum albumin, are, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21 st This is described in Edition, Troy, DB ed., Lipincott Williams and Wilkins, 2006, Part 5, Pharmaceutical Manufacturing, pp. 691–1092. See, in particular, pages 958–989, which are incorporated in their entirety by reference herein.

[0111] The binding molecules and NPC1-binding peptide conjugates described herein can be used in an inseparable or isolated form. Furthermore, the binding molecules and their NPC1-binding peptide conjugates can be used alone or in a mixture containing at least one other binding molecule or its NPC1-binding peptide conjugate. In other words, the binding molecules and NPC1-binding peptide conjugates can be combined and used as a pharmaceutical composition, for example, containing two or more of their binding molecules, two or more of their NPC1-binding peptide conjugates, one binding molecule and NPC1-binding peptide conjugate, and its variants. For example, different but complementary binding molecules and / or NPC1-binding peptide conjugates can be combined in a monotherapy to achieve a desired therapeutic effect. Alternatively, binding molecules and NPC1-binding peptide conjugates with identical activity can also be combined in a monotherapy to achieve a desired therapeutic or diagnostic effect. Optionally, the mixture further comprises at least one other therapeutic agent.

[0112] Another aspect of this disclosure relates to combination therapeutics, which comprise an NPC1-binding polypeptide and a pharmaceutically active moiety as described herein.

[0113] According to this aspect of the present disclosure, the pharmaceutically active moiety of a combination therapeutic agent may be any pharmaceutically active moiety known in the art. Suitable pharmaceutically active moieties include, but are not limited to, small molecule active moieties, nucleic acid molecules, antibodies, antibody-binding fragments, antibody derivatives, proteins or polypeptide fragments thereof, proteolytic chimeras (PROTACs), and their analogs and derivatives.

[0114] As used herein, the term “combination therapy” refers to the administration of two or more therapeutic agents, namely NPC1-binding polypeptides or NPC1-binding peptide conjugates containing them, in combination with an active pharmaceutical moiety. In some embodiments, the combination therapy is administered substantially simultaneously, for example, in a single capsule or other delivery vehicle containing a fixed ratio of the active ingredients. In some embodiments, the combination therapy is administered in multiple capsules or delivery vehicles, each containing the active ingredients. In some embodiments, the therapeutic agents of the combination therapy are administered sequentially, approximately simultaneously, or at different times. For example, in one embodiment, the NPC1-binding polypeptide described herein is administered as a neoadjuvant, i.e., before the administration of the pharmaceutically active moiety. In other embodiments, the NPC1-binding polypeptide is administered as standard adjuvant therapy, i.e., after the administration of the pharmaceutically active moiety. In all aspects, combination therapy provides beneficial effects of drug combinations in treating specific pathological conditions, for example, cancer, particularly early-stage, high-grade, and treatment-resistant cancers.

[0115] In any embodiment, the pharmaceutically active portion of the combination therapy is the cancer treatment agent. In some embodiments, the cancer treatment agent of the combination therapy is a chemotherapeutic agent. Suitable chemotherapeutic agents include alkylating agents (e.g., chlorambucil, cyclophophamide, CCNU, melphalan, procarbazine, thiotepa, BCNU, and busulfan), antimetabolites (e.g., methotraxate, 6-mercaptopurine, and 5-fluorouracil), anthracyclines (daunorubicin, doxorubicin, idarubicin, epirubicin, and mitoxantrone), antitumor antibiotics (e.g., bleomycin), monoclonal antibodies (e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab), and The cancer treatment agents include, but are not limited to, zumab, ibritumomab, panitumumab, rituximab, tocitumomab, and trastuxmab, platinum (e.g., cisplatin and oxaliplatin), or plant alkaloids (e.g., topoisomerase inhibitors, vinca alkaloids, taxanes (e.g., paclitaxel), and epipodophyllotoxin). In some embodiments, the cancer treatment agent is selected from cyclophosphamide, gemcitabine, vorinostat, temozolomide, bortezomib, carmustine, and paclitaxel.

[0116] In some embodiments, the cancer treatment agent in a combination therapy is an immune checkpoint inhibitor. Appropriate immune checkpoint inhibitors include, but are not limited to, CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor selected from pembrolizumab (Keytruda), nivolumab (Opdivo), and semiprimab (ribtayo). In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor selected from atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor such as ipilimumab (Yervoy).

[0117] In some embodiments, the cancer treatment agent in combination therapy is an epidermal growth factor (EGFR) inhibitor. Appropriate EGFR inhibitors include, but are not limited to, gefitinib, erlotinib, lapatinib, cetuximab, osimertinib, panitumumab, neratinib, vandetanib, nesitumumab, and dacomitinib.

[0118] In some embodiments, the cancer treatment agent in a combination therapy is an mTOR inhibitor. Suitable mTOR inhibitors include, but are not limited to, sirolimus, everolimus, temsirolimus, and everolimus.

[0119] Another aspect of this disclosure relates to a method for treating cancer in a subject. This method comprises the steps of selecting a subject having cancer, and administering to the subject a pharmaceutical composition containing an amount effective to treat the cancer, which contains any of the following: an NPC1-binding polypeptide as described herein, an NPC1-binding peptide conjugate comprising an NPC1-binding polypeptide as described herein, a polynucleotide encoding an NPC1-binding polypeptide or an NPC1-binding peptide conjugate, or any of the above-mentioned agents.

[0120] According to all of the methods described herein, “Subject” refers to any animal or human having a condition that would benefit from NPC1 inhibition. In one embodiment, the subject is a mammal. Exemplary mammalian subjects include, but are not limited to, humans, non-human primates, dogs, cats, rodents (e.g., mice, rats, guinea pigs), horses, cattle and dairy cows, sheep, and pigs.

