Drug delivery system for use in esophageal mucosa comprising agent effective in treatment or prevention of esophageal diseases

Abstract

The present invention relates to a drug delivery system for use in the esophageal mucosa, comprising at least one tablet-like preparation, in particular a film-shaped, foil-shaped or sheet-shaped preparation, comprising an agent effective in the treatment or prevention of esophageal diseases, a release mechanism, and a trigger mechanism, wherein the trigger mechanism is adapted to trigger the release of the tablet preparation by the release mechanism at a predetermined action site.

Description

Technical Field

[0001] The present invention relates to a drug delivery system comprising a medicament effective for treating or preventing esophageal diseases, particularly for application to the lumen, including but not limited to the esophageal mucosa, and for treating esophageal diseases, particularly Barrett's esophagus, esophageal stenosis and / or esophageal cancer. Background Art

[0002] Esophageal diseases include, for example, Barrett's esophagus or Barrett's disease. Barrett's esophagus (BE) is a premalignant condition characterized by the presence of intestinal metaplasia, which has specialized columnar epithelium interspersed with goblet cells, which are usually only found in the small and large intestines, and the columnar epithelium replaces the normal squamous epithelium in the distal part of the esophagus. The cells of Barrett's esophagus are divided into four categories: non-dysplasia (metaplasia), low-grade dysplasia, high-grade dysplasia, and adenocarcinoma. High-grade dysplasia and early adenocarcinoma can be treated with endoscopic mucosal resection, endoscopic submucosal dissection, radiofrequency ablation, or cryoablation. Advanced adenocarcinoma can be treated with surgical resection or palliative therapy. Patients with non-dysplastic mucosa are managed endoscopically with annual observation. The new 2022 guidelines of the American College of Gastroenterology recommend endoscopic eradication therapy for patients with BE and high-grade dysplasia and patients with BE and low-grade dysplasia. The European Society of Gastrointestinal Endoscopy recommends endoscopic eradication for persistent (≥6 months) low-grade dysplasia. In high-grade dysplasia, the risk of developing esophageal cancer is approximately 28% or higher per patient-year (Peters, Y., Al-Kaabi, A., Shaheen, NJ, et al., Barrett oesophagus. Nat Rev Dis Primers 5, 35 (2019)).

[0003] Esophageal cancer is the sixth most common cancer worldwide, causing an estimated 450,000 deaths each year. There are two distinct histological types of esophageal cancer: squamous cell carcinoma and adenocarcinoma. Esophageal squamous cell carcinoma is more common in East Asian and Middle Eastern countries, such as China, Iran, and Turkmenistan, while adenocarcinoma is more prevalent in Western countries. Over the past few decades, the incidence of adenocarcinoma has increased, while the incidence of squamous cell carcinoma has remained stable. Esophageal squamous cell carcinoma (ESCC) is one of the most lethal forms of human malignancy, characterized by late diagnosis, metastasis, treatment resistance, and frequent recurrence.

[0004] Drugs are delivered to the gastrointestinal lumen, especially the esophageal lumen, usually through endoscopically guided submembranous application. Topical applications of active ingredients include drug-coated esophageal stents or oral viscous drugs. Drugs currently under investigation involve orodispersible or orally disintegrating tablets, aerosols, or gel-like drugs with higher viscosity to increase contact time.

[0005] However, there are some challenges in applying active ingredients topically to the gastrointestinal membrane, especially the esophageal membrane. For example, it is difficult to topically apply a high dose of drug sufficient to achieve a therapeutically effective local concentration over a period of time. Possible reasons for too low concentrations at the site to be treated include degradation or inactivation of the drug by digestive secretions and enzymes, dilution by intestinal fluid, malabsorption, unavailability of prodrugs that require activation at the site to be treated, and too short a residence time at the site of action to effectively exert the drug's effect. When using liquid or gel-like drug delivery systems, short residence time at the site of action and / or too low local concentrations are particularly a problem. Therefore, high doses must be applied to achieve sufficient concentrations at the site to be treated. Higher applied doses of active ingredients are usually accompanied by an increase in intestinal absorption side effects and higher bioavailability; therefore, the dose of the active ingredient should be kept as low as possible.

[0006] It is known that BMP2 / 4 inhibitors are effective in treating Barrett's esophagus and are therefore effective in preventing esophageal adenocarcinoma. WO 2016 / 043577 discloses several BMP2 / 4 inhibitors, such as microRNA, SMAD inhibitors, intracellular protein phosphatases that interfere with BMP2 / 4 signaling, and extracellular molecules that bind to BMPs, which inhibit or enhance BMP activity. In addition, WU, JB, "Effects of siRNA-targeting BMP-2 on the abilities of migration and invasion of human liver cancer SMMC7721 cells and its mechanism", Cancer Gene Therapy, 2011, Vol. 18, pages 20-25, describes small interfering RNA (siRNA) against BMP-2. WO2018 / 193129 discloses several BMP2 / 4 inhibitors, including isolated, synthetic or recombinant antibodies that effectively inhibit BMP2 and BMP4 signaling. Inhibiting this signaling effectively restores the normal tissue lining of the esophagus and is therefore an effective treatment for Barrett's esophagus to prevent esophageal adenocarcinoma. Although these inhibitors are very effective, prior art liquid or gel-like drug delivery systems still show low concentrations at the site to be treated.

[0007] PD-1 (programmed cell death protein 1) is an immunosuppressive receptor, mainly expressed on activated T cells and B cells. Interactions with its ligands have been shown to weaken T cell responses in vitro and in vivo. Blocking the interaction between PD-1 and one of its ligands, PD-L1, has been shown to enhance tumor-specific CD8+T cell immunity, and therefore may help the immune system to clear tumor cells. The role of PD-1 in cancer has been confirmed in the literature. It is well known that the tumor microenvironment can protect tumor cells from effective immune destruction. PD-L1 has been shown to be expressed on many mouse and human tumors (and can be induced by IFNγ on most PD-L1 negative tumor cell lines), and is believed to mediate immune escape (Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505 (2003)). Blocking the PD-1 / PD-L1 interaction can enhance tumor-specific T cell immunity, thereby contributing to the immune system's clearance of tumor cells and the development of cancer immunotherapy.

[0008] Pembrolizumab is a humanized monoclonal antibody that targets PD-1, thereby blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2. Pembrolizumab is approved for the treatment of melanoma, Hodgkin's lymphoma, lung cancer, head and neck cancer, gastric cancer, urothelial carcinoma, cervical cancer, and breast cancer. Until recently, pembrolizumab was approved as a first-line treatment in combination with chemotherapy for the treatment of advanced or metastatic esophageal cancer. Each infusion of pembrolizumab is administered parenterally, resulting in systemic exposure, which is associated with serious side effects such as inflammation caused by autoimmune reactions. Antibodies against PD-1 represent a promising target for regional / local delivery, especially to the esophageal membrane, which may reduce side effects.

[0009] Paclitaxel belongs to the taxane class and is a chemotherapeutic agent currently used to treat a variety of cancers, including ovarian cancer, esophageal cancer, breast cancer, lung cancer, Kaposi's sarcoma, cervical cancer, and pancreatic cancer. Due to its low oral bioavailability, paclitaxel is usually administered systemically by intravenous injection, and its administration is accompanied by serious side effects, such as hair loss, heart problems, bone marrow suppression, numbness, allergic reactions, increased risk of infection, muscle pain, and diarrhea. Therefore, chemotherapeutic agents represent a promising target for regional / local delivery, especially to the esophageal membrane, which may reduce chemotherapy toxicity.

[0010] There remains a need for suitable drug delivery systems, particularly drug delivery systems for delivery to the esophagus, which can deliver agents effective for treating or preventing esophageal diseases for effective treatment while allowing for administration of the lowest possible dose to reduce side effects and / or delivering stable polynucleotides or polypeptides. Summary of the invention

[0011] Purpose of the Invention

[0012] An object of the present invention is to provide a drug delivery system capable of orally / topically administering an agent effective for treating or preventing esophageal diseases, for treating esophageal diseases, and having enhanced local efficacy.

[0013] Another object of the present invention is to provide a delivery system that allows the use of stable polynucleotides and polypeptides.

[0014] Another object of the present invention is to provide a delivery system that allows the use of agents effective in treating or preventing esophageal diseases such as Barrett's esophagus or esophageal cancer, such as adenocarcinoma, esophageal junction cancer or squamous cell carcinoma, at low doses, thereby minimizing potential side effects.

[0015] The objects of the invention are achieved by the subject matter of the independent claims. Preferred embodiments are the subject matter of the dependent claims.

[0016] Claimed Subject Matter

[0017] The present invention provides a drug delivery system for esophageal mucosa, comprising

[0018] at least one sheet-shaped preparation, in particular a film-shaped, foil-shaped or wafer-shaped preparation, comprising an active pharmaceutical ingredient;

[0019] Release agencies; and

[0020] Trigger mechanism, where

[0021] The trigger mechanism is suitable for triggering the release mechanism to release the preparation at a predetermined site of action, and wherein the release mechanism is suitable for releasing the preparation while moving along the esophageal mucosa,

[0022] wherein the drug delivery system further comprises a housing, wherein the housing contains a formulation, and wherein the housing comprises an aperture as part of a release mechanism, the aperture being configured to allow the formulation to exit the housing, and wherein the trigger mechanism is a retaining device that is part of or attached to the formulation such that the formulation spreads or unfolds as the dosage form moves down the esophageal mucosa and exits the housing through the aperture,

[0023] It is characterized in that the active pharmaceutical ingredient comprises an agent effective for treating or preventing esophageal diseases, preferably in combination with one or more additional active pharmaceutical ingredients.

[0024] In one embodiment, an agent effective for treating or preventing esophageal disease comprises or consists of an inhibitory polynucleotide, preferably an inhibitory polynucleotide in combination with a nucleic acid delivery system; an antibody; or an anti-proliferative agent.

[0025] In one embodiment, the inhibitory polynucleotide is selected from the group consisting of siRNA molecules, antisense oligonucleotides and aptamers.

[0026] In one embodiment, the inhibitory polynucleotide comprises or consists of an siRNA molecule or an antisense oligonucleotide and targets an RNA transcript encoding a BMP2 and / or BMP4 polypeptide (preferably a BMP2 and / or BMP4 polypeptide as shown in SEQ ID NO: 15 or SEQ ID NO: 16) or a portion thereof, or wherein the inhibitory polynucleotide comprises an aptamer that interferes with the activity of a BMP2 or BMP4 polypeptide (preferably a BMP2 or BMP4 polypeptide as shown in SEQ ID NO: 15 or SEQ ID NO: 16).

[0027] In one embodiment, the inhibitory polynucleotide comprises or consists of an siRNA molecule or an antisense oligonucleotide and targets an RNA transcript as shown in SEQ ID NO: 20 or SEQ ID NO: 21, or a portion thereof.

[0028] In one embodiment, the siRNA molecule comprises or consists of a double-stranded region, wherein the double-stranded region comprises a sequence as shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, preferably SEQ ID NO:18, or any other sequence comprising 80% or more sequence identity between the siRNA molecule and the target RNA transcript or a portion thereof, wherein preferably, the target RNA transcript or a portion thereof encodes a BMP2 and / or BMP4 polypeptide, preferably a BMP2 and / or BMP4 polypeptide as shown in SEQ ID NO:15 or SEQ ID NO:16.

[0029] In one embodiment, the siRNA molecule comprises or consists of a double-stranded region, wherein the double-stranded region comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand together form a double-stranded region, and the antisense strand is complementary to a target RNA transcript or a portion thereof as shown in SEQ ID NO: 20 or SEQ ID NO: 21. In one embodiment, the siRNA molecule does not comprise an overhang. In another embodiment, the siRNA molecule comprises an overhang of one or more nucleotides.

[0030] In one embodiment, the siRNA molecule comprises or consists of a double-stranded region, wherein the length of the double-stranded region is 15 to 30 base pairs, preferably 19 to 25 base pairs.

[0031] In one embodiment, the siRNA molecule comprises or consists of BMP2-siRNA 1, BMP2-siRNA 2 or BMP2-siRNA 3, preferably BMP2-siRNA 2, as shown in the following table:

[0032]

[0033] Each of the above sequences in the table contains an overhang of two nucleotides dTdT (deoxythymidine) or UU (uridine) attached to the 3' end of each strand.

[0034] In one embodiment, the nucleic acid delivery system is selected from the group consisting of liposomes, lipid bilayers, micelles, emulsions, cationic polymers and nanoparticles, preferably lipid nanoparticles or polymer nanoparticles.

[0035] In one embodiment, the antibody or binding fragment thereof is an isolated or recombinant or synthetic antibody or binding fragment thereof.

[0036] In one embodiment, the antibody or binding fragment thereof binds within, preferably binds to an epitope consisting of, the following residues:

[0037] a) residues 10-17, 45-56 and 69 of BMP4 (SEQ ID NO: 1),

[0038] b) residues 24-31, 57-68, 70-72, 89, 91, 101, 103, 104 and 106 of BMP4 (SEQ ID NO: 1), or

[0039] c) residues 34, 35, 39, 86-88, 90, 97, 98, 100, 102 and 109 of BMP4 (SEQ ID NO: 1)

[0040] Preferably, the binding of the antibody or fragment thereof is determined by epitope binning (surface plasmon resonance (SPR) sandwich cross-binding) and / or HADDOCK modeling.

[0041] In one embodiment, the antibody or binding fragment thereof that binds within residues 10-17, 45-56 and 69 of BMP4 specifically binds to at least Lys12, Arg15, Asp46 and Pro50 of BMP4 (SEQ ID NO: 1), or

[0042] The antibody or binding fragment thereof that binds within residues 24-31, 57-68, 70-72, 89, 91, 101, 103, 104, and 106 of BMP4 specifically binds to at least Asp30, Trp31, Leu66, and Lys101 of BMP4 (SEQ ID NO: 1), or

[0043] The antibody or binding fragment thereof that binds within residues 34, 35, 39, 86-88, 90, 97, 98, 100, 102, and 109 of BMP4 specifically binds to at least Ala34, Gln39, Ser88, Leu90, and Leu100 of BMP4 (SEQ ID NO: 1),

[0044] Preferably, the specific binding of the antibody or fragment thereof is determined by epitope binning (surface plasmon resonance (SPR) sandwich cross-binding) and / or HADDOCK modeling.

[0045] In one embodiment, the antibody or binding fragment thereof that binds to at least Lys12, Arg15, Asp46, and Pro50 of BMP4 comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:2, or a sequence that differs therefrom by no more than 1 amino acid, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:3, or a sequence that differs therefrom by no more than 1 amino acid, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:4, or a sequence that differs therefrom by no more than 1 amino acid, or

[0046] An antibody or binding fragment thereof that binds to at least Asp30, Trp31, Leu66, and Lys101 of BMP4 comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:5, or a sequence that differs therefrom by no more than 1 amino acid, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:6, or a sequence that differs therefrom by no more than 1 amino acid, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:7, or a sequence that differs therefrom by no more than 1 amino acid, or

[0047] The antibody or its binding fragment that binds to at least Ala34, Gln39, Ser88, Leu90 and Leu100 of BMP4 comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:8 or a sequence that differs from it by no more than 1 amino acid, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:9 or a sequence that differs from it by no more than 1 amino acid, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:10 or a sequence that differs from it by no more than 1 amino acid.

[0048] In one embodiment, the antibody or binding fragment thereof that binds to at least Lys12, Arg15, Asp46 and Pro50 of BMP4 comprises the amino acid sequence of SEQ ID NO: 11 or a sequence that is at least 70%, preferably 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical thereto, or

[0049] The antibody or binding fragment thereof that binds to at least Asp30, Trp31, Leu66 and Lys101 of BMP4 comprises the amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 70%, preferably 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical thereto, or

[0050] The antibody or binding fragment thereof that binds to at least Ala34, Gln39, Ser88, Leu90 and Leu100 of BMP4 comprises the amino acid sequence of SEQ ID NO:13 or a sequence that is at least 70%, preferably 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical thereto.

[0051] In one embodiment, the antibody or binding fragment thereof is a single chain antibody.

[0052] In one embodiment, the active pharmaceutical ingredient comprises two different antibodies or binding fragments thereof.

[0053] In one embodiment, the antibody or antibody fragment binds to PD-1, preferably human PD-1.

[0054] In one embodiment, the antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprises:

[0055] a. at least one CDR (complementarity determining region) selected from the group consisting of SEQ ID NO: 30, 31, 32, 36, 37 and 38, or a variant of any of said sequences; and / or

[0056] b. at least one CDR selected from the group consisting of SEQ ID NO: 33, 34, 35, 39, 40 and 41, or a variant of any of said sequences.

[0057] In one embodiment, the antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprises:

[0058] a. light chain CDR SEQ ID NO: 30, 31 and 32, or a variant of any of said sequences; and / or heavy chain CDR SEQ ID NO: 33, 34 and 35, or a variant of any of said sequences; or

[0059] b. Light chain CDR SEQ ID NOs: 36, 37 and 38, or a variant of any of said sequences; and / or heavy chain CDR SEQ ID NOs: 39, 40 and 41, or a variant of any of said sequences.

[0060] In one embodiment, the antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprises:

[0061] a. a heavy chain variable region comprising an amino acid sequence selected from the group consisting of:

[0062] i. SEQ ID NO: 26 or a variant thereof;

[0063] ii. SEQ ID NO: 28 or a variant thereof;

[0064] iii. amino acid residues 20 to 139 of SEQ ID NO:42 or a variant thereof; and

[0065] iv. an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% sequence identity to amino acid residues 20 to 139 of SEQ ID NO:42;

[0066] And further includes

[0067] b. a light chain variable region comprising an amino acid sequence selected from the group consisting of:

[0068] i. SEQ ID NO: 27 or a variant thereof;

[0069] ii. SEQ ID NO: 29 or a variant thereof;

[0070] iii. amino acid residues 20 to 130 of SEQ ID NO:44 or a variant thereof;

[0071] iv. amino acid residues 20 to 130 of SEQ ID NO:45 or a variant thereof;

[0072] v. amino acid residues 20 to 130 of SEQ ID NO:46 or a variant thereof; and

[0073] vi. an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% sequence identity to amino acid residues 20 to 130 of SEQ ID NO:44, 45 or 46.

[0074] In one embodiment, the antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprises:

[0075] a. A heavy chain comprising an amino acid sequence selected from the group consisting of:

[0076] i. amino acid residues 20 to 466 of SEQ ID NO:43 or a variant thereof; and

[0077] ii. amino acid residues 20 to 469 of SEQ ID NO:47 or a variant thereof; and

[0078] b. a light chain comprising an amino acid sequence selected from the group consisting of:

[0079] i. amino acid residues 20 to 237 of SEQ ID NO: 48 or a variant thereof;

[0080] ii. amino acid residues 20 to 237 of SEQ ID NO:49 or a variant thereof; and

[0081] iii. amino acid residues 20 to 237 of SEQ ID NO: 50 or a variant thereof.

[0082] In one embodiment, an antibody or antibody fragment that binds to PD-1, wherein the antibody or antibody fragment:

[0083] a. Binds to human PD-1 with a KD of about 100 pM or less;

[0084] b. binds to human PD-1 with a KD of about 30 pM or less;

[0085] c. binds to human PD-1 with about the same KD as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 44;

[0086] d. binds to human PD-1 with about the same KD as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 45;

[0087] e. About 7.5x10 5 1 / M·s or faster k assoc Binds to human PD-1;

[0088] f. About 1x10 6 1 / M·s or faster k assoc Binds to human PD-1;

[0089] g. About 2x10 -5 1 / s or slower k dissoc Binds to human PD-1;

[0090] h. About 2.7x10 -5 1 / s or slower k dissoc Binds to human PD-1;

[0091] i. About 3x10 -5 1 / s or slower k dissoc Binds to human PD-1; and / or

[0092] j. With IC of about 1 nM or lower 50 Block the binding of human PD-L1 or human PD-L2 to human PD-1.

[0093] In one embodiment, the antiproliferative agent is selected from the group consisting of: a taxane; a pyrimidine analog, and a platinum-based agent.

[0094] In one embodiment, the antiproliferative agent is a taxane selected from the group consisting of: paclitaxel ((2α,4α,5β,7β,10β,13α)-4,10-bis(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropionyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-yl-benzoate), docetaxel ((2R,3S)-4-acetoxy-2α-benzyloxy-13-[3-(N-tert-butoxycarbonyl)amino-2-hydroxy-3-phenyl]propionyl-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxotax-11-en-13α-yl ester); and cabazitaxel Paclitaxel is preferred.

[0095] In one embodiment, the antiproliferative agent is a pyrimidine analog, wherein the pyrimidine analog is a uracil analog, preferably 5-fluorouracil or capecitabine.

