Protein-antiviral compound conjugate

Abstract

This invention provides novel antibodies and antibody-drug conjugates that can neutralize multiple subtypes of influenza A virus and can be used as agents for the prevention or treatment of influenza A infection. [Solution] Compounds for the treatment of influenza-related diseases and disorders are provided, comprising antiviral compounds such as VX-787 and its derivatives, baloxavir and its derivatives, and baloxavir marboxil and its derivatives, as well as protein (e.g., antibody) drug conjugates thereof.

Description

[Technical Field]

[0001] (Government licensing rights) This invention was approved by the U.S. Department of Health and Human Services. This was carried out with government support under the awarded contract HHSO100201700020C. They possess certain rights in the invention.

[0002] (Cross-reference of related applications) This application is based on U.S. Provisional Application No. 62 / 965,7, filed on January 24, 2020, under Section 119 of the U.S. Patent Act. Claiming the benefits of U.S. Provisional Application No. 63 / 094,285, filed on October 20, 2020; These contents are incorporated fully herein by reference.

[0003] (Field) Provided herein are antiviral compounds and their protein conjugates, and A method for treating various diseases, disorders, and conditions, the antiviral compound and its The method involves administering a protein conjugate. [Background technology]

[0004] (background) Influenza has a long history characterized by waves of global pandemics, epidemics, resurgences, and major outbreaks. It is a highly contagious disease that is transmitted through contact. Despite annual vaccination efforts, Influenza infection results in significant morbidity and mortality rates.

[0005] Influenza viruses consist of three main types: A, B, and C. Influenza viruses require hemagglutinin, which is necessary for the virus to attach to and enter host cells. The antigens of two genes that encode surface glycoproteins: (HA) and neuraminidase (NA). They can be classified into subtypes based on allele variations in the sex domain.

[0006] Hemagglutinin is a globular hematopoietic molecule consisting of receptor binding sites (which are subject to frequent antigenic drift). The dodomain and the stem region (which is more conserved among various strains of influenza virus) It is a trimer glycoprotein containing two structural domains. The HA protein is a precursor. It is synthesized as a body (HA0), which then undergoes proteolytic processing to form two subunits. It produces halves (HA1 and HA2), which associate with each other to form a stem / spherical head structure. The HA1 peptide is involved in the attachment of viruses to the cell surface. The HA2 peptide is involved in endosomal It mediates the fusion of the virus and the cell membrane within the cell, and introduces ribonucleoprotein complexes into the cytoplasm. It forms a stem-like structure that enables the release of the body.

[0007] Currently, there are 18 subtypes (H1-H18) defined by the hemagglutinin protein. The 18 types of HA can be classified into two groups. Group 1 includes H1, H2, H5, It consists of H6, H8, H9, H11, H12, H13, H16, H17, and H18 subtypes, and Group 2 consists of H3, H4, H Includes subtypes 7, H10, H14, and H15.

[0008] Despite decades of research, it has not been found to broadly neutralize influenza A virus infection. It inhibits or reduces diseases caused by influenza A virus. There are no commercially available antibodies or antibody-drug conjugates (ADCs) for influenza A. To neutralize multiple subtypes of the virus and for the prevention or treatment of influenza A infection It is necessary to identify new antibodies and ADCs that can be used as pharmaceuticals. [Overview of the project]

[0009] (overview) Provided herein are compounds useful, for example, in antiviral therapy. In one embodiment, the compound is VX-787 and its derivatives, baloxavir and its Examples include derivatives, and / or baloxavir marboxil and its derivatives. In this case, the payload provided is as described herein (e.g., antiviral Compounds), linker-payloads (e.g., linker-antiviral compounds), and / or compounds Antibody-drug conjugate containing a conjugated anti-influenza antibody or its antigen-binding fragment It is a jugate.

[0010] In one embodiment, the provided compound has the following structure. [ka] (Here, L is the linker; BA is the binder; and k is an integer between 1 and 30).

[0011] In one embodiment, the following structure is provided. [ka] (Here, L is the linker; and RG is the reactive part.) or linker-payload having a pharmaceutically acceptable salt thereof (e.g., linker-anti-wheat It is an yl compound.

[0012] Another embodiment described herein is the payload described herein. Alternatively, to create compounds, linker-payloads, or antibody-drug conjugates and compositions. This is a method of production.

[0013] In another embodiment, provided herein are the subject matter as described herein. Methods for the treatment, prevention, reduction, or inhibition of diseases, disorders, or conditions associated with infection. The subject contains a payload as described herein (e.g., an antiviral compound), phosphorus Carr-payload (e.g., linker-antiviral compound), antibody-drug conjugate, or This is a method comprising administering an effective amount of a pharmaceutical composition. [Brief explanation of the drawing]

[0014] (Brief explanation of the drawing) [Figure 1] Figure 1A shows the intensity-weighted average linker-payload (e.g., linker-antiviral compound) loading measured by LC-MS for 11729-Q295-11. Figure 1B shows the intensity-weighted average linker-payload (e.g., linker-antiviral compound) loading measured by LC-MS for isotype control-Q295-11.

[0015] [Figure 2] Figure 2A shows the intensity-weighted average linker-payload (e.g., linker-antiviral compound) loading measured by LC-MS for 11729-HC-Cterm-11. Figure 2B shows the intensity-weighted average linker-payload (e.g., linker-antiviral compound) loading measured by LC-MS for isotype control-HC-Cterm-11.

[0016] [Figure 3] Figure 3 shows the ELISA sub-nanomolelic concentration-specific binding of 11729-HC-Cterm-11, 11729-HC-Nterm-11, 11729-LC-Cterm-11, and 11729-LC-Nterm-11 to influenza A-infected cells.

[0017] [Figure 4] Figure 4 shows a comparison of the antiviral efficacy against influenza A infection between 11729-HC-Cterm-11, 11729-LC-Cterm-11, 11729-LC-Nterm-11, mAb11729, isotype control antibodies, and isotype control-HC-Cterm-11.

[0018] [Figure 5] Figure 5 shows the in vitro stability of 11729-HC-Cterm-11 in human and monkey plasma after 72 hours (i.e., absence of linker-payload (e.g., linker-antiviral compound) loss; or absence of DAR reduction).

[0019] [Figure 6] Figure 6 shows a comparison of the antiviral efficacy against influenza A infection between 11729-HC-Cterm-11, isotype control antibodies, and isotype control-HC-Cterm-11.

[0020] [Figure 7] Figure 7 shows a comparison of the antiviral efficacy against influenza A infection between 11729-HC-Cterm-6, 11729-LC-Cterm-6, mAb11729, isotype control antibodies, isotype control-HC-Cterm-6 antibodies, and isotype control-LC-Cterm-6 antibodies.

[0021] [Figure 8]Figure 8 shows a comparison of the antiviral efficacy against influenza A infection between 11729-HC-Cterm-45a, 11729-LC-Cterm-45a, 11729-HC-Cterm-34, 11729-LC-Cterm-34, mAb11729, isotype control antibodies, and isotype control-antibody-drug conjugates.

[0022] [Figure 9] Figure 9 shows a comparison of the antiviral efficacy against influenza A infection between 5385-HC-Cterm-11, 5385-LC-Cterm-11, mAb11729, isotype control antibodies, and isotype control-antibody-drug conjugates.

[0023] [Figure 10] Figure 10 shows a comparison of the antiviral efficacy against influenza A infection between 11729-HC-Cterm-45d and mAb11729 and isotype control antibodies and isotype control-HC-Cterm-45d.

[0024] [Figure 11] Figure 11 shows the specific binding of 11729-HC-Cterm-45d to influenza A-infected cells. [Modes for carrying out the invention]

[0025] (Description of exemplary embodiments) Provided herein, for example, is a method for treating influenza infection in a subject. Useful compounds, compositions, and methods.

[0026] (definition) When referring to the compounds provided herein, unless otherwise indicated, the following terms shall apply: The following meanings apply. Unless otherwise defined, the technical and scientific terms used herein have the following meanings. All terms have the same meaning as those commonly used by those skilled in the art. If multiple definitions exist for a term, unless otherwise specified, these definitions shall prevail. To be preceded.

[0027] The term "influenza hemagglutinin," also known as "influenza HA," is, Virus attachment to host cells (via HA1 binding to α-2,3- and α-2,6-sialic acid) and Three types of structures found on the surface of influenza virions that mediate invasion (through changes in three-dimensional structure) It is a mer glycoprotein. HA has two structural domains: (high frequency of antigenic mutations) A spherical head domain containing a receptor binding site (received by various influenza viruses) It consists of stem regions (which are more conserved among species). Influenza HA is a precursor. It is synthesized as a body (HA0), which then undergoes proteolytic processing to form two subunits. It produces halves (HA1 and HA2), which associate with each other to form a stem / spherical head structure. Viral HA is the most variable antigen on the virus, but (18 subtypes are divided into two groups) The stems (HA2), which can be classified into groups, are highly conserved within each group. .

[0028] The amino acid sequence of full-length influenza HA is given as accession number FJ966082.1 in GenBan. Amino acid composition of influenza isolate H1 N1 A / California / 04 / 2009 provided to k The column illustrates this. The phrase "influenza-HA" refers to different influenza strains. Protein variants of influenza HA isolated from excipients, e.g., GQ149237.1, NC_002 This also includes 017, KM972981.1, etc. The phrase "influenza-HA" refers to recombinant influenza This also includes the HA or fragments thereof. This phrase is, for example, histidine tag, mouse or human Fc. This also includes influenza HA or fragments thereof coupled to a signal sequence.

[0029] The term "influenza infection" as used herein is also characterized as "flu". The term refers to a severe acute respiratory illness caused by the influenza virus. This phrase refers to respiratory infections, as well as high fever, headache, general throbbing and pain, fatigue and weakness, and certain symptoms. Examples include extreme fatigue, nasal congestion, sneezing, sore throat, chest discomfort, cough, shortness of breath, and bronchitis. This includes symptoms, including pneumonia and, in severe cases, death.

[0030] As used herein, “alkyl” refers to the monovalent saturated hydrocarbon radical moiety. The alkyl group is optionally substituted and can be linear, branched, or cyclic, i.e., cycloal It can be a kill. Alkyls are radicals having 1 to 20 carbon atoms. Nawachi, C 1-20 Alkyl; a radical having 1 to 12 carbon atoms, i.e., C 1-12 Alki A radical having 1 to 8 carbon atoms, i.e., C 1-8 Alkyl; 1 to 6 carbon atoms The radicals that possess, namely C 1-6 Alkyl; and radicals having 1 to 3 carbon atoms, Nawachi, C 1-3 Examples of alkyl groups include, but are not limited to, alkyl groups. These include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, and i-butyl. , pentyl portion, hexyl portion, cyclopropyl, cyclobutyl, cyclopentyl, and Examples include, but are not limited to, n-cyclohexyl. Examples include, but are not limited to, pentyl and i-pentyl. The hexyl portion is: n-hexyl is an example, but is not limited to it.

[0031] As used herein, "alkylene" refers to a divalent alkyl group. Unless otherwise specified, alkylenes contain 1 to 20 carbon atoms, but are not limited to these. The len group is optionally substituted with alkyl as described herein. In some embodiments, the alkylene is not substituted.

[0032] The notation for amino acids or amino acid residues whose stereochemistry is not specified is L-type, D-type. It is intended to include the amino acids, or racemic mixtures thereof.

[0033] As used herein, “haloalkyl” means alkyl as defined above. This refers to a halogen, where the alkyl is a halogen, such as fluorine (F), chlorine (Cl), bromine (Br), or comprising at least one substituent selected from iodine(I). Examples of haloalkyls include Examples include, but are not limited to, -CF3, -CH2CF3, -CCl2F, and -CCl3.

[0034] As used herein, “alkenyl” means at least two carbon atoms and one or more This refers to the monovalent hydrocarbon radical moiety containing a non-aromatic carbon-carbon double bond. They are optionally substituted and can be linear, branched, or annular. Then, radicals having 2 to 20 carbon atoms, i.e., C 2-20 alkenyl; radicals having 2 to 12 carbon atoms, i.e., C 2-12 alkenyl; radicals having 2 to 8 carbon atoms, rad ical, i.e., C 2-8 alkenyl; radicals having 2 to 6 carbon atoms, i.e., C 2-6 alkenyl; and radicals having 2 to 4 carbon atoms, i.e., C 2-4 alkenyl are exemplified but are not limited thereto. Examples of the alkenyl moiety include, but are not limited to, vinyl, propenyl, butenyl, and cyclohexenyl.

[0035] As used herein, "alkynyl" refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. The alkynyl is optionally substituted and can be linear, branched, or cyclic. Examples of alkynyl include radicals having 2 to 20 carbon atoms, i.e., C 2-20 alkynyl; radicals having 2 to 12 carbon atoms i.e., C 2-12 alkynyl; radicals having 2 to 8 carbon atoms, i.e., C 2-8 alkynyl; radicals having 2 to 6 carbon atoms, i.e., C 2-6 alkyn yl; and radicals having 2 to 4 carbon atoms, i.e., C 2-4 alkynyl are exemplified but are not limited thereto. Examples of the alkynyl moiety include, but are not limited to, ethynyl, propynyl, and b utynyl.

[0036] As used herein, "alkoxy" refers to a monovalent saturated hydrocarbon radical moiety Here, the hydrocarbon contains a single bond to an oxygen atom, and here, the radical is an acid It is located on an elementary atom, for example, in the case of ethoxy, it is CH3CH2-O·. Alkoxy substituents are It bonds to the compound it substitutes for via this oxygen atom of the alkoxy substituent. The 'C' is optionally substituted and is linear, branched, or cyclic, i.e., cycloalkoxy. It is possible. As alkoxys, those having 1 to 20 carbon atoms, i.e., C 1-20 Alkoxy; having 1 to 12 carbon atoms, i.e., C 1-12 Alkoxy; 1-8 pieces Having carbon atoms, that is, C 1-8 Alkoxy; having 1 to 6 carbon atoms. In other words, C 1-6 Alkoxy; and those having 1 to 3 carbon atoms, i.e., C 1-3 Arco Examples of xy include, but are not limited to, methoxy. Ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, i-butoxy Xy, pentoxy portion, hexoxy portion, cyclopropoxy, cyclobutoxy, cyclope Examples include, but are not limited to, antoxy and cyclohexoxy.

[0037] As used herein, "haloalkoxy" means an alcohol as defined above. This refers to xy, where the alkoxy is selected from halogens, for example, F, Cl, Br, or I. It includes at least one substituent.

[0038] As used herein, “aryl” refers to an aromatic compound in which the ring atom is a carbon atom. This refers to the monovalent radical portion. The aryl group is optionally substituted and can be monocyclic or polycyclic. For example, it can be a biring or triring. An example of the aryl portion is 6-2 Those having 0 ring carbon atoms, i.e., C 6-20 Aryl; having 6 to 15 ring carbon atoms. Suddenly, that is, C 6-15 Aryls, and those having 6 to 10 ring carbon atoms, i.e., C 6-10 Examples of aryls include, but are not limited to, . Phenyl, naphthyl, fluorenyl, azurenyl, anthrill, phenanthryl, and pyrethroid While "nil" is one example, it is limited to these.

[0039] As used herein, “arylalkyl” is a radical of an alkyl compound. This refers to the monovalent portion, where the alkyl compound is substituted with an aromatic substituent, that is, Furthermore, the aromatic compound contains a single bond to an alkyl group, and the radical is the alkyl group. It is located on the base. The arylalkyl group is linked to the exemplified chemical structure via the alkyl group. They combine. Arylalkyls have a structure, for example, [ka] (Here, B is an aromatic moiety, for example, aryl or phenyl) Yes, it is possible. The arylalkyl group is optionally substituted, i.e., an aryl group and / or Alkyl groups can be substituted as disclosed herein. An example of a kill is benzil, but it is not limited to this.

[0040] As used herein, "alkylaryl" is a radical of an aryl compound. This refers to the monovalent part, where the aryl compound is substituted with an alkyl substituent. In other words, the aryl compound contains a single bond to an alkyl group, and the radical is the aryl compound. It is located on the aryl group. The alkylaryl group is located on the aryl group, as exemplified in the chemical structure. It binds to the structure. Alkylaryls have a structure, for example, [ka] (Here, B is an aromatic moiety, for example, phenyl.) can be represented by A Killaryl is optionally substituted, i.e., the aryl group and / or alkyl group are They can be substituted as disclosed herein. Examples of alkylaryls include One example is Toluil, but it is not limited to this.

[0041] As used herein, "aryloxy" means that the ring atom is a carbon atom, and This refers to the monovalent radical of an aromatic compound in which a ring is substituted with an oxygen radical, that is, In other words, the aromatic compound contains a single bond to an oxygen atom, and the radical is on the oxygen atom. It is located in, for example, in the case of phenoxy, [ka] The aryloxy substituent is bonded to the compound it substitutes into via this oxygen atom. The aryloxy is optionally substituted. The aryloxy can be 6 to 20 units. A radical having a ring carbon atom, i.e., C 6-20 Aryloxy; a ring of 6 to 15 carbon atoms Those who possess, namely, C 6-15 Aryloxys, and those having 6 to 10 ring carbon atoms, In other words, C 6-10 Examples include, but are not limited to, aryloxy. Examples of the "shi" part include phenoxy, naphthoxy, and anthroxy, but It is not limited to them.

[0042] As used herein, "arylene" refers to an aromatic compound in which the ring atoms consist only of carbon atoms. It refers to the divalent part of the compound. Arylene can be arbitrarily substituted, and can be monocyclic or polycyclic, for example. It can be a biring or triring structure. Examples of arylene parts include 6 to 20. Those having a ring of carbon atoms, i.e., C 6-20 Arylene; having 6 to 15 ring carbon atoms things, that is, C 6-15 Arylenes, and those having 6 to 10 ring carbon atoms, i.e., C 6-10 Arrines are one example, but are not limited to them.

[0043] As used herein, "heteroalkyl" means a heteroatom in which one or more carbon atoms are present. Therefore, it refers to the substituted alkyl group. When used herein, it refers to the heteroalkenyl group. This refers to an alkenyl in which one or more carbon atoms are substituted by heteroatoms. When used in writing, "heteroalkynyl" refers to a group of one or more carbon atoms that are heteroatoms. This refers to the substituted alkynyl. Suitable heteroatoms include nitrogen, oxygen, and sulfur. Examples include, but are not limited to, heteroalkyls, heteroalkenyls, and heteroalkyls. The teloalkynyl is optionally substituted. An example of the heteroalkyl moiety is aminoalkynyl. Examples include, but are not limited to, lycyl, sulfonylalkyl, and sulfinylalkyl. It is not done. Examples of heteroalkyl parts include methylamino, methylsulfonyl, and methylamino. Chilsulfinyl can also be mentioned, but it is not limited to these.

[0044] As used herein, "heteroaryl" means a ring atom consisting of a carbon atom and at least A monovalent part is a radical of an aromatic compound containing one oxygen, sulfur, nitrogen, or phosphorus atom. This refers to a fraction. Examples of heteroaryl moieties include 5-20 ring atoms; 5-15 ring atoms; and 5 Examples include, but are not limited to, heteroaryl compounds having approximately 10 ring atoms. , has been arbitrarily replaced.

[0045] As used herein, "heterorialene" refers to a compound where one or more ring atoms of an aromatic ring are oxygenated. This refers to a divalent heteroaryl compound substituted with a sulfur, nitrogen, or phosphorus atom. Ren has been arbitrarily replaced.

[0046] As used herein, "heterocycloalkyl" means a compound of one or more carbon atoms. This refers to cycloalkyl groups that are substituted with atoms. Suitable heteroatoms include nitrogen, Examples include, but are not limited to, oxygen and sulfur atoms. Heterocycloalkyls are, It is arbitrarily substituted. Examples of heterocycloalkyl moieties include morpholinyl and piperix. Dinyl, tetrahydropyranil, pyrrolidinil, imidazolidinil, oxazolidinil, Examples include thiazolidinyl, dioxolanil, dithiolanil, oxanil, or thianil. However, it is not limited to these.

[0047] As used herein, "Lewis acid" refers to a molecule or ion that accepts a lone pair of electrons. This refers to the Lewis acid used in the methods described herein, which is not a proton. Lewis acids include non-metallic acids, metallic acids, hard Lewis acids, and soft Lewis acids. However, it is not limited to these. Lewis acids include aluminum, boron, iron, tin, Titanium, magnesium, copper, antimony, phosphorus, silver, ytterbium, scandium, ni Examples include, but are not limited to, Lewis acids of ckel and zinc. Examples include AlBr3, AlCl3, BCl3, methyl sulfide boron trichloride, BF3, and boron trifluoride. Methyl etherate, boron trifluoride methyl sulfide, boron trifluoride tetrahydrof Lan, dicyclohexylboron trifluoromethanesulfonate, iron(III) bromide, iron chloride ( III) Tin(IV) chloride, titanium(IV) chloride, titanium(IV) isopropoxide, Cu(OTf)2, CuCl2 CuBr2, zinc chloride, alkylaluminum halide (R n AlX 3-n (Here, R is hydroca Rubille)), Zn(OTf)2, ZnCl2, Yb(OTf)3, Sc(OTf)3, MgBr2, NiCl2, Sn(OTf)2, Ni( Examples include, but are not limited to, OTf)2 and Mg(OTf)2.

[0048] As used herein, "N-containing heterocycloalkyl" means having one or more carbon atoms It is substituted with a heteroatom, and at least one substituted heteroatom is a nitrogen atom. This refers to a certain cycloalkyl group. Besides nitrogen, suitable heteroatoms include oxygen and sulfur atoms. These are some examples, but are not limited to them. N-containing heterocycloalkyls can be optionally substituted. Examples of N-containing heterocycloalkyl moieties include morpholinyl and piperidinyl. Examples include pyrrolidinyl, imidazolidinyl, oxazolidinyl, or thiazolidinyl. However, it is not limited to these.

[0049] As used herein, the term "optionally substituted", when used to describe a radical moiety, e.g., optionally substituted alkyl, means that such a moiety is optionally bonded to one or more substituents. Examples of such substituents include halo, cyano, nitro, amino, hydroxyl, optionally substituted haloalkyl, aminoalkyl, hydroxyalkyl, azido, epoxy, optionally substituted heteroaryl, optionally substituted heterocycloalkyl,

Chemical Formula

[0050] As used herein, “binding agent” refers to a given binding partner, for example, an antigen and a specific Any molecule that can bind with the opposite sex, such as a protein, antibody, or its cleavage. It refers to one side.

[0051] As used herein, “linker” means a binder comprising one or more of the binders described herein. The compound, for example, is covalently linked to the payload or antiviral compound and the reinforcing agent. or (for example, via a reactive group) divalent, trivalent, Alternatively, it refers to the multivalent part.