[0121] In some embodiments, the subjects include a type of cancer characterized by cancer cells having increased macropinocytosis compared to corresponding non-cancerous cells. In some embodiments, the cancer is characterized by cancer cells having oncogenic mutations in H-ras, N-ras, or K-ras. In some embodiments, the subjects include cancers selected from pancreatic cancer, lung cancer, breast cancer, colon cancer, glioma, solid tumor, melanoma, glioblastoma multiforme, leukemia, renal cell carcinoma, hepatocellular carcinoma, prostate cancer, and myeloma.

[0122] In some embodiments, the subject has a type of cancer that is resistant to or has become resistant to therapeutic treatment for the primary cancer, for example, resistant to or resistant to chemotherapy, prior to administration of the NPC1-binding molecule or the pharmaceutical composition containing the NPC1-binding molecule. The step of administering the NPC1-binding molecule or the pharmaceutical composition containing the NPC1-binding molecule is carried out in an amount effective in resensitizing the cancer cells to therapeutic treatment for the primary cancer.

[0123] In some embodiments, a method for treating a subject having cancer further includes the step of administering a cancer treatment agent together with the NPC1-binding polypeptide, the NPC1-binding peptide conjugate, or a pharmaceutical composition comprising the same. Suitable cancer treatment agents that can be administered in combination with the NPC1 compositions described herein as a combination therapy are described above.

[0124] According to the methods described herein, an NPC1-binding molecule or a pharmaceutical composition containing an NPC1-binding molecule is administered systemically or topically, either alone or in combination with one or more cancer therapeutic agents. Appropriate systemic administration methods of the therapeutic agents and / or combination therapeutic agents disclosed herein include, but are not limited to, oral administration, topical administration, transdermal administration, parenteral administration, intradermal administration, intrapulmonary administration, intramuscular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, or administration by intranasal infusion, intracavitary infusion or intravesical infusion, intraocular administration, intraarterial administration, intrafocal administration, or application to the mucous membrane. In certain embodiments, the therapeutic agents of the methods described herein are delivered orally. Appropriate topical administration methods of the therapeutic agents and / or combination therapeutic agents disclosed herein include, but are not limited to, catheterization, implantation, direct injection, skin / perdermal application, portal vein administration to relevant tissue, or any other topical administration techniques, methods, or procedures commonly known in the art. The methods affecting drug delivery vary depending on the type of therapeutic agent and the type of cancer to be treated.

[0125] In the methods disclosed herein, the therapeutically effective dose of an NPC1-binding molecule or a pharmaceutically active ingredient comprising an NPC1-binding molecule, either alone or in combination with an oncological agent, is the amount that, when administered over a specific interval, achieves one or more therapeutic benchmarks (e.g., slowing or stopping tumor growth, tumor regression, cessation of symptoms, etc.). The NPC1-binding molecule or a pharmaceutically active ingredient comprising an NPC1-binding molecule, either alone or in combination with an oncological agent, for use in the methods disclosed herein, may be administered to a subject once or multiple times. In embodiments where the therapeutic composition is administered multiple times, it may be administered at predetermined intervals, such as daily, every other day, weekly, or monthly. Alternatively, the therapeutic composition may be administered at irregular intervals, as needed, for example, based on symptoms, the patient's health condition, etc. For example, the therapeutically effective dose may be administered once daily (qd) over 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 15 days. Optionally, the state of the cancer or its regression may be monitored during or after the procedure by, for example, multiparametric ultrasound (mpUS), multiparametric magnetic resonance imaging (mpMRI), and nuclear imaging (positron emission tomography [PET]) of the subject. The dosage of the therapeutic agent or combination therapy administered to the subject may be increased or decreased depending on the state of the cancer or the detected regression of the cancer.

[0126] Those skilled in the art can easily determine this amount based on each individual subject (e.g., the amount of compound needed to achieve a specific therapeutic benchmark in the subject being treated) or based on a population (e.g., the amount of compound needed to achieve a specific therapeutic benchmark in the average subject from a particular population). Ideally, the therapeutically effective dose should not exceed the maximum tolerable dose at which more than 50% of the subjects being treated experience side effects that prevent further drug administration.

[0127] The therapeutically effective amount for a subject can vary depending on various factors including the type and degree of the symptoms, the sex, age, weight, or general health of the subject, the method of administration, and the type of salt or solvate compound, variations in sensitivity to the drug, the specific type of disease, etc.

[0128] Another aspect of the present disclosure relates to a method of treating an infectious disease in a subject. The method includes the steps of selecting a subject having an infectious disease and administering to the subject an effective amount of an NCP1-binding polypeptide or an NPC1-binding peptide conjugate described herein for treating the infectious disease.

[0129] In some embodiments, the subject having an infectious disease has a filovirus. In some embodiments, the filovirus is Ebola virus or Marburg virus. Ebola and other filoviruses attach to and enter host cells via endocytosis. The internally transported virus localizes in the late endosome / lysosome and is cleaved by cysteine proteases. The cleaved Ebola glycoprotein acts as a ligand for NPC1. Inhibition of this interaction by an NPC1 inhibitor blocks viral infection. See, for example, Basu et al., 「Novel Small Molecule Entry Inhibitors of Ebola Virus」, J. Infect. Dis. 212(Suppl 2): S425-434 (2015), which is hereby incorporated by reference in its entirety. Thus, the NPC1-binding molecules described herein can be administered to a subject having or at risk of having a filovirus infection as a therapeutic means to inhibit, arrest the progression of, and / or reduce an infectious disease in the subject.