[0096] In one embodiment, the antiproliferative agent is a platinum-based agent, wherein the platinum-based agent is selected from the group consisting of: cisplatin ((SP-4-2)-diamminedichloroplatinum (II); DDP) or a salt thereof, carboplatin (diammineplatin (II)-cyclobutane-1,1-dicarboxylate) or a salt thereof, nedaplatin ( or its salt and oxaliplatin (Pt-(oxalate)-trans-I-diaminocyclohexane) or its salt. The platinum-based agent can be further selected from the group consisting of: triplatin tetranitrate (BBR3464) or its salt, phenanthriplatin (cis-[Pt(NH3)2-(phenanthridine)Cl]NO3) or its salt, picoplatin (picoplatin) or its salt and satraplatin ((OC-6-43)-bis(acetate-O)aminodichloro(cyclohexylamine)platinum; JM216) or its salt.

[0097] In one embodiment, the drug delivery system according to the present invention is used in therapy.

[0098] In one embodiment, the drug delivery system according to the present invention is used to treat or prevent esophageal diseases.

[0099] In one embodiment, the drug delivery system according to the present invention is used to treat or prevent esophageal diseases caused by or associated with immune system defects. In one embodiment, the drug delivery system according to the present invention is used to treat or prevent cancer.

[0100] In one embodiment, the drug delivery system according to the present invention is used to treat or prevent esophageal diseases, such as esophageal cancer.

[0101] In one embodiment, the drug delivery system according to the present invention is used to treat or prevent Barrett's esophagus, esophageal stenosis and / or esophageal cancer, such as adenocarcinoma, esophageal junction cancer or squamous cell carcinoma.

[0102] In one embodiment, the drug delivery system according to the invention is used in diagnosis, preferably wherein the active pharmaceutical ingredient is combined with a diagnostic marker.

[0103] In one embodiment, the drug delivery system according to the invention is used for in vitro diagnosis, preferably wherein the active pharmaceutical ingredient is combined with a diagnostic marker.

[0104] In one embodiment, diagnosis includes monitoring cellular uptake of an active pharmaceutical ingredient, monitoring the pathway of an active pharmaceutical ingredient within a tissue or organ, or monitoring treatment success, such as tumor size. DETAILED DESCRIPTION

[0105] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0106] It is noted that use of the indefinite article "a" or "an" means "one or more." Thus, for example, the term "esophageal disease" includes the incorporation of "an" and "more than one" esophageal disease.

[0107] As used herein, the terms "include" or "comprises" mean "including, but not limited to". The terms are intended to be open ended to specify the presence of any stated features, elements, integers, steps or components, but do not preclude the presence of adding one or more other features, elements, integers, steps, components or combinations thereof. The terms "include" or "comprises" therefore encompass the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the terms "include" or "comprises" used throughout the application, especially in the claims, may be replaced by the terms "consisting of" or "consisting essentially of".

[0108] Drug delivery systems comprising pharmaceutical formulations, but with different active pharmaceutical ingredients, and their uses are described in PCT / EP2015 / 002601, which is incorporated herein by reference in its entirety, in particular with respect to the invention according to PCT / EP2015 / 002601. Figure 8a , 8b In other words, the size, shape and composition of the housing, aperture, release and trigger mechanism and retaining device have been described in the references at least to a large extent.

[0109] The drug delivery system as described in PCT / EP2015 / 002601 is designed such that it comprises at least one sheet-like preparation, in particular a film-shaped, foil-shaped or sheet-shaped preparation, comprising an active pharmaceutical ingredient; a release mechanism; and a trigger mechanism, wherein the trigger mechanism is suitable for triggering the release mechanism to release the sheet-like preparation at a predetermined site of action, in particular the gastrointestinal tract. Figure 8a , 8b , 8c, the dosage form has an elongated strip-shaped preparation containing an active pharmaceutical ingredient. The preparation can be arranged in a compact state and an expanded state. The dosage form has a capsule device, such as a shell, which includes a hollow space for accommodating a compact preparation, the capsule device has a hole, and the first end of the preparation extends through the hole in the compact state, so that the preparation can be pulled out of the hollow space into the surrounding area of ​​the capsule, thereby converting the preparation from the compact state to the expanded state.

[0110] The drug delivery system according to the present invention is orally administered, and improves the local utilization rate of the medicament for the effective treatment or prevention of esophageal diseases contained in the preparation. This is in contrast to traditional oral administration systems (such as tablets or capsules), which are absorbed into the blood circulation through the gastrointestinal tract and only reach the site / position to be treated.

[0111] Because the medicament for effective treatment or prevention of esophageal disease is provided in the form of sheet preparation, particularly film-shaped, foil-shaped, thin sheet-shaped or strip-shaped preparation, local utilization is improved. This advantageously allows the sheet preparation (and the medicament for effective treatment or prevention of esophageal disease present therein) to be directly released to the position / position (treatment site) to be treated, such as esophageal mucosa. Therefore, preferably, the sheet preparation of large area is exposed to mucosa, i.e., exposed to esophageal mucosa, particularly exposed to the inner cavity, including but not limited to exposure to esophageal mucosa. When exposed to mucosa, the sheet preparation releases the medicament for effective treatment or prevention of esophageal disease. In addition, the preferred direct contact between mucosa and preparation causes the effective effect of the medicament for effective treatment or prevention of esophageal disease at the treatment site. Due to the direct delivery of medicament to the treatment site, compared with the use of conventional preparations such as suspensions or solutions, less medicament is required to cause systemic bioavailability reduction and concentration reduction in adjacent areas such as healthy areas. In addition, the effective effect of the medicament for effective treatment or prevention of esophageal disease by the membrane of the well-vascularized reabsorption site of the gastrointestinal tract (such as the small intestine or oral cavity) reduces side effects. Direct delivery to the treatment site also allows for a reduction in the dosage of the medicament contained in the formulation that is effective in treating or preventing esophageal disease, thereby advantageously further reducing side effects. Therefore, the drug delivery system of the present invention is particularly suitable for delivering all medicaments that are limited in therapeutic use due to high systemic toxicity or low systemic availability (e.g., due to high first-pass effect or loss / impairment of activity through the gastrointestinal tract).

[0112] Drug delivery system according to the present invention also advantageously allows relatively simple and discrete processing and simple, special space-saving storage.When compared with solution and gel, the medicament included in the effective treatment or prevention of esophageal disease in drug delivery system according to the present invention has improved stability, for example, under high heat and high humidity.No free water is usually left in drug delivery system according to the present invention, which further improves stability and reduces composition such as moldy or unavailable risk.Extra additives can be avoided, such as preservatives or other stabilizers, which is advantageous because known these additives can cause allergies or other side effects.

[0113] Furthermore, the destruction of the active pharmaceutical ingredient before reaching the intended site of action, for example by gastric acid and / or digestive enzymes, is advantageously minimized by the drug delivery system according to the present invention.

[0114] Release mechanism

[0115] Release mechanism relates to the mechanism that sheet preparation is expanded and released from capsule device (for example shell).Described shell contains sheet preparation of compact form.After trigger mechanism has started to release, described release mechanism releases preparation from shell.Described release mechanism preferably releases sheet preparation by can at least partly pull out shell of preparation.Therefore, sheet preparation is adjusted so that described sheet preparation can be expanded to predetermined degree by release mechanism.For example, described shell contains preparation of folded form, and described release mechanism expands preparation from its compact form (for example, from folded form) into its expanded form (for example, unfolded form).Therefore, described release mechanism causes the unfolding of preparation.In compact form, described preparation has less spatial range, for example, preparation is gathered together, coiled or wound or forms less spatial form in another way.This also allows to provide little dosage form, i.e. little shell, which makes oral intake of particularly drug delivery system more convenient for patient.In its expanded form, the surface area of ​​described sheet preparation increases by expansion, for example, by unfolding sheet preparation, particularly the surface area of ​​preparation containing the medicament of effective treatment or prevention of esophageal disease increases. Preferably, the surface area of ​​the preparation, particularly the surface area containing the medicament for effective treatment or prevention of esophageal diseases and contacting the esophageal mucosa, is in the order of magnitude of the esophageal mucosal surface area. When the shell moves downward along the esophageal mucosa, the preparation is released. For example, during the process of the patient swallowing the dosage form, the preparation is released from the shell through the hole. Therefore, the shell includes a hole as a part of the release mechanism, and the hole is configured to allow the preparation to leave the shell.

[0116] hole

[0117] In this respect, the hole is in the shell, i.e., forms an opening in the capsule device. In a preferred embodiment of the drug delivery system, the hole is formed as a slit. The slit is arranged so that the sheet preparation is released from the shell through the hole. This slit can be embodied in different arrangements and structures. For example, this hole is described in EP21175427.0, EP21175436.1, PCT / EP2015 / 002601 and PCT / EP2020 / 056934, and these patents are incorporated herein by reference in their entirety about the contents of the capsule device and hole.

[0118] Trigger mechanism / holding device

[0119] The drug delivery system comprises a trigger mechanism, wherein the trigger mechanism is suitable for triggering a release mechanism to release the sheet-like preparation at a predetermined action site, wherein the trigger mechanism is a holding device that is a part of the preparation or attached to the preparation.

[0120] Preferably, the preparation comprises a retaining device, and further preferably, the preparation comprises a retaining device positioned at one end of the preparation, and the retaining device particularly extends out of the shell through a hole. When the retaining device is fixed, the preparation can be taken out from the capsule device by pulling motion and / or force. The fixing of the retaining device is preferably realized by the retaining device being connected to a retainer. This retainer can be a rope member, such as a thick rope, a fine line or a tether. In a preferred embodiment, the retaining device is connected to an end of the preparation and an end of the thick rope, and the other end of the thick rope is fixed to an applicator, such as a retainer fixed to an applicator.

[0121] Preferably, the holding means is attached to the sheet-like preparation. Thus, the retainer, ie the string member or a part of the string member, forms the holding means. For example, one end of a thick string connected to the preparation forms the holding means.

[0122] Alternatively, the retaining device is adapted to be secured in the oral cavity, or the retaining device is adapted to be held in the hand during administration of the drug delivery system, such that the dosage form is spread and / or unfolded while the dosage form moves down the esophageal mucosa and exits the housing through the aperture.

[0123] In a preferred embodiment, a portion of the string member is connected to an end of the preparation, which extends out of the hole of the capsule device. Therefore, the holding device is formed by the extended end of the preparation and the string member connected thereto, and another portion of the string member serves as a retainer to retain the holding device so that it does not move when the preparation is swallowed, thereby generating a pulling force on the preparation, and when the capsule device moves down the esophagus, the pulling force pulls the preparation out of the capsule device.

[0124] It should be understood that the terms "site of action" and "site of application" as used herein are used interchangeably. In this regard, it should also be understood that "site of action" and "site of application" refer to the intended location of release of the formulation. In addition, an agent that is effective in treating or preventing esophageal disease and is released at the "site of action" or "site of application" may also exert its actual biochemical effect at another location in the body or at another location in the biochemical cycle, for example, during or after liver metabolism or when the agent reaches its target molecule. "Site of action" and "site of application" as used herein do not necessarily refer to the location of the biochemical, medical action of the active pharmaceutical ingredient.

[0125] Capsule device / housing

[0126] The drug delivery system according to the present invention also includes a shell, wherein the shell contains at least one sheet preparation, particularly film-shaped, foil-shaped or thin sheet-shaped preparation, the preparation comprises an effective treatment or prevention of esophageal disease medicament, and wherein the shell includes a hole as a part of a release mechanism, the release mechanism is configured to allow the preparation to leave the shell, so that when the dosage form moves downward along the esophageal mucosa and leaves the shell through the hole, the preparation is spread out or launched. The shell can be further prepared so that it protects the preparation from undesirable release. The shell is a capsule device, particularly with the shape of a capsule.

[0127] In a preferred embodiment, the shell includes a first half capsule shell and a second half capsule shell, and the capsule device is formed by sliding the first half capsule shell into the second half capsule shell to a mating position, so that the hole is formed in the mating position by overlapping the cross-section of the second half capsule shell with the opening located in the first half capsule shell.

[0128] In another embodiment, the two capsule halves are nested within each other, and the opening of the first capsule housing half is covered by a further provided overlapping wall portion, such as a patch or tape, attached to the first and / or second capsule housing half.

[0129] In another embodiment, the capsule halves are shaped like two nut shells and are positioned on top of each other to form a capsule. The hole is formed by a cutout, particularly in the edge of one of the two shells. Alternatively, the cutout may be formed on the edge of the two halves, which, when positioned and aligned on top of each other, form the hole.

[0130] In a preferred embodiment of the drug delivery system according to the invention, the shell is made of a material selected from the group consisting of hard gelatin, polymers, thermoplastics (e.g. Eudragit, etc.). In this respect, in particular, materials that have been successfully tested, used and / or authorized may be beneficial, for example for oral dosage forms.

[0131] For example, such a capsule device or housing is further described in EP21175427.0, EP21175436.1 and PCT / EP2020 / 056934, and the contents of these patents regarding the capsule device are incorporated herein by reference in their entirety.

[0132] Condition to be treated

[0133] The drug delivery system described herein is used in therapy. In one embodiment, it is applicable to the treatment and prevention of esophageal diseases, preferably esophageal diseases caused by or associated with immune system defects. In one embodiment, it is applicable to the treatment and prevention of cancer, preferably esophageal cancer. As used herein, "esophageal cancer" refers to cancers that originate or are present in the esophagus, including but not limited to squamous cell carcinoma, esophageal junction cancer (especially gastroesophageal junction cancer) and adenocarcinoma. In one embodiment, it is applicable to the treatment and prevention of Barrett's esophagus. In one embodiment, it is applicable to the treatment and prevention of esophageal adenocarcinoma. In one embodiment, it is applicable to the treatment and prevention of esophageal squamous cell carcinoma. In one embodiment, it is applicable to the treatment and prevention of esophageal stenosis.

[0134] In this application, the term "treatment and / or prevention" includes any means of improving a condition to be treated or preventing the condition to be treated from occurring. It also includes preventing the condition from getting worse and minimizing the severity of the condition.

[0135] An esophageal disease may be any disease or condition that interferes with the function or structure of the esophagus.

[0136] Esophageal disease may include a chronic inflammatory state, which may progress through a series of transforming dysplastic states before tumor development, which may also be caused by a combination of acid and bile reflux and may be associated with immune system defects. Esophageal disease also includes, but is not limited to, refractory esophageal disease, such as after first-line treatment. For example, refractory esophageal disease involves esophageal disease that is unsuccessful or inadequately treated, i.e., despite treatment, specific symptoms of esophageal disease persist. Refractory esophageal disease may also refer to recurrence after treatment. Therefore, in one embodiment, the present invention relates to the treatment of refractory esophageal disease, i.e., second-line treatment. For example, surgery or ablative therapy in cancer treatment may require post-treatment or second-line treatment with chemotherapeutic agents, sometimes in combination with radiotherapy to ensure complete destruction of cancerous tissue. Therefore, the present invention also includes second-line treatment for esophageal diseases (such as cancer).

[0137] In another embodiment, the present invention relates to first-line treatment or pre-treatment of esophageal disease, such as preventive treatment or adjuvant chemotherapy, followed by surgical therapy. For example, surgery or ablative therapy in cancer treatment may require first-line treatment or pre-treatment with a chemotherapeutic agent, such as neoadjuvant therapy, sometimes in combination with radiotherapy to reduce the tumor size to an operable size. Therefore, the present invention also includes first-line treatment or pre-treatment of esophageal disease (such as cancer), such as neoadjuvant therapy.

[0138] Preferred esophageal diseases include esophageal diseases caused by or associated with immune system defects. For example, the esophageal disease may be caused by or associated with reduced proliferation or activity of immune system cells, thereby disturbing esophageal homeostasis and promoting the development and / or progression of cancer, for example, due to mechanisms by which cancer cells evade immune surveillance.

[0139] One treatment option for cancer is ablation or surgery of the affected tissue, as an independent therapy or in combination with chemotherapy or radiotherapy, for example as a neoadjuvant therapy. "Neoadjuvant therapy" as used herein refers to chemotherapy and / or radiotherapy performed before ablation or surgery, with the goal of reducing the size of the tumor, preferably to an operable size. Ablation therapy or surgery may be accompanied by chemotherapy in the postoperative phase to avoid tumor spread and / or metastasis formation. Alternatively, chemotherapy as an independent therapy or in combination with radiotherapy (definitive chemoradiotherapy) is a treatment option, particularly in unresectable cancers.

[0140] Regardless of the time point in the treatment regimen, chemotherapy is usually administered systemically via parenteral administration, which is associated with high toxicity to patients. Therefore, new cancer treatment options with reduced toxicity are needed.

[0141] Barrett's esophagus is a condition in which the normal multilayered squamous epithelium is replaced by (specialized) columnar epithelium (i.e., metaplasia of intestinal or other columnar types). This process is thought to be the result of long-standing gastroesophageal reflux disease, which is most prevalent in middle-aged white men. In particular, specialized intestinal-type columnar metaplasia significantly increases the risk of developing esophageal adenocarcinoma. Esophageal adenocarcinoma is a highly malignant disease with a very poor prognosis that arises from the epithelial cells lining the esophagus. The incidence of Barrett's esophagus and esophageal adenocarcinoma is rapidly increasing, and the development of new prevention and treatment strategies is of vital importance.

[0142] The terms "Barrett's disease" and "Barrett's esophagus" are used interchangeably herein.

[0143] Barrett's esophagus does not have any specific symptoms, although patients with Barrett's esophagus may have symptoms related to gastroesophageal reflux disease. However, it does increase the risk of developing esophageal adenocarcinoma, a serious, potentially fatal form of esophageal cancer. Diagnosis of Barrett's esophagus can be accomplished using endoscopy, histology, and / or the use of biomarkers, such as described in US20120009597 A1.

[0144] Treatment of Barrett's esophagus without malignant features aims to reduce inflammation and epithelial-mesenchymal transition, including treatment with compounds to alleviate reflux or anti-reflux surgery. In the case of malignant degeneration, treatment is performed by endoscopic ablation therapy or surgical resection of the affected portion of the esophagus. Ablation therapy or surgery may be accompanied by neoadjuvant therapy and / or postoperative chemotherapy. As described herein, chemotherapy is highly toxic to patients due to its extra-gastrointestinal systemic delivery. Therefore, there is still a need in the art for new treatment options for esophageal cancer.

[0145] Esophageal squamous cell carcinoma (ESCC) originates from the epithelial cells lining the esophagus. Areas of high incidence include East to Central Asia, along the East African Rift Valley, and South Africa. There are many causes of ESCC that vary by region. Early studies in France associated smoking and alcohol abuse with a high incidence of ESCC, and other risk factors for ESCC include polycyclic aromatic hydrocarbons from various sources, hot foods, diet, and oral health. Esophageal squamous cell carcinoma is diagnosed by endoscopic biopsy. ESCC is the eighth most common cancer worldwide and has a poor prognosis due to the fact that diagnosis is often late. Therefore, the development of new prevention and treatment strategies is critical. In addition, esophageal squamous cell carcinoma is usually treated with neoadjuvant therapy followed by surgery or with radical chemoradiotherapy, which is highly toxic to patients. Therefore, there is still a need for new treatment options in this field.

[0146] The drug delivery system described herein can be used for therapy, preferably for the treatment of esophageal diseases. The esophageal diseases to be treated according to the present invention include but are not limited to esophageal diseases, wherein preferably, the esophageal diseases are caused or associated with immune system defects, or preferably cancer.

[0147] Preferably, the esophageal disease to be treated in the context of the present invention is Barrett's esophagus, in particular Barrett's esophagus at different stages, including metaplasia, low-grade and high-grade dysplasia. In another embodiment, the esophageal disease to be treated in the context of the present invention is preferably esophageal cancer, such as adenocarcinoma, esophageal junction cancer or squamous cell carcinoma, including but not limited to refractory cancer. In one embodiment, the treatment of esophageal cancer includes neoadjuvant therapy, i.e. surgery after chemotherapy. In one embodiment, the treatment of esophageal cancer includes the treatment of unresectable esophageal cancer, for example, by chemotherapy as an independent therapy without subsequent surgery or ablative therapy. In one embodiment, the treatment of esophageal cancer includes treating esophageal cancer by postoperative chemotherapy, i.e. chemotherapy after surgery. In another embodiment, the esophageal disease to be treated in the context of the present invention is preferably esophageal stenosis.