[0052] As used herein, “amide synthesis conditions” refers to, for example, carboxylic acids, activated carboxylic acids. To achieve amide formation through the reaction of ammonium acid or acyl halide with amines This refers to preferred reaction conditions. In some examples, amide synthesis conditions involve carboxylic acids and amines. This refers to reaction conditions suitable for achieving the formation of an amide bond between the two. In this process, the carboxylic acid is first converted to an activated carboxylic acid, and then the activated carboxylic acid Acids react with amines to form amides. Preferred conditions for achieving amide formation. This includes, but is not limited to, dicyclohexylcarbodiimide (DCC) and diisopropyl carbodiimide. Lubodiimide (DIC), (benzotriazole-1-yloxy)tris(dimethylamino)phospho Nium hexafluorophosphate (BOP), (benzotriazole-1-yloxy) tripyroxine Lidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazole-1 -Iloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bromo Tripyrrolidinophosphonium hexafluorophosphate (PyBrOP), O-(benzotriazon (Il-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O -(benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluorobole (TBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridin Um 3-oxide hexafluorophosphate (HATU), N-ethoxycarbonyl-2-ethoxy -1,2-Dihydroquinoline (EEDQ), N-ethyl-N'-(3-dimethylaminopropyl)carbodymic (EDC), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP) , 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), carbonyldiimidazole (CDI) , and 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpho Reactions between carboxylic acids and amines, including no-carbenium hexafluorophosphate (COMU). This includes, but is not limited to, methods that utilize reagents to achieve the desired result. In that example, the carboxylic acid is first converted to an activated carboxylic acid ester, and then the An activated carboxylic acid ester is treated with an amine to form an amide bond. One embodiment In this process, the carboxylic acid is treated with a reagent. The reagent deprotonates the carboxylic acid. Further activation of the carboxylic acid, followed by protonation by the deprotonated carboxylic acid. As a result of nucleophilic attack on the reagent, the product complex with the deprotonated carboxylic acid is formed. A body is formed. Thereafter, the activated carboxylic acid ester of a specific carboxylic acid is more sensitive to nucleophilic attack by an amine than before the carboxylic acid is activated. As a result, amide bond formation occurs. Therefore, the carboxylic acid is described as being activated. Exemplary reagents include DCC and DIC. than before the carboxylic acid is activated. As a result, amide bond formation occurs. Therefore, the carboxylic acid is described as being activated. Exemplary reagents include DCC and DIC. than before the carboxylic acid is activated. As a result, amide bond formation occurs. Therefore, the carboxylic acid is described as being activated. Exemplary reagents include DCC and DIC. than before the carboxylic acid is activated. As a result, amide bond formation occurs. Therefore, the carboxylic acid is described as being activated. Exemplary reagents include DCC and DIC.

[0053] As used herein, the term "residue" refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to the product of an amide coupling or peptide coupling to a suitable coupling partner of an amino acid or N-alkyl amino acid; where, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid or N-alkyl amino acid, resulting in a product in which the amino acid residue or N-alkyl amino acid residue is incorporated therein. As used herein, the term "residue" refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to the product of an amide coupling or peptide coupling to a suitable coupling partner of an amino acid or N-alkyl amino acid; where, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid or N-alkyl amino acid, resulting in a product in which the amino acid residue or N-alkyl amino acid residue is incorporated therein. As used herein, the term "residue" refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to the product of an amide coupling or peptide coupling to a suitable coupling partner of an amino acid or N-alkyl amino acid; where, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid or N-alkyl amino acid, resulting in a product in which the amino acid residue or N-alkyl amino acid residue is incorporated therein. As used herein, the term "residue" refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to the product of an amide coupling or peptide coupling to a suitable coupling partner of an amino acid or N-alkyl amino acid; where, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid or N-alkyl amino acid, resulting in a product in which the amino acid residue or N-alkyl amino acid residue is incorporated therein. As used herein, the term "residue" refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to the product of an amide coupling or peptide coupling to a suitable coupling partner of an amino acid or N-alkyl amino acid; where, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid or N-alkyl amino acid, resulting in a product in which the amino acid residue or N-alkyl amino acid residue is incorporated therein. As used herein, the term "residue" refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to the product of an amide coupling or peptide coupling to a suitable coupling partner of an amino acid or N-alkyl amino acid; where, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid or N-alkyl amino acid, resulting in a product in which the amino acid residue or N-alkyl amino acid residue is incorporated therein.

[0054] As used herein, "structural isomer" refers to a compound having the same molecular formula but a different chemical structure due to the way the atoms are arranged. Exemplary structural isomers include n-propyl and isopropyl; n-butyl, sec-butyl, and tert-butyl; and n-pentyl, isopentyl, and neopentyl, etc. As used herein, "structural isomer" refers to a compound having the same molecular formula but a different chemical structure due to the way the atoms are arranged. Exemplary structural isomers include n-propyl and isopropyl; n-butyl, sec-butyl, and tert-butyl; and n-pentyl, isopentyl, and neopentyl, etc. As used herein, "structural isomer" refers to a compound having the same molecular formula but a different chemical structure due to the way the atoms are arranged. Exemplary structural isomers include n-propyl and isopropyl; n-butyl, sec-butyl, and tert-butyl; and n-pentyl, isopentyl, and neopentyl, etc. As used herein, "structural isomer" refers to a compound having the same molecular formula but a different chemical structure due to the way the atoms are arranged. Exemplary structural isomers include n-propyl and isopropyl; n-butyl, sec-butyl, and tert-butyl; and n-pentyl, isopentyl, and neopentyl, etc.

[0055] A specific group, moiety, substituent, and atom are depicted using a wavy line that crosses a bond (singular or plural) to indicate the atom to which the group, moiety, substituent, atom is attached. For example, A specific group, moiety, substituent, and atom are depicted using a wavy line that crosses a bond (singular or plural) to indicate the atom to which the group, moiety, substituent, atom is attached. For example, For example,

Chemical formula

[0056] As used herein, the term "reactive linker" or the abbreviation "RL" means for example, [ka] Reactive group (where RG is a reactive group and SP is a spacer group) This refers to a monovalent group including ("RG") and a spacer group ("SP"). When used herein, A reactive linker may contain multiple reactive groups and multiple spacer groups. The spacer groups are Any divalent moiety that crosslinks the reactive group to another group, such as a payload (e.g., an antiviral compound). The reactive linker (RL) is the payload to which it is bound (e.g., antiviral). Along with the compound, synthetic precursors for the preparation of antibody conjugates as described herein Useful intermediates ("linker-payload" (LP) (e.g., linker-antiviral compound)) Provides, as used herein, the payload is an antiviral compound. The linker-payload incorporating these antiviral compounds is called "linker- It can be called an "antiviral compound." The reactive linker is another group, for example, an antibody. A functional group or functional part that can react with the reactive portion of a modified antibody or its antigen-binding fragment. It contains a reactive group. The reaction between the reactive group and an antibody, modified antibody, or its antigen-binding fragment results in The resulting portion, together with the linking group, is the "binder" of the conjugate described herein. The linker ("BL") portion is included. In one embodiment, the "reactive group" is an antibody or its anti A functional group or functional moiety that reacts with a cysteine ​​or lysine residue of the original binding fragment (e.g., Murray It is mid or N-hydroxysuccinimide (NHS) ester. In some cases, the opposite The functional group is, for example, [ka] This reacts with cysteine ​​residues on the antibody or its antigen-binding fragment, and then carbonizes it. Elementary-sulfur bonds, for example, [ka] (Here, Ab refers to an antibody or its antigen-binding fragment, and S is the functional group that is attached to Ab via it.) It forms a bond (referring to the sulfur atom on the cysteine ​​residue). In some cases, the reactive group is , functional groups, for example, [ka] This reacts with a lysine residue on the antibody or its antigen-binding fragment, forming an amide with it. Combining, for example, [ka] (Here, Ab refers to an antibody or its antigen-binding fragment, and NH refers to a functional group via Ab) (This refers to the NH atom on the lysine side chain residue that is bonded to it.)

[0057] As used herein, the term "biodegradable portion" means a substance that decomposes in vivo and is biodegradable. Non-toxic, biocompatible components that can be removed from the body through normal biological processes. This refers to the part that becomes biodegradable. In some embodiments, the biodegradable part lasts for about 90 days or less, or about 60 days or less. Below, or within approximately 30 days or less, completely or substantially decomposed in vivo, where the degree of decomposition is This is based on the percentage mass loss of the biodegradable portion, and complete decomposition is 100% This corresponds to mass loss. Examples of biodegradable parts include, but are not limited to, poly( ε-Caprolactone (PCL), Poly(3-Hydroxybutyrate) (PHB), Poly(Glycolic Acid) (PGA) , poly(lactic acid) (PLA), and copolymers thereof with glycolic acid (i.e., poly(D,L-lactate Examples include aliphatic polyesters such as docoglycolide (PLGA) (each of which is cited in the reference) The texts of Vert M, Schwach G, Engel R, and Coudane J are fully incorporated herein. References (1998) J Control Release 53(1-3):85-92; Jain RA's Literature (2000) Biomaterials 21(23 ):2475-2490; References by Uhrich KE, Cannizzaro SM, Langer RS, and Shakesheff KM (1999) Chemical Reviews 99(11):3181-3198; and Park TG's literature (1995) Biomaterials 16(15): (1123-1130).

[0058] As used herein, the terms “binding linker” or “BL” refer to a binding agent (e.g., For example, an antibody or its antigen-binding fragment) is used as a payload compound as described herein (e.g., VX-787 and its derivatives, and / or baloxavir and its derivatives (e.g., baloxavir Marboxyl), and optionally, any one or more side chain compounds that are linked, connected, or bonded to it. This refers to a divalent, trivalent, or polyvalent group or part of an antibody conjugate as described herein. A suitable binding linker for the gate utilizes the circulating half-life of the antibody conjugate. It is sufficiently stable, and at the same time, after the internalization of the antigen-mediated conjugate. The payload can be released. The linker is either cleavable or non-cleavable. It can be. Sclerolytic linkers are involved in intracellular metabolism after internalization, for example, hydrolysis, and filtration. Linkers are cleaved by natural or enzymatic reactions. Non-cleaving linkers are Phosphorus releases the attached payload via lysosomal degradation of the antibody after internalization. It is a linker. Suitable linkers include acid-unstable linkers and hydrolysis-unstable linkers. , enzyme-cleaving linkers, reductively unstable linkers, self-destructing linkers, and non-cleaving linkers Examples include, but are not limited to, peptides and glucurolinkers. Nido, succinimide-thioether, polyethylene glycol (PEG) units, hydrazone, Mar-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenes The product consists of or contains diloxycarbonyl (PABC) or para-aminobenzyl (PAB) units. Examples include, but are not limited to, those that use binders. The linker (BL) is a reactive group (RG) of the reactive linker (RL) and a binder, such as an antibody, a modified antibody, or It includes a portion formed by a reaction with the reactive portion of the antigen-binding fragment.

[0059] In some examples, BL is the following part: [ka] (Here, [ka] (This refers to the binding of an antibody or its antigen-binding fragment to cysteine.) This includes the following parts: [ka] (Here, [ka] (This is the binding of an antibody or its antigen-binding fragment to lysine.) Includes.

[0060] In some embodiments, the binder is an antibody or an antigen-binding fragment thereof. The antibody is It may be any form known to those skilled in the art.

[0061] As used herein, the term “antibody” refers to an antibody that specifically binds to or binds to a particular antigen. Any antigen-binding compound containing at least one complementarity-determining region (CDR) that specifically interacts with it. It refers to a complex of molecules or individuals. The term "antibody" refers to a complex of molecules interconnected by disulfide bonds. An immunoglobulin molecule contains four polypeptide chains: two heavy (H) chains and two light (L) chains. This includes a complete antibody molecule and its polymer (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain has a heavy chain variable region (HCVR or V in this specification). H (abbreviated as and includes the heavy chain constant region.) The heavy chain constant region is C H 1, C H 2, and C H It contains three domains, each of which is a light chain. Variable region (LCVR or V in this specification) L It includes the (abbreviated as) and the light chain steady region. The light chain steady region is one Domain (C L Includes 1). V H and V L The domain is a more advanced domain called the Framework Domain (FR). In addition to the highly variable regions called complementary determination regions (CDRs), which contain scattered areas where data is stored, It can be divided into smaller parts. Each V H and V L From the amino terminus to the carboxyl terminus The following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, with three CDRs and four CDs It consists of FR. In the various embodiments disclosed herein, The FR of an antibody (or its antigen-binding portion) suitable for the compound is identical to that of the human germline sequence. It may also be naturally or artificially modified. Amino acid consensus sequence This can be defined based on comparative analysis of two or more CDRs. The term "anti" as used herein refers to the anti- The term "body" also includes antigen-binding fragments of the complete antibody molecule. Terms such as "antigen-binding domain" or "antigen-binding portion," and "antigen-binding fragment" of an antibody, refer to antigens. It specifically binds to any naturally occurring, enzymatically obtained, or synthetically produced substance to form a complex. It comprises a polypeptide or glycoprotein that has been modified or is genetically modified. In one embodiment, The term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. The terms "antigen-binding fragment" or "antibody fragment" used herein refer to the following: This refers to one or more fragments of an antibody that retains the ability to bind to an antigen such as influenza HA. The antigen-binding fragment can be subjected to any suitable standard technique, such as proteolytic digestion or antibody-variable denaturation. Recombinant genetic engineering involving the manipulation and expression of DNA encoding the main and optionally constant domains. Using techniques, it can be obtained from complete antibody molecules. Such DNA is publicly known, or, for example, a commercial source, DNA library (e.g., phage-antibody library) DNA sequencing is readily available from (including) or can be synthesized. And, by manipulating using chemical or molecular biological techniques, for example, one or more variable and / or position the constant domain in a suitable configuration, or introduce codons and system It is possible to generate ine residues, modify, add, or delete amino acids. Non-limiting examples of antigen-binding fragments include: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv(scFv) molecule; (vi) dAb fragment; and (vii) amino acid mimicking the hypervariable region of an antibody. The smallest recognition unit consisting of acid residues (for example, a fragment containing CDR, or a CDR3 peptide, etc.) Examples include isolated CDRs, or restrictive FR3-CDR3-FR4 peptides. Other modified molecules, for example. Domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR transplant antibodies Body, Diabody, Triabody, Tetrabody, Minibody, Nanobody (for example, one Vale nanobodies, divalent nanobodies, etc.), small modular immunotherapy drugs (SMIPs), and shark variable IgNARs The domain is also included in the expression "antigen-binding fragment" as used herein. The original binding fragment typically contains at least one variable domain. The variable domain is an arbitrary subdomain. It may be an isopropyl or amino acid composition, and is usually adjacent to one or more framework sequences. Includes at least one CD-R that is either doing so or is in frame with it. H domain ga V L In the antigen-binding fragment associated with the domain, V H Domain and V L Domains are mutual Conversely, it may be any preferred configuration. For example, the variable region is a dimer, and V H -V H , V H -V L , or V L -V L It may contain dimers. Alternatively, the antigen-binding fragment of the antibody may be monolayer. Body V H or V L It may contain a domain. In one embodiment, antibody antigen binding cleavage One part consists of at least one variable domain covalently linked to at least one constant domain. It may contain . Variable and constant d The main non-restrictive and exemplary arrangement is: (i)VH -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1 - C H 2; (v) V H -C H 1 - C H 2 - C H 3; (vi) V H -C H 2 - C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1 - C H 2; (xii) V L -C H 1 - C H 2 - C H 3; (xiii) V L -C H 2 - C H 3; and (xiv) V L -C L are included. In any arrangement of the variable domain and the constant domain including any of the above exemplary arrangements, the variable domain and the constant domain may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region causes a flexible or semi-flexible connection between adjacent variable domains and / or constant domains in a single polypeptide molecule, and may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids. Further, the antibodies in this specification is more). Furthermore, the antibodies in this specification The original binding fragment may be a homodimer or heterodimer (or other multimer) of any of the above variable domain and constant domain arrangements that are non-covalently associated with each other and / or with one or more monomers V H or V L domains (e.g., by disulfide bonding). As with a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain can specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present disclosure using techniques routine in the art As with a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain can specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present disclosure using techniques routine in the art As with a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain can specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present disclosure using techniques routine in the art As with a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain can specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present disclosure using techniques routine in the art As with a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain can specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present disclosure using techniques routine in the art As with a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain can specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present disclosure using techniques routine in the art As with a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain can specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present disclosure using techniques routine in the art In certain embodiments described herein, the antibodies described herein are human antibodies In certain embodiments described herein, the antibodies described herein are human antibodies

[0062] Substitution of one or more CDR residues or deletion of one or more CDRs is also possible. Antibodies that can omit one or two CDRs for binding have been described in the scientific literature. Padlan et al. (1995 FASEB J. 9:1 33-139) analyzed the contact regions between an antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan et al. also found many antibodies in which one or two CDRs have no amino acids that contact the antigen (see also Vajdos et al., 2002 J Mol Biol 320:415-428) Padlan et al. (1The original binding fragment may be a homodimer or heterodimer (or other multimer) of any of the above variable domain and constant domain arrangements that are non-covalently associated with each other and / or with one or more monomers V Padlan et al. (1995 FASEB J. 9:1 33-139) analyzed the contact regions between an antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan et al. also found many antibodies in which one or two CDRs have no amino acids that contact the antigen (see also Vajdos et al., 2002 J Mol Biol 320:415-428) Padlan et al. (1995 FASEB J. 9:1

[0063] Based on previous studies, the CDR residue that does not come into contact with the antigen is located outside the Chothia CDR, specifically Kabat. From the region of CDR, it can be identified by molecular modeling and / or experimentally (for example) (However, residues H60-H65 in CDRH2 are often unnecessary). In combination, it is usually the corresponding position in the consensus of another human antibody sequence or such sequence. It is substituted with the amino acid occupying the position. The CDR to be substituted and the position of substitution within the amino acid are determined by experiment. They can also be selectively chosen. Experimental substitutions can be conservative or non-conservative substitutions. .

[0064] The fully human anti-influenza-HA monoclonal antibodies disclosed herein correspond to Compared to germline sequences, the framework of heavy and light chain variable domains and / or CDR regions It may contain one or more amino acid substitutions, insertions, and / or deletions. Such mutations are The amino acid sequences disclosed in the specification can be obtained, for example, from public antibody sequence databases. This can be easily confirmed by comparing it with a germline sequence. This disclosure is one or more One or more amino acids within the framework and / or CDR region are germline components from which the antibody originates. In the corresponding residue of the column, or in the corresponding residue of another human germline sequence, or in the corresponding germline The sequence has been mutated by a conserved amino acid substitution in a column residue (such a sequence change is described in this specification). In this document, the amino acid sequences disclosed herein are collectively referred to as "germline mutations." It comprises antibodies derived from any of the above and their antigen-binding fragments. Those skilled in the art will understand the information disclosed herein. Starting from heavy chain and light chain variable region sequences, one or more individual germline mutations or combinations thereof can be derived. Many antibodies and antigen-binding fragments can be easily produced, including in one embodiment. V H and / or V L Mutate all framework and / or CDR residues within the domain. Then, it returns to the residue found in the original germline sequence from which the antibody originated. And only specific residues, for example, the first 8 amino acids of FR1 or the last 8 amino acids of FR4. Mutant residues found only in the amino acid, or mutations found in CDR1, CDR2, or CDR3 Only the mutated residue is mutated to return to the original germline sequence. In another embodiment, 1 The above framework and / or CDR residues are used in different germline sequences (i.e., antibodies Mutations are introduced into the corresponding residues of a germline sequence (different from the germline sequence from which the gene originally originated). Furthermore, the antibodies of this disclosure, for example, have specific individual residues corresponding to specific germline sequences. While the base sequence is mutated, certain other residues that differ from the original germline sequence are maintained. It is either mutated or has been mutated into the corresponding residue in a different germline sequence, It may contain any combination of two or more germline mutations within the muwerk and / or CDR region. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations are used, for example. For example, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or activating properties. It is easy to test one or more desired characteristics, such as specific biological properties (depending on the case) and reduced immunogenicity. This can be tested. Antibodies and antigen-binding fragments obtained by this general method are within the scope of this disclosure. It is enclosed within the enclosure.

[0065] This disclosure relates to HCVR, LCVR, and / or CD disclosed herein, having one or more conservative substitutions. Fully human anti-influenza-HA monoclonal anti-influenza containing any variant of the R amino acid sequence This includes the body. For example, this disclosure includes, for example, HCVR, LCVR, and / or CDR disclosed herein. Conservative amino acids such as 10 or less, 8 or less, 6 or less, and 4 or less compared to any of the amino acid sequences. Anti-influenza-HA anti-influenza having HCVR, LCVR, and / or CDR amino acid sequences with substitutions Including the body.

[0066] As used herein, the term "human antibody" refers to a human germline immunoglobulin sequence. It is intended to include antibodies having variable and constant regions derived from this invention. The human mAbs of this disclosure are For example, CDRs, particularly CDR3, are not encoded by human germline immunoglobulin sequences. Mino acid residues (for example, by random or site-directed mutagenesis in vitro) This may include mutations introduced by somatic mutations in vivo. However, as used herein, the term "human antibody" refers to a different mammalian species (for example, It contains mAbs in which a CDR sequence derived from the germline of a mouse has been transplanted into a human FR sequence. Not illustrated. This term is used in non-human mammals or in the cells of non-human mammals. Includes recombinant antibodies. This term refers to antibodies isolated from or produced in human subjects. This term is not intended to include modified antibodies. Therefore, it does not contain naturally occurring antibodies that are normally present in naturally occurring, unmodified organisms.

[0067] As used herein, the term “recombinant” refers to, for example, DNA splicing and Techniques or methods known in the art, such as recombinant DNA technology including lancegenic expression. This refers to antibodies or their antigen-binding fragments produced, expressed, isolated, or obtained by [unspecified method / method]. This term refers to non-human mammals (transgenic non-human mammals (e.g., trans It is expressed in a cellular expression system (including a genic mouse) or in a cell (e.g., CHO cells), or This refers to antibodies isolated from recombinant combinatorial human antibody libraries. The term "recombinant human antibody" used here refers to an antibody prepared and expressed by recombinant means. All human antibodies produced or isolated, e.g., transfected into host cells Antibodies expressed using recombinant expression vectors, recombinant combinatorial human antibody libraries The antibody isolated from Lee is transgenic in relation to the human immunoglobulin gene. Antibodies isolated from animals (e.g., mice) (e.g., Taylor et al. (1992) Nucl. Acids) See Res. 20:6287-6295), or a split of the human immunoglobulin gene sequence to another DNA sequence. Prepared, expressed, produced, or isolated by any other means involving rising. It is intended to contain antibodies that are used in human germline immunoglobulins. Such recombinant human antibodies are intended to be used in human germline immunoglobulins. It has variable and constant regions derived from the robulin sequence. However, in one embodiment Therefore, such recombinant human antibodies induce mutagenesis in vitro (or the human Ig sequence). When using transgenic animals, undergo in vivo somatic mutagenesis. Therefore, the V of the recombinant antibody H and V L The amino acid sequence of the region is human germline V H and V L Distribution Derived from and related to the lineage, but naturally occurring within the human antibody germline repertoire in vivo. This is a sequence that may not be present. Human antibodies have two forms associated with hinge heterogeneity. It can exist in one form. In one form, immunoglobulin molecules are dimers of interchain heavy chain dis It contains stable 4-chain constructs of approximately 150-160 kDa, linked by rufidic bonds. Second form In this state, the dimer is not linked by interchain disulfide bonds, but rather by covalent bonds between the light chain and the heavy chain. A molecule of approximately 75-80 kDa is formed, composed of half the antibody. These forms have affinity Even after purification, separation is extremely difficult. Various intact IgG isotypes... The frequency of occurrence of the second form is not limited, but is associated with the hinge region isotype of the antibody. This is due to structural differences. Specifically, a single amino acid in the hinge region of the human IgG4 hinge. The substitution significantly increased the appearance of the second morphology to levels typically observed using human IgG1 hinges. It can be reduced (Angal et al. (1993) Molecular Immunology 30:105). This may be desirable in manufacturing, for example, to improve the yield of the desired antibody form. One or more mutations are in the hinge region, C H 2 regions, or C H It includes antibodies located within the three regions.