[0130] In some embodiments, the subject with the infection has a coronavirus. In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or Middle East respiratory syndrome coronavirus (MERS-CoV). Loss-of-function mutations in NPC1 have been shown to induce cholesterol synthesis, which combats coronavirus-mediated suppression of cholesterol synthesis (the whole is incorporated herein by reference, Daniloski et al., "Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells", Cell https: / / doi.org / 10.1016 / j.cell.2020.10.030 (2020)). Therefore, the NPC1-binding molecules described herein can be administered to subjects with or at risk of coronavirus infection as therapeutic means to inhibit infection, inhibit the progression of infection, and / or mitigate infection in a subject.

[0131] Suitable pharmaceutical compositions comprising an NPC1-binding molecule and / or its NPC1-binding peptide conjugate for administration to subjects with infectious diseases are described above.

[0132] Another aspect of this disclosure relates to a method for enhancing the endosomal release of a pharmaceutically active moiety in a subject where such enhancement is needed. In one embodiment, the method comprises administering to a subject an NPC1-binding peptide conjugate as described herein, i.e., an NPC1-binding peptide conjugate comprising a first NPC1-binding polypeptide moiety and a second moiety attached to the first moiety and comprising a pharmaceutically active moiety. In another embodiment, the method comprises administering to a subject a combination therapeutic agent as described herein, i.e., a combination therapeutic agent comprising an NPC1-binding polypeptide and a pharmaceutically active moiety.

[0133] According to this aspect of the present disclosure, the pharmaceutically active moiety may include, but is not limited to, any pharmaceutically active moiety known in the art, including, small molecule active moieties, nucleic acid molecule active molecules, antibodies or their bound fragments, antibody derivatives, proteins or polypeptide fragments, proteolytic chimeras (PROTACs), and their analogs and derivatives.

[0134] In any embodiment, the subject has a neurodegenerative disease, and the pharmaceutically active portion is suitable for treating said neurodegenerative disease. Exemplary neurodegenerative diseases include, but are not limited to, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease.

[0135] In any embodiment, the subject has amyotrophic lateral sclerosis (ALS), and the method comprises the step of administering an NPC1-binding peptide conjugate or an NPC1 combination therapeutic agent containing an ALS therapeutic agent to treat ALS in the subject. Suitable ALS therapeutic agents include, but are not limited to, glutamate blockers (e.g., riluzole, rilutek, and other derivatives), endaravone, Radicava, muscle relaxants (e.g., baclofen, tizanidine, and other derivatives), and their analogues and derivatives.

[0136] In any embodiment, a subject has Parkinson's disease, and the method comprises the step of administering an NPC1-binding peptide conjugate or an NPC1 combination therapeutic agent, which includes a Parkinson's disease therapeutic agent, to treat Parkinson's disease in the subject. Suitable therapeutic agents for treating Parkinson's disease include dopamine stimulants (e.g., carbidopa, levodopa, carbidopa-levodopa, entacapone, cabergoline, tolcapone, bromocriptine, amantadine, and other derivatives), dopamine agonists (e.g., pramipexole, mirapex, ropinirole, Requip, rotigotine, Neupro, apomorphine, apokaine), cognitive enhancers (rivastigmine, and other derivatives), anti-tremor agents (e.g., benzotropin, and other inducers). This includes, but is not limited to, benzotropic agents, MAOB inhibitors (selegiline, Zelapar, rasagiline, azilecto, safinamide, zadago, and other derivatives), catechol O-methyltransferase (COMT) inhibitors (e.g., entacapone, comtan, opicapon, ongentis, tolcapone, tasmal), anticholinergic agents (e.g., benzotropin, cogentine, trihexyphenidyl, and other derivatives), and their analogues and combinations.

[0137] In any embodiment, a subject has Huntington's disease, and the method includes administering an NPC1-binding peptide conjugate or an NPC1 combination therapeutic agent, which includes a Huntington's disease therapeutic agent, to treat Huntington's disease in the subject. Suitable therapeutic agents for treating the symptoms of Huntington's disease include, but are not limited to, motor control agents (e.g., tetrabenazine, xenazine, duetetrabenazine, Austedo, and other derivatives), antipsychotics (e.g., haloperidol, haldol, fluphenazine, risperidone, risperdal, olanzapine, zyprexa, quetiapine, seroquel, and other derivatives), chorea inhibitors (e.g., amantadine, Gocovri ER, Osmolex ER, levetiracetam, Keppra, Elepsia XR, Spritam, clonazepam, Klonopin, and other derivatives), as well as their analogues and derivatives.

[0138] In any embodiment, a subject has Alzheimer's disease, and the method comprises the step of administering an NPC1-binding peptide conjugate or an NPC1 combination therapeutic agent comprising an Alzheimer's disease therapeutic agent to treat Alzheimer's disease in the subject. Suitable therapeutic agents for treating Alzheimer's disease include, but are not limited to, cognitive enhancers (e.g., memantine, namenda, and other derivatives), cholinesterase inhibitors (e.g., donepezil, aricept, galantamine, razadyne, rivastigmine, exelon, and other derivatives), aducanumab, Aduhelm, and their analogues and derivatives.

[0139] In another embodiment, a method for enhancing the endosomal release of a pharmaceutically active moiety comprises the step of administering an NPC1-binding peptide conjugate or NPC1 combination therapeutic agent to a subject having an inflammatory condition, wherein the pharmaceutically active moiety of the NPC1-binding peptide conjugate or combination therapeutic agent is suitable for treating the inflammatory condition. Exemplary inflammatory conditions that can be treated according to this method include, but are not limited to, rheumatoid arthritis, atherosclerosis, macular degeneration, osteoporosis, immunoinflammation, nonimmune inflammation, nephritis, tuberculosis, multiple sclerosis, arthritis, chronic obstructive pulmonary disease (COPD), and Alzheimer's disease.