[0148] In a preferred embodiment of the present invention, the drug delivery system according to the present invention is applicable to the treatment of esophageal diseases, such as Barrett's esophagus, particularly different stages of Barrett's esophagus, including metaplasia, low-grade and high-grade dysplasia. In another preferred embodiment of the present invention, the drug delivery system according to the present invention is applicable to the treatment of esophageal cancer, such as adenocarcinoma, esophageal junction cancer or squamous cell carcinoma, including but not limited to refractory cancer. In one embodiment, the drug delivery system according to the present invention is applicable to the treatment of esophageal cancer by neoadjuvant therapy (i.e., surgery after chemotherapy). In one embodiment, the drug delivery system according to the present invention is applicable to the treatment of unresectable esophageal cancer, for example, by chemotherapy as an independent therapy, without the need for subsequent surgery or ablation therapy. In one embodiment, the drug delivery system according to the present invention is applicable to the treatment of esophageal cancer by postoperative chemotherapy, i.e., chemotherapy after surgery. In another embodiment, the drug delivery system according to the present invention is applicable to the treatment of esophageal stenosis.

[0149] The frequency of administration of the drug delivery device and the treatment period or application time point are not limited and may depend on the specific disease to be treated and / or the amount of active pharmaceutical ingredient of each drug delivery device. For example, the drug delivery device may be applied once a day or twice a day. If the drug delivery device is applied once a day, it is preferably applied at night to increase the patient's compliance. The drug delivery system of the present invention is preferably applied before going to sleep, i.e. after dinner and after oral hygiene. The treatment period may be between 7 days and 40 days, preferably 14 days to 30 days, more preferably 20 days to 28 days. Treatment may include a single treatment cycle or multiple cycles of the treatment period, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more treatment periods.

[0150] diagnosis

[0151] The drug delivery system described herein is also available for diagnosis. Diagnosis may include monitoring the uptake of the agent in the cell after administration or the approach of the agent into tissues, organoids, cell cultures or organs. Diagnosis may also include monitoring the success of treatment, such as the size of the tumor or the time period between the release of the agent from the drug delivery to the cell, tissue or organ uptake. For example, a preparation comprising an active pharmaceutical ingredient and a diagnostic marker can be administered to a cell, tissue, organ, cell culture or organ. The approach of the active pharmaceutical ingredient can be tracked by monitoring the diagnostic marker described herein. For purposes of the present invention, diagnosis may include in vivo and / or in vitro diagnosis.

[0152] For the purpose of diagnosis, the medicament for the effective treatment of esophageal diseases of the present invention can be combined with diagnostic markers. Any diagnostic marker suitable for in vivo or in vitro diagnosis can be used. For example, the medicament can be conjugated with a diagnostic marker or otherwise combined, such as embedded, included, compounded. Alternatively, one or more atoms or functional groups of the medicament can be replaced by a diagnostic marker. In principle, any known diagnostic marker can be used for diagnosis, including but not limited to radioisotopes, paramagnetic labels such as gadolinium or iron oxide, fluorophores, near infrared (NIR) fluorescent dyes or dyes, echo microbubbles, affinity labels (such as biotin, avidin, etc.), enzymes or any other suitable medicaments that can be detected by diagnostic imaging methods. In a specific non-limiting example, an agent effective in treating or preventing esophageal disease, such as an antibody of the present invention, can be conjugated to a near-infrared fluorescence (NIRF) imaging dye, such as, but not limited to, Cy5.5, Alexa680, Dylight680, or Dylight800, a radioisotope such as tritium (hydrogen-3), 11C (carbon), 13N (nitrogen), 15O (oxygen), 18F (fluorine), 32P (phosphorus), or 35S (sulfur). In another specific non-limiting example, an agent effective in treating or preventing esophageal disease, such as an siRNA or antisense oligonucleotide, can be conjugated to a fluorophore such as Atto633 (Figure 10). In one embodiment, an inhibitory polynucleotide of the present invention, such as an siRNA, an aptamer, or an antisense oligonucleotide, is used in combination with a diagnostic marker. In one embodiment, an antibody of the present invention is used in combination with a diagnostic marker. In one embodiment, an antiproliferative agent of the present invention is used in combination with a diagnostic marker. Suitable imaging systems for diagnosis, particularly in vivo diagnosis, include, but are not limited to, PET scanning and SPECT scanning involving radioisotopes.

[0153] Active pharmaceutical ingredients

[0154] As used herein, the term "active pharmaceutical ingredient" is used interchangeably with the term "active ingredient" or "API" and refers to an agent that is effective in treating or preventing esophageal disease.

[0155] The term "therapeutically effective dose" or "effective amount" refers to a dose or amount that produces the desired effect of administration. The exact dose or amount will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques. The term "therapeutically effective amount" is an amount that is effective to improve (the symptoms of) the disease. A therapeutically effective amount can be a "prophylactically effective amount" because prevention can be considered a therapy.

[0156] "An agent effective for treating or preventing esophageal disease" refers to a compound that alleviates, reduces, prevents, blocks or interferes with esophageal diseases or conditions. In particularly preferred embodiments, the agent effective for treating esophageal disease is an agent whose therapeutic application is limited by its high systemic toxicity, or is an agent with low systemic bioavailability, for example, due to a high first-pass effect and / or loss of activity due to passage through the gastrointestinal tract. Due to the local availability of the agent on the treatment side, the present invention enables or increases the therapeutic use of such an agent. An agent effective for treating or preventing esophageal disease refers to any type of compound, including but not limited to polynucleotides, such as inhibitory polynucleotides, such as siRNA molecules, antisense oligonucleotides, micro RNA (miRNA), antagomir or aptamers; small molecules; or polypeptides, such as antibodies or ligands. Where appropriate, the term also refers to a salt or any other derivative of an agent effective for treating or preventing esophageal disease.

[0157] "Sequence identity" or the language "identical sequence" is used to assess the similarity of two sequences, e.g., polynucleotide or polypeptide sequences; determined by calculating the percentage of identical residues when the two sequences are aligned for maximum correspondence between residue positions. Any known method can be used to calculate sequence identity; for example, computer software can be used to calculate sequence identity. Without wishing to be limited, sequence identity can be calculated by software such as BLAST-P or BLAST-N of the National Center for Biotechnology Information or any other suitable software known in the art. Importantly, the assessment of sequence similarity between a comparison sequence and a given sequence is determined over the full length of the comparison sequence. For example, the sequence of an siRNA molecule is a comparison sequence, and the sequence identity to a given sequence is determined by a BLAST-N program by a default algorithm for aligning two or more sequences. In another example, the antibody sequence as shown in SEQ ID NO: 11 is a comparison sequence, and the sequence identity to a given sequence is determined by a BLAST-P program by a default algorithm for aligning two or more sequences.

[0158] In one aspect of the present invention, the medicament for effectively treating or preventing esophageal diseases includes or is an inhibitory polynucleotide, preferably an inhibitory polynucleotide, such as siRNA molecules, miRNA, antagomir, antisense oligonucleotide and aptamer, more preferably siRNA molecules, antisense oligonucleotide or aptamer. In one embodiment, the length of the inhibitory polynucleotide is 10 to 100 nucleotides, preferably 15 to 50 nucleotides, more preferably 19 to 25 nucleotides. The polynucleotide can be "deoxyribonucleic acid" (DNA) or "ribonucleic acid" (RNA) or a derivative or modified form thereof. The polynucleotide can be double-stranded or single-stranded.

[0159] In a preferred embodiment, the agent effective for treating or preventing esophageal disease comprises, preferably consists of, or is an siRNA molecule.

[0160] As used herein, the term "small interfering RNA" or "siRNA" refers to exogenously synthesized RNA duplexes. Any method known in the art for producing siRNA can be used. Those methods are known to the skilled person. siRNA comprises or consists of two RNA strands, an antisense (or guide) strand and a sense (or passenger) strand. These molecules typically display 15-35 base pairs, preferably 19-25 base pairs, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 base pairs, and are typically prepared intracellularly by enzymatic cleavage of larger RNA duplexes. The siRNA may contain varying degrees of sequence complementarity to a target mRNA or RNA transcript in its antisense strand. Preferably, the siRNA molecules of the present invention have sufficient sequence identity and / or sequence complementarity to the target RNA transcript, preferably under physiological conditions, to interfere with, reduce, decrease, inhibit and / or block the expression and / or function of a polypeptide encoded by the target RNA transcript. Some siRNA molecules (but not all) include structures with overhangs. Overhangs are described as being advantageous and may be present at the 5' end or 3' end of any chain because they reduce the recognition of RNA enzymes. Some siRNA molecules have overhangs at both 3' ends of the chain, while others have overhangs only on one chain. Other siRNA molecules may be blunt-ended structures. Without being bound by any theory, it is said that these overhangs further enhance resistance to nuclease (RNase) degradation. As used herein, the term "overhang" or "tail" refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more continuous nucleotides of non-base pairing at the 3' end of one or both of the sense strand and the antisense strand. The term siRNA refers to a duplex of two independent chains, and also refers to a duplex of a single strand that may be formed (to form a double-stranded region) by a self-complementary sequence portion such as a hairpin structure.

[0161] The term "double-stranded region" as used herein refers to a region in which two complementary or partially complementary polynucleotides (eg, sense strands or antisense strands) form base pairs with each other.

[0162] The term "complementary" or "sequence complementarity" refers to hybridization or base pairing between nucleotides. Hybridization preferably refers to stringent hybridization conditions, such as washing in 1×SSC and 0.1% SDS for 1 h at 45° C., preferably 48° C., more preferably 50° C., in particular washing in 0.2×SSC and 0.1% SDS for 1 h. Base pairing may involve any base pairing, including but not limited to Watson / Crick base pairing.

[0163] The term "RNA transcript" or "mRNA transcript" includes, but is not limited to, a primary transcript, a pre-mRNA transcript, a transcript processing intermediate, a mature mRNA ready for translation, a transcript of one or more genes, or a nucleic acid from an mRNA transcript. Processing of the pre-mRNA transcript, and the possible use of alternative promoters and alternative polyadenylation sites, allows a single gene to produce many different mature RNAs by altering the splicing pattern in a process known as alternative splicing. Alternative splicing can also introduce or remove regulatory elements to affect the translation, localization, or stability of the mRNA. These alternatively spliced ​​mRNAs are translated into alternatively spliced ​​forms of proteins that contain amino acid sequences that differ from the corresponding wild-type or standard proteins produced by the normally spliced ​​mRNA.

[0164] In one embodiment, the inhibitory polynucleotide comprises an siRNA molecule that targets an RNA transcript or a portion thereof that encodes a polypeptide involved in the development of an esophageal disease or condition; or encodes a polypeptide that is expressed or whose expression is increased due to a disease or condition. The siRNA molecule has sufficient sequence complementarity and / or sequence identity to the target RNA transcript to inhibit, block, reduce or decrease the expression and / or function of the polypeptide encoded by the RNA transcript. In one embodiment, the polypeptide comprises or is a BMP2 or BMP4 polypeptide, preferably as shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0165] In one embodiment, the siRNA molecule comprises a double-stranded region, wherein the double-stranded region comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region together, and the antisense strand is complementary to a target RNA transcript encoding a BMP2 or BMP4 polypeptide. This complementarity is sufficient to inhibit, block, reduce or reduce the expression and / or function of BMP2 and / or BMP4. In one embodiment, the siRNA molecule has enough complementarity with an RNA transcript encoding BMP2 and / or BMP4, to inhibit, block, reduce or reduce the function of BMP2 and / or BMP4, such as BMP2 and / or BMP4 signal transduction, thereby restoring or providing the normal tissue lining of the esophagus or enhancing the formation of the normal tissue lining of the esophagus, and preventing or treating esophageal cancer. In a preferred embodiment, the RNA transcript is as shown in SEQ ID NO:20 or SEQ ID NO:21. Preferably, the siRNA molecule comprises a double-stranded region comprising a sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, preferably SEQ ID NO: 18, or any other sequence comprising 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity between the siRNA molecule and the target RNA transcript or a portion thereof. The length of the double-stranded region may be 15 to 35 base pairs, preferably 17 to 30 base pairs, more preferably 19 to 25 base pairs.

[0166] In one embodiment, the inhibitory polynucleotide comprises an siRNA molecule that targets an RNA transcript or a portion thereof as shown in SEQ ID NO: 20 or SEQ ID NO: 21. In one embodiment, the siRNA molecule comprises or consists of a double-stranded region, wherein the double-stranded region comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand together form the double-stranded region, and the antisense strand is complementary to a target RNA transcript or a portion thereof as shown in SEQ ID NO: 20 or SEQ ID NO: 21.

[0167] In one embodiment, the siRNA molecule does not show an overhang, i.e. it has a flat end. In another embodiment, the siRNA molecule shows at least one overhang. The overhang is believed to play a structural role in presenting a duplex to RISC. Each overhang can have one or more, preferably two, up to six continuous nucleotides. The nucleotides in the overhang can include nucleosides with nucleobases such as adenine, cytosine, guanine, thymine or uracil. Preferably, the overhang comprises two continuous nucleotides or is composed of two continuous nucleotides, preferably deoxyribonucleotides, such as deoxyribonucleotides comprising nucleoside deoxythymidine (dTdT). It is believed that the deoxyribonucleotides in the overhang confer nuclease resistance. Ribonucleotides can also be used at the overhang. The ribonucleotides can be complementary to the target transcript, or can include the nucleoside uridine, preferably UU.

[0168] In a preferred embodiment, the siRNA molecule consists of a double-stranded region comprising a sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, i.e., it has blunt ends. In another preferred embodiment, the siRNA molecule consists essentially of a double-stranded region comprising a sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, i.e., it consists of a double-stranded region and one or more consecutive overhang nucleotides (preferably two, up to six consecutive nucleotides), as described herein.

[0169] In a preferred embodiment, the siRNA molecule comprises, preferably consists of, BMP2-siRNA 1, BMP2-siRNA 2 or BMP2-siRNA 3 as shown in the following table:

[0170] Table 1

[0171]

[0172] Each of the above sequences in Table 1 comprises an overhang of two nucleotides dTdT (deoxythymidine) or UU (uridine) attached to the 3' end of each strand.

[0173] The siRNA molecule of the present invention can comprise chemically modified nucleotides or modified nucleotides. These modified nucleotides refer to non-standard nucleotides, including non-naturally occurring deoxyribonucleotides or ribonucleotides. This modification is introduced to increase or improve resistance to nucleases, intracellular uptake, target cell specificity and / or stability. Of course, introducing this modification will not affect the original inhibition, blocking, reduction or reduction activity of the siRNA molecule to the target RNA transcript. The modification can be introduced into the main chain, sugar unit and / or core base unit of the polynucleotide.

[0174] For example, the modification may be present in the backbone of the polynucleotide. For example, the phosphodiester bonds of the sense strand or antisense strand of the siRNA molecule may be completely or partially replaced by phosphorothioate or boron phosphate bonds to increase the stability of the siRNA molecule.

[0175] Another modification may be present in the sugar unit of the ribonucleotide of the siRNA molecule. For example, the ribose unit of the nucleoside may contain one or more bridged nucleic acids to increase the rigidity of the sugar unit, thereby increasing the binding affinity and stability of the siRNA. Examples include LNA (locked nucleic acid) or ENA (ethylene bridged nucleic acid). This modification can be introduced into the ribose unit of all or some nucleotides, preferably into the nucleotides at the 3' end or 5' end or both ends of the chain of the siRNA molecule.

[0176] For another example, the 2'-OH group of the ribose unit can be substituted by NH2, -NHR, -NR2, -COOR, -OR, -H, -F, -Cl, -Br, -I, -SH, -SR, -O-Me (or CH3, methyl), -2'-O-MOE (methoxyethyl), wherein R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc., as described in U.S. Patent No. 9,080,171 and U.S. Patent Application No. 2019 / 0024082, all of which are incorporated herein by reference. For example, the 2'-OH groups of the ribose units of the first and second nucleotides of the sense strand can be substituted by 2'-O-Me (methyl), or the 2'-OH group of the ribose unit of the second nucleotide of the antisense strand can be substituted by 2'-O-Me, or the 2'-OH of the ribose unit of the nucleotide containing guanine (G) or uridine (U) can be substituted by 2'-O-Me or 2'-F.

[0177] In another aspect of the present invention, the medicament for effectively treating or preventing esophageal diseases is an antisense oligonucleotide.Antisense oligonucleotides include polymers of deoxyribonucleotides or ribonucleotides.Generally, the antisense oligonucleotides are single-stranded molecules, which may contain complementary to their target mRNA or RNA transcripts to varying degrees.The antisense oligonucleotides have enough sequence complementarity with the target RNA transcripts to inhibit, block, reduce or reduce the expression and / or function of the polypeptide encoded by the RNA transcripts.The length of these molecules is generally shown as 15-35 nucleotides, preferably 19-25 nucleotides, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, and are generally exogenously synthesized.The antisense oligonucleotides of the present invention may include chemically modified nucleotides or modified nucleotides to increase the binding affinity and / or stability of the antisense oligonucleotides.The modification may be present in the main chain of the polynucleotides, and the sugar unit or the core base unit is disclosed in the context of the siRNA molecules herein.In one embodiment, the antisense oligonucleotides are morpholinos. Any method known in the art for producing antisense oligonucleotides can be used, including but not limited to exogenous chemical synthesis. Those methods are known to the skilled person. The term antisense oligonucleotide can also refer to an oligonucleotide present in a cell.

[0178] In one embodiment, the antisense oligonucleotide targets an RNA transcript encoding a BMP2 or BMP4 polypeptide, preferably a BMP2 or BMP4 polypeptide as shown in SEQ ID NO: 15 or SEQ ID NO: 16, or a portion thereof. In another embodiment, the antisense oligonucleotide comprises an antisense strand or consists of an antisense strand, wherein the antisense strand is complementary to a target RNA transcript as shown in SEQ ID NO: 20 or SEQ ID NO: 21, or a portion thereof. The complementarity between the antisense strand and the target RNA transcript is sufficient to inhibit, block, reduce or reduce the expression and / or function of BMP2 and / or BMP4. In one embodiment, the antisense strand has sufficient complementarity to an RNA transcript encoding a BMP2 and / or BMP4 polypeptide to inhibit, block, reduce or reduce the function of BMP2 and / or BMP4, such as BMP2 and / or BMP4 signaling, thereby restoring or providing a normal tissue lining of the esophagus or enhancing the formation of a normal tissue lining of the esophagus, and preventing or treating esophageal cancer.

[0179] In one embodiment, the antisense oligonucleotide comprises a sequence as shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, preferably SEQ ID NO: 23, or any other sequence comprising 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity between the complementary sequence of the antisense oligonucleotide molecule and the target RNA transcript. The length of the duplex between the antisense oligonucleotide and the target transcript may be 15 to 35 base pairs, preferably 17 to 30 base pairs, preferably 19 to 25 base pairs.

[0180] In another aspect of the present invention, the agent for the effective treatment or prevention of esophageal diseases is an aptamer. An aptamer is a single-stranded polynucleotide, typically comprising one or more double-stranded regions formed by self-complementary sequence portions such as hairpins. The aptamer specifically binds to a target polypeptide through a specific three-dimensional structure. In one embodiment, the binding of the aptamer interferes with the activity of a BMP2 or BMP4 polypeptide, preferably a BMP2 or BMP4 as shown in SEQ ID NO: 15 or SEQ ID NO: 16. Interference refers to inhibiting, blocking, reducing or lowering the activity of BMP2 and / or BMP4, such as BMP2 and / or BMP4 signal transduction. Preferably, the aptamer interferes with the activity of BMP2 and / or BMP4 by binding to the active site of BMP2 and / or BMP4, such as a receptor binding site for binding to a receptor on a target cell. In one embodiment, the binding of the aptamer to the BMP2 and / or BMP4 polypeptide inhibits, blocks, reduces or decreases the function of BMP2 and / or BMP4, such as BMP2 and / or BMP4 signaling, thereby restoring or providing the normal tissue lining of the esophagus or enhancing the formation of the normal tissue lining of the esophagus, and preventing or treating esophageal cancer. The binding usually occurs through non-covalent binding, such as electrostatic interactions, stacking of flat parts, shape complementation and / or hydrogen bonding. Any method for producing aptamers known in the art can be used. Those methods are known to technicians, including but not limited to SELEX (systematic evolution of ligands by exponential enrichment).

[0181] The inhibitory polynucleotides such as siRNA molecules or antisense oligonucleotides work in cells. Therefore, the inhibitory polynucleotides used with the drug delivery system of the present invention are advantageously introduced into esophageal cells. For the purpose of the present invention, it is useful to co-administer the inhibitory polynucleotides with a nucleic acid delivery system. The nucleic acid delivery system can increase or realize the intracellular delivery of inhibitory polynucleotides. The inhibitory polynucleotides can be compounded, connected, embedded, attached to the nucleic acid delivery system, or enclosed in the nucleic acid delivery system. For the purpose of the present invention, the nucleic acid delivery system includes but is not limited to liposomes, lipid bilayers, cationic polymers, micelles, emulsions or nanoparticles, such as lipid nanoparticles or polymer nanoparticles. Cationic polymers for delivering nucleic acids may include natural polymers such as chitosan, atelocollagen, cationic polypeptides, etc., and synthetic polymers such as poly (L-lysine), linear or branched polyethyleneimine (PEI), polycations based on cyclodextrin, dendrimers, etc.