[0068] As used herein, "isolated antibody" refers to other antibodies (Ab) with different antigen specificities. It is intended to refer to antibodies that substantially do not contain (for example, antibodies specifically targeting influenza-HA) The isolated antibody or fragment thereof that binds specifically binds to antigens other than influenza-HA. (Substantially does not contain the matching Ab). The antibodies described herein are isolated antibodies. This is also acceptable. The “isolated antibodies” used herein are identified and have a small amount of natural environment. It means antibodies separated and / or recovered from at least one component. For example, a small number of antibodies from an organism. Even if not present in all tissues, antibodies may be naturally present or naturally produced in some tissues. Alternatively, antibodies isolated or removed from cells are “isolated antibodies” for the purposes of this disclosure. Yes. Isolated antibodies also include antibodies in situ within recombinant cells. Isolated antibodies are , an antibody subjected to at least one purification or isolation step. According to one embodiment, Even if isolated antibodies are substantially free of other cellular material and / or chemical substances Good. The antibodies used herein are compared to the corresponding germline sequences from which the antibodies originated. One or more amino acid substitutions in the framework of the heavy chain and light chain variable domains and / or the CDR region. This may include, inserts, and / or deletions. Such mutations are disclosed herein. The amino acid sequence can be obtained, for example, from a germline sequence available from an official antibody sequence database. This can be easily confirmed by comparing it with the following. This disclosure is one or more frames One or more amino acids in the CDR region and / or the corresponding residue of the germline sequence from which the antibody originated. Based on, or on the corresponding residues of another human germline sequence, or the conserved germline residues of the corresponding germline residues The amino acid substitution has been mutated (such sequence changes are referred to herein as " "Germline mutations" (collectively referred to as "germline mutations"), which result in any of the amino acid sequences disclosed herein. This includes antibodies and their antigen-binding fragments. Those skilled in the art will understand the heavy and light chains disclosed herein. Starting from a variable region sequence, many include one or more individual germline mutations or combinations thereof. Antibodies and antigen-binding fragments can be easily produced.

[0069] The terms "blocking antibody," "neutralizing antibody," or "antagonist antibody" used herein are: This refers to an antibody whose binding to an antigen results in the inhibition of at least one biological activity associated with that antigen. It is intended that, for example, the antibody or antibody-drug conjugate of this disclosure will be used on host cells. It can prevent or block the attachment or entry of influenza. Furthermore, "neutralizing agent The body neutralizes the ability of pathogens to initiate and / or persist infection within the host, i.e. It is an antibody that can prevent, inhibit, reduce, prevent, or interfere with. Antibodies or antibody-drug conjugates retain their neutralizing ability through binding to influenza HA. If present, it can be called an "antibody that neutralizes influenza-HA activity." "Antibody" and "an antibody that neutralizes" or "antibody that neutralizes" The term "(odies that neutralize)" is used interchangeably herein. Antibodies can be obtained through appropriate formulation, or in association with active vaccination, or through diagnostic tools. It can be used alone or in combination with other antiviral agents as a preventative or therapeutic agent. It is possible. As used herein, "anti-influenza antibody" refers to an antigen (e.g., For example, its binding to HA is associated with at least one biological activity related to the influenza virus. This can refer to antibodies that cause inhibition.

[0070] The term "epitope" refers to a specific variable region within an antibody molecule, also known as a paratope. This refers to antigenic determinants that interact with the target antigen-binding site. A single antigen has multiple epitopes. It is possible. Therefore, different antibodies can bind to different parts of an antigen, and different biological processes. It may have a specific effect. The term "epitope" refers to an antigen to which B cells and / or T cells respond. This also refers to the area above. This also refers to the region of the antigen to which the antibody binds. B cell epitope This refers to discontinuous amino acids juxtaposed by the tertiary folding of consecutive amino acids or proteins. It can be formed from both acids. Epitopes formed from consecutive amino acids are Normally, it is retained when exposed to a denaturing solvent, but epithelium is formed by tertiary folding. Epitopes are usually lost when treated with denaturing solvents. Epitopes are usually unique in their spatial properties. The three-dimensional structure contains at least three, more commonly, at least five or eight to ten amino acids. Epitopes can be defined as structural or functional. Functional epitopes are usually structural. It is a subset of artificial epitopes and contains residues that directly contribute to the affinity of the interaction. The pitope may be three-dimensional, that is, it may be composed of nonlinear amino acids. In one embodiment, the epitope is an amino acid, a sugar side chain, a phosphoryl group, or a sulfate group. It may contain determinants, which are chemically active surface molecules such as honyl groups, and in some embodiments Therefore, it may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0071] The term "surface plasmon resonance" is used, for example, in the BIACORE® system (Pharmacia Bi). A biosensor matrix using osensor AB (Uppsala, Sweden and Piscataway, NJ) Real-time analysis of biomolecular interactions by detecting changes in protein concentration within the cell. This refers to the optical phenomenon that makes this possible.

[0072] Biolayer interferometry is a label-free technique for measuring biomolecular interactions. It has two surfaces: a layer of immobilized proteins at the tip of the biosensor and an internal reference layer. This is an optical analysis technique that analyzes the interference pattern of reflected white light. It can be measured in real time by detecting any change in the number of molecules bound to the tip. This causes a shift in the interference pattern (Abdiche, YN et al., Analytical Biochemist). ry, (2008), 377(2), 209-217). In one embodiment, "Real-time Biolayer" Using an interferometer-based biosensor (Octet HTX assay), a specific anti-influenza test was performed. The binding characteristics of the Enza HA antibody were evaluated.

[0073] The term "K" used in this specification D The term "equilibrium dissociation constant of a particular antibody-antigen interaction" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. This is intended to refer to...

[0074] As used herein, the term "cross-compete" means that an antibody that binds to an antigen and also binds to another antibody or This refers to an antibody or its antigen-binding fragment that inhibits or blocks the binding of that antigen-binding fragment. The phrase refers to bidirectional competition between two antibodies, that is, when the first antibody binds and the second antibody... This includes blocking the binding of the first antibody and the second antibody. In one embodiment, the first antibody and the second antibody Antibodies can bind to the same epitope. Alternatively, the first antibody and the second antibody can bind to one another. However, for example, steric hindrance may inhibit or block the binding of the second antibody, However, they can bind to overlapping epitopes. Cross-competition between antibodies is known in the art. This can be measured by methods such as real-time label-free biolayer interference assays. Cross-competition between two antibodies is self-binding (in this case, the first antibody and the second antibody are the same). Because it is an antibody, it is represented as the binding of a second antibody that falls below the background signal. This is possible. Cross-competition between two antibodies occurs, for example, in the baseline self-background The percentage of the second antibody below the bond (in this case, the first antibody and the second antibody are the same antibody) It can be expressed as a sum.

[0075] The terms "substantially identical" or "substantially identical" when referring to nucleic acids or fragments thereof are: With appropriate nucleotide insertions or deletions, it is optimally aligned with another nucleic acid (or its complementary strand). When each of them is incorporated in full herein by reference, WO 2016 / 100807 or US As discussed in issue 2016 / 0176953 A1, any well-known array identity algorithm, For example, when measured by FASTA, BLAST, or GAP, at least about 90%, more preferably or at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases This indicates rheotide sequence identity. It also indicates a nucleic acid molecule that has substantial identity with a reference nucleic acid molecule. In some cases, the polypeptide encoded by the reference nucleic acid molecule is the same as or substantially the same as the polypeptide encoded by the reference nucleic acid molecule. It may encode a polypeptide having an amino acid sequence similar to that of [the original molecule].

[0076] When applied to polypeptides, the term "substantial similarity" or "substantially similar" The phrase, for example, uses the default gap weights to create a program called GAP or BESTFIT. When optimally aligned by the microchip, the two peptide sequences exhibit at least 90% sequence identity. More preferably, sharing at least 95%, 98%, or 99% sequence identity. This means that, preferably, non-identical residue positions are different due to conservative amino acid substitutions. "Conservative amino acid substitution" refers to a substitution where amino acid residues have similar chemical properties (e.g., charge or sparseness). This is a substitution in which the amino acid residue is replaced by another amino acid residue having a side chain (R group) that is aqueous. Furthermore, conservative amino acid substitutions do not substantially alter the functional properties of a protein. If the amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity This can be adjusted upwards to correct for the conservative properties of the substitution. The means for this are well known to those skilled in the art (e.g., Pearson's literature (1994) Methods Mol. Biol. See 24: 307-331). Examples of amino acids with side chains having similar chemical properties include: 1) Aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) Aliphatic- Hydroxyl side chains: serine and threonine; 3) Amide-containing side chains: asparagine and glutamate 4) Aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) Basic side chains: ri Dinine, arginine, and histidine; 6) Acidic side chains: aspartic acid and glutamic acid, and 7) Sulfur-containing side chains: Examples include cysteine ​​and methionine. Preferred conserved amino acids The substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine. Nin, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine Alternatively, conservative substitutions are disclosed in the literature by Gonnet et al. (1992) Science 256: 1443 45. This is any change that has a positive value in the PAM250 log-probability matrix. A "existential" permutation is any change that has a non-negative value in the PAM250 log-probability matrix.

[0077] Polypeptide sequence similarity is typically measured using sequence analysis software. The protein analysis software analyzes various substitutions, deletions, and other modifications, including conservative amino acid substitutions. Similar sequences are matched using a similarity scale assigned to the ornament. For example, GCGSo Software is a closely related polypeptide, for example, homologous polypeptides derived from different species. Sequence homology or sequence identity between peptides, or between wild-type proteins and their mutants. To make a decision, you can use GAP and BESTFIT with default parameters. Which programs are included? For example, see GCG version 6.1. The polypeptide sequence is , using default or recommended parameters FASTA: GCG version 6.1 It is also possible to compare using RUM. FASTA (e.g., FASTA2 and FASTA3) is a query format. Alignment and percentage sequence of the highest overlap region between the column and the search sequence. It provides uniformity (Pearson's reference (2000), see above). The sequence has 12 gap-open penalties. Affingi using a gap extension penalty of 1 and 2, and a BLOSUM matrix of 62. We will compare them using the Smith-Waterman homology search algorithm, which employs CAP search. It is also possible to compare the sequence of the present invention with a database containing numerous sequences from various organisms. Another preferred algorithm in this case is a computer program that uses default parameters. The program is BLAST, particularly BLASTP or TBLASTN. For example, see the literature by Altschul et al. (1990) J. See Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402. .

[0078] The term "therapeutic effective dose" refers to the amount that is administered to produce the desired effect. This refers to [a specific condition]. The exact amount is determined by the purpose of treatment and can be administered by a person skilled in the art using known techniques. This can be verified (for example, Lloyd's literature (1999), the art, science, and technology of pharmaceutical compounding). (See Technology (The Art, Science and Technology of Pharmaceutical Compounding)) .

[0079] As used herein, the term "subject" means a disease or disorder such as a viral infection. Animals, preferably mammals, that require improvement, prevention, and / or treatment of [condition] The term refers to humans. The target is someone who may have an influenza infection, or who has an influenza infection. They are prone to developing influenza virus infections. The target group, or those who are "at high risk of contracting influenza virus infection," For individuals with immune system deficiency due to autoimmune diseases, immunosuppressive therapy is recommended (e.g., after organ transplantation). People receiving the treatment, those with human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), white blood cells People suffering from certain types of anemia that depletes or destroys the body's cells, or those undergoing radiation or chemotherapy. This includes people who are receiving treatment or suffering from inflammatory disorders. Furthermore, it also includes extremely young or elderly individuals. The target group is those at high risk: people who have had physical contact with an infected individual or who are in very close physical proximity to them. All of these have a high risk of developing influenza virus infection. Furthermore, the target group is... Because of proximity to the disease's epidemic, for example, the subjects live in densely populated cities, or in Because they are in very close proximity to individuals who have been confirmed or suspected of having the flu virus, Alternatively, the choice of occupation, for example, hospital staff, pharmaceutical researchers, travelers to infected areas, or frequently As someone who travels by plane, they are at risk of contracting influenza.

[0080] As used herein, “treat,” “treating,” or “ The term "treatment" refers to the administration of a therapeutic agent, such as a disclosed antibody, to a subject who needs it. This reduces or improves the severity of at least one symptom or sign of influenza infection. These terms refer to inhibiting the progression of a disease or the worsening of an infection. The positive prognosis, i.e., the therapeutic agent such as the disclosed antibody or antibody-drug conjugate. Administration of this drug may allow the subject to be immune to infection or to have a reduced viral titer. This includes cases where there is no viral titer at all. The therapeutic agent is administered to the target at a therapeutic dose. can.

[0081] The terms "prevent," "preventing," or "prevention" are open Influenza infection or influenza caused by administration of the indicated antibody or antibody-drug conjugate This refers to the inhibition of the manifestation of any symptoms or signs of influenza infection. This term refers to the virus Prevention of the spread of infection in exposed or at-risk individuals with influenza infection. Includes.

[0082] As used herein, “protective effect” refers to drugs such as antivirals or antiviral agents. Antibodies such as Luenza-HA antibody, or antibody-drug conjugates disclosed herein, The following: for example, increased survival after exposure to an infectious agent, decreased viral load, or infection The patient must demonstrate improvement in at least one symptom associated with the chromatin factor, or any one of the following: This can be demonstrated by any standard procedure known in the art to determine whether it is possible. It is possible.

[0083] As used herein, the terms "antiviral agent," "antiviral agent," and "antiviral compound" are used. The terms "antiviral compound" and "antiviral compound" The phrase describes treating, preventing, or improving a viral infection (e.g., influenza infection) in the subject. Applies to anti-infective drugs or therapies used for this purpose. ug) (or its synonyms "antiviral drug", "antiviral compound (anti-vi) The terms "antiviral compound" and "antiviral compound" are used by TAMI. FLU (registered trademark) (oseltamivir), RELENZA (registered trademark) (zanamivir), ribavirin, or This includes, but is not limited to, antferon-α2b. Antiviral drugs are used for influenza Contains the inhibitor. As used herein, "influenza inhibitor" refers to influenza. This refers to drugs used to inhibit the coronavirus infection, including oseltamivir. Not limited to the above. When used herein, polymerase inhibitors are used for influenza. This can refer to inhibitors of nucleic acid polymerases such as polymerase. An example polymerase The inhibitor is VX-787. While I don't wish to be bound by any particular theory, Influenza inhibitors work by targeting the influenza virus itself, Alternatively, by targeting host cells that may be targeted by the influenza virus, It is possible. For example, influenza inhibitors that target host cells can work within the cell. This can inhibit translation, thereby reducing virus replication.

[0084] The phrases "specifically bind" or "specifically bind to" refer to antibodies or their antigenic binding. This means that the composite fragment forms a complex with an antigen that is relatively stable under physiological conditions. The heterogeneous bond is at least about 1 × 10⁻⁶ -8 It can be characterized by an equilibrium dissociation constant less than or equal to M. (For example, a smaller K) D (This means a stronger bond). Two molecules are specifically bonded together. Methods for determining whether or not to do so are well known in the art, for example, equilibrium dialysis, table This includes face plasmon resonance, etc. As described herein, antibodies are influenza Real-time label-free testing on Octet® HTX biosensor, which specifically binds to Za-HA. It has been identified by biolayer interference assay. Furthermore, 1 in influenza-HA A multispecific antibody or influenza-HA that binds to one or more domains and one or more additional antigens. The bispecific compounds that bind to two different regions are still referred to herein as " It is considered an antibody that "specifically binds". In addition to neutralizing antibodies, it specifically binds to HA, but is not To prepare an antibody-drug conjugate using a neutralizing antibody within the scope of this disclosure. It can also do this. Such antibodies can, for example, deliver a payload to influenza-infected cells. It can function in this way.

[0085] The term "high affinity antibody" is derived from real-time label-free biolayer interference assays. For example, by Octet® HTX biosensor, or by surface plasmon resonance For example, when measured by BIACORE (trademark) or by solution affinity ELISA, at least tomo10 -8 M; preferably 10 -9 M; furt, 10 -10 M, more preferably 10 -11 M, More more preferably, 10 -12 M, K D The binding to influenza-HA is expressed as follows: This refers to mAbs that have affinity for each other.

[0086] "Slow dissociation rate", "K off " or "k d The phrase or term " " is not real-time Recognition biolayer interference assays, e.g., Octet® HTX biosensor, or table When determined by, for example, BIACORE(trademark), the surface plasmon resonance is 1 × 10⁻¹⁰. -3 s -1 Below is good Mashiku is 1 x 10 -4 s -1 This refers to antibodies that dissociate from influenza-HA at the following rate constants.

[0087] The "antigen-binding domain" or "antigen-binding portion" of an antibody as used herein, and the "anti- The terms "primordial binding fragment" refer to any naturally occurring fragment that specifically binds to an antigen to form a complex. Enzymatically obtained, synthesized, or genetically modified polypeptides or glycotans Contains protein.

[0088] In a specific embodiment, the antibody or antibody fragment of this disclosure is obtained by influenza-HA Antiviral drugs useful for treating infections caused by, antiviral linkers - Ligand such as payload, second anti-influenza antibody, or any other therapeutic portion Alternatively, it can be conjugated to therapeutic parts or other parts ("antibody-drug conjugate"). (or "immune conjugate").

[0089] As used herein, “sequential administration” means that each dose of the compound is administered at different times. Points, for example, different points separated by predetermined intervals (a few hours, a few days, a few weeks, or a few months). This means that it will be administered to the target on that day.

[0090] The terms “initial dose,” “secondary dose,” and “tertiary dose” are used as defined herein. This refers to the time series of compound administration. Therefore, the "initial dose" is the dose administered at the start of the treatment regimen. The dose administered (also called the "baseline dose"); the "secondary dose" is the initial dose. The first dose is the dose administered later; the "tertiary dose" is the dose administered after the secondary dose. The first dose, second dose, and tertiary dose all contain the same amount of the compound described herein. They can be obtained, but the frequency of administration can usually differ from one another.

[0091] As used herein, the term "immediate dose" refers to a series of multiple doses administered by an intermediary. This refers to the dose of the compound administered to the patient immediately before the next dose, in an order without a specified dose count. do.

[0092] As used herein, the term “payload” means having the desired biological effect. (For example, inhibiting influenza virus infection or replication), directly or via linkers A small molecule active component optionally conjugated to an antibody or its antigen-binding fragment via ( For example, it refers to antiviral compounds. The payload is 2,000 Da or less, 1,500 Da or less, or 900 It can be less than or equal to Da.

[0093] (Compound or payload) Provided herein are antiviral compounds or payloads. Any specific operation Although not bound by theory, antiviral compounds include (1) VX-787 and its derivatives; (2) baloxavir and its derivatives (e.g., baloxavir marboxil) are also included. In one embodiment, the antiviral compound is delivered to the cell as part of the conjugate. It is possible. In one embodiment, the antiviral compound targets, for example, the target element In or within cells, VX-787, baloxavir, and / or baloxavir marboxil, as The desired activity of each of these derivatives can be performed. Certain antiviral compounds The substance may have one or more additional activities.

[0094] In one embodiment, the structure of formula I is described herein: [ka] or a compound having a pharmaceutically acceptable salt thereof. In formula I, in some embodiments, ZZ is -OR 1 or -NHOH. In one embodiment, ZZ is -NHOH. In this case, ZZ is -OR 1 In equation I, ZZ is -OR 1 When this is the case, a useful R 1As a base, hydrogen and Beauty [ka] Examples include: One embodiment, R 1 is hydrogen. In one embodiment, R 1 teeth, [ka] In one embodiment, the compound is VX-787 or [ka] That is the case.

[0095] In one embodiment, the structure of formula II is described herein: [ka] or a compound having a pharmaceutically acceptable salt thereof. In one embodiment, R 1 is water It is prime. In one embodiment, R 1 This is -CH2OC(O)OCH3. In one embodiment, R 1 teeth, [ka] In one embodiment, the compound is baloxavir marboxil or [ka] In one embodiment, the compound is baloxavir or [ka] That is the case.

[0096] (Binder) Suitable binders for any of the conjugates provided in this disclosure include antibodies, Examples include viral receptors, or any other cell-binding or peptide-binding molecules or substances. However, it is not limited to these. The full-length amino acid sequence of an exemplary influenza HA is, It is listed in GenBank as accession number ACP44150.1.

[0097] Suitable binders include the surface proteins hemagglutinin (HA) and neuraminidase (NA). and antibodies that specifically bind to influenza virus proteins such as Matrix-2(M2) (e.g.) Examples include fully human antibodies and their antigen-binding fragments. In some embodiments, These binders regulate the interaction between the influenza virus and host cells. In one embodiment, the antibody or its antigen-binding fragment binds to mature hemagglutinin. In some embodiments, the antibody or its antigen-binding fragment is an HA0 hemagglutinin precursor. It binds to body proteins. Anti-influenza HA antibodies have high affinity for influenza viruses. It can bind to Rus HA. In one embodiment, the antibody herein blocks This is an antibody, which binds to influenza HA and inhibits the attachment of the virus to host cells. It can block entry and / or intrusion. In some embodiments, as specified herein These blocking antibodies can block the binding of the influenza virus to cells, however This can inhibit or neutralize the viral infectivity of host cells. Several embodiments In this context, blocking antibodies are useful in treating individuals suffering from influenza virus infection. It's possible. When antibodies are administered to a target that needs them, they can help control influenza in that target. It can reduce infections caused by viruses such as influenza. Using these, the target can These can reduce the amount of virus present. They can be used alone or to treat viral infections. As an adjunct therapy in conjunction with other therapeutic components or modalities known in the art for the purpose of It can be used in one embodiment. These antibodies are the stem of the viral HA. It can bind to epitopes in the region, the head region of the viral HA, or both. Furthermore, the identified antibodies are used prophylactically (before infection) to protect mammals from infection. It can be used, or to improve a previously established infection, or to treat an infection Used therapeutically (after infection has been established) to alleviate at least one related symptom. It is possible.

[0098] In one embodiment, the antibody is either a primary immunogen such as full-length influenza HA or recombinant. Immunized with a form of influenza HA or its fragments, and then with a secondary immunogen or influenza It is obtained from mice immunized with the immunoactive fragment of Luenza HA. ​​In one embodiment, The antibodies are immunized with the influenza vaccine composition, and then one or more recombinant HAs are produced. It is obtained from mice that have been booster-immunized with ptide. In one embodiment, the antibody is obtained from humans. Obtained. In one embodiment, the antibody is obtained from a mammal (e.g., a non-human mammal). In one embodiment, the antibody is obtained from a non-human primate.

[0099] The immunogen is a biologically active fragment and / or immunogenic fragment of influenza HA or this It may also be DNA encoding the active fragment. The fragment is the stem region (20) of the HA protein. Sui's paper, published online on February 22, 2009, in Nature Struct, and Mol. Biol.; 1 (See page 9) The HA protein head region may be obtained from a combination of these. .

[0100] Peptides are used for tagging or as carrier molecules, for example, keyhole limpet hemocyanin Modifications include the addition or substitution of specific residues for conjugation to (KLH). This can be done. For example, cysteine ​​can be attached to either the N-terminus or C-terminus of a peptide. They may also be added, or, for example, peptides for conjugation to KLH for immune use. A linker array may be added for manufacturing purposes.