[0140] Suitable anti-inflammatory agents for incorporation into NPC1-binding peptide conjugates or NPC1 combination therapies include nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, Advil, Motrin IB, naproxen sodium, Alive, and other derivatives), corticosteroids (e.g., prednisone and other derivatives), and conventional disease-modifying antirheumatic drugs (DMARDs) (e.g., methotrexate, Trexall, Otrexup, leflunomide, Arava, hydroxychloroquine, Plaquenil, sulfasalazine azulfite). This includes, but is not limited to, din and other derivatives, biological DMARDs (abatacept, orencia, adalimumab, humira, anakinra, kineret, certolizumab, cimzia, etanercept, enbrel, golimumab, simponi, infliximab, remicade, rituximab, rituxan, sarilumab, kevzara, tocilizumab, actemra, and other derivatives), targeted synthetic DMARDs (e.g., baricitinib, olumiant, tofacitinib, xeljanz, upadacitinib, rinvoq, and other derivatives), and their analogues and derivatives.

[0141] Further anti-inflammatory therapeutic agents for incorporation into NPC1-binding peptide conjugates or NPC1 combination therapies include, but are not limited to, statins (e.g., atorvastatin, lovastatin, simvastatin, pravastatin, and other derivatives), as well as other cholesterol agents (e.g., exetimibe, zetia, fenofibrate, gemfibrozil, and other derivatives), anticoagulants (e.g., aspirin and other derivatives), antithrombotic agents, and their analogues and derivatives.

[0142] In another embodiment, a method for enhancing the endosomal release of a pharmaceutically active portion comprises administering an NPC1-binding peptide conjugate or NPC1 combination therapeutic agent to a subject having a pharmaceutically active portion of the NPC1-binding peptide conjugate or combination therapeutic agent suitable for treating the pharmaceutically active portion. In any embodiment, the subject has a pharmaceutically active portion selected from osteoporosis or Paget's disease of bone.

[0143] In any embodiment, a subject has osteoporosis, and the method includes administering an NPC1 combination therapy agent, which includes an NPC1-binding peptide conjugate or an osteoporosis treatment agent, to treat the osteoporosis in the subject. Appropriate treatments for osteoporosis include, but are not limited to, bisphosphonates (e.g., alendronate, Binosto, Fosamax, ibandronate, Boniva, risedronate, Actonel, Atelvia, zoledronic acid, Reclast, Zometa, and other derivatives), denosumabs (e.g., Prolia, Xgeva, and other derivatives), hormone-related therapies (e.g., estrogen, raloxifene, Evista, testosterone, and other derivatives), bone-building drugs (e.g., teriparatide, Bonsity, Forteo, abaloparatide, Tymlos, romosozumab, Evenity, and other derivatives), as well as their analogues and derivatives.

[0144] In any embodiment, a subject has Paget's disease of bone, and the method comprises the step of administering an NPC1-binding peptide conjugate or an NPC1 combination therapeutic agent comprising a Paget's disease of bone therapeutic agent to treat Paget's disease of bone in the subject. Suitable Paget's disease of bone therapeutic agents include, but are not limited to, bisphosphonates (e.g., zoledronic acid, Reclast, Zometa, Pamidronate, Aredia, ibandronate, Boniva, and other derivatives), as well as oral bisphosphonates (e.g., Alendronate, Vinost, Risedronate, Actonel, Atelvia, and other derivatives), and their analogues and derivatives.

[0145] In another embodiment, a method for enhancing the endosomal release of a pharmaceutically active moiety comprises administering an NPC1-binding peptide conjugate or an NPC1 combination therapeutic agent to a subject having cancer, wherein the pharmaceutically active moiety of the NPC1-binding peptide conjugate or combination therapeutic agent is suitable for treating cancer. Pharmacoactive moieties known to treat cancer or available for treating cancer are described in detail above. In any embodiment, the subject has cancer associated with RAS pathway activation or hyperactivation (e.g., EGFR-induced cancer and PTEN-deficient cancer).

[0146] According to the methods described herein, NPC1-binding polypeptides, NPC1-binding peptide conjugates, or NPC1 combination therapies are administered systemically to treat various pathological conditions described herein (e.g., cancer, infections, neurodegenerative diseases, inflammatory conditions, and bone conditions) and / or to enhance endosomal release in subjects where such treatment is necessary. Appropriate systemic administration methods are disclosed above and include, but are not limited to, oral administration, topical administration, transdermal administration, parenteral administration, intradermal administration, intrapulmonary administration, intramuscular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, or administration by intranasal infusion, intracavitary infusion or intravesical infusion, intraocular administration, intraarterial administration, intrafocal administration, or application to mucous membranes.

[0147] The therapeutically effective dose of an NPC1-conjugated polypeptide, NPC1-conjugated peptide conjugate, or NPC1 combination therapy for treating the conditions described herein (e.g., cancer, infection, neurodegenerative diseases, inflammatory conditions, and bone conditions) and / or for enhancing the endosomal release of a pharmaceutically active portion is the amount that, when administered over a specific interval, achieves one or more therapeutic benchmarks (e.g., slowing or stopping infection, inhibiting infection, stopping symptoms, etc.). The NPC1-conjugated polypeptide, NPC1-conjugated peptide conjugate, or NPC1 combination therapy containing the same may be administered to the subject once or multiple times. In embodiments in which the therapeutic composition is administered multiple times, it may be administered at predetermined intervals, for example, daily, every other day, weekly, or monthly. Alternatively, the therapeutic composition may be administered at irregular intervals, for example, as needed based on symptoms, the patient's health condition, etc. For example, a therapeutically effective dose may be administered once daily for 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 15 days.

[0148] The therapeutically effective dose may vary depending on various factors, including the type and severity of symptoms, the subject's sex, age, weight, or overall health, the method of administration, as well as the type of salt or solvated compound, variations in drug sensitivity, and the specific type of filovirus infection. [Examples]

[0149] The following embodiments are provided to illustrate aspects of the present disclosure and are not intended to limit the scope of the present disclosure.