[0182] The inhibitory polynucleotides of the present invention may also comprise a diagnostic marker. Such a diagnostic marker may be attached to, for example conjugated to, any site of the polynucleotide, preferably the 3' and / or 5' end of the polynucleotide. Inhibitory polynucleotides including diagnostic markers such as antisense oligonucleotides or siRNA molecules may be used for the diagnosis described herein.

[0183] In another aspect of the present invention, the agent effective for treating or preventing esophageal disease is an antibody or a binding fragment thereof.

[0184] In one embodiment, the antibody or binding fragment thereof targets a polypeptide or portion thereof that is involved in the development of an esophageal disease or disorder, or a polypeptide that is expressed or whose expression is increased as a result of a disease or disorder.

[0185] In one embodiment, the target polypeptide encodes a BMP2 or BMP4 polypeptide, preferably as shown in SEQ ID NO: 15 or SEQ ID NO: 16. In another embodiment, the target polypeptide encodes PD-1. In a further embodiment, the target polypeptide encodes an EGF receptor, such as erb-b2 receptor tyrosine kinase 2.

[0186] As used herein, the term "BMP2" is used to refer to mature bone morphogenetic protein 2, preferably of human origin. The nucleotide sequence of human pro-BMP2 is publicly available with reference to GenBank Accession No. NM_001200. A portion of the amino acid sequence of mature BMP2 is shown herein as SEQ ID NO: 14. The residue numbers used herein refer to the positions of SEQ ID NO: 14.

[0187] As used herein, the term "BMP4" is used to refer to human mature bone morphogenetic protein 4. The nucleotide sequence of human pro-BMP4 is publicly available with reference to GenBank Accession No. NM_130851. A portion of the amino acid sequence of mature BMP4 is shown herein as SEQ ID NO: 1. The residue numbers used herein refer to the positions of SEQ ID NO: 1.

[0188] As used herein, the term "BMP4 signaling" refers to a BMP4 activation standard (phosphorylation of SMAD 1 / 5 / 8) assay to test the ability of BMP4 signaling, such as described in Shaifur Rahman et al., "TGF-β / BMP signaling and other molecular events: regulation of osteoblast genesis and bone formation", Bone Research 3, Article No.: 15005 (2015).

[0189] As used herein, the term "BMP2 signaling" refers to the ability of BMP2 to activate criterion (phosphorylation of SMAD 1 / 5 / 8).

[0190] As used herein, the term "wrist" of BMP4 refers to the region within the BMP4 protein that binds to type I receptors (eg, BMPR1a and BMPR1b).

[0191] As used herein, the term "knuckle" of BMP4 refers to the region within the BMP4 protein that binds to type II receptors (eg, BMPR2, ActRII, and ActRIIB).

[0192] In preferred embodiments, the epitope is located in the wrist within residues 10-17, 24-31, 45-72, 89, 91, 101, 103, 104 and 106 of BMP4 (SEQ ID NO: 1).

[0193] As used herein, the term "binding within" an epitope refers to an Ig-like molecule that binds to one, but preferably at least 2, 3, 5, 6, 7, 8 or more residues within a BMP4 epitope. In a preferred embodiment, the Ig-like molecule does not (substantially) bind to any other epitope of BMP4.

[0194] The term "binding" as used herein in the context of the interaction between an Ig-like molecule and an epitope refers to the ability of an Ig-like molecule to bind to an antigen via the immunoglobulin variable region of the Ig-like molecule with a dissociation constant, KD, of 1x10 -6 M or smaller, preferably 1x10 -7M or smaller, preferably 1x10 -8 M or smaller, preferably 6x10 -9 M or smaller, preferably 3x10 -9 M or smaller, preferably 2x10 -9 M or smaller.

[0195] As used herein, an Ig-like molecule that "specifically binds" a certain epitope refers to an Ig-like molecule that binds to the certain epitope, but preferably does not (substantially) bind to another epitope.

[0196] As used herein, the term "does not substantially bind" an epitope means that it does not bind or does not bind to the epitope with high affinity, i.e., with an affinity of 1x10 -6 M or higher, more preferably 1x10 -5 M or higher, more preferably 1x10 -4 M or higher, more preferably 1x10 -3 M or higher, even more preferably 1x10 -2 or higher KD binds to the epitope.

[0197] As used herein, the term "antigen binding portion" (or "antigen binding fragment thereof" or "antigen binding fragment" or "binding fragment" or "binding fragment thereof" or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., bind to an epitope within a BMP4 or PD-1 protein). Typically, a "binding fragment" or "antibody fragment" retains at least 10% of the binding activity of the parent when the activity is expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. It has been shown that the antigen binding function of an Ig-like molecule can be achieved by fragments of a full-length antibody. The antibody fragment can be obtained by manipulating naturally occurring antibodies, or can be obtained using recombinant methods. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a partial hinge region {see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rded. 1993]; (iv) a Fd fragment consisting of a VH domain and a CH1 domain; (v) a Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody, (vi) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, a single variable domain and two constant In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be linked by a synthetic linker using recombinant methods, which enables them to form a single protein chain in which the VL region and the VH region are paired to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 243:423-426). USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. In a non-limiting example, the antibody fragment may be a single domain antibody (single-domain antibody) derived from a natural source. The term single domain antibody may also refer to its multivalent presentation form. Heavy chain antibodies of camelid origin lack light chains, so their antigen binding sites consist of one domain called VHH.Single domain antibodies, called VNARs, have also been observed in sharks; other single domain antibodies can be engineered based on human heavy or light chain sequences. As used herein, "single domain antibodies" include antibodies isolated directly from VL, VH, VHH or VNAR repositories of any source by phage display or other display technologies, as well as antibodies produced by further modifying such single domain antibodies by humanization, affinity maturation, stabilization, dissolution (e.g., camelization) or other methods of antibody engineering. The present invention also encompasses homologs, derivatives or fragments that retain or improve the antigen binding function and specificity of single domain antibodies. The structure of single domain antibodies is well known to those skilled in the art. Single domain antibodies contain a single immunoglobulin domain that retains an immunoglobulin fold; most notably, only three CDRs form an antigen binding site. However, not all CDRs are necessary for binding to antigens. For example, and without wishing to be limiting, one, two or three CDRs may contribute to the binding and recognition of antigens by the single domain antibodies of the present invention. The CDRs of single-domain antibodies are referred to herein as CDR1, CDR2 and CDR3, and are numbered based on Kabat (Kabat et al., 1991). The single-domain antibodies may be of camelid origin and may therefore be based on camelid framework regions; alternatively, the CDRs may be transplanted to the framework regions of other antibody domains, such as, but not limited to, VNAR, human VH or human VL framework regions. In another alternative, the above CDRs may be transplanted to the framework regions of other types of antibody fragments (Fv, scFv, Fab). The present invention also encompasses diabodies. "Diabodies" are small antibody fragments with two antigen binding sites. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to pair between two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161 and Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0198] Preferred antibodies of the present invention are disclosed in WO 2016 / 043577 and WO 2018 / 193129, which are incorporated herein in their entirety.

[0199] Preferably, the antibody or its binding fragment according to the present invention is bound to residues 10-17, 45-56 and 69 of BMP4 (SEQ ID NO: 1), preferably in conjunction with an epitope consisting of these residues. The epitope comprises a hydrophobic groove, which is believed to be important for BMP4 specific binding. The advantage of the antibody or its binding fragment according to this embodiment is that the antibody or its binding fragment has low affinity to other members of the BMP family and is very effective in specifically inhibiting BMP4 signal transduction. Preferably, the antibody or its binding fragment is substantially not bound to BMP2, BMP5, BMP6 or BMP7. Another advantage is that the antibody or its binding fragment does not inhibit BMP2-mediated signal transduction, thereby reducing or even avoiding adverse side effects during in vivo use. More preferably, the antibody or its binding fragment specifically binds to at least one residue selected from the group consisting of Lys10, Asn11, Lys12, Asn13, Cys14, Arg15, Arg16 and His17 of BMP4, at least one residue selected from the group consisting of Gly45, Asp46, Cys47, Pro48, Phe49, Pro50, Leu51, Ala52, Asp53, His54, Leu55 and Asn56 of BMP4, and Ser69 of BMP4. Preferably, the antibody or its binding fragment binds to more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, preferably 16 residues thereof. In a highly preferred embodiment, the antibody or its binding fragment specifically binds to at least Lys12, Arg15, Asp46 and Pro50 of BMP4.

[0200] In a preferred embodiment, the antibody or binding fragment thereof according to the present invention is a single-chain antibody. In a preferred embodiment, the antibody or binding fragment thereof comprises a heavy chain CDR2 consisting of an amino acid sequence of SEQ ID NO:3 or a sequence that differs therefrom by no more than 2 amino acids, a heavy chain CDR3 consisting of an amino acid sequence of SEQ ID NO:4 or a sequence that differs therefrom by no more than 1 amino acid, and further preferably a heavy chain CDR1 consisting of an amino acid sequence of SEQ ID NO:2 or a sequence that differs therefrom by no more than 1 amino acid. In a highly preferred embodiment, the antibody or binding fragment thereof comprises an amino acid sequence of SEQ ID NO:11.

[0201] In another embodiment, the antibodies or binding fragments thereof according to the present invention bind within residues 24-31, 57-68, 70-72, 89, 91, 101, 103, 104 and 106 of BMP4 (SEQ ID NO: 1), preferably binding to an epitope consisting of these residues. This region represents a "hydrophobic pocket" within the wrist epitope of BMP4. The advantage of antibodies or binding fragments thereof that bind to this region is that these antibodies or binding fragments thereof have very high affinity for BMP4 and BMP2 and are also able to effectively inhibit BMP4 and BMP2 signaling. Preferably, the antibody or its binding fragment specifically binds to at least one residue selected from the group consisting of Ser24, Asp25, Val26, Gly27, Trp28, Asn29, Asp30, Trp31 of BMP4; at least one residue selected from the group consisting of Ser57, Thr58, Asn59, His60, Ala61, Ile62, Val63, Gln64, Thr65, Leu66, Val67 and Asn68 of BMP4; at least one residue selected from the group consisting of Val70, Asn71 and Ser72 of BMP4; at least one residue selected from the group consisting of Tyr103 and Gln104 of BMP4; and Met89, Tyr91, Lys101 and Met106 of BMP4. Preferably, the antibody or its binding fragment binds to more than 9, 10, 11, 12, 13, preferably 14 residues thereof. In a highly preferred embodiment, the antibody or binding fragment thereof specifically binds to Asp30, Trp31, Leu66 and Lys101 of BMP4.

[0202] In a preferred embodiment, the antibody or its binding fragment is a single-chain antibody. In a preferred embodiment, the single-chain antibody or its binding fragment is capable of binding to the "hydrophobic pocket" region within the above-mentioned BMP4 wrist epitope. In a preferred embodiment, the antibody or its binding fragment according to the present invention comprises a heavy chain CDR3 consisting of an amino acid sequence of SEQ ID NO:7 or a sequence that differs from it by no more than 1 amino acid. Without wishing to be bound by theory, it is believed that this CDR3 is important for the binding interaction with the hydrophobic pocket of the BMP4 wrist. Preferably, the antibody or its binding fragment further comprises a heavy chain CDR1 consisting of an amino acid sequence of SEQ ID NO:5 or a sequence that differs from it by no more than 1 amino acid, and a heavy chain CDR2 consisting of an amino acid sequence of SEQ ID NO:6 or a sequence that differs from it by no more than 1 amino acid. In a highly preferred embodiment, the antibody or its binding fragment comprises an amino acid sequence of SEQ ID NO:12.

[0203] In another embodiment, the antibody or its binding fragment of the present invention binds to residues 34, 35, 39, 86-88, 90, 97, 98, 100, 102 and 109 of BMP4 (SEQ ID NO: 1), preferably binding to an epitope consisting of these residues. This region represents the so-called BMP4 "joint" epitope. Antibodies or their binding fragments that specifically bind to residues in this region also have high affinity for BMP4, but in addition have high affinity for BMP2, and slightly lower affinity for BMP5 and BMP6. Preferably, the antibody or its binding fragment specifically binds to Ala34, Gln39, Ser88, Leu90 and Leu100 of BMP4.

[0204] In a preferred embodiment, the antibody or its binding fragment is a single-chain antibody. In a preferred embodiment, the antibody or its binding fragment capable of binding to the joint as described above comprises a heavy chain CDR1 consisting of an amino acid sequence of SEQ ID NO:8 or a sequence that differs by no more than 1 amino acid, and a heavy chain CDR2 consisting of an amino acid sequence of SEQ ID NO:9 or a sequence that differs by no more than 1 amino acid. Without wishing to be bound by theory, it is believed that these CDRs are important for the binding interaction with the joint of BMP4. The antibody or its binding fragment preferably further comprises a heavy chain CDR3 consisting of an amino acid sequence of SEQ ID NO:10 or a sequence that differs by no more than 1 amino acid. In a preferred embodiment, the antibody or its binding fragment comprises an amino acid sequence of SEQ ID NO:13.

[0205] In a preferred embodiment, an antibody or its binding fragment is used that binds, preferably binds, to an epitope consisting of the hydrophobic pocket of the BMP4 wrist. These antibodies or their binding fragments inhibit the signaling of BMP2 and BMP4. Such antibodies or their binding fragments are disclosed on pages 22-24 of WO2016 / 042050. Preferably, the antibody specifically binds to at least one residue selected from the group consisting of Ser24, Asp25, Val26, Gly27, Trp28, Asn29, Asp30, Trp31 of BMP4 (SEQ ID NO: 1); at least one residue selected from the group consisting of Ser57, Thr58, Asn59, His60, Ala61, Ile62, Val63, Gln64, Thr65, Leu66, Val67 and Asn68 of BMP4 (SEQ ID NO: 1); at least one residue selected from the group consisting of Val70, Asn71 and Ser72 of BMP4 (SEQ ID NO: 1); at least one residue selected from the group consisting of Tyr103 and Gln104 of BMP4 (SEQ ID NO: 1); and Met89, Tyr91, Lys101 and Met106 of BMP4 (SEQ ID NO: 1). Preferably, the antibody or binding fragment thereof binds to more than 9, 10, 11, 12, 13, preferably 14 residues thereof. In a highly preferred embodiment, the antibody or binding fragment thereof specifically binds to Asp30, Trp31, Leu66 and Lys101.

[0206] In a preferred embodiment, the antibody or its binding fragment is a single-chain antibody. In a preferred embodiment, the antibody or its binding fragment according to the present invention comprises a heavy chain CDR3 consisting of an amino acid sequence of SEQ ID NO:7 or a sequence that differs from it by no more than 1 amino acid. Without wishing to be bound by theory, it is believed that this CDR3 is important for the binding interaction with the hydrophobic pocket of the BMP4 wrist. Preferably, the antibody or its binding fragment further comprises a heavy chain CDR1 consisting of an amino acid sequence of SEQ ID NO:5 or a sequence that differs from it by no more than 1 amino acid, and a heavy chain CDR2 consisting of an amino acid sequence of SEQ ID NO:6 or a sequence that differs from it by no more than 1 amino acid. In a highly preferred embodiment, the antibody or its binding fragment comprises an amino acid sequence of SEQ ID NO:12.

[0207] In a preferred embodiment, the present invention provides heterologous or homologous multimeric molecules with increased antigen affinity for antigens and / or increased inhibition of BMP signaling. Therefore, the present invention provides multimeric antibodies comprising at least one, more preferably at least two antibodies that bind to the above-mentioned BMP4 wrist hydrophobic pocket, or multimeric antibodies comprising at least one, more preferably at least two antibodies that bind to the above-mentioned BMP4 joint epitope.

[0208] In another preferred embodiment, the antibody or its binding fragment may have a sequence substantially identical to SEQ ID NO: 11, 12 or 13. The substantially identical sequence may contain one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations of a reference sequence may produce a mutant peptide having no substantial changes in physiological, chemical or functional properties compared to the reference sequence; in this case, the reference sequence and the mutant sequence will be considered to be substantially identical polypeptides.

[0209] Conservative amino acid mutations used herein may include additions, deletions or substitutions of amino acids; in a non-limiting example, conservative amino acid mutations are conservative amino acid substitutions. Conservative amino acid substitutions are defined herein as substitutions of one amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge or polarity).

[0210] Conservative amino acid substitutions used herein can replace another amino acid of the same group with an alkaline, neutral, hydrophobic or acidic amino acid. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pK value greater than 7, which is usually positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R) and lysine (Lys or K). The term "neutral amino acid" (also referred to as "polar amino acid") refers to a hydrophilic amino acid whose side chain is uncharged at physiological pH, but has at least one bond in which a pair of electrons shared by two atoms is closer to one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N) and glutamine (Gln or Q). The term "hydrophobic amino acids" (also referred to as "non-polar amino acids") refers to amino acids that exhibit a hydrophobicity greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A) and glycine (Gly or G).

[0211] "Acidic amino acids" refer to hydrophilic amino acids with a side chain pK value of less than 7, which are generally negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

[0212] Substantially identical sequences of the present invention may be at least 70% identical; in another example, substantially identical sequences may be at least 70, 71, 72, 73, 74, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical at the amino acid level to the sequences described herein. Importantly, substantially identical sequences retain the activity and specificity of the reference sequence. As known to those skilled in the art, amino acid residues of an antibody or binding fragment thereof, particularly within the framework region, may be mutated (substituted or deleted) without affecting the functional properties (antigen recognition and binding) of the antibody or binding fragment thereof.

[0213] Standard assays for assessing the binding ability of Ig-like molecules to one or more epitopes are known in the art, including, for example, ELISA, Western blot, flow cytometry, and RIA. The binding kinetics (e.g., binding affinity) of the antibody can also be assessed by standard assays known in the art, such as by ELISA, Scatchard, and Biacore analysis. Preferably, the affinity KD of the Ig-like molecule for BMP4 is less than 1590 pM when measured using surface plasmon resonance analysis. -1 Preferably, the KD is less than 1000 pM -1 , more preferably less than 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 750, 700, 650, 635, 600, 575, 513, 393, 100, 91, 75, 50, 32, 25, 10 pM -1 .

[0214] In addition, it is described that the Ig-like molecules of the present invention inhibit BMP4 signaling to the same extent as Noggin. This can be explained by the wide accessibility of Noggin contact points, which covers both BMPR1a and BMPR2 epitopes. The C-terminal finger-like region of Noggin is responsible for blocking BMPR2 binding, while the N-terminal clip-like region binds to the BMPR1a epitope. This significant structural accessibility is conserved in BMP antagonists and can explain their lack of BMP specificity. Assays for determining whether Ig-like molecules compete with Noggin are known in the art. In a preferred embodiment, such an assay includes a surface plasmon resonance (SPR) sandwich cross-binding or "epitope binning" assay, preferably as described in Examples 7 and 8 on pages 48-52 of WO 2016 / 043577.

[0215] The binding epitope of the antibody can be determined by standard methods known in the art, including but not limited to "epitope binning" experiments (surface plasmon resonance (SPR) sandwich cross-binding), and preferably verified by subsequent HADDOCK modeling, for example, by using the HADDOCK software (High Ambiguity Driven Protein-Protein Docking) as described in Examples 7, 8 and 9 on pages 48-55 of WO 2016 / 043577 (Domininguez et al., 2003), which is incorporated herein by reference.

[0216] Specifically, in "epitope binning" or surface plasmon resonance (SPR) sandwich cross-binding assays, a ligand is bound to a control molecule immobilized on a support such as a chip. After ligand binding, the ligand bound to the control molecule contacts a second molecule. If the epitope of the second molecule on the ligand is still accessible, the second molecule will only bind to the bound ligand. Therefore, if the control molecule and the second molecule compete for binding to the ligand, binding will not occur because they share the same epitope. For purposes of the present invention, the control molecule can be a first antibody of the present invention, the ligand can be an antigen such as BMP4, and the second molecule can be a second antibody of the present invention that is different from the first antibody.

[0217] Mutation experiments can be performed using a mutant variant of a second molecule (such as an antibody) to further validate the model or confirm the binding to certain epitopes. Mutation variants can be produced by any method known in the art, including but not limited to site-directed mutagenesis by overlap extension PCR.