[0101] The specific anti-influenza antibodies, anti-influenza-HA antibodies, or ADCs used herein are , possessing antiviral activity, as determined, for example, by in vitro or in vivo assays. It can bind to influenza-HA and neutralize its activity. Certain anti-influenza antibodies, anti-influenza-HA antibodies, or ADCs can be used in vitro or When determined by in vivo assay, it can bind to HA but has neutralizing activity. It does not bind to influenza-HA, and its activity, and consequently, the virus to host cells. This specification neutralizes the attachment and / or entry of and the subsequent viral infection. The ability of the antibody or ADC is determined by a binding assay or activity assay as described herein. It can be measured using any standard method known to those skilled in the art.

[0102] Antibodies or ADCs specific to influenza-HA do not contain additional labels or portions. It is not necessary, or these may contain a label or portion at the N-terminus or C-terminus. Good. In one embodiment, the label or portion is biotin. In a binding assay... The position of the label (if any) determines the orientation of the peptide relative to the surface to which it is bound. It can be determined. For example, if the surface is coated with avidin, the N-terminus The biotin-containing peptide is configured such that the C-terminus of the peptide is distal to the surface. The label will be oriented. In one embodiment, the label is a radionuclide, a fluorescent dye, or MRI It may be a detectable label. In one embodiment, such a labeled antibody is used in imaging It can be used in diagnostic assays including guagreases. In one embodiment, additional parts The part is a peptide tag. In one embodiment, the ADC contains an antibody heavy chain, and the antibody heavy The chain further includes a peptide tag at its C-terminus. In one embodiment, the ADC comprises an antibody heavy chain. Furthermore, the antibody heavy chain contains a peptide tag (e.g., pentapeptide) at its C-terminus, where The peptide tag is linked to the linker-payload (for example, via transglutaminase). This is a pentapeptide sequence LLQGA (for use when conjugating). One embodiment In this, the ADC contains two antibody heavy chains, and each antibody heavy chain has a peptide tag at its C-terminus. Furthermore, it includes. In one embodiment, the ADC comprises two antibody heavy chains, and each antibody heavy chain The C-terminus further contains a peptide tag, where the peptide tag is a pentapeptide sequence LL This is QGA.

[0103] In one embodiment, the antibody includes a light chain. In one embodiment, the light chain is kappa It is a light chain. In one embodiment, the light chain is a lambda light chain. In one embodiment The antibody contains a heavy chain. In some embodiments, the heavy chain is IgA. In some embodiments, the heavy chain is IgD. In some embodiments, the heavy chain is IgE. In some embodiments, the heavy chain is IgG. In some embodiments, The heavy chain is IgM. In some embodiments, the heavy chain is IgG1. In some embodiments, the heavy chain is IgG2. In some embodiments, the heavy chain is IgG3. In some embodiments, the heavy chain is IgG4. In some embodiments, The heavy chain is IgA1. In some embodiments, the heavy chain is IgA2.

[0104] In some embodiments, the antibody is an antibody fragment. In some embodiments The antibody fragment is the Fv fragment. In some embodiments, the antibody fragment is the Fab fragment. In some embodiments, the antibody fragment is the F(ab')2 fragment. In some embodiments, the antibody fragment is the Fab' fragment. The fragment is an scFv(sFv) fragment. In some embodiments, the antibody fragment is an scFv-Fc fragment.

[0105] In some embodiments, the antibody is a monoclonal antibody. In this example, the antibody is a polyclonal antibody. In some embodiments, the antibody is It is a bispecific antibody containing a first antigen-binding domain and a second antigen-binding domain. .

[0106] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. .

[0107] In one embodiment, the antibody is glued to one or more heavy chain positions numbered 295 in the EU numbering system. Contains a glutamine residue. In this disclosure, this position is glutamine 295 or Gln295 or Q295. It is called. Those skilled in the art will know that this is a conserved glutamine residue in the wild-type sequences of many antibodies. They will be aware of this. The identification of residue Q295 includes those described herein. This can be easily achieved with standard sequence alignment tools. Other useful implementations In one embodiment, the antibody can be modified to include a glutamine residue. In this case, the antibody contains one or more N297Q mutations. The antibody is formulated to contain glutamine residues. The techniques for modifying columns are within the scope of the skills of those skilled in the art (e.g., the literature of Ausubel et al., Current (See Protoc. Mol. Biol.) In one embodiment, the antibody comprises an antibody heavy chain, and the antibody The heavy chain further includes a peptide tag at its C-terminus. In one embodiment, the antibody comprises an antibody heavy chain. Furthermore, a peptide tag, such as a transglutaminase recognition sequence, is attached to the C-terminus of the antibody heavy chain. or further comprising a pentapeptide tag, wherein the peptide tag is a pentapeptide sequence It is LLQGA.

[0108] (Preparation of human antibodies) Methods for producing human antibodies using transgenic mice are well known in the art. Using such publicly known methods in connection with this disclosure, specifically binding to influenza-HA It is possible to produce a matching human antibody. Using an immunogen containing one of the following, Antibodies against influenza HA can be produced. In one embodiment, this specification The antibodies in this context are full-length native influenza HA (e.g., GenBank accession). (See number FJ966082.1), or live attenuated virus or inactivated virus, or the protein It is obtained from mice immunized with DNA encoding the substance or a fragment thereof. Alternatively, Fluenza-HA protein or its fragments are produced and modified using standard biochemical techniques. It can be used as an immunogen. In one embodiment, the immunogen is recombinantly produced This is an influenza-HA protein or a fragment thereof. In one embodiment described herein In this embodiment, the immunogen may be an influenza virus vaccine. In one embodiment, one or more additional immunization injections may be administered. The virus contains one or more influenza virus strains, or hemagglutinin derived from these strains. It can include, for example, Protein Sciences' H1 A / New Caledonia / 20 / 1999, H5 A / I Indonesia / 05 / 2005, H3 A / Victoria / 361 / 2011, H7 A / Netherlands / 219 / 2003, if This refers to H9 A / Hong Kong / 1073 / 1988, or influenza B virus strains B / Victoria / 2 / 87, B / South See Chang / 3451 / 93, B / Singapore / 11 / 1994, B / Florida / 4 / 2006, or B / Yamagata / 16 / 88. In one embodiment, the additional immunization injection is a 1:1 mixture of influenza strains or It may contain a 1:1 mixture of hemagglutinin derived from the strain. In one embodiment, the immunogen is , E. coli or any other eukaryote or mammal, for example, Chinese hamster Recombinant influenza HA peptide expressed in ovarian (CHO) cells, or influenza It could even be the virus itself.

[0109] VELOCIMMUNE® technology for producing monoclonal antibodies (e.g., US 6,596,5 See Article 41, Regeneron Pharmaceuticals, VELOCIMMUNE® (registered trademark), or any other publicly available information Using this method, against influenza-HA having a human variable region and a mouse constant region First, high-affinity chimeric antibodies are isolated. VELOCIMMUNE® technology is used when mice react to antigen bites. In response to stimulation, endogenous mice produce antibodies containing the human variable region and the mouse constant region. It has a genome that includes human heavy and light chain variable regions functionally linked to a constant region locus. This involves the creation of transgenic mice, which encode the variable regions of the antibody's heavy and light chains. The DNA is isolated and functionally ligated to the DNA encoding the human heavy chain and light chain constant regions. Then, The DNA is expressed in cells capable of expressing fully human antibodies.

[0110] Typically, VELOCIMMUNE® mice are administered the target antigen, and lymphoid cells (for example) Next, B cells are collected from a mouse expressing the antibody. These lymphoid cells are then fused with a myeloma cell line. By combining them, immortal hybridoma cell lines are prepared, and such hybridoma cell lines are screened. Hybridoma cells that are lean and selective, and produce antibodies specific to the target antigen. Identify the strain. Isolate the DNA encoding the variable regions of the heavy and light chains, and select the desired heavy and light chains. Such antibody proteins can be linked to specific isotypes of constant regions. It can be produced in cells, for example, CHO cells. Alternatively, antigen-specific chimeric antibodies or light chains. Furthermore, directly isolating the DNA encoding the variable domain of the heavy chain from antigen-specific lymphocytes. can.

[0111] First, a high-affinity chimeric antibody possessing both a human variable region and a mouse constant region is isolated. Each of these is fully incorporated by reference in WO 2016 / 100807 or US 2016 / 0176953 A1. As described, antibodies are selected to have desirable characteristics including affinity, selectivity, and epitopes. Characterize and select the mouse steady region. Replace the mouse steady region with the desired human steady region as described herein. Fully human antibodies, such as wild-type or modified IgG1 or IgG4, are produced. The constant region can vary considerably depending on the specific application, but high affinity antigen binding and target specificity are important. Its distinguishing feature is its presence in the variable region.

[0112] (biological equivalent) The anti-influenza-HA antibodies and antibody fragments described herein are the same as those of the antibodies described. It has an amino acid sequence that differs from the amino acid sequence, but retains the ability to bind to influenza HA. It contains proteins. When such mutant antibodies and antibody fragments are compared to the parent sequence... , including the addition, deletion, or substitution of one or more amino acids, but the biological activity of the antibody described and They exhibit essentially equivalent biological activity. Similarly, the antibody-coding DNA sequences disclosed herein are When compared to the given sequence, it includes one or more additions, deletions, or substitutions of nucleotides, but this An antibody or antibody fragment that is essentially biologically equivalent to the antibody or antibody fragment in the specification. It includes sequences that correspond to other biologically equivalent anti-influenza-HA antibodies and antibody fragments. Each of these is fully incorporated by reference in WO 2016 / 100807 or US 2016 / 0176953 A1 It is as stated in the issue.

[0113] (Biological characteristics of antibodies) Generally, antibodies in this specification function by binding to influenza HA. For example, this specification provides a bio-layer interferometer based on real-time biolayer interferometry. When measured by a sensor (Octet HTX assay) or by surface plasmon resonance, 1 K less than 0 nM D At (for example, at 25°C or 37°C), antibodies that bind to influenza HA and antibodies against the It is a proto-binding fragment. In one embodiment, the antibody or its antigen-binding fragment is, for example, Each is fully incorporated by citation in WO 2016 / 100807 or US 2016 / 0176953 A1. Using the assay format described, or substantially similar assays, surface plasmons can be detected. When measured by sound, the levels were approximately less than 5 nM, less than 2 nM, less than 1 nM, less than 500 pM, and less than 250 pM. K is full, approximately, or less than approximately 100 pM. D Then, it binds to influenza-HA.

[0114] Non-limiting, exemplary in vitro assays for measuring binding activity are, each cited. As fully incorporated by Example 3 of WO 2016 / 100807 or US 2016 / 0176953 A1 In Example 3 of WO 2016 / 100807 or US 2016 / 0176953 A1, influenza- The binding affinity and dissociation constant of anti-influenza-HA antibodies for HA are real-time bio Determined by a layer interferometer-based biosensor (Octet HTX assay). WO 20 Examples 4 and 5 of US 16 / 100807 or US 2016 / 0176953 A1 use a neutralization assay. The infectivity of one strain from a diverse group of influenza viruses was determined. WO 2016 / 100807 In Example 6 of US 2016 / 0176953 A1, a specific antibody was found to affect the complement-dependent cells of virus-infected cells. It has been shown to transmit cystic cystopathy (CDC) in vitro. WO 2016 / 100807 or US 2016 / 0 Examples 7 and 10 of 176953 A1 demonstrate that the specific antibodies of this disclosure are either prophylactic or therapeutic. When administered, it demonstrated that it could neutralize influenza A infection in vivo. It is.

[0115] Furthermore, each of the following is provided herein by reference and is fully incorporated herein: Assay form defined in WO 2016 / 100807 or US 2016 / 0176953 A1. The results were obtained by surface plasmon resonance at 25°C using the formula, or by a substantially similar assay. The dissociation half-life is over 100 minutes (t 1 / 2) Antibodies that bind to influenza HA and their antigen binding. It is a fragment. In one embodiment, the antibody or antigen-binding fragment in this specification is, for example, Each of these is incorporated in full herein by reference by WO 2016 / 100807 or US 2 Assay formats defined in 016 / 0176953 A1 (e.g., imAb capture or antigen capture format) The measurement was performed by surface plasmon resonance at 25°C using the formula (), or by a substantially similar assay. At times exceeding approximately 200 minutes, 300 minutes, 400 minutes, 500 minutes, 600 minutes, 700 minutes, and 800 minutes. Over, approximately over 900 minutes, or approximately over 1000 minutes 1In one embodiment, Furthermore, the antibodies or antigen-binding fragments in this specification have a dissociation half-life of more than 300 minutes (t 1 / 2) inf It binds to Luenza HA. ​​In one embodiment, the antibodies used herein are from monkeys and mice. When tested, it showed a dissociation half-life approximately 1.5 to 2 times longer compared to the control antibody labeled as I mAb. This leads to an increase.

[0116] Also provided herein is the infection of a host cell with the influenza virus. An antibody or antigen-binding fragment thereof that neutralizes sex. In some embodiments, the antibody is For example, WO 2016 / 100807 or US 2016 / 0176, each of which is fully incorporated by citation. Microneutralization assays as shown in Examples 4 and 5 of 953 A1, or substantially similar assays In the case of sieve, ICs in the range of approximately 1.6 nM to approximately 130 nM 50 And various representative Group 1 influenza Nzavirus (H1N1 A / Puerto Rico / 08 / 1934; H5N1 A / Vietnam / 1203 / 2004; H1N1 A Cali California / 07 / 2009; H1N1 A / Wisconsin / 1933; H1N1 A / Brisbane / 59 / 1997, H9N2 A Hong Kong / 33982 / 2009, H13N6 a / Seagull / Maryland / 704 / 1977, and H16N3 A / Beachbird / Delau It exhibits neutralizing efficacy against (Ea / 172 / 2006). In one embodiment, it shows neutralizing efficacy against the host cell. Antibodies or their antigen-binding fragments that neutralize the infectivity of the influenza virus have an IC5 of less than 130 nM. A value of 0 indicates neutralizing efficacy.

[0117] Furthermore, this specification provides for EC in the range of approximately 20 nM to approximately 66 nM. 50Complement-dependent in infected cells An antibody or its antigen-binding fragment that mediates cytotoxicity (each of which is completely combined by reference) (See Example 6 of WO 2016 / 100807 or US 2016 / 0176953 A1). In one embodiment In this case, the antibody or its antigen-binding fragment has an EC of less than 66 nM. 50 So, the complement-dependent cells of infected cells It mediates harm.

[0118] This specification describes an increase in protection or in vivo treatment of type A compared to a control antibody. This is an anti-influenza-A HA antibody that neutralizes influenza infection. Specific antibodies are preventive. When administered pre-infection or therapeutically (after infection), it shows neutralization; each of these is cited in the reference. For a more fully integrated implementation, see Example 7 of WO 2016 / 100807 or US 2016 / 0176953 A1. I want to.

[0119] In one embodiment, the following is provided herein: a substance that specifically binds to influenza HA. This is an isolated recombinant antibody or its antigen-binding fragment, wherein the antibody or its fragment is The following characteristics: (a) It is a fully human monoclonal antibody; (b) It can be used in surface plasmon resonance assays. When measured by 10 -9 Dissociation constant less than M (K D ) to bind to influenza HA; (c ) Dissociation half-life (t) in the range of approximately 370 minutes to over 1000 minutes 1 (d) (d) ICs of approximately 1.6 nM to approximately 130 nM 50 in Group 1 influenza A influenza virus selected from H1N1, H5N1, H9N2, H13N6, and H16N3 (e) To demonstrate neutralization of illus; (e) EC of approximately 20 nM to approximately 66 nM 50 And, influenza virus infected cells (f) When administered before or after viral administration This demonstrates protection, as shown by increased survival in animal models of influenza virus infection. To indicate two or more of the things.

[0120] In this specification, antibodies possess two or more of the biological characteristics described above or any combination thereof. It can possess. Other biological characteristics of antibodies as described herein are as described herein. An overview of this disclosure, including the examples of its application, will be obvious to those skilled in the art.

[0121] (Amino acid and nucleotide sequences of the heavy and light chain variable regions) In some embodiments, the linker-payload or the payload is conjugated The antibody or its antigen-binding fragment can be an antibody that targets influenza HA. Exemplary influenza HA antibodies are, for example, each of which is fully described herein by reference. This can be found in WO 2016 / 100807 or US 2016 / 0176953 A1, which are incorporated into the whole. In some embodiments, influenza HA antibodies are heavy chain complementation determinations including SEQ ID NO: 20. Region (HCDR)-1; HCDR2 including SEQ ID NO: 22; HCDR3 including SEQ ID NO: 24; Light chain phase including SEQ ID NO: 28 Complementary Determination Region (LCDR)-1; LCDR2 containing SEQ ID NO: 30; and LCDR3 containing SEQ ID NO: 32. In one embodiment, the influenza HA antibody has a heavy chain variable region including SEQ ID NO: 18 (HCV Includes a light chain variable region (LCVR) including R) and Sequence ID No. 26. In any of the embodiments described above. Influenza HA antibodies, without causing impairment of antibody function or binding, bind to a certain site. It can be prepared by site-directed mutagenesis involving the insertion of a lutamine residue. For example, In any of the embodiments described above, the influenza HA antibody is Asn297Gln(N297Q) mutant. It may contain differences. Such antibodies with the N297Q mutation are transglutamin It can approach the -se, and therefore conjugate the payload or linker-payload. It contains one or more additional native glutamine residues in its variable region that can be gated. This is possible. In one embodiment, the antibody comprises HCVR and has a peptide at the C-terminus of HCVR The dotag further includes. In one embodiment, the antibody comprises HCVR and the C-terminus of HCVR Further comprising a peptide tag, wherein the peptide tag is the pentapeptide sequence LLQGA In one embodiment, the antibody comprises two HCVRs, and each HCVR has a peptide at its C-terminus. Further includes a cydotag. In one embodiment, the antibody comprises two HCVRs, and each H The CVR further includes a peptide tag at its C-terminus, where the peptide tag is a pentapeptide. The sequence is LLQGA.

[0122] Table 1 shows the heavy and light chain variable regions and CDRs of the selected anti-influenza HA antibodies. This shows the nucleic acid sequence identifier. The corresponding nucleic acid sequence identifiers are shown in Table 2. Table 1: Amino acid sequence identifiers [Table 1] * The mAb contains one or more mutations in its constant region. Table 2: Nucleic acid sequence identifiers [Table 2] * The mAb contains one or more mutations in its constant region.

[0123] The binding linker is added to specific amino acids within the antibody or antigen-binding molecule. It can be bound to a binder, such as an antibody or antigen-binding molecule. This embodiment of the present disclosure Examples of amino acid additions that can be used in relation to this include, for example, lysine (for example) See also US No. 5,208,020; US No. 2010 / 0129314; Hollander et al., Bioconjugate Chem., 20 08, 19:358-361; WO 2005 / 089808; US 5,714,586; US 2013 / 0101546; and US 2012 / See issue 0585592), cysteine ​​(e.g., US 2007 / 0258987; WO 2013 / 055993; WO 201 3 / 055990; WO 2013 / 053873; WO 2013 / 053872; WO 2011 / 130598; US 2013 / 010154 See No. 6; and US No. 7,750,116), selenocysteine ​​(e.g., WO 2008 / 122039; and Hof (See er et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formil Glycine (e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al.) References, Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and the literature by Rabuka et al., Nat. Pro See Tocols, 2012, 10:1052-1067, non-natural amino acids (e.g., WO 2013 / 068874 and WO (See issue 2012 / 166559), and also include acidic amino acids (see, for example, WO issue 2012 / 05982). Linkers conjugate antigen-binding proteins by attaching to carbohydrates. It is also possible to do so (for example, US 2008 / 0305497, WO 2014 / 065661, Ryan et al., Food & Agriculture). iculture Immunol., 2001, 13:127-130, and the literature by Jeger et al., Angew Chem Int Ed Engl., (See 2010, 49:9995-9997).

[0124] In some cases, the binder is an antibody or antigen-binding molecule, and the antibody is a lysine residue. It is bound to the linker via a group. In some embodiments, it binds to an antibody or antigen. The molecule is bound to the linker via a cysteine ​​residue.

[0125] The linker is transmitted via transglutaminase-based chemical enzymatic conjugation. It can be conjugated to one or more glutamine residues (for example, the literature by Jeger et al.) Angew Chem Int Ed Engl., 2010, 49:9995-9997 and Dennler et al., Bioconjugate C See hem. 2014, 25:569-578). For example, in the presence of transglutaminase, One or more glutamine residues in the body can be coupled to a primary amine compound. For example, transglutaminase-mediated coupling can be used with antibody drugs. Includes a payload or linker-payload that directly provides an object conjugate. Primary amine compounds are later used for further compounding toward the synthesis of antibody-drug conjugates. This also includes linkers and spacers functionalized with reactive groups that can react. Antibodies containing glutamine residues are either isolated from natural sources or contain one or more glutamine residues. It can be modified as follows: Glutamine residue in antibody polypeptide chain (glutaminyl modification) Techniques for artificially producing antibodies or antigen-binding molecules are within the scope of the skills of those skilled in the art. In this embodiment, the antibody is aglycosylated.

[0126] In one embodiment, the antibody or glutaminyl-modified antibody or antigen-binding molecule is small At least one polypeptide chain sequence contains at least one glutamine residue. In this embodiment, the antibody, glutaminyl-modified antibody, or antigen-binding molecule each contains one Gln29 It comprises two heavy chain polypeptides having 5 or Q295 residues. In a further embodiment, an antibody Alternatively, glutaminyl-modified antibodies or antigen-binding molecules may have one or more glutamyl atoms at a site other than heavy chain 295. Contains min residues. Included herein are those carrying the N297Q mutation described herein. This is the antibody described in this section.

[0127] In one embodiment, the antibody or glutaminyl-modified antibody or antigen-binding molecule is the antibody weight The antibody chain comprises a chain and further comprises a peptide tag at the C-terminus of the antibody heavy chain. In one embodiment, the antibody The body or glutaminyl-modified antibody or antigen-binding molecule contains an antibody heavy chain, and the antibody heavy chain The C-terminus further contains a peptide tag, where the peptide tag is a pentapeptide sequence LL QGA is used in one embodiment. It contains two antibody heavy chains, and each antibody heavy chain further contains a peptide tag at its C-terminus. In one embodiment, the antibody or glutaminyl-modified antibody or antigen-binding molecule is two antibodies The antibody comprises a weight chain, and each antibody weight chain further comprises a peptide tag at its C-terminus, wherein the peptide The ptidotag is the pentapeptide sequence LLQGA.

[0128] (Linker) In one embodiment, the linker L portion of the conjugate described herein is A portion that covalently links the combination to the payload compound described herein, for example, This is the divalent portion. In other examples, linker L is a binder as described herein. A trivalent or polyvalent moiety is covalently linked to the linker compound. A suitable linker is, for example, If the content of each is fully incorporated herein by reference, antibody-drug conjugate Antibody-drug conjugates and immunotoxins; edited by Phillips, GL; Spr inger Verlag: New York, 2013; Antibody-Drug Conjugates; Ducry, L. (ed.); Humana Press, 2013; Antibody-Drug Conjugates ); Edited by Wang, J., Shen, W.-C., and Zaro, JL; Springer International Publishing, 2 It can be found at 015. In one embodiment, the linker-pay described herein The linker L portion of the load contains a binder in divalent form on the payload compound described herein. The payload compounds described herein can be covalently linked. It is a jointly connected part. In other examples, the linker-payroll described herein The linker L portion of the code is a trivalent or polyvalent portion, and the binder is as described herein. The payload described herein can be covalently linked to the load compound. This is the portion covalently linked to the compound. The payload compound is the chemical of formulas I and II above. The compound contains these residues after binding to or incorporation of linker L, The linker payload is a binder such as an antibody or its antigen-binding fragment. Further binding can be performed to form an antibody-drug conjugate. Those skilled in the art will know The payload portion has specific functional groups that are favorable for linking to the linker and / or binder. They would be aware of this. For example, in one embodiment, there is no linker, and payload The code is directly bound to the binder. In another embodiment, the payload contains carbon It contains an acid, and the binder contains lysine, in this case, each carboxylic acid and lysine is, It is involved in amide bond formation, which allows the payload residue to be directly attached to the binder residue. The functional group can also include hydroxyl (for example, baloxavir, and its derivatives, e.g., , baloxavir marboxyl derivatives), and carboxylic acids (e.g., VX-787 and its derivatives) This includes those that take the form of an ester when linked to L, as seen in [example].