[0150] Example 1 - NPC1 is upregulated in KRas tumor tissue. To explore the association between mutant KRas tumor tissue and NPC1 expression, pancreatic cancer tissue was analyzed because these samples are predominantly mutant KRas (approximately 97%). NPC1 expression in tumor tissue was found to be higher compared to normal adjacent tissue (Figure 1A). Furthermore, Kaplan-Meier analysis revealed that patients with high NPC1 expression had poor survival rates (Figure 1B). To test whether NPC1 inhibition has an in vitro growth inhibitory effect, itraconazole, an NPC1-targeting tool compound, was used. Dose-dependent inhibition of cell proliferation by itraconazole was observed in mutant KRas CRC cells (Figure 2).

[0151] For drug discovery, we established a robust set of biomarkers for NPC1 inhibition. As previously demonstrated in fibroblasts, siRNA-mediated NPC1 knockdown and small molecule-mediated NPC1 inhibition resulted in cholesterol accumulation in endosomes. NPC1 was confirmed to play a necessary role in cholesterol transport in mutant KRas cancer cell lines (Figure 3A). Furthermore, autophagy flux inhibition has been shown to be a consequence of NPC1 inhibition. Using immunofluorescence and biochemical analysis of the autophagy marker LC3B, it was confirmed that the autophagy flux is blocked by NPC1 knockdown and small molecule inhibition (Figures 3B and 3C).

[0152] Example 2 - NPC1 monobody for treating cancer Unfortunately, available small-molecule NPC1 tool compounds can easily pass through the cell membrane and are therefore not specific for targeting cancer cells. Consequently, we developed a novel molecular entity to selectively target NPC1 within cancer cells.

[0153] Interestingly, it was discovered that the monobody, a small synthetic binding protein, is internally translocated via macropinocytosis by mutant Ras cancer cell lines. For this reason, we created an NPC1-specific monobody that is internally translocated by mutant Ras-expressing cancer cells to facilitate binding to and inhibition of NPC1 within the endosomal compartment.

[0154] Screening of two monobody libraries under intellectual property rights generated the NPC1 monobodies described herein, i.e., binding molecules having any single amino acid sequence from SEQ ID NO: 32 to 43, which exhibited strong target binding at 2.5 nM. The generated monobodies showed no difference in NPC1 binding ability under cholesterol-depleted or cholesterol-loaded conditions. Furthermore, the NPC1 monobodies showed stronger binding affinity to NPC1 at acidic pH levels similar to those encountered in endosomes (pH 5-6) compared to a more neutral environment (pH 7.5) (Figures 4-6).

[0155] A major advantage of monobodies compared to antibodies is their lower manufacturing cost. However, if the monobodies need to be refolded or aggregated during production, the manufacturing cost can exceed that of antibodies. However, the monobodies described herein do not require refolding and exhibit little to no aggregation during production. Using both imaging-based and biochemical approaches, the monobodies described herein were confirmed to inhibit NPC1 in cell culture. NPC1-targeted monobody clones N23 (SEQ ID NO: 36) and N34 (SEQ ID NO: 40) showed the greatest NPC1 inhibition by endosomal cholesterol accumulation (Figure 7). Figure 7B DLD1 cells (Ras mutation, colon) were treated with the top monobody hits and cholesterol was measured in the Philippines. By targeting the cholesterol-binding domain of NPC1, our preliminary data show that the two monobodies (N23 and N34) improve cholesterol capture and induce vesicle disruption (Figures 7A-7C).

[0156] To demonstrate that NPC1-targeted monobodies have specificity for mutant Ras cancer cells, each monobody was observed to induce LC3B accumulation in mutant KRas-inducible HeLa cell lines (Figure 8). NPC1-targeted monobody clones N23 and N34 did not induce LC3B accumulation in HeLa cells (Figure 8A, macropinocytosis-negative), but showed efficacy in HeLa KRasV12 cells (Figure 8B, macropinocytosis-positive). When candidate monobodies were compared in a CRC cell proliferation assay, N34 showed a proliferation inhibitory effect, while N23 did not show significant inhibition (Figure 9).

[0157] The macropinocytosis-dependent uptake was confirmed in CRC cell lines. Wild-type KRas CRC cells (HCA7) are macropinocytosis-negative and do not take up the N34 monobody (Figure 10A; left column of the image). However, mutant KRas CRC cells (DLD-1 and HCT-116) are macropinocytosis-positive and efficiently transport the N34 monobody into their cells (Figure 10A; center and right columns of the image). Therefore, N34 shows dose-dependent LC3B accumulation in HCT-116 cells but not in HCA7 cells (Figure 10B). This suggests that macropinocytosis-dependent NPC1 inhibition by the NPC1-targeting monobody. Finally, it was shown that when the N34 monobody was injected intratumorally into xenografts, the monobody was taken up by CK8-positive tumor cells (Figure 11). N34-positive tumor cells showed cholesterol accumulation compared to non-targeted control monobody (FN). Furthermore, in another study, two hours after injection, the N34-positive regions of xenografts showed active accumulation of cholesterol and LC3B compared to the N34-negative regions (Figure 12). Hyperactivation of ERK was also observed upon NPC1 knockdown in vitro (Figure 13A). This observation was confirmed in vivo using an N34 monobody (Figure 13B). Treatment of NPC1 knockdown cells with dacomitinib, a selective and irreversible EGFR inhibitor, blocked ERK hyperactivation, suggesting that ERK hyperactivation upon NPC1 inhibition may be a result of EGFR activation (Figure 14). This is further supported by the observation that EGFR activation was observed upon NPC1 inhibition with N34 in vivo (Figure 15).