[0218] The antibodies of the present invention can be produced by any method known in the art, including but not limited to the methods described in WO 2016 / 043577, which is incorporated herein in its entirety, in particular page 26, line 15 to page 33, line 28.

[0219] In another embodiment of the antibody or fragment thereof targeting a polypeptide or portion thereof involved in the development of an esophageal disease or disorder, or targeting a polypeptide expressed due to a disease or disorder or whose expression is increased due to a disease or disorder, the antibody or fragment thereof targets a polypeptide encoding PD-1, preferably human PD-1.

[0220] In some embodiments, the antibody or antibody fragment blocks the binding of PD-L1 and / or PD-L2 to PD-1, preferably blocks the binding of human PD-L1 and / or human PD-2 to human PD-1.

[0221] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is from a particular species or belongs to a particular antibody class or subclass, while the remainder of the chain is from another species or belongs to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0222] The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric antibody that contains minimal sequences from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), in which residues from the hypervariable regions of the receptor are replaced by residues from the hypervariable regions of non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates, with the desired specificity, affinity and ability. In some cases, the Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues not found in the receptor antibody or the donor antibody. These modifications are to further improve the performance of the antibody. Typically, humanized antibodies will include substantially all of at least one, typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0223] The term "hypervariable region" as used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining region" or "CDR" defined by sequence alignment, such as residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain (see Kabat et al., 1991, Sequences of proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) and / or those residues from a "hypervariable loop" (HVL) as defined herein, e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain (see Chothia and Leskl, 1987, J. Mol. Biol. 196:901-917). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0224] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human, and / or an antibody prepared using any of the techniques for preparing human antibodies disclosed herein. This definition specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0225] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially uniform antibody population, i.e., the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, contrary to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The modifier "monoclonal" means that the characteristic of an antibody is obtained from a substantially uniform antibody population, and is not interpreted as requiring antibodies to be produced by any specific method. For example, the monoclonal antibody used according to the present invention can be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or can be prepared by a recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al., 1991, Nature 352:624-628 and Marks et al., 1991, J. MoI. Biol. 222:581-597. Monoclonal antibodies herein specifically include "chimeric" antibodies.

[0226] Preferred antibodies of the invention are disclosed in WO 2008 / 156712, which is incorporated herein in its entirety, especially pages 6-11.

[0227] In one embodiment, the PD-1 antibody or antibody fragment of the present invention includes an antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprising:

[0228] a. at least one CDR (complementarity determining region) selected from the group consisting of SEQ ID NO: 30, 31, 32, 36, 37 and 38, or a variant of any of said sequences; and / or

[0229] b. at least one CDR selected from the group consisting of SEQ ID NO: 33, 34, 35, 39, 40 and 41, or a variant of any of said sequences.

[0230] In one embodiment, the antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprises:

[0231] a. light chain CDR SEQ ID NO: 30, 31 and 32, or a variant of any of said sequences; and / or heavy chain CDR SEQ ID NO: 33, 34 and 35, or a variant of any of said sequences; or

[0232] b. Light chain CDR SEQ ID NOs: 36, 37 and 38, or variants of any of said sequences; and / or heavy chain CDR SEQ ID NOs: 39, 40 and 41, or variants of any of said sequences.

[0233] In one embodiment, the antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprises:

[0234] a. a heavy chain variable region comprising an amino acid sequence selected from the group consisting of:

[0235] i. SEQ ID NO: 26 or a variant thereof;

[0236] ii. SEQ ID NO: 28 or a variant thereof;

[0237] iii. amino acid residues 20 to 139 of SEQ ID NO:42 or a variant thereof; and

[0238] iv. an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% sequence identity to amino acid residues 20 to 139 of SEQ ID NO:42;

[0239] and further includes

[0240] b. a light chain variable region comprising an amino acid sequence selected from the group consisting of:

[0241] i. SEQ ID NO: 27 or a variant thereof;

[0242] ii. SEQ ID NO: 29 or a variant thereof;

[0243] iii. amino acid residues 20 to 130 of SEQ ID NO:44 or a variant thereof;

[0244] iv. amino acid residues 20 to 130 of SEQ ID NO:45 or a variant thereof;

[0245] v. amino acid residues 20 to 130 of SEQ ID NO:46 or a variant thereof; and

[0246] vi. an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% sequence identity to amino acid residues 20 to 130 of SEQ ID NO:44, 45 or 46.

[0247] Specifically, in one embodiment, the present invention comprises an antibody or antigen-binding fragment comprising: a heavy chain variable region comprising SEQ ID NO: 26 or a variant thereof; and / or a light chain variable region comprising SEQ ID NO: 27 or a variant thereof.

[0248] In one embodiment, the invention comprises an antibody or antigen-binding fragment comprising: a heavy chain variable region comprising SEQ ID NO: 28 or a variant thereof; and / or a light chain variable region comprising SEQ ID NO: 29 or a variant thereof.

[0249] In one embodiment, the invention comprises an antibody or antigen-binding fragment comprising: a heavy chain variable region comprising amino acid residues 20 to 139 of SEQ ID NO: 42 or a variant thereof; and / or a light chain variable region comprising amino acid residues 20 to 130 of SEQ ID NO: 44 or a variant thereof.

[0250] In one embodiment, the invention comprises an antibody or antigen-binding fragment comprising: a heavy chain variable region comprising amino acid residues 20 to 139 of SEQ ID NO: 42 or a variant thereof; and / or a light chain variable region comprising amino acid residues 20 to 130 of SEQ ID NO: 45 or a variant thereof.

[0251] In one embodiment, the invention comprises an antibody or antigen-binding fragment comprising: a heavy chain variable region comprising amino acid residues 20 to 139 of SEQ ID NO: 42 or a variant thereof; and / or a light chain variable region comprising amino acid residues 20 to 130 of SEQ ID NO: 46 or a variant thereof.

[0252] In one embodiment, the invention comprises an antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% sequence identity to amino acid residues 20 to 139 of SEQ ID NO:42; and / or a light chain variable region comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% sequence identity to amino acid residues 20 to 130 of SEQ ID NO:44, 45 or 46.

[0253] In one embodiment, the antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprises:

[0254] a. A heavy chain comprising an amino acid sequence selected from the group consisting of:

[0255] i. amino acid residues 20 to 466 of SEQ ID NO:43 or a variant thereof; and

[0256] ii. amino acid residues 20 to 469 of SEQ ID NO:47 or a variant thereof; and

[0257] b. a light chain comprising an amino acid sequence selected from the group consisting of: i. amino acid residues 20 to 237 of SEQ ID NO: 48 or a variant thereof;

[0258] ii. amino acid residues 20 to 237 of SEQ ID NO:49 or a variant thereof; and

[0259] iii. amino acid residues 20 to 237 of SEQ ID NO: 50 or a variant thereof.

[0260] In one embodiment, the present invention provides an isolated antibody or antibody fragment that binds to human PD-1, comprising: a heavy chain comprising amino acid residues 20 to 466 of SEQ ID NO: 43 or a variant thereof, and / or a light chain comprising amino acid residues 20 to 237 of SEQ ID NO: 48 or a variant thereof.

[0261] In one embodiment, the present invention provides an isolated antibody or antibody fragment that binds to human PD-1, comprising: a heavy chain comprising amino acid residues 20 to 466 of SEQ ID NO: 43 or a variant thereof, and / or a light chain comprising amino acid residues 20 to 237 of SEQ ID NO: 49 or a variant thereof.

[0262] In one embodiment, the present invention provides an isolated antibody or antibody fragment that binds to human PD-1, comprising: a heavy chain comprising amino acid residues 20 to 466 of SEQ ID NO: 43 or a variant thereof, and / or a light chain comprising amino acid residues 20 to 237 of SEQ ID NO: 50 or a variant thereof.

[0263] In one embodiment, the present invention provides an isolated antibody or antibody fragment that binds to human PD-1, comprising: a heavy chain comprising amino acid residues 20 to 469 of SEQ ID NO: 47 or a variant thereof, and / or a light chain comprising amino acid residues 20 to 237 of SEQ ID NO: 48 or a variant thereof.

[0264] In one embodiment, the present invention provides an isolated antibody or antibody fragment that binds to human PD-1, comprising: a heavy chain comprising amino acid residues 20 to 469 of SEQ ID NO: 47 or a variant thereof, and / or a light chain comprising amino acid residues 20 to 237 of SEQ ID NO: 49 or a variant thereof.

[0265] In one embodiment, the present invention provides an isolated antibody or antibody fragment that binds to human PD-1, comprising: a heavy chain comprising amino acid residues 20 to 469 of SEQ ID NO: 47 or a variant thereof, and / or a light chain comprising amino acid residues 20 to 237 of SEQ ID NO: 50 or a variant thereof.

[0266] In one embodiment, the variant of the antibody or antibody fragment that binds to PD-1, preferably human PD-1, of the present invention may contain up to three, ie, one, two or three conservative amino acid substitutions.

[0267] In one embodiment, the antibody or antibody fragment that binds to PD-1, preferably human PD-1, further comprises:

[0268] a. a human heavy chain constant region or a variant thereof, wherein the variant comprises up to 20, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conservative amino acid substitutions; and / or

[0269] b. a human light chain constant region or a variant thereof, wherein the variant comprises up to 20, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conservative amino acid substitutions.

[0270] In one embodiment, the human heavy chain constant region of the antibody or antibody fragment that binds to PD-1, preferably human PD-1, of the present invention may comprise a γ4 or γ1 human heavy chain constant region or a variant thereof, wherein the variant comprises up to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conservative amino acid substitutions.

[0271] In one embodiment, the PD-1 binding antibody or antibody fragment of the present invention may:

[0272] a. Binds to human PD-1 with a KD of about 100 pM or less;

[0273] b. binds to human PD-1 with a KD of about 30 pM or less;

[0274] c. binds to human PD-1 with about the same KD as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 44;

[0275] d. binds to human PD-1 with about the same KD as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 45;

[0276] e. About 7.5x10 5 1 / M·s or faster k assoc Binds to human PD-1;

[0277] f. About 1x10 6 1 / M·s or faster k assoc Binds to human PD-1;

[0278] g. About 2x10 -5 1 / s or slower k dissoc Binds to human PD-1;

[0279] h. About 2.7x10 -5 1 / s or slower k dissoc Binds to human PD-1;

[0280] i. About 3x10 -5 1 / s or slower k dissoc Binds to human PD-1; and / or

[0281] j. With IC of about 1 nM or lower 50 Block the binding of human PD-L1 or human PD-L2 to human PD-1.

[0282] KD, k assoc and k dissoc The value can be measured using any available method. In a preferred embodiment, the dissociation constant is measured using biointerferometry (e.g., the ForteBio Octet method described in, for example, Example 2 on page 45 of WO 2008 / 156712). In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance (e.g., Biacore) or Kinexa.

[0283] Furthermore, in any embodiment, the antibodies or antibody fragments of the present invention can block the binding of human PD-L1 or human PD-L2 to human PD-1, IC 50 The blockade of ligand binding can be measured using any method known in the art, and the IC 50 , for example, the FACS or FMAT (fluorescence microvolume assay technology) methods described in the "Ligand Blockade" section of Example 2, e.g., WO 2008 / 156712, pages 46-47.

[0284] The present invention also includes antibodies or antibody fragments that compete with any antibody of the present invention targeting PD-1 (preferably human PD-1) for a binding epitope on PD-1 and have one of the following characteristics:

[0285] a. Binds to human PD-1 with a KD of about 100 pM or less;

[0286] b. binds to human PD-1 with a KD of about 30 pM or less;

[0287] c. binds to human PD-1 with about the same KD as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 44;

[0288] d. binds to human PD-1 with about the same KD as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 45;

[0289] e. About 7.5x10 5 1 / M·s or faster k assoc Binds to human PD-1;

[0290] f. About 1x10 6 1 / M·s or faster k assoc Binds to human PD-1;

[0291] g. About 2x10 -5 1 / s or slower k dissoc Binds to human PD-1;

[0292] h. About 2.7x10 -5 1 / s or slower k dissoc Binds to human PD-1;

[0293] i. About 3x10 -5 1 / s or slower k dissoc Binds to human PD-1; and / or

[0294] j. With IC of about 1 nM or lower 50 Block the binding of human PD-L1 or human PD-L2 to human PD-1.

[0295] In one embodiment, the antibody or antibody fragment of the present invention that binds to PD-1, preferably human PD-1, may be:

[0296] a. chimeric antibodies or fragments thereof;

[0297] b. human antibodies or fragments thereof;

[0298] c. a humanized antibody or fragment thereof; and / or

[0299] d. An antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 and diabody.

[0300] In one embodiment, the antibody or antibody fragment of the present invention that binds to PD-1, preferably human PD-1, can increase the activation of T cells.

[0301] In one embodiment, the antibody or antibody fragment that binds to human PD-1 is a monoclonal and / or humanized antibody or a fragment of a monoclonal and / or humanized antibody. In one embodiment, the antibody or antibody fragment that binds to human PD-1 is a chimeric antibody or a chimeric antibody fragment. In one embodiment, the antibody that binds to human PD-1 is pembrolizumab (Keytruda) or a fragment thereof. In another embodiment, the antibody that binds to the EGF receptor, in particular to ERBB2, is trastuzumab (Herceptin) or a fragment thereof.

[0302] The preparation of chimeric, human, humanized or monoclonal antibodies and methods of antibody purification are known in the art and can be prepared as described, for example, in WO 2008 / 156712, which is incorporated herein in its entirety, particularly with respect to pages 27-31.

[0303] In another preferred aspect of the present invention, the agent effective for treating or preventing esophageal disease is an antiproliferative agent.

[0304] As used herein, "antiproliferative agent" refers to a substance that prevents, blocks, reduces, decreases or inhibits cell proliferation and / or cell growth (especially of cancer cells). The term also relates to agents that are active in preventing, reducing, reducing or inhibiting the spread of cells (especially malignant cells) to surrounding tissues. Thus, the antiproliferative agent can interfere with the formation of metastases. The term can also relate to agents that kill cells (especially cancer cells). The agents include, but are not limited to, chemotherapeutic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, plant alkaloids, plant taxanes, platinum-based agents or steroid hormones. The antiproliferative agents described herein are known in the art and can be prepared according to any known method. The antiproliferative agents described herein are also commercially available.

[0305] In one embodiment, the anti-proliferative agent is selected from the group consisting of a taxane; a pyrimidine analog, and a platinum-based agent.

[0306] Taxanes belong to the class of diterpenes. They were originally identified from natural sources, such as plants of the genus Taxus. Due to their poor water solubility, the pharmaceutical formulation of taxanes is difficult. In principle, any taxane can be used for the purposes of the present invention, including but not limited to isolated natural taxanes, naturally modified taxanes, such as semisynthetic taxanes or first and / or second generation taxanes. Taxanes are mitotic inhibitors, also known as spindle toxins. Because they prevent the depolymerization of microtubules, they inhibit the cell division process by disrupting microtubule function. In one embodiment, the taxane is selected from the group consisting of paclitaxel, docetaxel and cabazitaxel, preferably paclitaxel. In one embodiment, the taxane is present in a carrier, such as a liposome, preferably a cationic liposome.

[0307] Pyrimidine analogs are heterocyclic organic compounds based on pyrimidine. Pyrimidine analogs are used in cancer therapy due to their anti-metabolic activity. Specifically, pyrimidine analogs act as structural units of DNA and interfere with the production of DNA by incorporating chemically altered nucleotides into growing DNA chains or by exhausting the supply of deoxynucleotides required for DNA proliferation and DNA replication. They interrupt normal cell division and interfere with tumor growth because tumor cells spend more time in cell division than other cells. Therefore, tumor cells are particularly affected by pyrimidine analogs. In a preferred embodiment, the pyrimidine analog is a uracil analog, preferably 5-fluorouracil or capecitabine. In one embodiment, the pyrimidine analog is present in a nucleic acid vector system as described herein.

[0308] Platinum-based agents are coordination complexes of platinum, used to treat almost half of cancer patients. Without being bound by any theory, platinum-based agents cause crosslinking of DNA, and the crosslinking formed inhibits DNA repair and / or DNA synthesis. In a preferred embodiment, the platinum-based agent is selected from the group consisting of cisplatin or its salt, carboplatin or its salt, nedaplatin or its salt and oxaliplatin or its salt. The platinum-based agent can be further selected from the group consisting of triplatinum tetranitrate or its salt, phenanthreneplatin or its salt, picoplatin or its salt and satraplatin or its salt. The platinum-based agent can also include a platinum salt, preferably a cisplatin salt, a carboplatin salt or an oxaliplatin salt.

[0309] The present invention provides a drug delivery system, which includes the medicaments described herein for effective treatment or prevention of esophageal diseases. The drug delivery system described herein may also include one or a combination of (e.g., two or more different) medicaments for effective treatment or prevention of esophageal diseases described herein, or other medicaments for effective treatment or prevention of esophageal diseases.

[0310] For example, the inhibitory polynucleotides, antibodies or antibody fragments or antiproliferative agents of the invention can be combined with treatments that are considered standard of care for cancer. The rationale for such a combination is that the combination will induce or promote an initial clinical response to standard of care treatment, induce a durable clinical response and long-term immune control of the disease.

[0311] In one embodiment, treatment using the inhibitory polynucleotides, antibodies, or antibody fragments of the present invention can be combined with chemotherapy. Chemotherapy will cause cancer cell death, thereby increasing the release of tumor antigens. This increase in tumor antigen availability can lead to synergy with treatment using the inhibitory polynucleotides, antibodies, or antibody fragments of the present invention. Non-limiting examples are provided by combining inhibitory polynucleotides, antibodies, or antibody fragments, or antiproliferative agents with antiproliferative agents of the present invention. In one embodiment, treatment using antiproliferative agents of the present invention can be combined with chemotherapy (i.e., an antiproliferative agent different from the antiproliferative agents of the present invention) to provide a synergistic effect.

[0312] In one embodiment, treatment using the inhibitory polynucleotides, antibodies or antibody fragments or antiproliferative agents of the invention can be combined with radiation therapy. Radiation therapy induces cancer cell death and increases tumor antigen presentation and the availability of activated immune cells. In another embodiment, treatment using the inhibitory polynucleotides, antibodies or antibody fragments or antiproliferative agents of the invention can be combined with surgery to remove cancer cells from a subject.

[0313] Additional Active Pharmaceutical Ingredients (API)

[0314] The API in this dosage form may be administered together with an additional API.

[0315] In addition to the medicament effective for treating or preventing esophageal diseases, the additional API that may be present is referred to herein as an "additional active pharmaceutical ingredient" or "additional active ingredient" or "additional API". In principle, any additional pharmaceutically active agent may be used that enhances or increases the efficacy of the medicament effective for treating or preventing esophageal diseases. Such additional API may be selected by the skilled person according to his or her general knowledge depending on the condition to be treated and / or prevented.

[0316] For example, the present invention also includes immunoconjugates comprising an antibody or antibody fragment of the present invention linked to a therapeutic agent such as a bacterial toxin, an antiproliferative agent, or a radiotoxin. Non-limiting examples of cytotoxic agents include paclitaxel, cytochalasin B, mitomycin, etoposide, and vincristine or other antimetabolites, alkylating agents, antibiotics, and antimitotic agents. As used herein, "immunoconjugate" refers to an antibody or fragment thereof conjugated to a therapeutic moiety such as a bacterial toxin, a cytotoxic drug, an antiproliferative agent, or a radiotoxin. The toxic moiety can be conjugated to the antibody of the present invention using methods available in the art.

[0317] In some embodiments, the inhibitory polynucleotides, antibodies or antibody fragments or antiproliferative agents of the present invention can be combined with a second therapeutic agent or treatment. In one embodiment, the inhibitory polynucleotides, antibodies or antibody fragments or antiproliferative agents of the present invention can be combined with cancer treatment, which includes the use of recombinant cytokines or secretory immune factors or other antiproliferative agents, such as taxanes, pyrimidine analogs (e.g., fluoropyrimidine analogs) or platinum-based agents. Non-limiting examples of combinations include combining inhibitory polynucleotides, antibodies or antibody fragments or antiproliferative agents of the present invention with recombinant IL-2, chemotherapeutic agents or recombinant EFNα2. Recombinant IL-2 enhances the growth of T cells in cancer patients. Recombinant EFNα2 inhibits cancer cell growth, but also increases the expression of inhibitory ligands of PD-1 on cancer cells, antigen presenting cells and other somatic cells in patients receiving treatment. The inhibitory polynucleotides, antibodies or antibody fragments or antiproliferative agents of the present invention can be combined with other cytokines that are considered useful for treating cancer.