[0129] In one embodiment, the linker is stable under physiological conditions. The linker is cleavable, for example, in the presence of an enzyme or within a certain pH range or value. The payload portion can be released even without it. In some embodiments, the linker - includes an enzyme-cleavable moiety. Examples of enzyme-cleavable moieties include peptide bonds and esters. Examples include, but are not limited to, hydrazones, disulfide linkages, and disulfide linkages. In some embodiments, the linker includes a cathepsin-cleaving linker.

[0130] In some embodiments, the linker includes a non-cutting portion. In this context, the non-cutting linker is [ka] or derived from the residue. In some embodiments, non-cleavable linker-payload The residues are, [ka] or a positional isomer thereof. In some embodiments, the non-cleaving linker is [ka] or derived from the residue. In some embodiments, non-cleavable linker-payload The residues are, [ka] or a positional isomer thereof. In one embodiment, the linker is maleimidecyclohexa It is either a carboxylate or 4-(N-maleimidomethyl)cyclohexanecarboxylic acid (MCC). In these structures, [ka] This indicates binding with the binder. In some of these structures, [ka] For example, a binder having an azide or alkyne functional group and a complementary alkyne or azide functional group. These show the click chemical residues produced by the reaction of the linker-payload containing the group. In other examples within the structure, [ka] For example, a Michael addition reaction involving one or more binders (cysteine) and maleimide functional groups. Divalent sulfur produced by reaction with one or more linkers or linker-payloads having This indicates "do". In these structures, in other examples, [ka] For example, as understood by those skilled in the art, one or more binders lysine and activating or inactivating them. Reaction with one or more linkers or linker-payloads having carboxyl functional groups This shows the amide bond that is formed. In one embodiment, [ka] For example, as understood by those skilled in the art, one or more binders lysine and activated carboxy A reaction with one or more linkers or linker-payloads having syl functional groups results in the formation of This shows a mido bond.

[0131] In some embodiments, a preferred linker is a single binder, such as an antibody. Examples include those that are chemically bound to the two cysteine ​​residues, but are not limited to these. Yes. Such linkers are destroyed as a result of the conjugation process of antibodies. It can mimic the function of a disulfide bond.

[0132] In some embodiments, the linker contains one or more amino acids. Preferred amino acids Examples include natural, unnatural, standard, non-standard, protein-constitutive, and non-protein-constitutive. and L- or D-α-amino acids are examples. In some embodiments, the linker is Lanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, f Phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine , glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, if or citrulline, their derivatives, or any combination thereof (e.g., dipeptides, t Includes lipeptides, oligopeptides, polypeptides, etc. In one embodiment, One or more side chains of the ano acid are linked to the following side chain groups. In some embodiments, phosphorus KAR is the amino acid valine and citrulline (e.g., divalent-Val-Cit- or divalent-VCit-). It contains or is a peptide consisting of the amino acid. In some embodiments, Linka - refers to the amino acid alanine and alanine, i.e., divalent -AA- or the amino acid It is a peptide. In some embodiments, the linker is the amino acid glutamate. It is a peptide containing or composed of amino acids, i.e., -EA- and alanine. In some embodiments, the linker is the amino acids glutamic acid and glycine, that is, The peptide contains -EG- or consists of the amino acid. In some embodiments The linker contains the amino acids glycine and glycine, i.e., -GG- or the amino It is a peptide composed of acids. In some embodiments, the linker is a glucose molecule of amino acids. A compound containing tamin, valine, and citrulline, i.e., -QV-Cit- or -QVCit- or the It is a peptide composed of amino acids. In some embodiments, the linker is an amino acid Glutamic acid, valine, and citrulline, i.e., containing -EV-Cit- or -EVCit- or a peptide consisting of the amino acid. In some embodiments, the linker is A peptide containing or comprising the amino acid-GGGGS-. Several embodiments In this, the linker is a peptide containing the amino acid -GGGGG- or composed of the amino acid. In some embodiments, the linker contains the amino acid -GGGGK- or the amino It is a peptide composed of acids. In some embodiments, the linker is the amino acid-GFGG- It contains or is a peptide consisting of the amino acid. In some embodiments, Linka - comprises the amino acids lysine, valine, and citrulline, i.e., -KVCit- or the It is a peptide composed of amino acids. In some embodiments, the linker is an amino acid It is a peptide containing -KVA- or consisting of the amino acid. In some embodiments, The linker is a peptide containing or composed of the amino acid-VA-. In any of the embodiments, and throughout this disclosure, as will be understood by those skilled in the art. Standard three-letter or one-letter amino acid notation is used. Examples of one-letter amino acid notation are shown below. For example, G represents glycine, K represents lysine, S represents serine, V represents valine, and alanine. Examples include A, which represents [something], and F, which represents phenylalanine.

[0133] In some embodiments, the linker includes a self-destructing group, which is known to those skilled in the art. It can be any such group. In a particular embodiment, the self-destructing group is p-amino It is benzyl (PAB) or its derivatives. A useful derivative is p-aminobenzyl oxy Carbonyl (PABC) is an example. Those skilled in the art will know that the self-destructing group is the remaining raw material of the linker from the payload. They are likely aware that they can perform a chemical reaction that releases offspring.

[0134] In some embodiments, the linker is: [ka] And, Here: SP 1 It is a spacer; SP 2 It is a spacer; [ka] is one or more bonds with the binder; [ka] is one or more connections with the payload; Each AA is an amino acid residue; and n is an integer between 0 and 10.

[0135] SP 1 The spacer is (AA) n A portion or residue is attached to the binder (BA) or a reactive group residue bound to BA. This is the connecting part. Suitable SP 1 Alkylene or polyether can be used as a spacer. Examples include, but are not limited to, those including, or both of the end of the spacer, For example, the spacer portion bonded to BA or AA during the chemical synthesis of the conjugate A portion derived from the reactive part used for coupling antibodies or AA to a spacer. It can be minutes. In one embodiment, n is 0, 1, 2, 3, or 4 (that is, (For example, when n is 0, AA does not exist). In a specific embodiment, n is 2. In one embodiment, n is 3. In a specific embodiment, n is 4.

[0136] In some embodiments, SP 1 The spacer contains alkylene. Several implementations In this manner, SP 1 The spacer is C 5-7 Contains alkylene. In some embodiments, SP 1 The spacer contains polyether. In some embodiments, SP 1 Spacer It contains ethylene oxide polymers such as polyethylene glycol (PEG). The polymer unit of eng glycol is generally -(OCH2CH2) p -(Here, p is an integer from 1 to 100) It can be expressed as -(OCH2CH2)2- or -OCH2CH2-OCH2CH2- or PEG2. It is also possible to do so. In one embodiment, polyethylene glycol is PEG1. In one example, polyethylene glycol is PEG2. In another example, Polyethylene glycol is PEG3. In one demonstration, polyethylene glycol It is PEG4. In one example, polyethylene glycol is PEG5. In one example, polyethylene glycol is PEG6. In another example, Polyethylene glycol is PEG7. In one embodiment, polyethylene glycol It is PEG8. In one example, polyethylene glycol is PEG9. For example, polyethylene glycol is PEG 10 In one example, Polyethylene glycol is PEG 11 For example, polyethylene glyco The PEG 12In one example, polyethylene glycol is PEG 13 That is One example is polyethylene glycol, PEG 14 In one embodiment, Polyethylene glycol is PEG 15 In one example, polyethylene Glycols are PEG 16 In one example, polyethylene glycol is PEG 17 In one example, polyethylene glycol is PEG 18 It is a certain implementation. In this context, polyethylene glycol is PEG 19 In one example, Tylene glycol is PEG 20 For one example, polyethylene glycol is PEG 21 In one example, polyethylene glycol is PEG 22 That is. For example, polyethylene glycol is PEG 23 In one example, Polyethylene glycol is PEG 24 For example, polyethylene glyco The PEG 25 In one example, polyethylene glycol is PEG 26 That is One example is polyethylene glycol, PEG 27 In one embodiment, Polyethylene glycol is PEG 28 In one example, polyethylene Glycols are PEG 29 In one example, polyethylene glycol is PEG 30 In one example, polyethylene glycol is PEG 31 It is a certain implementation. In this context, polyethylene glycol is PEG 32 In one example, Tylene glycol is PEG 33 For one example, polyethylene glycol is PEG 34 In one example, polyethylene glycol is PEG 35 That is. For example, polyethylene glycol is PEG 36 In one example, Polyethylene glycol is PEG 37 For example, polyethylene glyco The PEG 38 In one example, polyethylene glycol is PEG 39 That is One example is polyethylene glycol, PEG 40 In one embodiment, Polyethylene glycol is PEG 41 In one example, polyethylene Glycols are PEG 42 In one example, polyethylene glycol is PEG 43 In one example, polyethylene glycol is PEG 44 It is a certain implementation. In this context, polyethylene glycol is PEG 45 In one example, Tylene glycol is PEG 46 For one example, polyethylene glycol is PEG 47 In one example, polyethylene glycol is PEG 48 That is. For example, polyethylene glycol is PEG 49 In one example, Polyethylene glycol is PEG 50 For example, polyethylene glyco The PEG 51 In one example, polyethylene glycol is PEG 52 That is One example is polyethylene glycol, PEG 53 In one embodiment, Polyethylene glycol is PEG 54 In one example, polyethylene Glycols are PEG 55 In one example, polyethylene glycol is PEG 56 In one example, polyethylene glycol is PEG 57 It is a certain implementation. In this context, polyethylene glycol is PEG 58 In one example, Tylene glycol is PEG 59 For one example, polyethylene glycol is PEG 60 In one example, polyethylene glycol is PEG 61 That is. For example, polyethylene glycol is PEG 62 In one example, Polyethylene glycol is PEG 63 For example, polyethylene glyco The PEG 64 In one example, polyethylene glycol is PEG 65 That is One example is polyethylene glycol, PEG 66 In one embodiment, Polyethylene glycol is PEG67 In one example, polyethylene Glycols are PEG 68 In one example, polyethylene glycol is PEG 69 In one example, polyethylene glycol is PEG 70 It is a certain implementation. In this context, polyethylene glycol is PEG 71 In one example, Tylene glycol is PEG 72 For one example, polyethylene glycol is PEG 73 In one example, polyethylene glycol is PEG 74 That is. For example, polyethylene glycol is PEG 75 In one example, Polyethylene glycol is PEG 76 For example, polyethylene glyco The PEG 77 In one example, polyethylene glycol is PEG 78 That is One example is polyethylene glycol, PEG 79 In one embodiment, Polyethylene glycol is PEG 80 In one example, polyethylene Glycols are PEG 81 In one example, polyethylene glycol is PEG 82 In one example, polyethylene glycol is PEG 83 It is a certain implementation. In this context, polyethylene glycol is PEG 84 In one example, Tylene glycol is PEG 85For one example, polyethylene glycol is PEG 86 In one example, polyethylene glycol is PEG 87 That is. For example, polyethylene glycol is PEG 88 In one example, Polyethylene glycol is PEG 89 For example, polyethylene glyco The PEG 90 In one example, polyethylene glycol is PEG 91 That is One example is polyethylene glycol, PEG 92 That is the case.

[0137] In some embodiments, SP 1 Spacers are: [ka] And, Here: RG' is the reactive group residue after the reaction between the reactive group RG and the binder; [ka] This is a bond with a binder; [ka] (AA) n It is a combination with; n is an integer between 0 and 10; and b is an independent integer between 1 and 92.

[0138] The reactive group RG can form one or more bonds with the binder, any known to those skilled in the art. It can be a reactive group. The reactive group RG has within its structure that it reacts with a binder (for example, an anti- The body and its cysteine ​​or lysine residues, or the azide moiety, for example, PEG-N3 functionalized anti The body and one or more glutamine residues; or the amino portion, for example, with a PEG-NH2 functionalized antibody and one or more (by reacting with the glutamine residue) to form the antibody-drug conjugate described herein. This is a region that includes a part that can perform the action. After conjugation to the binder, the reactive group is It becomes a reactive group residue (RG'). An example of a reactive group is a halo that can react with a binder. Acetyl, isothiocyanate, succinimide, N-hydroxysuccinimide, or ma Examples include, but are not limited to, those containing the reimide portion.

[0139] SP 2 Spacers are used if present (AA) n This is the part that connects to the payload. As for suitable spacers, SP 1 The items listed above are examples of spacers. , but not limited to these. 2 As a spacer, alkylene or poly This includes, but is not limited to, substances containing reether or both. SP 2 Spec The end of the spacer, for example, the portion of the spacer directly coupled to the payload or AA, During the chemical synthesis of the gate, the payload or AA is used in the SP 2 Coupling to the spacer It can be a part derived from the reactive part used for the purpose. In some examples... SP 2 The end of the spacer, for example, the SP directly coupled to the payload or AA. 2 Spacer part The minutes involve coupling the payload or AA to the spacer during the chemical synthesis of the conjugate. These can be reactive residues used for the purpose of causing a reaction.

[0140] In some embodiments, SP 2 If a spacer is present, -NH-(p-C6H4)-CH2- -NH-(p-C6H4)-CH2OC(O)-, amino acids, dipeptides, tripeptides, oligopeptides, [ka] and selected from the group consisting of any combination thereof. In one embodiment, each [ka] This is the coupling with the payload, and each [ka] (AA) n It is either a combination with or, if n=0, does not exist.

[0141] In the above equation, each (AA) n This is an amino acid or optionally p-aminobenzyloxycal It is a bonyl residue (PABC). n can be 0; in that case, (AA) n It does not exist. PABC If present, preferably only one PABC is present. Preferably, the PABC residue is present. If present, it is located near the payload (AA) n It is bound to the terminal AA in the base. Suitable amino acids include natural, unnatural, standard, non-standard, protein-constitutive, and non-protein amino acids. Examples include crystalline composition and L- or D-α-amino acids. In some embodiments, AA These are alanine, valine, leucine, isoleucine, methionine, tryptophan, and pheny Lualanine, proline, serine, threonine, cysteine, tyrosine, asparagine, guar Glutamic acid, aspartic acid, glutamic acid, lysine, arginine, histidine, or Citrulline, its derivatives, or any combination thereof (e.g., dipeptides, tripeptides) Includes (butyrates and oligopeptides, etc.). In one embodiment, one or more amino acids The chain is linked to the following side chain groups. In some embodiments, n is 2. One embodiment of this, (AA) n This is valine-citrulline. In some embodiments , (AA) n This is citrulline-valine. In some embodiments, (AA) n is, valine- It is alanine. In some embodiments, (AA) n It is alanine-valine. A few embodiments, (AA) n It is valine-glycine. In some embodiments (AA) n It is glycine-valine. In some embodiments, (AA) n is, valine- It is citrulline-PABC. In some embodiments, (AA) n This is citrulline-valine-PA BC. In some embodiments, n is 3. In some embodiments, (A A) n This is glutamate-valine-citrulline. In some embodiments, (AA) n teeth , glutamine-valine-citrulline. In some embodiments, (AA) n is, It is valine-alanine. In some embodiments, (AA) n is lysine-valine-cy It is Torlin. In some embodiments, n is 4. (AA) n It is glutamate-valine-citrulline-PAB. In some embodiments, (AA) n It is glutamine-valine-citrulline-PABC. Those skilled in the art can express PABC as follows: Construction: [ka] It is likely recognized as a p-aminobenzyloxycarbonyl residue containing the PABC residue. The base has been shown to facilitate the cleavage of specific linkers in vitro and in vivo. For example, in one embodiment, cleavage of PABC results in a carboxylate or carboxylic acid portion. minutes (that is, each of them, [ka] ) remains intact along with the antiviral compound or the rest of the payload. In this embodiment, each [ka] PA is the binding of the antiviral compound (e.g., payload) to the rest of the compound. Those skilled in the art will understand PA B is p-aminobenzyl (i.e., -NH-(p-C6H4)-CH2- or [ka] It is likely recognized as a divalent residue of ). In one embodiment, the PAB residue is a specific ri It has been shown to facilitate the cleavage of ligatures in vitro and in vivo. For example, in a certain real In terms of implementation, [ka] By cleavage, the alkoxide or hydroxyl portion (i.e., respectively) [ka] ) remains intact along with the rest of the antiviral compound (e.g., payload). In one embodiment, each [ka] This involves binding to the antiviral compound or the rest of the payload.

[0142] (Linker-payload) In one embodiment, the linker-payload is coupled to the linker, and the above formula I and Any specific compound or Peyro that is encompassed by one or more of the formulas in Formula II This includes, herein, the linker described herein is the antibody or so described herein. It includes a portion that is reactive with the antigen-binding fragment. In certain embodiments, the linker is the above The carboxyl or hydroxyl atoms in one or more of formulas I and II are bonded to each other. In one embodiment, the linker-payload has the following structure. [ka] or a pharmaceutically acceptable salt thereof, where L is an embodiment disclosed herein. The linker is one of the embodiments described herein; and RG is an embodiment disclosed herein. It is a reactive part described in one of the following. In one embodiment, the linker-payroll The code is, [ka] or a salt that is acceptable as a medicine: where SP 1 and SP 2 If it exists, Honmyo The spacer base is described in any of the embodiments disclosed in the details; RG is the present invention Reacts with an antibody or its antigen-binding fragment described in any of the embodiments disclosed in this document. This is a part of the sex; each AA is described in any of the embodiments disclosed herein. It is an amino acid; and n is an integer between 1 and 10.

[0143] In one embodiment, provided herein is: [ka] It is a compound selected from the group consisting of (i.e., linker-payload).

[0144] (Conjugate / Antibody-drug conjugate (ADC)) Provided herein are therapeutic components for treating influenza virus infection. For example, human anti-influenza conjugated with toxoids or antiviral drugs. This is a HA monoclonal antibody (i.e., ADC). The antibody binds to its target. It is possible to link the therapeutic agent to the antibody at any position along the antibody, as far as possible. In this embodiment, the therapeutic agent is a second different antibody against influenza-HA or its ADC. It is also possible. In one embodiment, the antibody is a drug specific to virus-infected cells. It can be conjugated. It can be conjugated with anti-influenza-HA antibodies. The types of therapeutic components that can be performed depend on the disease to be treated and the desired therapeutic effect to be achieved. This will be taken into consideration. In one embodiment, provided herein are antibodies or This is the antigen-binding fragment, where the antibody is of formula I and / or II as described herein. It is conjugated with one or more compounds. In one embodiment, it is an anti-influenza anti The body or its antigen-binding fragment is one of each of the embodiments disclosed herein. It is conjugated to the payload via the linker described in one of the links.

[0145] In one embodiment, the antibody-drug conjugate has the following structure [ka] It has, where Ab is an anti-influenza antibody or its antigen-binding fragment; and L is phosphorus. A is a cart; and P is an antiviral compound or payload. In one embodiment, Ab P is an anti-influenza antibody or its antigen-binding fragment; and P is an influenza inhibitor. In one embodiment, Ab is an anti-influenza antibody or its antigen-binding fragment. Furthermore, P is a polymerase inhibitor. In one embodiment, Ab is an anti-influenza anti It is the body or its antigen-binding fragment; and P is VX-787, its derivatives, or its residues. In this embodiment, Ab is an anti-influenza antibody or its antigen-binding fragment; and P is The compound is baloxavir, its derivatives, or its residues. In one embodiment, Ab is an anti-hemagglutamic acid. It is a rutinin antibody or its antigen-binding fragment; and P is an antiviral compound. In this example, Ab is an anti-influenza antibody or its antigen-binding fragment; and P is an anti-influenza antibody. It is a Rus compound. In one embodiment, Ab is an anti-hemagglutinin antibody or its antigen-binding agent. It is a fragment; and P is an influenza inhibitor. In one embodiment, Ab is an anti-hemorrhagic agent. It is a glutinin antibody or its antigen-binding fragment; and P is a polymerase inhibitor. In any of the embodiments, Ab is an anti-influenza antibody or its antigen binding cleavage. A fragment or an anti-hemagglutinin antibody or its antigen-binding fragment, wherein the antibody is as described above. It is conjugated to the compound of formula I. In any embodiment of this paragraph, Ab This refers to anti-influenza antibodies or their antigen-binding fragments or anti-hemagglutinin antibodies or The antigen-binding fragment is, here, the antibody is conjugated to the compound of formula II above. In one embodiment, Ab is an anti-hemagglutinin antibody or its antigen-binding fragment. Furthermore, P is VX-787, its derivatives, or its residues. In one embodiment, Ab is an anti-H It is a maglutinin antibody or its antigen-binding fragment; and P is baloxavir, its derivatives, or This is the residue. In any of the embodiments of this paragraph, k is an integer from 1 to 30. In one embodiment, the herein provides an antibody or its antigen-binding fragment, [ka] ADC conjugated to a compound or a pharmaceutically acceptable salt thereof, and here in, L is a linker as described herein; and RG is a reactive part as described herein. It is minutes. In one embodiment, what is provided herein is a conjugated transformation Mixed [ka] (Here, L is a linker as described herein.) This is the ADC selected from the available options.

[0146] In one embodiment, the following structure is provided herein: [ka] An ADC compound having, where L and BA are as described elsewhere in this specification. Yes, k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, k is 1 The range is ~2, 1~3, 2~3, 2~4, 3~4, or 1~4. In one embodiment, the above The compound conjugated to -L-BA is one or more compounds of the above formulas I and / or II. Including, where BA is the binder; L is the linker; and k is 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10. In one embodiment, conjugate to -L-BA as described above. The compound comprises one or more compounds of the above formulas I and / or II, where BA is a binder. And L is a linker, and k is in the range of 1-2, 1-3, 2-3, 2-4, 3-4, or 1-4. It is enclosed.

[0147] In one embodiment, provided herein, [ka] An ADC compound selected from the group consisting of TIFF2026076189000063.tif249170, where BA is an antibody or its antigen-binding fragment. And k is an integer from 1 to 30. In one embodiment, k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, k is 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10 , or in the range of 9 to 10. In any of the embodiments of this paragraph, if k is greater than 1 , BA is for the conjugation of the payload and / or linker-payload. It is assumed that the above cysteine, lysine, and / or glutamine residues are included. For example The above description of ADC indicates that one or more cysteine, lysine, and / or glutamine residues of BA are present. If it includes an Eload and / or Linker-Payload (for example, when k ≥ 1), then 1 or more. The drug-to-antibody ratio (DAR) is assumed. Binding from BA to -S- and / or from BA to -NH- is assumed. Each involves the binding of the binder (BA) to the cysteine ​​or transglutamated glutamine residue of BA. The bond is shown. And the bond from BA-S- and / or BA-NH- to carbon is shown, or This indicates linkage to a linker as described elsewhere in this specification. Therefore, the cysteine ​​of BA Sulfur derived from the residue and / or nitrogen derived from the transglutamated glutamine residue of BA are Multiple payloads and / or linker-payloads can be conjugated (e.g., BA) The parentheses are drawn to indicate that DAR ≥ 1. In one embodiment, B A is an antibody or an antigen-binding fragment thereof as described herein.