[0158] Example 3 - NPC1-binding peptide conjugate enhances endosomal escape of the pharmaceutically active portion. A split GFP assay was developed to measure protein endosomal escape. GFP1-10, lacking the 11β domain necessary for fluorescence, was stably expressed in mutant Ras PDAC MIA PaCa-2 cells. Endosomal escape of GFP11β is required for a positive signal. NPC1-targeted monobodies and control monobodies were co-delivered with the free GFP11β domain. Fluorescence was observed with the NPC1-targeted monobodies, but not with the unbound control monobodies (FN) (Figure 16).

[0159] Endosomal escape was also observed in small molecule assays (Figure 17). Calcein is a membrane-impermeable liquid-phase uptake marker that is semi-quenched when in close proximity to other calcein molecules within a vesicular compartment, but dequenched upon intracellular release and molecular diffusion, resulting in increased cellular fluorescence. Similar to the GFP1-10 split assay, co-delivery of the NPC1-targeted moiety resulted in endosomal escape, but this was not observed with the control monobody, and cellular fluorescence was increased. The NPC1-targeted monobody showed increased escape when paired with nanoparticles, which may be advantageous for improved nanoparticle cargo escape (Figures 18A-18B). The NPC1-targeted monobody demonstrated the ability to induce escape from small molecules, biologics, and nanoparticles.

[0160] While preferred embodiments have been described and explained in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, etc., can be made without departing from the spirit of the disclosure, and that these will therefore be deemed to be within the scope of the disclosure as defined in the appended claims.

Claims

1. A Niemann-Pick disease type C1 (NPC1) binding polypeptide containing a fibronectin type III (FN3) domain, The FN3 domain includes β-chains A to G, each separated by corresponding loops AB, BC, CD, DE, EF, and FG, respectively. The conjugated polypeptide is (1) FG loop amino acid sequence of SEQ ID NO:10, BC loop amino acid sequence of SEQ ID NO:18, and CD loop amino acid sequence (N34) of SEQ ID NO:26; or (2) The FG loop amino acid sequence of SEQ ID NO:6, the BC loop amino acid sequence of SEQ ID NO:19, and the CD loop amino acid sequence of SEQ ID NO:23 (N23); or (3) FG loop amino acid sequence of SEQ ID NO: 5, BC loop amino acid sequence of SEQ ID NO: 18, and CD loop amino acid sequence (N22) of SEQ ID NO: 23; or (4) The FG loop amino acid sequence of SEQ ID NO:7, the BC loop amino acid sequence of SEQ ID NO:18, and the CD loop amino acid sequence (N24) of SEQ ID NO:24; or (5) FG loop amino acid sequence of SEQ ID NO:8, BC loop amino acid sequence of SEQ ID NO:18, and CD loop amino acid sequence (N26) of SEQ ID NO:25; or (6) The FG loop amino acid sequence of SEQ ID NO:9, the BC loop amino acid sequence of SEQ ID NO:18, and the CD loop amino acid sequence of SEQ ID NO:26 (N31); or (7) FG loop amino acid sequence of SEQ ID NO:11, BC loop amino acid sequence of SEQ ID NO:20, and CD loop amino acid sequence (N35) of SEQ ID NO:24; or (8) FG loop amino acid sequence of SEQ ID NO:12, BC loop amino acid sequence of SEQ ID NO:21, and CD loop amino acid sequence (N38) of SEQ ID NO:27; or (9) FG loop amino acid sequence of SEQ ID NO:13, BC loop amino acid sequence of SEQ ID NO:20, and CD loop amino acid sequence (C45) of SEQ ID NO:28; or (10) FG loop amino acid sequence of SEQ ID NO:2, BC loop amino acid sequence of SEQ ID NO:15, and DE loop amino acid sequence of SEQ ID NO:14 (N8); or (11) FG loop amino acid sequence of SEQ ID NO:3, BC loop amino acid sequence of SEQ ID NO:16, and DE loop amino acid sequence of SEQ ID NO:22 (N16); or (12) FG loop amino acid sequence of SEQ ID NO:4, BC loop amino acid sequence of SEQ ID NO:17, and DE loop amino acid sequence of SEQ ID NO:30 (N18) including, Niemann-Pick disease type C1 (NPC1) binding polypeptide.

2. The conjugated polypeptide according to claim 1, wherein β-chains A to G comprise the amino acid sequences of VSDVPRD (A), SLLISWD (B), YYRITYGET (C), QEFTV (D), TATIS (E), GVDYTITVYA (F), and PISINYRT (G) of SEQ ID NO:

1.

3. The conjugated polypeptide according to claim 1, wherein the β-chain A is an amino acid sequence (aa1-7 of SEQ ID NO: 1) which is VSDVPRD, or a variant amino acid sequence thereof, comprising a variant amino acid sequence which includes one or more substitutions selected from D3S, R6T, and D7K.

4. The binding polypeptide according to claim 1, comprising one or more of the following: β-chain B, which includes the amino acid sequence YYRITYGET (aa31-39 of SEQ ID NO: 1), or a variant amino acid sequence thereof, and which includes a variant amino acid sequence containing Y31H and / or one of R33V, R33D, or R33F; The amino acid sequence that is QEFTV (SEQ ID NO: 1 aa46-50), or a variant amino acid sequence thereof, comprising a variant amino acid sequence containing an amino acid substitution selected from one of E47T and E47K, and / or one of T49K and T49A, β-chain D; and β-chain F containing the amino acid sequence GVDYTITVYA (aa65-74 of SEQ ID NO: 1), or a variant amino acid sequence including the A74T amino acid substitution.

5. The conjugated polypeptide according to any one of claims 1 to 4, wherein the FN3 domain comprises the FG loop amino acid sequence of SEQ ID NO:10, the BC loop amino acid sequence of SEQ ID NO:18, and the CD loop amino acid sequence (N34) of SEQ ID NO:

26.