[0318] In a specific embodiment, an antibody or binding fragment thereof for use in the present invention, such as an antibody targeting a polypeptide encoding PD-1, preferably an antibody that binds to human PD-1, such as pembrolizumab (Keytruda), or a fragment thereof, or an antibody that binds to an EGF-receptor, preferably trastuzumab, or a fragment thereof, is combined with a platinum-based antiproliferative agent and / or a fluoropyrimidine-based antiproliferative agent for the treatment of an esophageal disease or condition. In one embodiment, the platinum-based antiproliferative agent is, for example, cisplatin. In one embodiment, the fluoropyrimidine-based antiproliferative agent is 5-fluorouracil and / or capecitabine. In one embodiment, the esophageal disease or condition is esophageal cancer, such as esophageal squamous cell carcinoma, preferably progressive or metastatic esophageal squamous cell carcinoma, more preferably esophageal squamous cell carcinoma with a tumor expressing PD-L1, or esophageal adenocarcinoma / esophageal junction cancer, preferably progressive or metastatic esophageal adenocarcinoma, more preferably esophageal adenocarcinoma with a tumor expressing PD-L1, which may be HER-2 positive or HER-2 negative. In preferred embodiments, the treatment is first-line treatment.

[0319] preparation

[0320] In a preferred embodiment of the drug delivery system according to the present invention, the sheet-like preparation is a sheet or is formed into a sheet. The term "sheet" used herein refers to a sheet-like object comprising a plurality of layers for encapsulating an agent effective for treating or preventing esophageal diseases.

[0321] Such a sheet can conform to the irregular surface contours of the intended site of action, in particular the irregular surface contours of the esophageal mucosa, in particular after the sheet absorbs the moisture contained in the esophageal mucosa. In addition, the sheet-like preparation of the dosage form according to the present invention can be gellable or swellable.

[0322] In a preferred embodiment of the drug delivery system according to the present invention, the thickness of the sheet-like preparation is 0.01 mm to 2 mm, preferably 0.03 mm to 1 mm, preferably 0.05 mm to 0.1 mm. This is conducive to providing a sheet-like preparation with a relatively small thickness.

[0323] In a preferred embodiment of the drug delivery system according to the present invention, the area of ​​the sheet preparation is 0.5 to 25 cm 2 , preferably 1 to 10 cm 2 .

[0324] The sheet preparation can have different shapes. In particular, the sheet preparation can have a circular, triangular, quadrilateral or polygonal shape. In one embodiment, the hole is adapted to the corresponding shape of the preparation.

[0325] In a preferred embodiment of the drug delivery system according to the present invention, the sheet-shaped preparation containing a pharmaceutical agent effective for treating or preventing esophageal diseases, particularly a film-shaped, foil-shaped or thin sheet-shaped preparation, contains a pharmaceutical agent effective for treating or preventing esophageal diseases, and its drug content is 0.0001 to 50% by weight, preferably 0.001 to 25% by weight, and most preferably 0.01 to 10% by weight.

[0326] The sheet-like preparation containing an agent effective for treating or preventing esophageal diseases may have a single-layer or multi-layer structure, wherein at least one layer (preferably the first layer) contains an agent effective for treating or preventing esophageal diseases.

[0327] In a preferred embodiment, the sheet preparation has a multilayer structure of multiple layers, wherein at least the first layer contains an agent effective for treating or preventing esophageal diseases, and wherein at least another layer contains at least one additional active pharmaceutical ingredient, which is the same or different agent effective for treating or preventing esophageal diseases, or is not an agent effective for treating or preventing esophageal diseases, such as a steroid.

[0328] In a preferred embodiment, the layer containing the agent effective for treating or preventing esophageal disease and / or the further layer containing the additional active pharmaceutical ingredient comprises a polymer, preferably a film-forming polymer.

[0329] The polymer in the layer can be used only as a carrier for the medicament and / or additional API for the effective treatment or prevention of esophageal disease, or it can be used as a reservoir for the above-mentioned substances. Such a layer can release the medicament and / or additional active pharmaceutical ingredient for the effective treatment or prevention of esophageal disease under the action of a fluid. The medicament and / or additional API for the effective treatment or prevention of esophageal disease can be released immediately or in a controlled release manner.

[0330] In a preferred embodiment of the drug delivery system according to the present invention, the sheet-like preparation comprises at least a first layer containing an agent effective for treating or preventing esophageal diseases and / or another layer containing an agent effective for treating or preventing esophageal diseases and / or an additional API, wherein the at least one first layer and / or the another layer is an adhesive layer.

[0331] In a preferred embodiment of the drug delivery system according to the present invention, at least one first layer containing the active ingredient and / or the further layer containing the active ingredient comprises a polymer, preferably a film-forming polymer, wherein the polymer is a water-dispersible and / or decomposable and / or water-disintegrable film-forming polymer.

[0332] The polymer for the first layer containing the active substance and / or for the further layer containing the active substance can be selected from the group comprising polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl acetate, polyethylene glycol, polyethylene oxide polymers, polyurethanes, polyacrylic acid, polyacrylates, polymethacrylates, poly(methyl vinyl ether-maleic anhydride), starch, starch derivatives, natural gums, alginates, pectins and gelatins, pullulan, gel-forming proteins, chitosan, agar, agarose, carrageenan, xanthan gum, tragacanth gum, dextran and cellulose ethers such as ethylcellulose, hydroxyethylcellulose, propylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylethylcellulose, cellulose acetate, povidone and copovidone. In a preferred embodiment, the polymer is polyvinyl alcohol, preferably polyvinyl alcohol 18-88.

[0333] The polymers can be used alone or in combination with one another to produce a sheet preparation for a dosage form according to the invention which has the desired properties, such as adhesion, release or disintegration properties.The sheet preparation according to the invention can consist of a single polymer layer.

[0334] In addition, the sheet preparation used in the dosage form according to the present invention can have a two-layer or multi-layer structure, wherein at least one layer contains an effective agent for treating or preventing esophageal diseases, optionally in combination with a nucleic acid delivery system and / or other excipients for stabilizing the effective agent for treating or preventing esophageal diseases. It is also possible that multiple layers contain an effective agent for treating or preventing esophageal diseases or additional APIs.

[0335] In a preferred embodiment of the drug delivery system according to the present invention, the sheet-like preparation comprising an antiproliferative agent comprises a monolayer structure or consists of a monolayer structure, wherein the (preferably first) layer contains an antiproliferative agent, preferably coated with an antiproliferative agent, preferably paclitaxel. The layer comprises a polymer, preferably a film-forming polymer, wherein the polymer is a water-dispersible and / or decomposable and / or water-disintegratable film-forming polymer. The polymer is a polymer as described herein, preferably polyvinyl alcohol, preferably polyvinyl alcohol 4-88 or polyvinyl alcohol 18-88. The layer also comprises additives as described herein, such as a plasticizer, preferably glycerol and a surfactant, preferably lauryl alcohol, such as ethoxylated lauryl alcohol. Such surfactants are commercially available, including but not limited to Brij TM L23. The purpose of surfactants is to provide further stability by reducing hydrolysis and adsorbing undesirable substances.

[0336] In a further preferred embodiment of the drug delivery system according to the present invention, the sheet-like preparation comprising an inhibitory polynucleotide comprises a monolayer structure or is composed of a monolayer structure, wherein the (preferably first) layer contains an inhibitory polynucleotide, preferably coated with an inhibitory polynucleotide, preferably siRNA, antisense oligonucleotide or aptamer, optionally in combination with a nucleic acid delivery system. The layer comprises a polymer, preferably a film-forming polymer, wherein the polymer is a water-dispersible and / or decomposable and / or water-disintegratable film-forming polymer. The polymer is a polymer as described herein, preferably polyvinyl alcohol, preferably polyvinyl alcohol 18-88. The layer also comprises an additive as described herein, such as a plasticizer, preferably glycerol and / or a stabilizer, preferably sucrose.

[0337] In a further preferred embodiment of the drug delivery system according to the present invention, the sheet-like preparation comprising the antibody comprises a monolayer structure or is composed of a monolayer structure, wherein the (preferably first) layer contains the antibody, preferably coated with the antibody. The layer comprises a polymer, preferably a film-forming polymer, wherein the polymer is a water-dispersible and / or decomposable and / or water-disintegratable film-forming polymer. The polymer is a polymer as described herein, preferably polyvinyl alcohol, preferably polyvinyl alcohol 18-88.

[0338] In another preferred embodiment of the drug delivery system according to the present invention, the sheet-like preparation containing the agent effective for treating or preventing esophageal diseases comprises at least one first inactive ingredient layer containing no active pharmaceutical ingredient.

[0339] In a preferred embodiment of the drug delivery system according to the present invention, the sheet-shaped preparation, particularly the film-shaped, foil-shaped or sheet-shaped preparation, comprising the active pharmaceutical ingredient comprises at least one other active ingredient-free layer containing no active pharmaceutical ingredient.

[0340] In a preferred embodiment of the drug delivery system according to the present invention, the first inactive ingredient layer and / or at least one other inactive ingredient layer is a water-insoluble layer, which preferably comprises a water-insoluble substance selected from ethylcellulose and / or a combination of ethylcellulose and other water-insoluble substances, a hydrophobic plasticizer, especially triethyl citrate, and / or a dye and / or an aromatic and / or a flavoring agent.

[0341] In particular, the use of ethylcellulose may be beneficial due to its properties, including good processability, biocompatibility, and water insolubility.

[0342] In a preferred embodiment of the drug delivery system according to the present invention, the first inactive ingredient layer and / or at least one other inactive ingredient layer is an adhesive layer of a desired thickness.

[0343] The adhesive layer may be a mucoadhesive polymer selected from the group consisting of cellulose derivatives such as hydroxypropylcellulose, starch and starch derivatives, polyvinyl alcohol, polyethylene oxide, polyethylene, polypropylene, polyacrylic acid and polyacrylate derivatives, polyvinyl pyrrolidone, povidone, copovidone, sodium alginate, gelatin, xanthan gum, carrageenan, pectin, dextran, lectin, chitosan, pullulan and mixtures thereof.

[0344] Additionally, or alternatively, the adhesive layer may comprise a solvent selected from the group consisting of water, ethanol, methanol, acetone, organic solvents and mixtures thereof.

[0345] In addition, the preparation may also contain additives such as colorants, fragrances, flavoring agents, preservatives, antioxidants, penetration enhancers, solubilizers, disintegration accelerators, pore formers, lubricants, stabilizers and mixtures thereof. In particular, the following substances qualify as additives: lubricants, lubricants, glidants, binders, additional active ingredients, disintegrants, antioxidants, chelating agents, coating agents, flow agents, preservatives, fillers, surfactants, plasticizers, stabilizers and pigments. In addition, additives may be selected from the following group: pore formers, penetration enhancers, solubilizers, emulsifiers, including polyethoxylated sorbitan fatty acid esters, ethoxylated fatty alcohols and lecithin; plasticizers, including polyethylene glycol, glycerol and other polyols, higher alcohols such as dodecanol, undecanol or octanol, sorbitol, mannitol and other sugar alcohols, dexpanthenol and triglycerides; fillers, including highly dispersed silicon dioxide, titanium dioxide, zinc oxide, chalk and starch; colorants; sweeteners and flavorings; humectants; preservatives ; pH regulators and antioxidants; disintegration accelerators; penetration enhancers that promote reabsorption of the active pharmaceutical ingredient into the mucosa, such as cellular uptake, such as fatty acids and their salts and fatty acid esters, preferably saturated fatty acids such as caprylic acid (C8), capric acid (C10), octadecanoic acid (C18), or unsaturated fatty acids such as oleic acid (C18), salcaprozate (SNAC) or its salts, terpenes, glycolipids, medium chain triglycerides, synthetic waxes such as isopropyl myristate, branched fatty alcohols such as Eutanol Urea, polypropylene glycol, dimethyl sulfoxide, azone, azone analogs, polyols such as propylene glycol, tocopherol or essential oils such as menthol. A preferred plasticizer is glycerol.

[0346] The sheet preparation may further comprise at least one taste-masking additive. This advantageously masks the active pharmaceutical ingredient of bitterness or other unpleasant tastes, but is also conducive to accelerating the onset of the active pharmaceutical ingredient. The taste-masking additive is well known to those skilled in the art. This taste-masking additive can especially include sugar alcohols and sodium bicarbonate selected from mannitol, sorbitol, xylitol, maltitol, lactitol, erythritol, threitol and isomalt.

[0347] In particular, the additives may increase the local availability of the active ingredient, such as penetration enhancers.

[0348] According to a preferred embodiment, the drug delivery system according to the present invention, particularly the sheet preparation, is intended to achieve the delayed release of active ingredients. The medicament for the effective treatment or prevention of esophageal disease is preferably released within a period of 4 hours, preferably within a period of 6 hours, and most preferably within a period of 8 hours. In order to achieve the delayed release of active ingredients in the case of a double or multilayer preparation, at least one layer in the layer containing the medicament for the effective treatment or prevention of esophageal disease, particularly a polymer layer, has the delayed release of active ingredients.

[0349] For delayed release of the active ingredient, the film-shaped medicament is preferably formulated as a slowly dissolving or slowly disintegrating film, which completely disintegrates or dissolves only after a few hours. Preferably, they completely disintegrate or dissolve only after 4 hours, preferably only after 6 hours, even most preferably only after 8 hours or even only after 24 hours.

[0350] In particular, the agent effective for treating or preventing esophageal diseases and the optional additional API are released over a period of 15 minutes to 24 hours, 2 hours to 24 hours, 3 hours to 12 hours, 4 hours to 8 hours or 5 hours to 6 hours.

[0351] The sheet preparation can be prepared by a person skilled in the art by a basically known method, for example by coating an inert support with a liquid composition comprising a polymer, an agent effective for treating or preventing esophageal diseases / additional active pharmaceutical ingredients and optional additives and solvents, by a method involving, for example, a doctor blade (e.g., solvent casting), a spray processor or an extrusion processor. The film layer obtained in this way is dried. For a multilayer sheet preparation, one or more coatings can be applied to an existing film layer in the same manner, or can be manufactured separately and then laminated.

[0352] In the process of preparing the preparation, it may be necessary to consider the temperature sensitivity, pH sensitivity, enzyme stability and / or solubility of the medicament used for the effective treatment or prevention of esophageal disease. Of course, in the whole production process of the drug delivery device, the specific properties of the active pharmaceutical ingredient should also be considered. Therefore, in addition, considering the required low dose, an impregnation method can be used. In this preferred method of the present invention, only the solution containing the medicament for the effective treatment or prevention of esophageal disease is applied, such as spraying or dripping on the polymer film, and finally it is dried. These methods are known to the technician and are described in the embodiments provided herein.

[0353] In another example, an agent effective in treating or preventing esophageal disease can be incorporated and thereby embedded in a polymer film, such as by solvent casting.

[0354] In all these methods, attention should be paid to the solvent used and the drying conditions. As a very good drying method, freeze drying can be used.

[0355] In addition, depending on the stability of the incorporated effective therapeutic or preventive agent for esophageal disease, it is also conceivable to melt extrude the polymer and the effective therapeutic or preventive agent for esophageal disease, for example, as described in Example 5 herein. Alternatively, the solution containing the effective therapeutic or preventive agent for esophageal disease can be applied to the polymer film by inkjet method.

[0356] In one embodiment, the formulation is prepared so that the agent effective in treating or preventing esophageal disease is present only in certain portions of the membrane, which would allow for tailored treatment of the mucosa in only the designated areas.

[0357] Alternatively, and preferably, the first area of ​​the sheet preparation can contact with esophageal mucosa, and the second area of ​​the sheet preparation can contact with oral mucosa. In this way, esophageal mucosa can be treated with the medicament effectively treating or preventing esophageal diseases, and oral mucosa is treated with the second medicament, additional API for effectively treating or preventing esophageal diseases or is untreated, or additives are released to oral mucosa. Especially, flavorings and / or local anesthetics can be released, particularly in order to increase or reduce the generation of saliva and / or make the application of the drug delivery system more pleasant and / or suppress the impulse of vomiting. Alternatively, the first area of ​​the sheet preparation can contact with esophageal mucosa, and the second area of ​​the sheet preparation can contact with the mucosa of the upper stomach (such as cardia or cardia and fundus). Therefore, it is possible to locally treat esophagus and part of the stomach.

[0358] In another preferred embodiment, the drug delivery system, particularly the capsule device, includes a sinker device. The sinker device is configured to provide negative buoyancy to the capsule device. In the experiment of the inventor who supported the discovery of this preferred embodiment, it was found that reducing buoyancy, such as by increasing the mass of the capsule device, led to the reliability of the mechanical process of expanding the preparation from a compact state to an extended state. In the case of a strip preparation, the unwinding of the preparation from a compact state (wherein the strip preparation is wound around the winding shaft) to an extended state is significantly promoted and more effective. The potential problem of this preferred embodiment is that it is observed that the transition of the preparation from a compact state to an extended state is sometimes incomplete. Although the present invention has improved the efficiency of expansion or unwinding by providing a spacing between the opening and the preparation, the sinker device also improves the efficiency of expansion. It is believed that the capsule device, even if swallowed correctly by the patient in the presence of water or an aqueous solution, will not be completely filled with water, but bubbles will sometimes remain in the capsule device. Air contributes to buoyancy, and the sinker device assists in resisting the buoyancy effect by assisting in replacing air or by using a material denser than water using gravity. Further details about the sedimentation section can be inferred from WO2020 / 183005, which is incorporated herein by reference.

[0359] Preferred embodiments of the drug delivery system according to the present invention are suitable for application to the nasopharyngeal mucosa.

[0360] When the sheet-like preparation releases the medicament effective for treating or preventing esophageal diseases locally and / or over an extended period of time, optionally together with an additional API, the therapeutic response can be improved, in particular the local effect of the medicament effective for treating or preventing esophageal diseases can be increased by, for example, a penetration enhancer. Such penetration enhancers are known in the art. In addition, the necessity for systemic administration can be reduced, in particular due to the spatial extension of the area of ​​action.

[0361] In a preferred embodiment of the drug delivery system according to the present invention, the area and / or surface area of ​​the sheet-like preparation is 0.5 to 25 cm 2 , preferably 2 to 25 cm 2 , preferably 5 to 25 cm 2 , preferably 5 to 15 cm 2 , preferably greater than 0.5 cm 2 , and preferably less than 40cm 2 Preferably, the ratio of the length of the sheet to the width of the sheet is 40:1 to 400:1, or preferably 60:1 to 300:1, or preferably 80:1 to 200:1. The width may be the average width of the sheet, for example measured perpendicularly to the length of the sheet. The ratio may be the ratio of the length and the circumference of the sheet, in particular the average value of the sheet, wherein in the case of a strip-shaped sheet, the circumference may be, for example, twice the width of the sheet.

[0362] In some embodiments of the drug delivery system according to the present invention, the sheet preparation is in the solid state, particularly when it is in compact form and / or after it has just released. This can beneficially enhance, realize or promote some of the above-mentioned advantages. Especially, when it is in the solid state before release, this can improve storability. Especially, when the medicament of described effective treatment or prevention esophageal disease is in the solid state after it releases, this can enhance and / or realize its targeting and / or sustained release. In addition, or alternatively, in some embodiments of the drug delivery system according to the present invention, the sheet preparation is suitable for dissolving immediately, after delay, in a time-controlled mode or under a certain stimulation after its release, for example biodegradable. This can beneficially enhance, realize or promote some of the above-mentioned advantages. Especially, this can improve the convenience of user, because the sheet preparation does not need to be removed.

[0363] Additionally, or alternatively, in certain embodiments of the dosage form according to the invention, the sheet is adapted to dissolve, e.g. biodegrade, preferably in a time-controlled manner, e.g. within one hour, or within one to two hours, or within one to five hours, or within one to twelve hours, or within one to twenty-four hours. This improves user convenience, as the sheet does not need to be removed.

[0364] Applicator / Retainer

[0365] In one embodiment, an applicator with a retainer is used to assist in swallowing the capsule device in combination with a drinking cup. The applicator combined with the drinking cup allows the patient to take the drug delivery system, just like drinking water from a bottle. Therefore, the applicator is mounted on the drinking cup as an access port (mouthpiece). The drug delivery system is located in the retainer of the applicator. When drinking, the liquid of the drinking cup rinses the internal applicator and retainer, which can release the preparation from the retainer and deliver it to the patient's mouth, and then the patient swallows it. The rope member is a retainer and is wrapped around the retainer. The rope member is also connected to the retainer and the end of the preparation extending through the hole. Therefore, during drinking, when the preparation leaves the retainer, the retainer is unwound until it is tightened. Then a force is applied to the preparation to pull the preparation out of the capsule.