[0148] In one embodiment of the ADC described herein, Ab or BA is a transglutamin. It is a ze-modified antibody or its antigen-binding fragment. In one embodiment, Ab or BA is a conjugate Transglutaminase modification containing at least one glutamine residue used in the process It is an antibody or its antigen-binding fragment. In one embodiment, Ab or BA is a conjugation A transglutaminase-modified antibody containing at least two glutamine residues used for This is the antigen-binding fragment. In one embodiment, Ab or BA is used in the conjugation. A transglutaminase-modified antibody or the one containing at least three glutamine residues It is an antigen-binding fragment. In one embodiment, Ab or BA is used for conjugation. A transglutaminase-modified antibody or its antigen containing at least four glutamine residues It is a binding fragment. In one embodiment, Ab or BA is a small amount available for conjugation. A transglutaminase-modified antibody containing at least one glutamine residue, or its antigen binding. It is a fragment. In one embodiment, Ab or BA is available for conjugation. Transglutaminase-modified antibodies or their antigen-binding fragments, each containing two glutamine residues. In one embodiment, Ab or BA is available for conjugation, at least A transglutaminase-modified antibody containing three glutamine residues or its antigen-binding fragment. In one embodiment, Ab or BA is used for conjugation, at least four It is a transglutaminase-modified antibody containing a glutamine residue or its antigen-binding fragment. In one embodiment, Ab or BA is a transglutaminase-modified antibody or its antigen-binding cleavage. It is a single unit, where the conjugation is at two Q295 residues; and k is 2 In one embodiment, Ab or BA is a transglutaminase-modified antibody or its anti- This is the original binding fragment, where the conjugation is at two Q295 residues in the EU numbering system. It is a transylvanic compound containing an antibody heavy chain. A suglutaminase-modified antibody or its antigen-binding fragment, where the conjugation is , located at the C-terminus of the heavy chain; and k is 2. In one embodiment, Ab or BA This is a transglutaminase-modified antibody containing an antibody heavy chain or its antigen-binding fragment, and here The conjugation is via glutamine. In one embodiment, Ab or BA is a transglutaminase-modified antibody containing an antibody heavy chain or its antigen-binding fragment. Here, the conjugation is mediated by glutamine, and k is 2. In this embodiment, Ab or BA is a transglutaminase-modified antibody containing an antibody heavy chain or the This is an antigen-binding fragment, where the conjugation is in the LLQGA sequence at the C-terminus of the antibody heavy chain. This is mediated by glutamine. In one embodiment, Ab or BA contains an antibody heavy chain. Lance glutaminase-modified antibody or its antigen-binding fragment, where conjugation The interaction is via glutamine in the LLQGA sequence at the C-terminus of the antibody heavy chain; and k is 2. In one embodiment, Ab or BA is a transglutaminase-modified antibody or its antigen-binding agent. This is a fragment, where the conjugation occurs at two Q295 residues and two N297Q residues. It is such that; and k is 4. In one embodiment, Ab or BA is transglutamin -ase-modified antibody or its antigen-binding fragment, where conjugation is the EU numbering system This is in the two Q295 residues and the two N297Q residues; and k is 4. In one embodiment In this specification, Ab or BA is mAb11729 as described herein.

[0149] In one embodiment, Ab or BA is a transglutaminase-modified antibody or its antigen-binding agent. This is a fragment, where the conjugation is at two Q295 residues; and DA R is a) approximately 2.0; b) greater than 0 to approximately 12.0; c) between approximately 0.5 and approximately 8.0; d) approximately 0.5 e) Is it approximately 6.0?; f) Is it approximately 1.0 to 4.0?; g) Is it approximately 1.0 or 2.0? (i) is present or (h) is approximately 2.0. In one embodiment, Ab or BA is transglutaminase A modified antibody or its antigen-binding fragment, where the conjugation is defined as 2 of the EU numbering system. It is at one Q295 residue; and the DAR is a) approximately 2.0; or b) greater than 0 to approximately 12.0 c) Is it approximately 0.5 to approximately 8.0?; d) Is it approximately 0.5 to approximately 6.0?; e) Is it approximately 1.0 to approximately 4.0?; f) g) is about 1.0 or about 2.0; or h) is about 2.0. In one embodiment Ab or BA is a transglutaminase-modified antibody containing an antibody heavy chain or its antigen-binding fragment. And here the conjugation is at the C-terminus of the heavy chain; and DAR is a) Is it approximately 2.0?; b) Is it greater than 0 to approximately 12.0?; c) Is it between approximately 0.5 and approximately 8.0?; d) Is it between approximately 0.5 and e) Is it approximately 6.0?; f) Is it approximately 1.0 to approximately 4.0?; g) Is it approximately 1.0? (or h) is approximately 2.0. In one embodiment, Ab or BA is a transg A lutaminase-modified antibody or its antigen-binding fragment, where the conjugation is a gu This is mediated by luthamine. In one embodiment, Ab or BA is a trans containing an antibody heavy chain. A suglutaminase-modified antibody or its antigen-binding fragment, where the conjugation is , mediated by glutamine; and DAR is a) approximately 2.0; b) greater than 0 to approximately 12.0 c) Is it approximately 0.5 to approximately 8.0?; d) Is it approximately 0.5 to approximately 6.0?; e) Is it approximately 1.0 to approximately 4.0?; f) g) is about 1.0 or about 2.0; or h) is about 2.0. In one embodiment Ab or BA is a transglutaminase-modified antibody containing an antibody heavy chain or its antigen-binding fragment. Here, the conjugation is glutamine in the LLQGA sequence at the C-terminus of the antibody heavy chain. This is done via a transglutamate. In one embodiment, Ab or BA is a transglutamate containing an antibody heavy chain. A minase-modified antibody or its antigen-binding fragment, where conjugation is the antibody weight It is via glutamine in the LLQGA sequence at the C-terminus of the chain; and the DAR is a) approximately 2.0 b) Is it greater than 0 to approximately 12.0?; c) Is it between approximately 0.5 and approximately 8.0?; d) Is it between approximately 0.5 and approximately 6.0?; e) f) Approximately 1.0 to approximately 4.0; g) Approximately 1.0 or approximately 2.0; or h) Approximately 2.0 In one embodiment, Ab or BA is a transglutaminase-modified antibody or its anti- This is the original binding fragment, where the conjugation consists of two Q295 residues and two N297Q residues. It is in the range of; and DAR is a) about 2.0; b) greater than 0 to about 12.0; c) about 0. d) Is it 5 to approximately 8.0?; e) Is it approximately 0.5 to approximately 6.0?; f) Is it approximately 1.0 to approximately 4.0?; g) Is it approximately 2.0, 3.0, or 4.0?; g) Is it approximately 1.0?; h) Is it approximately 2.0?; i) It is approximately 3.0; or j) approximately 4.0. In one embodiment, Ab or BA is transgluta A minase-modified antibody or its antigen-binding fragment, where the conjugation is EU numbered This is in two Q295 residues and two N297Q residues of the system; and the DAR is a) approximately 2.0 a) Is it greater than 0 to approximately 12.0? c) Is it between approximately 0.5 and approximately 8.0? d) Is it between approximately 0.5 and approximately 6.0? e ) is approximately 1.0 to 4.0; f) is approximately 1.0 or 2.0; g) is approximately 1.0; or h) is approximately 2.0 In one embodiment, Ab or BA is a transglutaminase-modified antibody or its anti- This is the original binding fragment, where the conjugation consists of two Q295 residues and two N297Q residues. It is in the range of; and DAR is a) about 2.0; b) greater than 0 to about 12.0; c) about 0. d) Is it 5 to approximately 8.0?; e) Is it approximately 0.5 to approximately 6.0?; f) Is it approximately 1.0 to approximately 4.0?; g) Is it approximately 2.0, 3.0, or 4.0?; g) Is it approximately 1.0?; h) Is it approximately 2.0?; i) j) is approximately 3.0; or j) is approximately 4.0. In one embodiment, Ab or BA is as described herein. This is the mAb11729.

[0150] In one embodiment of the ADC described herein, Ab or BA is an anti-influenza antibody. or its antigen-binding fragment. In one embodiment, Ab or BA is anti-influenza A. It is an antibody or an antigen-binding fragment thereof. In one embodiment, Ab or BA is an anti-influenza agent. It is an antibody of group 1 or an antigen-binding fragment thereof. In one embodiment, Ab or BA is an anti It is an influenza H1 antibody or its antigen-binding fragment. In one embodiment, Ab or BA is This is an anti-influenza A group 2 antibody or its antigen-binding fragment. In one embodiment, Ab or BA is an anti-influenza H3 antibody or its antigen-binding fragment. In one embodiment Ab or BA is an anti-influenza B antibody or its antigen-binding fragment. In one embodiment... The ADC then conjugates the payload with an anti-influenza anti-influenza agent via a linker. The antibody-drug conjugate comprises the body or an antigen-binding fragment thereof, wherein the antibody-drug conjugate is polymerase Basic protein 2 (PB2) (VX-787), polymerase acid protein (PA) (baloxavir and / (or baloxavir marboxil), and / or polymerase basic protein 1 (PB1) It binds to and / or inhibits them. In one embodiment, the ADC is via the linker The payload contains an anti-influenza antibody or its antigen-binding fragment conjugated, Here, the antibody-drug conjugate, when measured by ELISA, has a concentration of at least 4.0 × 1 0 -9 M, at least 3.5 × 10 -9 M, or at least 3.0 × 10 -9 Polymerase basicity due to affinity of M It binds to and / or inhibits protein 2(PB2)(VX-787). In one embodiment, A DCs are conjugated in the payload via a linker with anti-influenza antibodies or other The antibody-drug conjugate comprises an antigen-binding fragment, wherein the antibody-drug conjugate is measured by ELISA. At that time, at least 4.0 × 10 -9 M, at least 3.5 × 10 -9 M, or at least 3.0 × 10 -9 M affinity It binds to polymerase basic protein 1 (PB1) and / or inhibits it. In one embodiment, the ADC receives an anti-influenza agent conjugated to the payload via a linker. The product comprises an Enza antibody or its antigen-binding fragment, wherein the antibody-drug conjugate is Immun When measured by oSpot® analysis, at least 2.5 × 10⁻⁶ -9 M, at least 2.0 × 10 -9 M, or at least 1.5 × 10 -9 M IC 50 polymerase basic protein 2 (PB2) (VX-787) It binds to and / or inhibits it. In one embodiment, the ADC connects to the linker via the linker. This comprises an anti-influenza antibody conjugated with an Erode or its antigen-binding fragment, Thus, when the antibody-drug conjugate was measured by ImmunoSpot® analysis... , at least 2.5 × 10 -9 M, at least 2.0 × 10 -9 M, or at least 1.5 × 10 -9 M IC 50 de po It binds to and / or inhibits the remerase basic protein 1 (PB1).

[0151] (Method for preparing compounds or payloads, and linker-payloads) The compounds provided herein are prepared by any method that is obvious to those skilled in the art, and It can be separated or obtained. Exemplary preparation methods are described in detail in the following examples. In one embodiment, the compounds provided herein are commercially available or Typically, it can be prepared according to schemes A to C: (Scheme A. Exemplary preparation scheme) [ka] (Scheme B1. Exemplary preparation scheme) [ka] (Scheme B2. Exemplary preparation scheme) [ka] (Scheme B3. Exemplary preparation scheme) [ka]

[0152] In the above exemplary preparation scheme A (see J. Med. Chem. 2014, 57, 6668), R 1 teeth It is described in relation to Formula I. In Scheme A, maleic anhydride and 1,3-cyclohe After Diels-Alder cycloaddition with xadiene, endo-A1 was stirred under basic conditions. Therefore, we can provide epimerized trans-A2. Curtius rearrangement and benzylalco A3 is provided by trapping with a rod. A4 is provided by hydrogenation. 2, Treatment with 4-dichloropyrimidine and chiral separation provide A6 via intermediate A5. This is done. Suzuki coupling by substituted azaindole boronic acid esters and subsequent desorption. Through this process, compounds of formula I, including VX-787 and its derivatives, are produced.

[0153] In the above example preparation schemes B1-B3 (see OPRD 2019, 23, 1298), R 1 is Equation I It is described in relation to I. In scheme B1, protecting B1 and providing B2 is Yes, it is possible. Then, B2 is reduced to provide B3, and then the hydroxyl is replaced with methoxy. By doing so, B4 is provided. B5 can be esterified to provide B6. B6 is Bo Treatment with c-hydrazine yields pyridone B7, which is then deprotected under acidic conditions to obtain B8. This can provide a disubstituted hydrazine. The combination of B4 and B8 in the presence of a Lewis acid provides a disubstituted hydrazine. Rc-B9 is provided. By nitrogen deprotection and Pd-mediated cyclization, rac-B10 is provided. Separation of rac-B10 via the formation of radidodiastereomers, followed by hydrolysis, leads to B12 This occurs.

[0154] In Scheme B2, B13 is orthometallated and quenched with DMF to provide an aldehyde. This undergoes intramolecular cyclization with a carboxylic acid to provide B14. B14 is then converted with thiophenol. By processing, B15 can be provided. Reduction of B15 provides B16. (Three-ring type) Rufid B17 is obtained by treating B16 under acidic conditions. B18 is produced by reduction of B17. To be served.

[0155] In Scheme B3, baloxavir and its derivatives are provided by the combination of B12 and B18. It is done. By deprotection, baloxaviric acid B19 is provided. Baloxaviric acid B19 is re-alkylated. By converting it, B20 can be provided.

[0156] The linker-payloads described herein are typically as shown in Scheme C. It can be synthesized by a series of coupling steps: (Scheme C. Exemplary preparation scheme) [ka]

[0157] In the above exemplary preparation scheme C, R 1 This is as described in relation to Equation I. In scheme C, VX-787 and its derivatives are treated with a linker supporting a leaving group (LG). Therefore, a linker-payload (e.g., linker-(VX-787)) is provided.

[0158] The conjugates described herein are linked to the linker-payloads described herein. This is combined with a binder, for example, the antibody described herein, under standard conjugation conditions. It can be synthesized by coupling (for example, as quoted herein) See Doronina et al., Nature Biotechnology 2003, 21, 778, for a complete integration. ). If the binder is an antibody, the antibody is used via one or more cysteine ​​or lysine residues of the antibody. It may be coupled to the linker-payload. The linker-payload is, for example, The antibody is then exposed to a reducing agent, such as dithiothrelitol, to break the disulfide bonds of the antibody. The antibody is then purified, for example, by gel filtration, and subsequently subjected to a suitable reaction. By processing with a linker-payload containing a maleimide group, It can be coupled to cysteine ​​residues (see, for example, exemplary preparation scheme C). (See image). Suitable solvents include, but are not limited to, water, DMA, DMF, and DMSO. No. Linker-Peyrow contains a reactive group, such as an activated ester or an acid halide group. The compound can be coupled to the lysine residue of the antibody. Suitable solvents include water. Examples include, but are not limited to, DMA, DMF, and DMSO. Conjugates are, for example, , including size exclusion chromatography, dialysis, and ultrafiltration / diafiltration, known proteins It can be purified using a filtration technique.

[0159] The binder, for example, the antibody, is conjugated by a click chemistry reaction. It is also possible. In some embodiments of the click chemistry reaction, linker-pay Rhode is a reactive group that can undergo a positional isomeristic 1,3-addition cycloaddition reaction with azide, for example For example, it contains alkynes. Such preferred reactive groups are described above. The antibody is one or more. It contains an azide group. Examples of such antibodies include azide-polyethylene glycol. Examples include antibodies functionalized with a base. In one embodiment, such a functionalized antibody is In the presence of the enzyme transglutaminase, at least one glutamine residue, for example, Antibodies containing the Gln295 chain are induced by treatment with a primary amine compound. In this embodiment, such functionalized antibodies are less likely to be produced in the presence of the enzyme transglutaminase. An antibody having at least one glutamine residue, for example, the heavy chain Gln297, is subjected to a primary amine compound. It is induced by treatment. An example of such an antibody is the Asn297Gln(N297Q) mutant. Examples include: In one embodiment, such a functionalized antibody is transglutaminase-transglutaminase In the presence of the enzyme, at least two glutamine residues, e.g., heavy chain Gln295 and heavy chain Gln29 Antibodies containing 7 are induced by treatment with a primary amine compound. One example is the Asn297Gln(N297Q) mutant. In one embodiment, the antibody is Two or four glutamine residues in total, as described in this paragraph, are used for two heavy chains. It has.

[0160] In one embodiment, such a functionalized antibody is in the presence of the enzyme transglutaminase. Below, an antibody having at least one glutamine residue, for example, heavy chain Gln295, is subjected to primary amino acids. It is induced by treatment with a compound or peptide tag. In one embodiment, Functionalized antibodies such as transglutaminase can detect at least one glutaminase in the presence of the enzyme transglutaminase. Antibodies having a tamine residue, for example, the heavy chain Gln297, are tagged with a primary amine compound or peptide tag. It is induced by treatment. An example of such an antibody is the Asn297Gln(N297Q) mutant. Examples include: In one embodiment, such a functionalized antibody is transglutaminase-transglutaminase In the presence of the enzyme, at least two glutamine residues, e.g., heavy chain Gln295 and heavy chain Gln29 Antibodies containing 7 are induced by treatment with a primary amine compound or peptide tag. An example of such an antibody is the Asn297Gln(N297Q) mutant. In one embodiment, In this context, the antibody is used to target a total of two or four glutamine residues as described in this paragraph. It has two heavy chains like this.

[0161] In one embodiment, the functionalized antibody or antigen-binding molecule includes an antibody heavy chain, and the antibody heavy chain The C-terminus of further comprises a peptide tag. In one embodiment, a functionalized antibody or antigen-binding component The child comprises an antibody heavy chain, and further comprises a peptide tag at the C-terminus of the antibody heavy chain, where the The peptide tag is the pentapeptide sequence LLQGA. In this embodiment, a functionalized antibody or The antigen-binding molecule contains two antibody heavy chains, and each antibody heavy chain has a peptide tag at its C-terminus. Furthermore, it includes: In one embodiment, the functionalized antibody or antigen-binding molecule comprises two antibody heavy chains. Furthermore, each antibody heavy chain further includes a peptide tag at its C-terminus, where the peptide tag This is the pentapeptide sequence LLQGA.

[0162] In one embodiment, the antibody contains two glutamine residues, one in each heavy chain. In certain embodiments, the antibody contains a Q295 residue in each heavy chain. Further embodiments In this case, the antibodies are 1, 2, 3, 4, 5, 6, 7, 8, or more gluta It contains glutamine residues. These glutamine residues are present in the heavy chain, light chain, or both the heavy and light chains. These glutamine residues are either wild-type residues or modified residues. This can be done. The antibody can be prepared by standard techniques.

[0163] Those skilled in the art will know that antibodies are often glycosylated at residue N297, near residue Q295, in the heavy chain sequence. They will recognize that it has been converted. Glycosylation at residue N297 occurs at residue Q295. It can interfere with lanceglutaminase (see Dennler et al., above). Therefore, In a convenient embodiment, the antibody is not glycosylated. In another embodiment, The body is deglycosylated or aglycosylated. In a specific embodiment, antibody weight The chain has an N297 mutation. In other words, the antibody no longer has an asparagine residue at position 297. It has mutated so that it does not have N297Q mutation. In a particular embodiment, the antibody heavy chain has N297Q mutation. It has mutations. Such antibodies are site-specific, removing or inactivating glycosylated sequences. By targeted mutagenesis, or by any interfering glycosylation site or any other interfering It is prepared by site-directed mutagenesis, which involves inserting glutamine residues into locations distant from the structure. Such antibodies can be isolated from natural or artificial sources.

[0164] Subsequently, an antibody that does not interfere with glycosylation is reacted with a primary amine compound, or The process is carried out by this method. In one embodiment, an aglycosylated antibody is reacted with a primary amine compound. To produce glutaminyl-modified antibodies, either by rinsing or processing with them. In one embodiment, Deglycosylating antibodies are reacted with or treated with primary amine compounds to produce gluta This produces Minil-modified antibodies.

[0165] Primary amines form covalent bonds with glutamine residues in the presence of transglutaminase. It can be any primary amine. Useful primary amines are specified herein. As described (see, for example, Exemplary Preparation Scheme C). Transglutaminase is, It can be any transglutaminase that is considered suitable by those skilled in the art. In one embodiment, transglutaminase is used to remove free axylamine from a primary amine compound. It catalyzes the formation of isopeptide bonds between the mino group and the acyl group on the side chain of the glutamine residue. It is an enzyme. Transglutaminase is a protein-glutamine-γ-glutamyltran enzyme. Also known as spherase. In a particular embodiment, transglutaminase is It is classified as EC 2.3.2.13. Transglutaminase is considered suitable for use in the following cases. It can be derived from the source of intent. In one embodiment, transglutaminase It is a microorganism. Useful transglutaminases are found in Streptomyces moba. Streptomyces mobaraense, Streptomyces cinnamonenum Streptomyces griseo-carneu (Streptomyces cinnamoneum) m), Streptomyces lavendulae, and Bacillus subtilis (Baci It is isolated from *Illus subtilis*. It is a non-microbial organism containing mammalian transglutaminase. Transglutaminases, for example, non-microbial transglutaminases combined with cofactors Transglutaminase can also be used. In one embodiment, transglutaminase is used in the present invention. It can be produced by any technique deemed suitable by those skilled in the art, or by any technique deemed suitable by those skilled in the art. It can be obtained from any source deemed suitable by certain embodiments. In this context, transglutaminase is obtained from commercial sources.

[0166] In one embodiment, a glutaminyl-modified antibody is reacted with a reactive linker-payload. Either allow it to form an antibody-linker-payload conjugate or process it in this way. The reaction may proceed under conditions that are considered favorable by those skilled in the art. In one embodiment, In this case, the glutaminyl-modified antibody and the linker-payload compound Under conditions suitable for forming a bond between the reactive linker and the payload compound, Allow contact. Suitable reaction conditions are well known to those skilled in the art.

[0167] (Pharmaceutical compositions and therapeutic methods) Provided herein are methods for treating and preventing diseases, illnesses, or disorders, One or more of the compounds disclosed herein, for example, the formulas provided herein. A method comprising administering one or more of the compounds in therapeutic or prophylactic effective amounts. Diseases, disorders, and / or illnesses include those associated with viral infections as described herein. These are some examples, but are not limited to them.

[0168] The compounds described herein may be used alone or in combination with one or more additional (adjunctive) therapeutic agents. It can be administered. One or more additional therapeutic agents may be administered to the compound described herein. It can be administered immediately before, simultaneously with, or immediately after. This disclosure is described herein. A pharmaceutical composition comprising any of the listed compounds in combination with one or more additional therapeutic agents, and This includes a therapeutic method that involves administering such a combination to a subject who needs it.