6. The conjugated polypeptide according to claim 1, wherein the FN3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40, 36, 32-35, 37-39, and 41-43.

7. The conjugated polypeptide according to claim 6, comprising the amino acid sequence of SEQ ID NO:

40.

8. A first portion comprising the conjugated polypeptide according to any one of claims 1 to 7, A second portion is connected to the first portion and is selected from a pharmaceutically active portion, a diagnostic portion, a half-life extension portion, a delivery vehicle, a prodrug, a second binding molecule, a polymer, and an unbinding protein. A conjugate containing Niemann-Pick disease type C1 (NPC1) conjugated peptide.

9. The NPC1-binding peptide conjugate according to claim 8, wherein the second portion is a pharmaceutically active portion.

10. The NPC1-binding peptide conjugate according to claim 9, wherein the pharmaceutically active portion is one of a small molecule, a nucleic acid molecule, an antibody or its antigen-binding fragment, an antibody derivative, a protein or its polypeptide fragment, and a proteolytic chimera (PROTAC).

11. The NPC1-binding peptide conjugate according to claim 10, wherein the nucleic acid molecule is an siRNA, an aptamer, a miRNA, an immunostimulant oligonucleotide, a splice-switching oligonucleotide, or a guide RNA.

12. The NPC1-binding peptide conjugate according to claim 9, wherein the pharmaceutically active portion is a cancer treatment agent or an immunomodulator.

13. The aforementioned cancer treatment agent is selected from antimetabolites, alkaloids, alkylating agents, mitotic inhibitors, antitumor antibiotics, DNA binding agents, toxins, antiproliferative agents, DNA antagonists, radionuclides, thermoblisters, proteolytic chimeras (PROTACs), and nucleic acid inhibitors. The alkaloid is selected from the group consisting of duocalmycin, docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, vindesine, and their analogs and derivatives. The alkylating agent is selected from the group consisting of busulfan, improsulfan, pigosulfan, benzodepa, carbocon, metsuredepa, uredepa, altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, chlorambucyl, chloranafadin, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide HCl, melphalan, nobemeviquin, perphosphamide, phenesterine, prednimustine, trophosphamide, uracil mustard, carmustine, chlorozotosine, fotemustine, lomustine, nimustine, semustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitractol, pipobromane, temozolomide, and their analogs and derivatives. The antitumor antibiotic is selected from the group consisting of acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, calitiamycin, carbicin, cardinophylline, chromomycin, dactinomycin, daunorubicin, 6-diazo-5-oxo-l-norleucine, doxorubicin, epirabicin, idarubicin, menogalil, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, pirarubicin, plicamycin, porphyromycin, puromycin, pyrrolobenzodiazepine, streptonigrin, streptozocin, tubercidine, dinostatin, zolubicin, and their analogs and derivatives. The antimetabolite is selected from the group consisting of SN-38, denopterin, edatrexate, mercaptopurine (6-MP), methotrexate, pyritrexime, pteropterin, pentostatin (2'-DCF), tomdex, trimethrexate, cladridine, fludarabine, thiamiprine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, floxuridine, fluorouracil, gemcitabine, tegafur, hydroxyurea, urethane, and their analogs and derivatives. The antiproliferative agent is selected from the group consisting of acegraton, amsacrin, bisanthren, camptothecin, dephosphamide, demecolsin, diaziquan, diflomotecan, eflornithine, eriptinium acetate, etogluside, etopside, fenretinide, gallium nitrate, hydroxyurea, lamelarin D, ronidamin, miltefosine, mitogluzone, mitoxantrone, mopidamol, nitracrin, pentostatin, fenamet, 2-ethyl-hydrazide podophyllate, procarbazine, razoxane, sobuzoxane, spirogermanium, teniposide, tenuazonic acid, triadiquan 2,2',2''-trichlorotriethylamine, and their analogs and derivatives. The mitotic inhibitor is selected from the group consisting of auristatin, mytansinoids, drastatin, tubulsin, taxane, eposilone, vinca alkaloids, and their analogs and derivatives. The immunomodulator is a macrophage type 1 stimulant. The immunomodulator is a macrophage type 2 stimulant. The immunomodulator is a T-cell stimulant. The immunomodulator is a dendritic cell stimulant, or The immunomodulator is a neutrophil stimulant. The NPC1-binding peptide conjugate according to claim 12.

14. (i) The macrophage type 1 stimulant is selected from the group consisting of paclitaxel, colony-stimulating factor-1 (CSF-1) receptor antagonist, IL-10 receptor antagonist, Toll-like receptor (TLR)-2 agonist, TLR-3 agonist, TLR-4 agonist, TLR-7 agonist, TLR-8 agonist, and TLR-9 agonist, (ii) The macrophage type 2 stimulant is selected from the group consisting of IL-33, IL-4 receptor agonists, glucocorticoids, IL-10 receptor agonists, and IL-1 receptor agonists. (iii) The T cell stimulant is an interferon gene stimulator (STING) agonist, (iv) The dendritic cell stimulant is selected from the group consisting of CpG oligonucleotides, imiquimod, camptothecin, colchicine, podophyllotoxin, and derivatives thereof, and / or (v) The neutrophil stimulant is recombinant granulocyte colony-stimulating factor protein (filgrastim) or pegylated recombinant granulocyte colony-stimulating factor protein. The NPC1-binding peptide conjugate according to claim 13.

15. The NPC1-binding peptide conjugate according to claim 8, wherein the second portion of the conjugate is a delivery vehicle or a diagnostic portion, or The NPC1-binding peptide conjugate according to claim 9, wherein the pharmaceutically active portion is connected to a delivery vehicle.