[0366] Such an applicator and drinking cup are described, for example, in PCT / EP2020 / 056927, the contents of which are incorporated herein by reference in their entirety with respect to the applicator, drinking cup and thread. Such a retainer is further described, for example, in EP21175427.0 and EP21175436.1, the contents of which are incorporated herein by reference in their entirety with respect to the retainer.

[0367] In a preferred embodiment, the retainer is wound around the support structure of the holder, and one end of the retainer is attached to the support structure and the other end is connected to the dosage of the capsule device. Therefore, the capsule device is positioned and retained inside the holder of the applicator. When the patient swallows the dosage form, the retainer begins to unwind from the support structure. The applicator and the support structure have a cylindrical shape so that the support structure fits into the applicator and is particularly mounted therein in a rotatable form so that the retainer can be unwound from the structure by rotating the structure.

[0368] This is particularly advantageous if the dosage form is to be administered regularly, particularly daily, since administration of the capsule device can be accomplished without professional assistance.

[0369] In a preferred embodiment of the drug delivery system according to the present invention, the release mechanism comprises a retainer, which is preferably a thin wire, wherein the thin wire is expandable from a compact form to an expanded form and is connected to one end of the dosage form extending from the capsule device. BRIEF DESCRIPTION OF THE DRAWINGS

[0370] Exemplary embodiments of the invention will be described in more detail below with reference to the accompanying drawings and samples, from which further features, advantages and embodiments will become apparent.

[0371] Figure 1a ,1b Each shows a schematic diagram of a capsule device of a drug delivery system.

[0372] Figure 2 A schematic diagram of the formulation in partially expanded form is shown.

[0373] Figure 3 A schematic diagram of one end of a dosage form connected to a retainer for pulling the dosage form out of a capsule device is shown.

[0374] Figure 4 A schematic diagram of the formulation as a three-layer sheet is shown.

[0375] Figure 5a , 5b Each shows a schematic diagram of a capsule device having an aperture formed by overlapping wall portions or by telescoping two capsule half shells into one another.

[0376] Figure 6 A schematic semi-transparent view of a drug delivery system is shown.

[0377] Figure 7 A schematic diagram of an applicator is shown having a holder and a retainer wrapped around the holder with a drug delivery system located inside the holder.

[0378] Figure 8a , 8b Each shows before swallowing the drug delivery system ( Figure 8a ) and period ( Figure 8b ), Schematic diagram of a patient taking a drug delivery system using an applicator and a drinking cup.

[0379] FIG. 9 shows the drug delivery system before swallowing ( Figure 9a ) and after ( Figure 9b ), a schematic diagram of a particular embodiment of a drug delivery system including a sinker for administration by a patient using an applicator and a drinking cup.

[0380] Fig.10a The structure of the fluorescent dye Atto633 is shown. Fig.10b The structure of ATTO633 and the chemical bonding to RNA are shown: the dotted line indicates the attachment site of the RNA.

[0381] Fig.11 HPLC results with calibration samples, blank membrane samples (without paclitaxel) and API loaded membrane samples (with paclitaxel) are shown. Shown are milliabsorbance units (mAU) versus retention time in minutes.

[0382] Fig.12The effect of the membrane on RNA stability is shown. RNA was analyzed on a 20% polyacrylamide gel. "RNA stock" refers to pure synthetic RNA, "F" refers to the membrane, "RNA1" refers to RNA added after the membrane was dissolved, and "RNA" refers to RNA applied to the dry membrane.

[0383] Fig.13 The reproducibility of stability results obtained with RNA synthesized with commercial siRNA is shown. "RNA stock" refers to the pure commercial RNA sample, "F+RNA" refers to the RNA applied on the dry membrane.

[0384] Fig.14 The storage stability of RNA on dry films is shown. "1" refers to the analysis performed directly after the sample was dried, and "2" refers to the analysis of the dried sample after keeping it at 4°C for 4 days.

[0385] Fig.15 The storage stability of RNA on the membrane after solubilization is shown. "1" refers to the analysis directly after solubilization, "2" refers to the analysis of the solubilized sample after 4 days at 4°C, and "3" refers to the analysis of the solubilized sample after 4 days at 4°C in the presence of an RNase inhibitor. The boxes indicate locations where the RNA is slightly degraded as shown in column 2, and this degradation can be prevented in the presence of an RNase inhibitor as shown in column 3.

[0386] Fig.16 The stability of antisense BMP2 RNA on the membrane under various application conditions is shown. "1" refers to the stock solution, i.e., the RNA solution before application to the membrane, "2" refers to the sample in which the membrane was in a wet state when RNA was applied to the membrane, "3" refers to the sample in which the membrane was in a dry state when RNA was applied to the membrane, "4" refers to the sample in which water was applied to the membrane and RNA was applied after the membrane was dissolved, "5" refers to the sample in which water was applied to the membrane, "6" refers to the sample in which RNA was applied to the membrane after the membrane was dissolved, and "7" refers to the sample after the membrane was dissolved. For samples 2 to 6, 0.5 cm 2 The membrane has been dissolved in 75 μl of water.

[0387] FIG. 17 shows the stability of Atto633-labeled antisense BMP2 RNA compared to unlabeled antisense BMP2 RNA on the membrane under various applied conditions. Fig.17a Ethidium bromide staining of RNA. The boxes indicate the positions of RNA on the gel. Fig.17bThe fluorescence of the labeled RNA is shown. "1" refers to the sample in which the membrane was in a dry state when the labeled RNA was applied to the membrane, "2" refers to the sample in which the membrane was in a wet state when the labeled RNA was applied to the membrane, "3" refers to the sample of the labeled RNA, "4" refers to the sample in which the membrane was in a dry state when the unlabeled RNA was applied to the membrane, "5" refers to the sample in which the membrane was in a wet state when the unlabeled RNA was applied to the membrane, and "6" refers to the sample of the unlabeled RNA. For samples 2 to 6, 0.5 cm 2 The membrane has been dissolved in 75 μl of water.

[0388] Figure 18 shows the stability of labeled and unlabeled BMP2 RNA after application to membranes and subsequent storage at 4°C for 10 days. Fig.18a The fluorescence of the labeled RNA is shown. Fig.18b Ethidium bromide staining of RNA is shown. "1" refers to the sample to which labeled BMP2 RNA was applied and the membrane was dissolved, and "2" refers to the sample to which labeled BMP2 RNA was not applied. For sample 1, 0.5 cm 2 The membrane has been dissolved in 75 μl of water.

[0389] Fig.19 The state of the initial product before drying is shown. Fig.19 is a graphic representation of the liposome size measurement results.

[0390] Fig. 20 The state of liposomes after drying on different membrane compositions is shown. Panel A shows the size measurement curves of liposomes after drying (black, dark grey and grey curves) and the comparison with the liposome size before drying (light grey curve). Different membrane compositions were used.

[0391] Figure 1a A schematic diagram of a drug delivery system 1 having a capsule device 2 having a first capsule shell half 2a and a second capsule shell half 2b, which are nested with each other to form a hole 3. A dosage form 4 is shown in its compact form within the capsule 2, with one end 4a extending out of the hole 3. The arrow indicates the direction of movement of the dosage form 4 as it is pulled out of the capsule 2, i.e., the first capsule shell half 2a, through the hole 3. Figure 1a In FIG. 1 , it is shown that the hole 3 is formed laterally to the central axis A of the capsule 2 and is arranged in the first half capsule shell 2a. Figure 2 In b, it is shown that the hole 3 is formed along the central axis A of the capsule 2 and is arranged in the first half capsule shell 2a.

[0392] Figure 2 A schematic diagram of a partially expanded form of the formulation 4 is shown. The formulation 4 is drawn to have a sheet-like shape. The central area of ​​the formulation 4 is indicated by a dotted line, so Figure 2Basically, the end 4a of the preparation 4 protruding from the hole 3 of the capsule device 2 and the still slightly coiled end 4b of the preparation 4 are shown. The coiled end 4b represents a compact form of the preparation 4. At the end 4a extending through the hole 3, the retaining device 5 is shown to have a patch-like shape. The retaining device 5 includes a strip 5a. The strip 5a is Figure 2 In the embodiment shown, it is used as a connector to connect the holding means 5 to the end 4a of the preparation 4. Alternatively, the end 4a of the preparation 4 is directly connected to the retainer, such as a thin wire. In such an embodiment, the holding means 5, 5a is formed by the retainer itself.

[0393] Figure 3 This preferred embodiment is shown. The end 4a of the preparation 4 is shown to have a sheet-like shape, and the retainer 6 overlaps the end of the preparation 4 with a length d to form a retaining device 5, 5a. The connection between the retainer 6 and the end 4a of the preparation 4 is such that when the retainer 6 is tightened to pull the preparation 4 out of the capsule device 2, for example by swallowing the dosage form 1, the pulling force can be transmitted through the connection.

[0394] Figure 4 Formulation 4 is shown divided into several layers. Figure 4 In the embodiment shown, the preparation is a sheet comprising three different layers 7. A top layer 7a is formed as an adhesive layer, a middle layer 7b contains an agent effective in treating or preventing esophageal diseases, and Figure 4 The lowest drawn layer in the graph shows a protective layer, for example a water protective layer.

[0395] Figure 5a , 5b Each shows a schematic diagram of a capsule device 2 of a drug delivery system 1 having a hole 3 formed by overlapping wall portions 9 or by telescoping two capsule half shells 2a, 2b. Figure 5a As shown, the first half capsule shell 2a is slit on the second half capsule shell, as shown by the dotted line. The second half capsule shell includes a recess 8. By partially sliding the two half bodies 2a, 2b against each other, the first half capsule shell 2a partially covers the recess 8 of the second half capsule shell 2b. Then, a wall portion 9 is further provided to cover the remaining open space formed by the recess 8, so that the hole 3 is formed as an opening, through which the preparation 4 can leave the shell 2.

[0396] Alternatively, Figure 5b An embodiment is shown in which the two capsule shell half 2a, 2b overlap in the joined position to such an extent that the opening 3 is formed by a particularly cylindrical wall of the first capsule shell half, which overlaps the opening 10 of the second capsule shell half 2b.

[0397] Figure 6A schematic semi-transparent view of a drug delivery system 1 is shown. The first and second capsule shell halves 2a, 2b are joined in a joined position, thereby forming a hole 3 by covering the opening 10 of the second capsule shell half 2b in this position. The end 4a of the formulation 4 is shown extending through the hole 3. The drug dosage form 1 further includes a sinker element 11, which is located in the first capsule shell half 2a. The sinker extends from the first capsule shell half 2a into the second capsule shell half 2b, while the groove 11a extends from the outside of the capsule device 2 into the inner space to position the sinker 11 and prevent the sinker from moving within the capsule. Figure 6 In FIG. 4 , the preparation 4 is shown to be located below the sedimentation 11. The groove prevents the sedimentation 11 from sliding into the preparation 4.

[0398] Figure 7 A schematic diagram of an applicator 12 is shown, the applicator having a holder 13 and a retainer 6 wrapped around the holder 13, while the drug delivery system 1 is located inside the holder 13. The applicator and the holder preferably have a cylindrical shape. The capsule device 2 is located inside the holder 13, with the first half 2a pointing towards the applicator cover 12a. The cover 12a is removed for use. Figure 7 In the illustrated embodiment, the capsule device 2 further comprises a sedimentation portion 11 located in the first half capsule shell 2a and a formulation 4 located in the second half capsule shell 2b. Figure 7 In the embodiment shown, the first half capsule shell 2a is additionally pressed toward the bottom of the applicator 12. Thus, the curved retainer 13a is located above the capsule device 2. The retainer 13a is curved so that its shape matches the shape of the first half capsule shell 2a. Pressing the capsule 2 into the retainer 13 is achieved by a compression spring 14, one end of which is attached to the cover 12a of the applicator 12 and the other end is attached to the curved retainer 13a. A drying element 15 is located inside the applicator 12, at the cover 12a of the applicator 12. This prevents the preparation 4 from being damaged by moisture. The applicator 12 does not necessarily include a curved retainer 13, a drying element 15 or a compression spring 14.

[0399] Figure 8a , 8b Each shows before swallowing the drug delivery system ( Figure 8a ) and period ( Figure 8b ), a schematic diagram of a patient taking the drug delivery system 1 using the applicator and drinking cup. Figure 8a A patient is shown receiving an application of a drug delivery system as described herein comprising a capsule device 2. A drinking cup 16 is filled with a liquid and an applicator 12 is attached to the cup 16. The applicator 12 comprises a retainer 6 and a drug delivery system 1, which further comprises a capsule device 2 connected to a formulation 4, and the formulation 4 is at least partially coiled inside the capsule device 2. Figure 8bIt is illustrated that when a patient swallows the dosage form 1, the dosage form 1 is then delivered to the stomach through the esophagus. The retainer 12 pulls the preparation 4 out of the capsule device 2. The preparation 4 then spreads along the esophagus, thereby delivering the active ingredient of the dosage form 1 to the esophageal mucosa.

[0400] Reference Symbols List

[0401] 1 Drug delivery system

[0402] 2 Capsule device

[0403] 2a First half capsule shell

[0404] 2b Second half capsule shell

[0405] 3 holes

[0406] 4 Preparation

[0407] 4a End of the preparation extending through the hole

[0408] 4b Coiled end of the preparation

[0409] 5. Holding device

[0410] 5a Band

[0411] 6 Retainer

[0412] 7th floor

[0413] 7a Adhesion layer

[0414] 7b Layer containing active pharmaceutical ingredient

[0415] 7c Protective layer

[0416] 8 concavity

[0417] 9 Wall section

[0418] 10 Opening

[0419] 11 Settlement elements

[0420] 11a Groove

[0421] 12 Applicator

[0422] 12a Applicator cap

[0423] 13 Retainer

[0424] 13a Bent retainer

[0425] 14 Spring

[0426] 15 Drying element

[0427] 16 Drinking cup

[0428] List of abbreviations:

[0429] hPD-1.08 Mouse monoclonal anti-hPD-1 antibody

[0430] hPD-1.09 Mouse monoclonal anti-hPD-1 antibody

[0431] 109A-H Humanized IgG1 09A heavy chain sequence with zero back mutations

[0432] 409A-H Humanized IgG4 09A heavy chain sequence with zero antibody framework region back mutations

[0433] K09A-L-11 Humanized 09A-κ sequence, whose framework originally had a CDR1 of 11aa length

[0434] K09A-L-16 Humanized 09A-κ sequence, whose framework originally had a 16aa length of CDR1

[0435] K09A-L-17 Humanized 09A-κ sequence, whose framework originally had a CDR1 of 17aa length

[0436] Example

[0437] Example 1: Preparation of polymer film containing paclitaxel

[0438] A "base polymer mixture" was prepared with the ingredients and amounts shown in Table 2, without the addition of paclitaxel. After the membrane was prepared, the membrane was coated with paclitaxel.

[0439] Table 2

[0440]

[0441] The polymer films were prepared using a solvent casting technique according to the following protocol:

[0442] Solvent Casting Technology:

[0443] 1. Preparation of polymer mixture

[0444] 2. Preparation of film laminates

[0445] 3. Cutting wet laminate

[0446] 4. Coating wet laminates

[0447] 5. Dry at room temperature

[0448] The resulting films were flexible, bubble-free, optically uniform, and exhibited a consistent film surface.

[0449] In addition, the content of paclitaxel in the membranes was analyzed. For this purpose, five 1 cm squares were cut at random locations on the membranes. 2 of round sample and dissolved in HPLC analysis was performed in L23-buffer / methanol (MeOH) with Hypersil ODS150x4.6mm as stationary phase and acetonitrile / phosphate buffer as mobile phase. HPLC results are shown in Fig.11 Comparison of the results of the API-loaded membrane samples with those of the calibration samples confirmed that paclitaxel can be stably prepared in the membrane used in the drug delivery device of the present invention.

[0450] In addition, the content of paclitaxel on the membrane is shown in Table 3.

[0451] Table 3

[0452]

[0453] *Standard deviation, **Relative standard deviation

[0454] The results in Table 3 show that 1 cm 2 Membrane loadings of about 0.2 μg are possible. It must be noted that the standard deviation and the maximum value of the drug loading are determined by the process technology and in principle do not represent any limitations.

[0455] Paclitaxel is insoluble in water and has poor absorbability. However, the inventors were able to surprisingly show stable loading of paclitaxel on a membrane that can be used in the drug delivery system of the present invention. In addition, in the art, chemotherapy, parenteral administration of paclitaxel requires 220 mg / m 2 The high dose of paclitaxel is given at three-week intervals and is associated with side effects such as alopecia, nausea, vomiting, diarrhea, changes in blood counts, neuropathy, and myalgia. Regional and / or local administration of paclitaxel by the drug delivery system of the present invention can improve local administration to the target site, thereby increasing the value of the drug, particularly in the treatment of esophageal diseases.

[0456] Example 2: Preparation of RNA-containing polymer membranes

[0457] In order to test the stability of RNA as an active pharmaceutical ingredient in the drug delivery system of the present invention, the following sequence was synthesized: 5'UGC GCAGAA UGA GAU GAG UUG 3' (SEQ ID NO: 25)

[0458] A "base polymer mixture" was prepared using the ingredients and amounts shown in Table 4, without the addition of paclitaxel.

[0459] Table 4

[0460] Total proportion PVA 18-88 18％ glycerin 2％ Dem.water 80％

[0461] The polymer films were prepared using a solvent casting technique according to the following protocol:

[0462] Solvent Casting Technology

[0463] 1. Preparation of "Base Polymer Mixture"

[0464] 2. Sterilization

[0465] 3. Preparation of film laminates

[0466] 4. Drying

[0467] 5. Cutting (A = 0,25cm 2 )

[0468] 6. Apply RNA Sample to Polymer Membrane

[0469] 7. Dry the final sample

[0470] ! Points 3-7 under sterile conditions!

[0471] The stability of RNA on the membrane was analyzed using 20% ​​denaturing polyacrylamide (PAA) gel. Fig.12 As shown, the membrane had no effect on the stability of the RNA. The experiment was repeated with commercially available siRNA. The inventors observed comparable stability to that of a commercial siRNA targeting IL-6 (Thermo Fisher). Fig.13 ).

[0472] The stability of RNA on the membrane before and after membrane dissolution was further analyzed. Storage of dried samples on the membrane had no effect on RNA stability, e.g. Fig.14 As shown, the drug delivery device including RNA as the active pharmaceutical ingredient is storage stable. Storage after film dissolution showed slight degradation of RNA in the solvent, which could be reversed by adding RNase inhibitors ( Fig.15 ).

[0473] The results showed that RNA remained stable after being coated into the membrane and under various storage conditions, and therefore could be used as an active pharmaceutical ingredient in drug delivery systems, thus providing a new therapeutic option for drug delivery systems, especially in the treatment of esophageal diseases.

[0474] Example 3: Stability analysis of BMP2 antisense RNA on membrane under different application conditions and storage conditions

[0475] In order to test the stability of BMP2 antisense RNA as an active pharmaceutical ingredient in the drug delivery system of the present invention, the following sequence was synthesized:

[0476] 5'-AUU UCG AGU UGG CUG UUG C-3' (SEQ ID NO: 23), wherein an overhang consisting of two UUs (uridine nucleotides) is linked to the 3' end.

[0477] This sequence was used for stability experiments in both unlabeled and labeled form (labeled with the dye Atto633 attached to the 3' end to visualize the RNA during preparation (Figure 10)).

[0478] Membranes containing BMP2 antisense RNA were prepared as described in Example 2, except that PVA 4-88 was used instead of PVA 18-88.

[0479] like Fig.16 and Fig.16 As shown in the legend of , the stability of antisense BMP2 RNA was analyzed under various applied conditions. 10 pmol of each probe was loaded on a 20% denaturing polyacrylamide gel. The gel was run at 140 volts for 3 hours and then stained with SYBR gold for 20 minutes.

[0480] As shown in Figure 17 and the legend of Figure 17, the stability of the labeled antisense BMP2 RNA was analyzed under various applied conditions, compared with unlabeled antisense BMP2 RNA. In lanes 1 to 5, 5 pmol of probe and in lanes 4 to 6, 10 pmol of each probe were loaded on a 20% denaturing polyacrylamide gel. The gel was run at 140 volts for 3 hours and then stained with ethidium bromide for 30 minutes ( Fig.17a ), or by exciting with light at a wavelength of 630 nm and measuring emission at 675 nm ( Fig.17b ). Fig.17b The band of BMP2 RNA in Fig.17a Because the unlabeled BMP2 RNA has no fluorescent marker, the unlabeled BMP2 RNA band is Fig.17b Not visible in.