[0169] Suitable additional therapeutic agents include antiviral drugs, such as a second antiviral compound or Examples include payloads, autoimmune therapies, hormones, biologics, or monoclonal antibodies. These may be, but are not limited to, the following. In some embodiments, the auxiliary therapeutic agent is Antiviral drugs, anti-inflammatory drugs (e.g., corticosteroids or nonsteroidal anti-inflammatory drugs), Antibodies that specifically bind to influenza HA, influenza vaccines, nutritional supplements (e.g.) For example, antioxidants, and palliative therapies to treat influenza infections: It can be selected. In one embodiment, the anti-inflammatory agent is a corticosteroid and non It is selected from the group consisting of steroidal anti-inflammatory drugs. In one embodiment, the nutritional supplement is , an antioxidant. Preferred therapeutic agents include the antiviral compounds described herein or We can also list any acceptable salts or derivatives of the payload as pharmaceuticals, but these Not limited to the above. In some embodiments, the auxiliary therapeutic agents described herein are As an antibody-drug conjugate, compound, or pharmaceutical composition, it can be administered via various routes of administration. It is administered. For example, an adjunct therapeutic agent can be administered orally. An example of an antiviral drug that would be administered in this way is oseltamivir. In this embodiment, oseltamivir is an antibody-drug conjugate, a compound, or a pharmaceutical combination. It is administered before the administration of the product. In some embodiments, oseltamivir is administered to the antibody- It is administered simultaneously with the administration of a drug conjugate, compound, or pharmaceutical composition. Depending on the application, oseltamivir may be an antibody-drug conjugate, a compound, or a pharmaceutical composition. It is administered after the administration of the substance. In some embodiments, the antiviral drug is an anti-type A virus. Influenza drugs or anti-influenza B drugs (e.g., antibodies or their antigen-binding portions), for example Antibodies that specifically bind to influenza A HA or specifically bind to influenza B HA It is an antibody that does this.

[0170] In some embodiments of the methods described herein, multiple doses are described herein. Compounds (or compounds described herein and additional therapeutic agents referred to herein) A pharmaceutical composition containing any combination of the above is administered to the subject over a specified period of time. This method according to this embodiment of the Disclosure applies to a subject with multiple doses as specified herein. This disclosure includes administering the compounds described herein in sequence. A single initial dose of the compound, followed by one or more secondary doses of the compound, and optionally thereafter The present disclosure includes a method comprising sequentially administering one or more tertiary doses of the compound. Examples of dosages include 50 mg / kg, 49 mg / kg, 48 mg / kg, 47 mg / kg, 46 mg / kg, 45 mg / kg, and 44 mg / kg. mg / kg, 43mg / kg, 42mg / kg, 41mg / kg, 40mg / kg, 39mg / kg, 38mg / kg, 37mg / kg, 36mg / kg, 3 5mg / kg, 34mg / kg, 33mg / kg, 32mg / kg, 31mg / kg, 30mg / kg, 29mg / kg, 28mg / kg, 27mg / kg, 26mg / kg, 25mg / kg, 24mg / kg, 23mg / kg, 22mg / kg, 21mg / kg, 20mg / kg, 19mg / kg, 18mg / kg , 17mg / kg, 16mg / kg, 15mg / kg, 14mg / kg, 13mg / kg, 12mg / kg, 11mg / kg, 10mg / kg, 9mg / kg , 8mg / kg, 7mg / kg, 6mg / kg, 5mg / kg, 4mg / kg, 3mg / kg, 2mg / kg, 1mg / kg, 0.9mg / kg, 0.8m g / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, and While 0.05 mg / kg is an example, it is not limited to this value.

[0171] In one embodiment, the amounts of the compound contained in the initial, secondary, and / or tertiary doses are: In one embodiment, they differ from one another during the course of treatment (for example, they are adjusted upward or downward as needed). In this case, two or more (e.g., 2, 3, 4, or 5) doses are given at the start of the treatment regimen as "Roady It is administered as a "single dose," followed by subsequent doses administered at a lower frequency (for example, It is administered as a "maintenance dose."

[0172] In some exemplary embodiments of this disclosure, each secondary and / or tertiary dose is the same as the immediately preceding dose. From the amount for 1 to 26 weeks (for example, 1, 1 1 / 2, 2, 21 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7 , 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2 , 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 2 2, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 (2 weeks or longer) It will be administered later.

[0173] A method according to this aspect of the disclosure involves administering to a patient any number of secondary and / or tertiary doses of compounds. This may include administering a single secondary dose to the patient. For example, in one embodiment, only a single secondary dose may be administered to the patient. It is administered to. In other embodiments, two or more (for example, 2, 3, 4, 5, 6, 7, 8, or so) A secondary dose (more than) is administered to the patient. Similarly, in one embodiment, a single tertiary dose Only the dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7) A tertiary dose of 8, or more, is administered to the patient. The administration regimen is tailored to the specific target. For life, indefinitely, or until such treatment is no longer therapeutically necessary. It can be carried out until it is no longer beneficial.

[0174] In embodiments including multiple secondary doses, each secondary dose is administered at the same frequency as the other secondary doses. It may be administered as follows: For example, each secondary dose may be given to the patient 1-2 weeks after the previous dose. It may be administered 1 to 2 months later. Similarly, in embodiments including multiple tertiary doses, each Each tertiary dose may be administered at the same frequency as other tertiary doses. For example, each tertiary dose It may be administered to the patient 2 to 12 weeks after the previous dose. In one embodiment, The frequency with which secondary and / or tertiary doses are administered to patients varies throughout the course of the treatment regimen. It is possible. The frequency of administration may also be determined by the physician according to the individual patient's needs after clinical examination. It may be adjusted during the course of treatment.

[0175] This disclosure states that loading doses 2-6 are administered at a first frequency (e.g., once a week, once every two weeks). The medication is administered to the patient at intervals of 3 weeks, 1 month, 1 month every 2 months, etc., followed by 2 or more maintenance doses. This includes a dosage regimen in which the dose is administered to the patient at a lower frequency. For example, in this embodiment of the present invention According to this, if the loading dose is administered once a month, the maintenance dose is 1 every 6 weeks. It may be administered to the patient once every two months, once every three months, or so on.

[0176] This disclosure relates to the compounds and / or conjugates described herein, for example, formulas I and II Pharmaceutical compositions of compound antibody-drug conjugates, for example, the compounds described herein Substances, their salts, stereoisomers, positional isomers, polymorphs, and pharmaceutically acceptable carriers and diluents The composition includes, and / or excipients. Examples of suitable carriers, diluents, and excipients include: , a buffer for maintaining the appropriate composition pH (e.g., citrate buffer, succinate buffer) Buffers, acetate buffer, phosphate buffer, lactate buffer, oxalate buffer, etc. (,), carrier protein (e.g., human serum albumin), physiological saline, polyol (e.g., Trehalose, sucrose, xylitol, sorbitol, etc.), surfactants (for example, por (e.g., resorbate 20, polysorbate 80, polyoxolate), antimicrobial agents, and antioxidants. Examples include, but are not limited to, these agents.

[0177] In one embodiment, a compound or payload, linker-payload, ADC, or These compositions may be provided via a different route of administration. In one embodiment, a combination The drug can be administered via subcutaneous, intradermal, intramuscular, oral, intravenous, intraperitoneal, inhalation, and nasal passages. Selected from the group. In one embodiment, the route of administration of the composition is orally. In this embodiment, the route of administration of the composition is intravenous. In one embodiment, the administration of the composition The route of administration is intraperitoneal. In one embodiment, the route of administration of the composition is inhalation. In this embodiment, the route of administration of the composition is intranasal.

[0178] In some cases, the diseases described herein are those related to infection in the subject. A method for treating, preventing, alleviating, or inhibiting a disorder or condition, wherein the subject is given formula I and / or compounds of II, linker-payloads as described herein, and / or as described herein. The effective amount or therapeutic effective amount of ADCs, combinations thereof, or these pharmaceutical compositions as described above. A method comprising administering a drug. In some embodiments, the infection is caused by a viral infection. It is a disease. In some embodiments, the infection is an influenza virus infection. In some embodiments, the infection is influenza A virus infection. In that embodiment, the infection is caused by influenza B virus. In this case, the infection is caused by influenza A virus infection and influenza B virus infection. It is a stain. In one embodiment, the deployment of an unconjugated payload to a target. Side effects associated with administration include the administration of a conjugated payload or ADC to an equivalent target. It is reduced compared to [the other option].

[0179] The compounds disclosed herein are used in relation to the antibody-drug conjugate described herein. A treatment for influenza in a subject involves administering an effective amount of a cereal, compound, or pharmaceutical composition. It can also be used to treat, prevent, mitigate, or inhibit dandruff infection. In some embodiments, In this context, influenza infection is caused by infection with the influenza A virus. In some embodiments, influenza infection is caused by influenza A of group 1. It is caused by the virus. In some embodiments, influenza infection It is caused by the influenza A H1 virus. In some embodiments, Influenza infection is caused by influenza A virus of group 2. In some cases, influenza infection is caused by influenza A H3 virus. It is caused by Rus. In some embodiments, influenza infection is not It is caused by a known or unknown influenza virus. In some embodiments, Influenza infection is caused by infection with the influenza B virus. In some embodiments, influenza infection is caused by the influenza A virus. It is caused by infection with influenza B virus. In some embodiments In this context, influenza infection is caused by influenza A virus infection, Group 1 influenza A Influenza infection, influenza A H1 infection, influenza A infection of group 2, influenza A Influenza H3 infection, unknown or unidentified influenza virus, influenza B virus It is caused by a Rus infection, or any combination thereof. [Examples]

[0180] (Examples) Provided herein are VX-787 and its derivatives, baloxavir and its derivatives, and Roxavir marboxyl and its derivatives, their protein conjugates, and disease A method for treating ailments, disorders, and diseases, comprising VX-787, baloxavir, and baloxavir A method comprising administering marboxil and its conjugates.

[0181] (Example 1: Linker-payload synthesis) VX-787 and baloxavir as payloads for delivery by anti-hemagglutinin antibodies It was used to test the effects of various linkers in combination with VX-787 or baloxavir. The linker-payload was synthesized as shown below (linker-(VX-787)): Substance 6 and compound 11; linker-baloxavir: compound 15). All solvents used were left as they were. I used and purchased it from either Sigma Aldrich or Fisher Scientific. 1 The H spectrum is The data was recorded using Varian Inova 300 MHz and 500 MHz NMR spectrometers. The chemical shift (δ) was used for analysis. The NMR solvent used should be reported in ppm, and the s-singular, d-double, t-tripular, and q-quadular lines should be used. dd - double line of double lines, dt - double line of triple lines, dq - double line of quadruple lines, and m - multiple lines. The coupling constant (J) was reported in Hertz (Hz). Chromatographic purity was expressed in Chr. omolith® FastGradient RP-18e analytical column (50 x 2 mm, Merck KGaA, P / N 1.520) Using the analytical HPLC methods described in 07.0001) and below, an Agilent 110 equipped with a 6130 Quadrupole LC / MS was used. Determined by 0, 1260 Infinity, or 1200 Series LC / MS systems: injection volume 2-10 μL; flow rate 1 mL / min; 5-95% acetonitrile in water, 4 minutes; Agilent diode array detector, λ=254 nm; room temperature. Low-resolution mass spectrometry using an electrospray ionization source. The procedure is performed using a Gilent system, and analysis is performed using either a single quadrupole or an ion trap mass detector. Ta.

[0182] Compound 6 was synthesized from VX-787 as described in Scheme 1 below.

[0183] (Scheme 1) [ka] Compound 2: Compound 2 was prepared using PCT Int. Appl., 2014145090. tert-butyl((S) -1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentane-2- Il(amino)-3-methyl-1-oxobutan-2-yl)carbamate 1 (700 mg, 1.46 mmol) is mixed with CH3C The reaction mixture was dissolved in a mixture of N / H2O / TFA (3:1:1 = v / v / v, 6 mL / 2 mL / 2 mL). The reaction mixture was left at room temperature until 7 PM. The mixture was stirred and monitored by LC-MS. After concentration in vacuum, crude product 2 (0.5 g salt) was prepared. It was used directly in the next process without further purification. MS(ESI, pos.): C 18 H 29 N5O4 Calculated value: 379.2; Measured value: 380.2 (M+H).

[0184] Compound 4: Compound 2 (100 mg, 0.263 mmol), 6-(Fmoc-amino)caproic acid 3 (93 mg, 0.263 mmol) , 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-Ox Sidehexafluorophosphate (HATU, 200 mg, 0.526 mmol), and 1-hydroxy-7-aza Benzotriazole (HOAt, 35 mg, 0.263 mmol) was dispensed into two oven-dried drum vials. It was added. Then, anhydrous DMF (2 mL) was added, and the reaction mixture was allowed to mature at ambient temperature for 5 minutes. Then, via syringe, N,N-diisopropylethylamine (DIEA, 137 μL, 0.789 mmol) was administered. The solution was added dropwise. The homogeneous yellow solution was stirred under nitrogen at room temperature for 2 hours, and the reaction was terminated by LC / MS. Monitoring was performed using a 50g C18 Aq Gold column (gradient elution: 10-95°C in water). The solutions were purified for 20 minutes with %MeCN (both containing 0.05% acetic acid). The pure fractions were combined and dried. When frozen on ice and freeze-dried, title compound 4 becomes a white solid (120 mg, 65%). Obtained. MS: C 39 H50 Calculated value of N6O7: 714.3; measured values: 715.3 (M+H), 737.3 (M+Na).

[0185] Compound 5: Compound 4 (39.4 mg, 0.055 mmol) and 4-dimethylaminopyridine (DMAP, 5 mg, 0.03 mmol) Add 9 mmol) to a stirred suspension of VX-787 (22 mg, 0.055 mmol) in anhydrous THF (6 mL) under argon at room temperature. Then, anhydrous THF of N,N'-dicyclohexylcarbodiimide (DCC, 17 mg, 0.083 mmol) was added. (2 mL) of the solution was added dropwise to the reaction mixture. After stirring for 16 hours, the mixture was evaporated to dryness, and the residue was 3 mL. The crude material was dissolved in mL of DMSO. 50 g of the crude material was eluted onto a C18 Aq Gold column (gradient elution: 10-95% MeCN in water). Both were purified with 0.05% AcOH. The product fractions were combined and frozen on dry ice. Upon drying, the title compound 5 was obtained as a white solid (38 mg, 63%). MS (ESI, pos.) : C 59 H 67 F2N 11 Calculated value of O8: 1095.5; measured value: 1096.4 (M+H).

[0186] Compound 6: 5% piperidine (0.8 mL) in DMF is mixed with N,N-dimethyl compound 5 (33 mg, 0.0301 mmol) Add the solution to a stirred solution of formamide (DMF, 1 mL) under argon at ambient temperature for 30 minutes, and the resulting solution The liquid was stirred. The completion of the reaction was confirmed by LC / MS. The reaction solution was then removed via the ISCO system. Using a 30g C18 Aq. Gold column (gradient elution: 10-95% MeCN in water, 0.05% acetic acid on both sides, 30 minutes) The product was purified directly. The fractions containing the product were combined and frozen on dry ice, then freeze-dried overnight. Upon drying, title compound 6 was obtained as an off-white solid (22 mg, 85%). MS: C 44 H 57 F2N 11 Calculated value of O6: 873.4; measured value: 874.4 (M+H). [ka]

[0187] Compound 11 was synthesized from VX-787 as described in Scheme 2 below.

[0188] (Scheme 2) [ka] Compound 8: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 1.99g, 8.0 (5 mmol) p-aminobenzyl alcohol in dichloromethane (19 mL) and methanol (7.6 mL) It was added to a solution of (0.99 g, 8.05 mmol) at room temperature. After stirring for 5 minutes, Fmoc-valine-citrulline was added. 7 (2.0 g, 4.03 mmol) was added all at once, and the resulting solution was stirred for 18 hours. The volatile substances were then removed. Remove in the air, pulverize the residue with ether (20 mL), then add ether (20 mL), ethyl acetate (20 mL), And when washed sequentially with ether (20 mL), the title compound 8 (2.2 g, 98% yield) becomes a pale yellow solid. This was obtained by doing so. MS(ESI, pos.): C 33 H 39 Calculated value of N5O6: 601.29; measured value: 602.3 (M+H).

[0189] Compound 2: In a 20 mL vial, Fmoc-valine-citrulline-PAB(OH) 8 (2.0 g, 3.33 mmol) is dissolved in DMF. Dissolve in 5% piperidine in (10 mL) and stir at room temperature for 1 hour. Remove the precipitate by filtration. The filtrate was prepared using a gradient elution method with 5-95% MeCN / H2O (both containing 0.05% TFA) and 100g C18 Aq. It was refined with rum. The pure fractions were combined, frozen, and freeze-dried. The refined fraction was freeze-dried. When repeated on a solid, the title compound 2 (0.98g, 61% yield) turns a fluffy off-white color. It was obtained as a solid. MS(ESI, pos.): C 18 H 29 Calculated value of N5O4: 379.22; Measured value: 380.2 (M+H) .

[0190] Compound 9: DIEA (40 μL, 0.22 mmol) is converted to valine-citrulline-PAB(OH) * TFA salt 2 (100 mg, 0.2 mmol) l) Add to a solution of Fmoc-PEG8-NHS ester (167 mg, 0.22 mmol) in anhydrous DMF (2 mL) and stir for 45 minutes. Mixed. The reaction mixture was monitored by LC / MS, and gradient elution of 5-95% MeCN / H2O (both 0.05%) was observed. Purified using a 100g C18 Aq column (containing %AcOH). The pure fractions were combined and frozen. When freeze-dried, the title compound 8 (145 mg, 71%) becomes a fluffy, off-white solid. This was obtained by doing so. MS(ESI, pos.): C 52 H 76 N6O 15 Calculated value: 1024.54; measured value: 1025.5 (M+H).

[0191] Compound 10: A solution of DCC (18.6 mg, 0.09 mmol) in dichloromethane (3 mL) at room temperature is mixed with Fmoc-PEG8- Phosphate-citrulline-PAB(OH) 9 (61.8 mg, 0.06 mmol), VX-787 (24 mg, 0.06 mmol), and DMAP (7. 2 mg (0.06 mmol) of anhydrous dichloromethane (12 mL) was added dropwise to a suspension at room temperature and stirred for 16 hours. The volatile substances were removed in a vacuum, and the residue was obtained by gradient elution of 5-95% MeCN / H2O (both containing 0.05% AcOH). The solution was purified using a 50g C18 Aq column. The pure fractions were combined, frozen, and freeze-dried. The title compound 10 was obtained as a fluffy, off-white solid (50 mg, 51% yield). MS(ESI, pos.): C 72 H 93 F2N 11 O 16 Calculated value: 1405.68; measured value: 1406.6 (M+H).

[0192] Compound 11: 5% piperidine in DMF (0.8 mL) is added to anhydrous DMF (1.6 mL) of Compound 10 (50 mg, 0.035 mmol). After adding to solution L and stirring for 40 minutes, gradient elution of 5-95% MeCN / H2O (both containing 0.05% AcOH) was performed. The solution was purified using a 50g C18 Aq column. The pure fractions were combined, frozen, and freeze-dried. When this is done, the title compound 11 (43 mg, 95% yield) is obtained as a fluffy, off-white solid. MS(ESI, pos.): C 57 H 83 F2N 11 O 14 Calculated value: 1183.61; measured value: 1184.6 (M+H). [ka]

[0193] (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl)-7-hydo Roxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]tri The synthesis of compound 15 from azine-6,8-dione 13 is described in scheme 3 below.

[0194] (Scheme 3) [ka] Compound 12: Thionyl chloride (11.8 mg, 0.1 mmol) is converted to Fmoc-Cap-valine-citrulline-PAB(OH) 4(6 The solution was added to a 1 mL solution of anhydrous dichloromethane (1.2 mg, 0.086 mmol) at room temperature and stirred for 1 hour. After the starting material is consumed, volatile substances are removed in a vacuum, and the residue is gradient-eluted to 5-95% MeC Purified using a 30g C18 Aq column with N / H2O (both containing 0.05% AcOH). Pure fraction When the two are combined, frozen, and freeze-dried, the title compound 12 turns into a fluffy off-white color. It was obtained as a solid (38 mg, 60% yield). MS (ESI, pos.): C 39 H 49 Calculated value of ClN6O6: 732.3; Measured value: 733.3 (M+H).

[0195] Compound 14: K2CO3 (28 mg, 0.20 mmol) and NaI (6 mg, 0.041 mmol) are combined with (R)-12-((S)-7,8-diflu Oro-6,11-dihydrodibenzo[b,e]thiepin-11-yl)-7-hydroxy-3,4,12,12a-tetra Hydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione 13 (20mg) DMF solution (1 m³) in Fmoc-Cap-valine-citrulline-PAB-Cl (40 mg, 0.052 mmol) and Fmoc-Cap-valine-citrulline-PAB-Cl (40 mg, 0.052 mmol) Add to (1) and heat the mixture at 65°C for 40 minutes. Cool the reaction solution to room temperature and add 5-60% MeCN / H2O( Both were purified using a 30g C18 Aq column (both containing 0.05% acetic acid). The pure fractions were combined. When frozen and freeze-dried, the title compound 14 becomes a fluffy, off-white solid (4 It was obtained as 0 mg (83% yield). MS (ESI, pos.): C 63 H 67 F2N9O 10 Calculated value of S: 1179.5; measured value. The value is 1180.3 (M+H).

[0196] Compound 15: A solution of 1M TBAF in THF (21 μL, 0.021 mmol) is mixed with Compound 14 (25 mg, 0.021 mmol) in THF (1 The solution was added dropwise and stirred at room temperature for 1.5 hours. The volatile substances were removed under vacuum, and the residue was collected. Purified using a 30g C18 Aq column with ~60% MeCN / H2O (both containing 0.05% acetic acid). When the exquisite fractions are combined, frozen, and freeze-dried, the title compound 15 (14 mg, 70%) becomes fluffy. It was obtained as an off-white solid. MS(ESI, pos.): C 48 H 57 Calculated value for F2N9O8S, 95 7.4; Measured value 958.3 (M+H). [ka]

[0197] (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl)-7-hydo Roxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]tri The synthesis of compound 18 from azine-6,8-dione (13) is described in scheme 4 below.

[0198] (Scheme 4) [ka] Compound 17: Thionyl chloride (9 μL, 0.122 mmol) is mixed with Mal-cap-Val-Cit-PAB-OH(16) (35 mg, 0.061 mmol). The mmol of the reaction was added to a 3 mL suspension of anhydrous DCM, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then subjected to vacuum. The solution is concentrated, azeotropically dried with toluene (4 mL), and then, without further purification, proceeded to the next step. Used in MS(ESI, pos.): C 26 H 39 Calculated value of ClN6O6: 590.3; measured value: 591.3 M+H).

[0199] Compound 18: K2CO3 (41.5 mg, 0.3 mmol) and sodium iodide (9 mg, 0.06 mmol) are (R)-12- ((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl)-7-hydroxy-3,4 ,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8 - DMF of Zion (13) (23 mg, 0.048 mmol) and Mal-cap-Val-Cit-PAB-Cl (17) (35 mg, 0.06 mmol) It was added to 2 mL of solution. The reaction mixture was heated at 60°C for 1 hour, then cooled to room temperature, and 5-95% MeCN was added. The solution was purified using a 30g C18 Aq column with H2O (both containing 0.05% AcOH). The pure fraction was then extracted. When combined and freeze-dried, compound 18 (28 mg, 57%) becomes a fluffy, off-white solid. It was obtained as follows: MS(ESI, pos.): C 52 H 57 F2N9O 10 Calculated value of S: 1037.4; Measured value: 1038.3 (M+H) . [ka]

[0200] (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl)-7-hydo Roxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]tri The synthesis of compound 23 from azine-6,8-dione (13) is described in scheme 5 below.