16. (i) The delivery vehicle is selected from the group consisting of nanoparticles, polymer-based particles, and lipid-based particles, and / or (ii) The diagnostic portion is selected from the group consisting of a fluorescent dye, a radioisotope, a contrast agent suitable for imaging, a radionuclide having a chelating agent, and a photosensitizer. NPC1-binding peptide conjugate according to claim 15

17. An isolated polynucleotide encoding an NPC1-binding polypeptide according to any one of claims 1 to 7 or an NPC1-binding peptide conjugate according to claim 8.

18. A vector comprising the isolated polynucleotide described in claim 17.

19. A host cell comprising the vector according to claim 18.

20. A conjugated polypeptide according to any one of claims 1 to 7, an NPC1-conjugated peptide conjugate according to any one of claims 8 to 16, an isolated polynucleotide according to claim 17, or a vector according to claim 18, Pharmaceutical carriers and A pharmaceutical composition containing [the specified substance].

21. A conjugated polypeptide according to any one of claims 1 to 7, The pharmaceutically active part and A combination therapy that includes this.

22. The combination therapeutic agent according to claim 21, wherein the pharmaceutically active portion is selected from the group consisting of a small molecule, a nucleic acid molecule, an antibody or its antigen-binding fragment, an antibody derivative, a protein or its polypeptide fragment, a proteolytic chimera (PROTAC), and a cancer therapeutic agent.

23. The cancer treatment agent is a chemotherapy agent, The combination therapeutic agent according to claim 22, which is an immune checkpoint inhibitor, or an epidermal growth factor (EGFR) inhibitor, or an mTOR inhibitor.

24. (i) The chemotherapeutic agent is selected from the group consisting of cyclophosphamide, gemcitabine, vorinostat, temozolomide, bortezomib, carmustine, and paclitaxel, and / or (ii) The immune checkpoint inhibitor is selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. The combination therapeutic agent according to claim 23.

25. A pharmaceutical composition according to claim 20 for use in a method for treating cancer in a subject, wherein the method comprises the following steps: A step of administering to a subject with cancer an amount of the pharmaceutical composition effective in treating the cancer. A pharmaceutical composition containing [the specified substance].

26. The cancer is characterized by cancer cells having increased macropinocytosis compared to the corresponding non-cancerous cells, or The aforementioned cancer is characterized by cancer cells having oncogenic mutations in H-ras, N-ras, or K-ras. The pharmaceutical composition according to claim 25.

27. ​​The pharmaceutical composition according to claim 25 or 26, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, glioma, solid tumor, melanoma, glioblastoma multiforme, leukemia, renal cell carcinoma, hepatocellular carcinoma, prostate cancer, or myeloma.

28. The above method is a step of administering a cancer treatment agent together with the pharmaceutical composition. A pharmaceutical composition according to any one of claims 25 to 27, further comprising:

29. The pharmaceutical composition according to claim 28, wherein the cancer treatment agent is a chemotherapeutic agent, an immune checkpoint inhibitor, or an epidermal growth factor (EGFR) inhibitor or an mTOR inhibitor.

30. (i) The chemotherapeutic agent is selected from the group consisting of cyclophosphamide, gemcitabine, vorinostat, temozolomide, bortezomib, carmustine, paclitaxel, mitoxantrone, and capecitabine, or (ii) The immune checkpoint inhibitor is selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. The pharmaceutical composition according to claim 29.

31. The above method is a step of administering the pharmaceutical composition together with radiotherapy. A pharmaceutical composition according to any one of claims 25 to 30, further comprising the above.

32. A pharmaceutical composition for use in a method for treating an infectious disease in a subject, comprising a conjugated polypeptide according to any one of claims 1 to 7 or an NPC1-conjugated peptide conjugate according to any one of claims 8 to 16, wherein the method comprises the following steps: A step of administering to a subject with an infectious disease an amount of the pharmaceutical composition effective in treating the infectious disease. A pharmaceutical composition containing [the specified substance].

33. The pharmaceutical composition according to claim 32, wherein the infection is caused by a filovirus or coronavirus.

34. (i) The filovirus is the Ebola virus or the Marburg virus, and / or (ii) The coronavirus is either Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) or Middle East Respiratory Syndrome Coronavirus (MERS-CoV), The pharmaceutical composition according to claim 33.

35. For use in methods that enhance the endosomal release of the pharmaceutically active portion in subjects where such enhancement is necessary. A first portion comprising the conjugated polypeptide according to any one of claims 1 to 7, A second portion is connected to the first portion and includes a pharmaceutically active portion. A pharmaceutical composition comprising an NPC1-binding peptide conjugate containing, or An NPC1-binding polypeptide according to any one of claims 1 to 7, The pharmaceutically active part and A combination therapeutic agent comprising the following steps: The process of administering the pharmaceutical composition or the combination therapeutic agent to the subject. A pharmaceutical composition or combination therapeutic agent containing [the specified substance].

36. The pharmaceutical composition or combination therapeutic agent according to claim 35, wherein the pharmaceutically active portion is selected from the group consisting of a small molecule, a nucleic acid molecule, an antibody or its antigen-binding fragment, an antibody derivative, a protein or its polypeptide fragment, and a proteolytic chimera (PROTAC).

37. The subject has a neurodegenerative disease, and the pharmaceutically active portion is suitable for treating the neurodegenerative disease, The subject has an inflammatory state, and the pharmaceutically active portion is suitable for treating the inflammatory state. The subject has a bone pathology condition, and the pharmaceutically active portion is suitable for treating the bone pathology condition, or The subject has cancer, and the pharmaceutically active portion is suitable for treating the cancer. The pharmaceutical composition or combination therapeutic agent according to claim 35 or 36.

38. The neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease. The aforementioned inflammatory condition is rheumatoid arthritis or atherosclerosis. The bone condition is osteoporosis or Paget's disease of bone, and / or The aforementioned cancer is associated with RAS pathway activation. The pharmaceutical composition or combination therapeutic agent according to claim 37.

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