[0481] After the dried membranes were stored at 4°C for 10 days, the stability of the labeled antisense BMP2 RNA was analyzed by comparing it to the unlabeled antisense BMP2 RNA. The probes were loaded on a 20% denaturing polyacrylamide gel. The gel was run at 140 volts for 3 hours and then stained with ethidium bromide for 30 minutes ( Fig.18b ), or by exciting with light at a wavelength of 630 nm and measuring emission at 675 nm ( Fig.18a ). Fig.18a The band of BMP2 RNA labeled in Fig.18b The unlabeled BMP2 RNA band is shown in Figure 2.

[0482] The results can be summarized as follows:

[0483] After applying BMP2 RNA to the membrane, no differences were observed between the test and stock samples in each individual experiment. This was also true for the sensitivity-enhancing marker samples.

[0484] BMP2 RNA is not degraded by the membrane.

[0485] The slight bending of the bands in the gel can be explained by the presence of polymers in the gel.

[0486] At every time point tested in the experiments, the polymer did not negatively affect the stability of the RNA.

[0487] Even after storage of the dried membrane at 4°C for 10 days, no RNA degradation was observed.

[0488] The above results indicate that the drug delivery system of the present invention is suitable for administering RNA, which provides a new therapeutic option for drug delivery systems.

[0489] Example 4: Encapsulation of RNA into liposomes

[0490] Liposome preparation

[0491] Liposome preparation was performed according to the thin layer hydration (TLH) method, which is based on the protocol of Bangham [1]. For this purpose, a chloroform solution containing an appropriate amount of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was placed in a glass tube and dried under a stream of nitrogen. For complete drying, the sample was incubated overnight under vacuum.

[0492] The dried lipid film was dissolved in 1 mL of Tris-HCl (pH 8) containing 250 mM sucrose (1.66 mg / mL).

[0493] Next, the vesicles were frozen eight times in liquid nitrogen and thawed at 54° C. Homogenization was performed using an extrusion method through a 100 nm polycarbonate membrane.

[0494] Liposomal encapsulation of RNA

[0495] For the experiments, the RNA sequence 5'-AUU UGG UUG CUU-3' (SEQ ID NO: 23) was used because it is part of a siRNA that was found to be an effective siRNA for BMP-2 in the literature by Wu et al. [2]. The RNA used for this work was purchased from biomers.net GmbH and was HPLC pure grade.

[0496] For encapsulation, 7.5 nmol of RNA[3] was used and then redissolved with 150 μL of liposome solution. 100 μL of ethanol containing 10 mM CaCl2 was slowly dripped into the mixture of liposomes and RNA at room temperature while mixing continuously. The sample was then dialyzed against liposome buffer at 4°C overnight.

[0497] Purification of liposome-encapsulated RNA

[0498] Magnetic particles are used to separate free RNA from liposome-encapsulated RNA. These particles are coated with a silica layer and have quaternary aminoethyl groups on the surface[4]. Due to the positive charge generated on the surface, free RNA binds through the negative backbone[4] and can therefore be separated from the liposome-encapsulated RNA.

[0499] This protocol was inspired by the publication of Ye and Beverly [4]. All separations were performed at room temperature. For one sample of liposomes, in a first step, 20 μL and 5 μL of each resuspended magnetic particles were added to a reaction tube. After 2 min, the supernatant was removed therefrom on a magnetic separator. For purification and equilibration of the particles, 200 μL of liposome buffer was added to the particles and incubated for 2 min. After 2 min of magnetic separation, the supernatant can be removed. Subsequently, the liposomes are added to a reaction vessel containing 20 μL of the magnetic particle suspension and incubated for 30 s after resuspension. After 2 min of separation, the liposome sample can be removed. The procedure is repeated with a reaction tube containing 5 μL of the equilibrated magnetic particle suspension.

[0500] Size determination

[0501] Dynamic light scattering (DSL) was used to determine the size of the liposomes. Before use in the drug delivery system, 100 μL of a 1:100 diluted sample was placed in a microcuvette with a 10 mm channel length. After a two-minute equilibration period, measurements were performed at 25°C. Water was used as a standard (refractive index 1.33; absorbance 0.001; viscosity 0.833).

[0502] Encapsulation efficiency

[0503] If the RNA concentration in the purified liposomes is determined and compared to the amount of material used, the encapsulation efficiency (EE) can be calculated (Equation 1).

[0504]

[0505] n(end): amount of material after purification of liposomes from free RNA.

[0506] n(start): amount of substance used for liposome preparation.

[0507] Quant-itTM RiboGreen was used to determine the concentration of RNA. The RNA used for encapsulation was also used to prepare the standard set.

[0508] For this experiment, when only RNA outside the liposome was measured, TE buffer consisting of 10 mM Tris-HCl and 1 mM EDTA at pH 7.5 was used. When total RNA was measured, 2% TritonX-100 was added to the TE buffer.

[0509] The standard series of liposomes was diluted 1:500. The diluted liposome samples and the stock RNA solution (1 μmol / L) were used to prepare a standard series (0, 6, 30, 50, 100, 150 nmol / L), each with a volume of 100 μL. The samples were also diluted 1:500. Subsequently, 100 μL of the 1:200 diluted dye was added to each standard or sample. After brief mixing, the samples were incubated in the dark for 3 minutes. Fluorescence measurements were performed at an excitation wavelength of approximately 480 nm. Emission was measured at 525 nm. To determine the encapsulated RNA, the difference between the amount of substance with and without TritonX-100 was taken. The results are used to calculate EE (Formula 2 below).

[0510] Application to drug delivery devices

[0511] For the application of liposome-encapsulated RNA, only about 1 cm 2 The membrane is of the size required for the analysis.

[0512] Table 5: Composition of polymer mixtures used for preparing membranes by solvent casting technique.

[0513]

[0514]

[0515] For drying, approximately 40 pmol of liposome-encapsulated RNA was applied to the membrane. The membrane was dried in a desiccator under vacuum for 3 h. After drying, the membrane was stored in a refrigerator at 4°C.

[0516] To analyze the dried liposomes, each membrane was solubilized with 100 μL of deionized water. Subsequently, the retention of RNA and the size of the liposomes were determined.

[0517] For measurement by DLS, 100 μL of a 1:10 dilution of the dissolved film was used in a microcuvette. Using water as a standard, only the viscosity needed to be adjusted, which was determined to be 2.4 mPa*s using a rotational rheometer.

[0518] To determine retention, the concentration of RNA was determined using Quanti-it™ RiboGreen reagent in the absence of Triton X-100 in the sample. In this case, the solubilized membranes were diluted 1:84.

[0519] Since a small amount of RNA is still present outside the liposome after separation, it must be subtracted from the measured amount of RNA. Subsequently, the retention can be calculated according to Equation 2. Here, n(outside) is the measured amount of RNA material outside the liposome after drying, minus the RNA that was already outside the liposome before drying, and n(inside, sol) is the amount of encapsulated RNA material measured before drying.

[0520]

[0521] Table 6: Average size values, PDI and EE

[0522]

[0523] result

[0524] After the preparation step for encapsulating RNA, the average size of the liposomes was 191.7 nm (Table 6). For the same batch, an EE of 10.72% could be determined, so 0.804 nmol was encapsulated. While 0.804 nmol was encapsulated, 120 pmol remained outside the liposomes. This had to be taken into account when determining retention. Drying was then performed in three different ways, and the changes in liposome size and RNA retention were subsequently compared. Fig. 20 a shows a graph of the size determination of liposomes after drying and rehydration. The light grey curve for the liposomes before drying is inserted for comparison. The curve for the liposomes dried on a membrane without added sucrose is shown in grey. After rehydration, the average size of the dried liposomes (187 nm) is slightly smaller than before drying ( Fig. 20 a). Both the black curve sample and the dark grey curve sample were dried on a membrane containing sucrose. In addition, the liposome buffer was added to the sample of the black curve at the same volume fraction as a protection buffer before applying it to the membrane for drying. For both samples, a clear reduction in the liposome diameter could be measured ( Fig. 20 a).

[0525] For the retention of RNA after drying the liposomes, all samples reached values ​​above 95%. This is a much better retention than the control group, which had a retention of 90.255%, and slightly higher than that previously published by Wolkers et al. [5]. For the samples dried on membranes without sucrose, no RNA release was measured. The other two samples dried on membranes containing sucrose also showed very good retention of 95.13% and 96.13% (with protection buffer).

[0526] Retention in all experiments was higher than in the control experiments, and the liposomes generally remained within the size range recommended for at least systemic therapy [3].

[0527] Example 5: Preparation of polymer films by melt extrusion

[0528] -Choose a suitable polymer matrix: Choose a polymer matrix that is compatible with the active ingredient you intend to use. Typical polymers are polyvinyl alcohol (PVA), cellulose ethers and polyethylene glycol (PEG), combined with a suitable plasticizer.

[0529] - Active ingredient preparation: The active ingredient is usually prepared into a suitable formulation (eg, as a powder or granules).

[0530] - Mixing components: Mix the polymer matrix and active ingredient in a mixer to form a homogeneous mixture.

[0531] - Extrusion: The mixture is fed into an extruder where it melts at high temperature and is forced through a die. The die forms a film which is deposited on a cooling plate.

[0532] - During the melt extrusion process, after the polymer compound is forced through the die, the extruded film may undergo a rolling process to further optimize its thickness and properties. Depending on the specific requirements of the film, the rolling process may include cold rolling or hot rolling.

[0533] -Post-processing: After the extrusion process, the extruded film is cut into the desired size and shape. The film is then usually further processed, such as drying, coating or lamination, to improve its physical and pharmaceutical properties.

[0534] References

[0535] 1. Trucillo P, Campardelli R, Reverchon E. Liposomes: From Bangham to Supercritical Fluids. Processes. August 21, 2020; 8(9): 1022.

[0536] 2.Wu JB,Fu HQ,Huang LZ,Liu AW,Zhang JX.Effects of siRNA-targetingBMP-2on the abilities of migration and invasion of human liver cancerSMMC7721 cells and its mechanism.Cancer Gene Ther.January2011;18(1):20-5.

[0537] 3.Somiya M,Yamaguchi K,Liu Q,Niimi T,Maturana AD,Iijima M,et al.One-step scalable preparation method for non-cationic liposomes with high siRNAcontent.International Journal of Pharmaceutics.July2015;490(1-2):316-23.

[0538] 4.Ye G,Beverly M.The use of strong anion-exchange(SAX)magneticparticles for the extraction of therapeutic siRNA and their analysis byliquid chromatography / mass spectrometry.Rapid Communications in MassSpectrometry.2011;25(21):3207-15.

[0539] 5.Wolkers WF,Oldenhof H,Tablin F,Crowe JH.Preservation of driedliposomes in the presence of sugar and phosphate.Biochimica et BiophysicaActa(BBA)-Biomembranes.March 2004;1661(2):125-34.

[0540] Sequence Listing:

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

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[0554]

Claims

1. A drug delivery system for esophageal mucosa, comprising at least one sheet-shaped preparation, in particular a film-shaped, foil-shaped or sheet-shaped preparation, comprising an active pharmaceutical ingredient; Release agencies; and Trigger mechanism, where The trigger mechanism is suitable for triggering the release mechanism to release the preparation at a predetermined action site, and wherein the release mechanism is suitable for releasing the preparation while moving along the esophageal mucosa, wherein the drug delivery system further comprises a housing, wherein the housing contains the formulation, and wherein the housing comprises an aperture as part of the release mechanism, the aperture being configured to allow the formulation to exit the housing, and wherein the trigger mechanism is a retaining device that is part of or attached to the formulation such that the formulation spreads or unfolds when the dosage form moves down the esophageal mucosa and exits the housing through the aperture, Features The active pharmaceutical ingredient comprises an agent effective for treating or preventing esophageal disease, preferably in combination with one or more additional active pharmaceutical ingredients.

2. The drug delivery system of claim 1, wherein the agent effective for treating or preventing esophageal disease comprises an inhibitory polynucleotide, preferably an inhibitory polynucleotide in combination with a nucleic acid delivery system; an antibody; or an antiproliferative agent.

3. The drug delivery system according to claim 2, wherein the inhibitory polynucleotide is selected from the group consisting of small interfering RNA (siRNA) molecules, antisense oligonucleotides and aptamers.

4. according to the drug delivery system described in claim 2 or 3, wherein the inhibitory polynucleotide comprises siRNA molecule or antisense oligonucleotide, and the RNA transcript or its part of the BMP2 or BMP4 polypeptide of target coding, preferably as shown in SEQ ID NO:15 or SEQ ID NO:16, or wherein the inhibitory polynucleotide comprises targeting as shown in SEQ ID NO:20 or SEQ ID NO:21 RNA transcript or its part siRNA molecule or antisense oligonucleotide, or wherein the inhibitory polynucleotide comprises interference BMP2 or BMP4 polypeptide, preferably as shown in SEQ ID NO:15 or SEQ ID NO:16 BMP2 or BMP4 polypeptide active fit.

5. The drug delivery system according to claim 3 or 4, wherein the siRNA molecule comprises: (a) a double-stranded region comprising a sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, preferably SEQ ID NO: 18, or any other sequence comprising 80% or more sequence identity between the siRNA molecule and a target RNA transcript or a portion thereof encoding a BMP2 or BMP4 polypeptide, preferably a BMP2 or BMP4 polypeptide as shown in SEQ ID NO: 15 or SEQ ID NO: 16, (b) a double-stranded region, wherein the double-stranded region comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand together form a double-stranded region, and the antisense strand is complementary to the target RNA transcript as shown in SEQ ID NO: 20 or SEQ ID NO: 21 or a portion thereof, and / or (c) BMP2-siRNA 1, BMP2-siRNA 2 or BMP2-siRNA 3 as shown in the following table: Each of the above sequences in the table contains an overhang of two nucleotides dTdT (deoxythymidine) or UU (uridine) attached to the 3' end of each strand.

6. The drug delivery system according to claim 2: i) wherein the antibody or binding fragment thereof binds within the following residues: a) residues 10-17, 45-56 and 69 of BMP4 (SEQ ID NO: 1), b) residues 24-31, 57-68, 70-72, 89, 91, 101, 103, 104 and 106 of BMP4 (SEQ ID NO: 1), or c) residues 34, 35, 39, 86-88, 90, 97, 98, 100, 102, and 109 of BMP4 (SEQ ID NO: 1); ii) wherein the antibody or binding fragment thereof that binds to at least Lys12, Arg15, Asp46 and Pro50 of BMP4 comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:2, or a sequence that differs therefrom by no more than 1 amino acid, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:3, or a sequence that differs therefrom by no more than 1 amino acid, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:4, or a sequence that differs therefrom by no more than 1 amino acid, or wherein the antibody or binding fragment thereof that binds to at least Asp30, Trp31, Leu66 and Lys101 of BMP4 comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:5 or a sequence that differs therefrom by no more than 1 amino acid, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:6 or a sequence that differs therefrom by no more than 1 amino acid, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:7 or a sequence that differs therefrom by no more than 1 amino acid, or wherein the antibody or binding fragment thereof that binds to at least Ala34, Gln39, Ser88, Leu90 and Leu100 of BMP4 comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 8 or a sequence that differs therefrom by no more than 1 amino acid, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 9 or a sequence that differs therefrom by no more than 1 amino acid, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 10 or a sequence that differs therefrom by no more than 1 amino acid; and / or iii) wherein the antibody or binding fragment thereof that binds to at least Lys12, Arg15, Asp46 and Pro50 of BMP4 comprises the amino acid sequence of SEQ ID NO: 11 or a sequence that is at least 70%, preferably 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical thereto, or wherein the antibody or binding fragment thereof that binds to at least Asp30, Trp31, Leu66 and Lys101 of BMP4 comprises the amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 70%, preferably 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical thereto, or The antibody or its binding fragment that binds to at least Ala34, Gln39, Ser88, Leu90 and Leu100 of BMP4 comprises the amino acid sequence of SEQ ID NO:13 or a sequence that is at least 70%, preferably 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical thereto.

7. The drug delivery system according to claim 2, wherein the antibody comprises: i) an antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprising: a. at least one CDR (complementarity determining region) selected from the group consisting of SEQ ID NO: 30, 31, 32, 36, 37 and 38, or a variant of any of said sequences; and / or b. at least one CDR selected from the group consisting of SEQ ID NO: 33, 34, 35, 39, 40 and 41, or a variant of any of said sequences; ii) an antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprising: a. light chain CDR SEQ ID NO: 30, 31 and 32, or a variant of any of said sequences; and / or heavy chain CDR SEQ ID NO: 33, 34 and 35, or a variant of any of said sequences; or b. light chain CDR SEQ ID NO: 36, 37 and 38, or a variant of any of said sequences; and / or heavy chain CDR SEQ ID NO: 39, 40 and 41, or a variant of any of said sequences; iii) an antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprising: a. a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: i. SEQ ID NO: 26 or a variant thereof; ii. SEQ ID NO: 28 or a variant thereof; iii. amino acid residues 20 to 139 of SEQ ID NO:42 or a variant thereof; and iv. an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% sequence identity to amino acid residues 20 to 139 of SEQ ID NO:42; and further includes b. a light chain variable region comprising an amino acid sequence selected from the group consisting of: i. SEQ ID NO: 27 or a variant thereof; ii. SEQ ID NO: 29 or a variant thereof; iii. amino acid residues 20 to 130 of SEQ ID NO:44 or a variant thereof; iv. amino acid residues 20 to 130 of SEQ ID NO:45 or a variant thereof; v. amino acid residues 20 to 130 of SEQ ID NO:46 or a variant thereof; and vi. an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% sequence identity to amino acid residues 20 to 130 of SEQ ID NO: 44, 45 or 46; and / or iv) an antibody or antibody fragment that binds to PD-1, preferably human PD-1, comprising: a. A heavy chain comprising an amino acid sequence selected from the group consisting of: i. amino acid residues 20 to 466 of SEQ ID NO:43 or a variant thereof; and ii. amino acid residues 20 to 469 of SEQ ID NO:47 or a variant thereof; and b. a light chain comprising an amino acid sequence selected from the group consisting of: i. amino acid residues 20 to 237 of SEQ ID NO: 48 or a variant thereof; ii. amino acid residues 20 to 237 of SEQ ID NO:49 or a variant thereof; and iii. amino acid residues 20 to 237 of SEQ ID NO: 50 or a variant thereof.

8. The drug delivery system according to claim 2 or 7, wherein the antibody comprises an antibody or an antibody fragment, which: a. With a K of about 100 pM or less D Binds to human PD-1; b. With a K of about 30 pM or less D Binds to human PD-1; c. with a K of about the same as that of an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 44 D Binds to human PD-1; d. with a K of about the same as that of an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 45 D Binds to human PD-1; e. At about 7.5 x 10 5 1 / M·s or faster k assoc Binds to human PD-1; f. At about 1x 10 6 1 / M·s or faster k assoc Binds to human PD-1; g. About 2x 10 -5 1 / s or slower k dissoc Binds to human PD-1; h. At about 2.7 x 10 -5 1 / s or slower k dissoc Binds to human PD-1; i. About 3x 10 -5 1 / s or slower k dissoc Binds to human PD-1; and / or j. With IC of about 1 nM or lower 50 Block the binding of human PD-L1 or human PD-L2 to human PD-1.

9. The drug delivery system according to claim 2, wherein the antiproliferative agent is selected from the group consisting of: a taxane, preferably selected from the group consisting of paclitaxel, docetaxel and cabazitaxel, more preferably paclitaxel; a pyrimidine analog, preferably a uracil analog, more preferably 5-fluorouracil or capecitabine; and a platinum-based agent, preferably selected from the group consisting of cisplatin or a salt thereof, carboplatin or a salt thereof, nedaplatin or a salt thereof and oxaliplatin or a salt thereof.

10. A drug delivery system according to any preceding claim for use in therapy.

11. A drug delivery system according to any one of the preceding claims for use in the treatment or prevention of an esophageal disease, preferably wherein the esophageal disease is caused by or is associated with an immune system defect such as cancer.

12. A drug delivery system according to any one of the preceding claims for use in the treatment or prevention of Barrett's esophagus, esophageal stenosis and / or esophageal cancer, such as adenocarcinoma, esophageal junction cancer or squamous cell carcinoma.

13. The drug delivery system according to any one of claims 1 to 9, for use in diagnosis, preferably wherein the active pharmaceutical ingredient is combined with a diagnostic marker.

14. Use of a drug delivery system according to any one of claims 1 to 9 for in vitro diagnosis, preferably wherein the active pharmaceutical ingredient is combined with a diagnostic marker.

15. The use according to claim 14 or the drug delivery system according to claim 13, wherein the diagnosis comprises monitoring the cellular uptake of the active pharmaceutical ingredient, monitoring the pathway of the active pharmaceutical ingredient in a tissue or organ, or monitoring the success of treatment, such as tumor size.

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