[0201] (Scheme 5) [ka] Compound 20: DIEA (41 μL, 0.236 mmol) is (R)-12-((S)-7,8-difluoro-6,11-dihydrodi Benzo[b,e]thiepin-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxa Dino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione(13) (30 mg, 0.044 mmol) and chemical Compound 19 (28.5 mg, 0.059 mmol) was added to a chloroform (0.5 mL) solution. The reaction mixture was heated at room temperature for 0.5 After stirring for a certain amount of time, the compound was purified using a 4g silica gold column with 0-5% MeOH / DCM. 20 (31 mg, 75%) was obtained as a colorless solid. MS(ESI, pos.): C 30 H 29 Calculated value of F2N7O7S2 701.2; measured value 702.1 (M+H).

[0202] Compound 21: Triphenylphosphine (28.8 mg, 0.11 mmol) is added to Compound 20 (31 mg, 0.044 mmol). It was added to a 10:1 THF / H2O (0.88 mL) solution. After stirring at room temperature for 24 hours, the reaction mixture was concentrated to dryness. When purified using a 5.5g C18 Aq column with 5-95% MeCN / H2O (both containing 0.05% AcOH), Compound 21 (20 mg, 68%) was obtained as a fluffy, off-white solid. MS(ESI, pos.): C 30 H 31 Calculated value for F2N5O7S2: 675.2; measured value: 676.1 (M+H). Compound 23: HATU (28.5 mg, 0.075 mmol) and DIEA (13 μL, 0.075 mmol) are combined with Compound 21 (20 mg, 0. It was added to a 1.0 mL solution of DMF containing 0.30 mmol) and Mal-cap-Val-OH(22) (27.5 mg, 0.089 mmol). After stirring, the reaction mixture is prepared using 30g C with 5-95% MeCN / H2O (both containing 0.05% AcOH). The compound was purified using an 18 Aq column. The pure fractions were combined and freeze-dried to obtain compound 23 (4.5 mg, 16 %) was obtained as a fluffy, off-white solid. MS(ESI, pos.): C 45 H 51 F2N7O1 Calculated value for 1S2: 967.3; measured value: 968.2 (M+H). [ka]

[0203] (Scheme 6) [ka] Compound 24: Triethylamine (17 μL, 0.12 mmol), Trifluoromethanesulfonic anhydride (20 μL, 0.12 mmol) and DMAP (0.7 mg, 0.006 mmol) were added to (R)-12-((S)-7,8-difluoro-6,11 -dihydrodibenzo[b,e]thiepin-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H- [1,4]Oxadino[3,4-c]Pyrido[2,1-f][1,2,4]Triadin-6,8-dione(13)(29mg, 0.06mm The solution was added to a 2 mL solution of DCM at 0°C. After stirring for 1 hour, the reaction mixture was concentrated under vacuum, and the residue was removed. When purified using a 4g silica gold column with ethyl acetate / hexane, compound 24 (24mg, 66 %) was obtained as a yellowish solid. MS(ESI, pos.): C 25 H 18 Calculated value of F5N3O6S2: 615.1; actual value Measured value 616.0 (M+H).

[0204] Compound 25: Triflate 24 (24 mg, 0.04 m³) in anhydrous 1,4-dioxane (1 mL) in a microwave tube. A mol (mol) was added, and ammonium hydroxide (0.4 mL) was added. The reaction mixture was heated at 80°C for 48 hours. The solvent was removed under reduced pressure, and the residue was treated with 5-95% MeCN / water (both containing 0.05% AcOH). The compound was purified using a 15.5g C18 Aq column. The pure fractions were combined and freeze-dried to obtain the compound. 25 (18.3 mg, 75%) was obtained as a fluffy, yellowish solid. MS(ESI, pos.): C 24 H 20 F The calculated value for 2N4O3S was 482.1, and the measured value was 483.1 (M+H). [ka]

[0205] (Scheme 7) [ka] Compound 26: In a vial, under argon, add triflate 24 (24.4 mg, 0.04 mg) in anhydrous DMF (1 mL). mmol) was added, along with LiCl (5.2 mg, 0.12 mmol), Pd(PPh3)4 (2.3 mg, 0.002 mmol), and Et3SiH (19 μL (0.12 mmol) was added. The reaction mixture was heated at 65°C for 2 hours. 5-95% MeCN / H2O (both 0 When purified using a 15.5g C18 Aq column with 0.05% AcOH, the title compound 26 (4.6mg, 25 %) was obtained as a fluffy, off-white solid. MS(ESI, pos.): C 24 H 19 F2N3O3 Calculated value of S: 467.1; Measured value: 468.1 (M+H). [ka]

[0206] (Scheme 8) [ka] Compound 27: A solution of DIEA and 0.8 M H2S in THF (90 μL, 0.072 mmol) was mixed with Triflate 24 (15 mg). It was added to a 0.024 mmol (0.024 mmol) DMF (1 mL) solution. After stirring for 1 hour, the volatile substances were removed under vacuum. The residue was then analyzed using a 5.5g C18 Aq column with 5-95% MeCN / H2O (both containing 0.05% AcOH). It was purified using [method]. When the pure fractions were combined and freeze-dried, the title compound 27 (7.0 mg, 58%) was obtained. It was obtained as a fluffy, off-white solid. MS(ESI, pos.): C 24 H 19 Total F2N3O3S2 Calculated value: 499.1; Measured value: 500.1 (M+H). [ka]

[0207] (Scheme 9) [ka] Compound 28 was analyzed according to the literature procedure in Bioconjugate Chemistry (2016), 27(10), 2549-2557. It was made.

[0208] Compound 29: DCC (57 mg, 0.227 mmol), Compound 28 (90 mg, 0.185 mmol), VX-787 (74 mg, 0.18 The mixture was added to an anhydrous DCM (8 mL) solution of 5 mmol) and DMAP (22 mg, 0.185 mmol). The mixture was left at room temperature for 3 hours. The mixture was stirred. The solid was filtered off, and the filtrate was concentrated under vacuum. The residue was then treated with 5-95% MeCN / H2O (both). Both methods were purified using a Teledyne ISCO equipped with 100g C18 Aq (containing 0.05% AcOH). When the elegant fractions are combined and freeze-dried, the title compound 29 (120 mg, 75%) becomes fluffy off It was obtained as a white solid. MS(ESI, pos.): C 40 H 40 F2N6O 14 Calculated value: 866.3; measured value The value is 867.3 (M+H).

[0209] Compound 30: Nitrogen is passed through a solution of Compound 29 (120 mg, 0.138 mmol) in THF (10 mL) for 10 minutes. By rinsing, it was deoxygenated. Zinc powder (180 mg, 2.76 mmol) and ammonium formate 26 mg of nium (0.414 mmol) was added to this solution. The reaction mixture was deoxygenated again for 5 minutes and then heated to 60°C. The mixture was heated for 3.5 hours. The reaction mixture was cooled to ambient temperature, the solid was removed by filtration, and the filtrate was vacuum-sealed. When concentrated in the solution, title compound 30 (110 mg, 100%) is obtained, and this is further purified. It was not used in the next step. MS(ESI, pos.): C 40 H 42 F2N6O 12 Calculated value: 836.3; measured value: 837. 3 (M+H).

[0210] Compound 31: Oxalyl chloride (26 μL, 0.304 mmol) and DMF (2 μL) are mixed with Fmoc-N-amide-PEG8 acid ( It was added to a 100 mg (0.152 mmol) solution of anhydrous DCM (5 mL). After stirring for 30 minutes, the volatile substance was thoroughly mixed. When removed in the air, Fmoc-N-amide-PEG8-COCl was obtained. In another vial, compound 30 ( Dissolve 110 mg (0.138 mmol) in anhydrous THF (3 mL), add DIEA (53 μL, 0.304 mmol), then Fmoc- A solution of N-amide-PEG8-COCl in THF (4 mL) was added. After 1 hour, the solvent was removed under reduced pressure, and the residue was removed. This is a Teledy with 100g C18 Aq using 5-95% MeCN / H2O (both containing 0.05% AcOH). The product was purified using ISCO. The fractions containing the pure product were combined, frozen, and freeze-dried. Then, the title compound 31 (86 mg, 53% yield based on recovered starting material) was fluffy off-white It was obtained as a white solid. MS(ESI, pos.): C 74 H 89 F2N7O 23 Calculated value: 1481.6; measured value The value is 1482.6 (M+H).

[0211] Compound 32: Compound 31 (86 mg, 0.058 mmol) in a 12 mL solution of MeOH at 0°C, with 0.1 M NaOMe in MeOH. The solution (1.16 mL, 0.116 mmol) was added, and the reaction mixture was stirred at 0°C for 1 hour. Dowex® resin The reaction mixture was quenched by adding [a certain substance]. After filtering out the resin and concentrating the filtrate, Compound 32 was obtained and used in the next step without purification.

[0212] Compound 33: Compound 32 (0.058 mmol) in DMF (3 mL) mixed with 5% piperidine solution in DMF (1.5 mL) Add the following, stir the reaction mixture for 45 minutes, then inject it into a Teledyne ISCO 50g C18 Aq column. Elution was performed with 5-95% MeCN / H2O (both containing 0.05% AcOH as a modifier). Pure fraction When the ingredients are combined, frozen, and freeze-dried, the title compound 33 (32 mg, 49% after two steps) becomes fluffy. It was obtained as a fluffy, off-white solid. MS(ESI, pos.): C 53 H 73 F2N7O 18 Calculated value , 1133.5; measured value 1134.4 (M+H).

[0213] Compound 34: A solution of Compound 33 (32 mg, 0.028 mmol) in THF (2 mL) and water (1 mL) at room temperature, A 0.025 mM LiOH solution (1.1 mL, 0.028 mmol) was added, and the reaction mixture was stirred for 2 hours. Volatile substances Remove under reduced pressure, and the residue is obtained by gradient elution of 5-95% MeCN / H2O (both using 0.05% AcOH as a modifier). Purified using a Teledyne ISCO equipped with a Gemini 30×150mm column (which uses [a specific technology / method]). When the mixture is combined, frozen, and freeze-dried, the title compound 34 (26 mg, 82%) becomes fluffy. It was obtained as a white solid. MS(ESI, pos.): C 52 H 71 F2N7O 18 The calculated value is 1119.5; Measured value: 1120.4 (M+H). [ka]

[0214] (Scheme 10) [ka] Compound 35 was purified according to the literature procedure in Bioconjugate Chemistry (2016), 27(10), 2549-2557. It was made.

[0215] Compound 36: EDC-HCl (67 mg, 0.350 mmol) and DMAP (34 mg, 0.278 mmol) in CH2Cl2 (16 mL) It was added to a mixture of X-787 (100 mg, 0.250 mmol) and alcohol 37 (125 mg, 0.250 mmol). (Ambient) After stirring at room temperature for 22 hours, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with H2O (20 mL). The aqueous layer was extracted with three 20 mL portions of ethyl acetate. The combined organic layer was then dissolved in a 0.5 N HCl (aqueous) solution (20 Wash with (mL), then with saturated NaHCO3 (aqueous) solution (20 mL), and finally with brine (20 mL). Afterward, it was dried over Na2SO4, filtered, and concentrated in vacuum. 45-100% acetate in hexane. By chromatography on 12g silica gel using chill, compound 58b (149) was purified. (mg, 67% yield, 82% purity) is obtained and used in the next step without further purification. Used. MS(ESI, pos.): C 41 H 42 F2N6O 14 Calculated value: 880.27; measured value: 881.30 (M+H).

[0216] Compound 37: Argon is passed through a solution of Compound 36 (35 mg, 0.0397 mmol) in THF (1.5 mL) for 5 minutes. By ringing it, it was deoxygenated. Powdered Zn (102 mg, 1.56 mmol), then, Ammonium formate (12 mg, 0.0.190 mmol) was added. Argon was passed through the mixture for another 3 minutes. The reaction mixture was bubbled, and then heated in a pipe block at 60°C under argon for 24 hours. The reaction mixture was filtered, and the solid was washed with several parts of THF. The filtrate was concentrated under vacuum, and then hexacrystalline phosphate was added. Chromatography on 4g silica gel, eluting with 40-100% ethyl acetate in the solution. Upon purification, compound 37 (14 mg, 42%) was obtained as a pale yellow solid. MS (ESI, pos.): C 41 H 44 F2N6O 12 Calculated value: 850.30; measured value: 851.30 (M+H).

[0217] Compound 38: Oxalyl chloride (8 μL, 0.0933 mol) and DMF (2 μL) are mixed with Fmoc-N-amide-PEG8-acid (3 It was added to a 0 mg, 0.0452 mmol anhydrous CH2Cl2 (1 mL) solution. The resulting pale yellow solution was cooled to ambient temperature. The mixture was stirred for 1 hour, and then concentrated under vacuum. Three 1 mL portions of anhydrous CH2Cl2 were added to the resulting residue. The following were added and concentrated after each addition. In a separate vial, iPr2NEt (16 μL, 0.0918 mmol) was prepared Compound 37 (20 mg, 0.0235 mmol) was added to a CH2Cl2 (200 μL) suspension. Acid acidification was performed on this mixture. A solution of (0.0452 mmol) of the substance in CH2Cl2 (400 μL) was added dropwise. After stirring at ambient temperature for 1 hour, the mixture was... The product was concentrated in vacuum. The crude product was dissolved in DMSO and packed into a 5.5g C18 Aq column, and both Elution was performed with 20-100% MeCN in H2O containing 0.05% HOAc as the solvent. A picture containing the pure product was obtained. When the mixture is combined and freeze-dried, compound 38 (7.3 mg, 21% yield) is obtained as a white solid. MS(ESI, pos.): C 75 H 91 F2N7O 23 Calculated value: 1495.61; measured value: 1496.60 (M+H).

[0218] Compound 39: 0.1M NaOMe / MeOH solution (13 μL, 0.0013 mmol) is mixed with Compound 38 (2.0 mg, 0.00134 mmol). It was added to a 400 μL solution of anhydrous methanol at 0°C. After stirring in an ice bath for 4 hours, the reaction mixture was 0. The solution was neutralized with 2M HCl / MeOH solution (6.5 μL, 0.0013 mmol) and concentrated under vacuum. CH2Cl2 (0.5 mL) The mixture was added three times, and concentrated after each addition. The resulting residue was dissolved in anhydrous DMF (0.2 mL). The mixture was then treated with 10% piperidine / DMF solution (0.2 mL). After 30 minutes, the reaction mixture was collected in a 5.5 g C18 Aq column. The sample was packed into a container and eluted with 0-100% MeCN in H2O containing 0.1% TFA in both solvents. When the fractions containing the compound were combined and freeze-dried, the TFA salt of compound 39 (1.1 mg, 71%) was found to be white. It was obtained as a solid. MS(ESI, pos.): C 52 H 73 F2N7O 17 Calculated value: 1105.50; Measured value: 1106.5 0 (M+H). [ka]

[0219] (Scheme 11) [ka] Compound 41a: General Procedure A: Fmoc-Phe-OSu(40a) (107 mg, 0.22 mmol) and Val-Cit-PABA TF In a solution of A salt (2) (99 mg, 0.2 mmol) in DMF (1.5 mL), N,N-diisopropylethylamine (105 μL, (0.6 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes, and then injected into a 50 g C18 Aq. column. The solution was then eluted using a gradient of 5-95% MeCN:H2O (both containing 0.05% AcOH). The pure fraction was then combined. When mixed, frozen, and freeze-dried, the title compound 41a (23 mg, 14%) becomes a fluffy off-white It was obtained as a white solid. MS(ESI, pos.): C 42 H 48 Calculated value of N6O7: 748.4; measured value: 749 .3 (M+H).

[0220] Compound 41b: General Procedure B: Fmoc-Asp(Oallyl)-OH(40b) (83mg, 0.21mmol), Val-Cit-PA A solution of BA.TFA salt (2) (99 mg, 0.2 mmol) and HOAt (41 mg, 0.3 mmol) in DMF (1.5 mL) is mixed with NMM (66 μL). (0.6 mmol) and EDCI (48 mg, 0.25 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and then... Inject into a Teledyne ISCO 30 g C18 Aq. column and add 5-95% MeCN / H2O (both containing 0.05% AcOH). Extracted using (containing). The pure fractions were combined, frozen, and freeze-dried to obtain the title compound 41b. (85 mg, 56%) was obtained as a fluffy, off-white solid. MS(ESI, pos.): C 40 H 48 Calculated value of N6O9: 756.3; measured value: 757.4 (M+H).

[0221] Compound 41c: Prepared using general procedure B with Fmoc-His(Trt)-OH(40c) (130 mg, 0.21 mmol). Prepared as a fluffy, off-white solid. Yield = 146 mg (74%). MS (ESI, pos.) : C 58 H 60 Calculated value for N8O7: 980.5; measured value: 981.4 (M+H).

[0222] Compound 41d: Fmoc-Glu(O-allyl)-OSu(40d) (51 mg, 0.1 mmol) is used in a general procedure A. Prepared as a fluffy, off-white solid. Yield = 25 mg (33%). MS(ESI, po s.): C 27 H 26 Calculated value of N2O8: 506.2; measured value: 507.2 (M+H).

[0223] Compound 41e: A solution of Val-Cit-PABA (380 mg, 1.0 mmol) at 0°C in anhydrous DMA (5 mL) is mixed with FmocLeuOH (40e) (424 mg, 1.2 mmol), HOAt (170 mg, 1.2 mmol), EDC-HCl (240 mg, 1.2 mmol), and N-methionine Lumorpholine (220 μL, 2.0 mmol) was added. After stirring in an ice bath for 2.5 hours, the reaction mixture was heated with H2O ( The solution was diluted to 35 mL. The product was recovered by vacuum filtration and then analyzed with H2O (5 mL), followed by LC-MS analysis. Continue adding 10-20 mL portions of ethyl acetate until it is shown that <5% of FmocLeuOH remains. It was washed three times. After drying overnight under vacuum, the white solid was crushed into a powder and then under vacuum. Upon further drying, compound 41e (616 mg, 86%) was obtained. MS(ESI, pos.): C 39 H 50 N6O7 Calculated value: 714.4; Measured value: 715.4 (M+H).

[0224] Compound 41f: Val-Cit-PABA (200 mg, 0.527 mmol) and FmocArg(Pbf)OH( at 0°C in anhydrous DMF (5 mL) A mixture of 40f) (410 mg, 0.632 mmol) and HOAt (86 mg, 0.632 mmol) contains N-methylmorpholine (90 μg). L (0.818 mmol) and EDC-HCl (120 mg, 0.626 mmol) were added. The reaction mixture was stirred in an ice bath for 3 hours. Then, pack a 100g C18 column and add 10-60% M H2O containing 0.05% HOAc to both solvents. Elution was performed using eCN. The fractions containing the pure product were combined and freeze-dried to obtain compound 41f. (345 mg, 65%) was obtained as a white solid. MS(ESI, pos.): C 52 H 67 N9O 10 Calculated value of S, 10 09.47; Measured value 1010.40 (M+H).

[0225] Compound 42a: General procedure A: VX-787 (11 mg, 0.026 mmol), DMAP (3.2 mg, 0.026 mmol), and DCC (8 mg, 0.039 mmol) in a 3 mL solution of Fmoc-Phe-Val-Cit-PAB-OH (41a) (20 mg, 0.026 mmol) in DCM. It was added to the mixture. After stirring for 5 hours, an additional VX-787 (4 mg) was added, and the reaction mixture was stirred for 16 hours. The residue was then concentrated in a vacuum. The residue was dissolved in DMSO (1 mL) and gradient eluted with 5-95% MeCN / H2O (both). The solutions were purified using Teledyne ISCO on a 15.5g C18 Aq. column (both containing 0.05% AcOH). When the pure fractions were combined, frozen, and freeze-dried, the title compound 42a (10 mg) was recovered. Based on the material composition, a 50% yield was obtained as a fluffy, off-white solid. MS(ES) I, pos.): C 62 H 65 F2N 11 Calculated value of O8: 1129.5; measured value: 1130.4 (M+H). Unreacted 41a (7mg) was also measured. I collected it.

[0226] Compound 42b: General procedure B: EDCI (13.2 mg, 0.069 mmol) and Compound 41b (35 mg, 0.046 mmol) VX-787 (18.5 mg, 0.046 mmol) and DMAP (5.6 mg, 0.046 mmol) were added to a DCM (6 mL) solution. The reaction mixture was stirred at room temperature for 2 hours. An additional 6 mg of VX-787 was added, and the reaction mixture was stirred overnight. Remove the medium under reduced pressure, dissolve the residue in DMF (1.5 mL), and add 5-95% MeCN / H2O (both 0.05% TF). The pure fraction was purified using a Teledyne ISCO equipped with a 30g C18 Aq column (containing A). When combined and freeze-dried, the title compound 42b (23 mg, 44%) turns a fluffy off-white color. It was obtained as a solid. MS(ESI, pos.): C 60 H 65 F2N 11 O 10 Calculated value: 1137.5; Measured value: 1138.4 (M+H).

[0227] Compound 42b': Pd(PPh3)4 (2.4 mg, 0.002 mmol) and phenylsilane (1 drop) are combined with compound 42b (23 mg It was added to a 0.020 mmol THF (0.8 mL):DMF (0.4 mL) solution, and the reaction mixture was stirred for 30 minutes. When the substance is removed in a vacuum, compound 42b' is obtained, and this is then used in the next step without purification. I used it.

[0228] Compound 42c: When synthesized according to the general procedure B using compound 41c (39 mg, 0.04 mmol), Table The compound 42c (30 mg, 55%) was obtained as a fluffy, off-white solid. MS(ESI , pos.): C 78 H 77 F2N 13 Calculated value of O8: 1361.6; measured value: 1362.4 (M+H).

[0229] Compound 42d: When synthesized according to general procedure A using compound 41d (15.4 mg, 0.02 mmol), The title compound 42d (13 mg, 57%) was obtained as a fluffy, off-white solid. MS(ES) I, pos.): C 61 H 67 F2N 11 O 10 Calculated value: 1151.5; measured value: 1152.4 (M+H).

[0230] Compound 42d': Pd(PPh3)4 (1.3 mg, 0.001 mmol) and phenylsilane (2 μL, 0.0165 mmol) Add compound 42d (13 mg, 0.011 mmol) to a THF (1.5 mL) / DMF (0.5 mL) solution and stir the reaction mixture for 20 minutes. Mixed. After removing volatile substances in a vacuum, compound 42d' was obtained, which was then purified. It was not available and was used in the next step.

[0231] Compound 42e: Fmoc-Leu-Val-Cit-PABA(41e) (89mg, 0.125mmol), DMAP (30mg, 0.245mmol) and EDC-HCl (48 mg, 0.250 mmol) in a 3:1 THF:DMA (3 mL) solution of VX-787 (100 mg, 0.250 mmol) The reaction mixture was added. The reaction solution was stirred at ambient temperature for 2 hours, and then concentrated in a vacuum to remove THF. The remaining DMA solution was packed into a 50g C18 Aq column, and 5% HOAc was added to both solvents in H2O. Elution was performed with ~100% MeCN. The fraction containing the product was freeze-dried, and then both solvents were used. Chromatography on a 30g C18 Aq column, eluting with 50-100% MeCN in H2O containing 0.05% HOAc. The compound was re-purified by fluoroscopy. After freeze-drying, the title compound 42e (37 mg, 24%) was analyzed by LC-MS to obtain a result of ~9. It was obtained as a white solid that was 0% pure. Without further purification of the product, the next step was taken. Used in the process. MS(ESI, pos.): C 59 H 67 F2N 11 Calculated value of O8: 1095.51; Measured value: 1096.50 (M+H) .

[0232] Compound 42f: DMAP (8 mg, 0.0655 mmol) and EDC-HCl (12 mg, 0.0626 mmol) are combined with compound 41f (31 mg It was added to a 3:1 THF:DMA (760 μL) solution of (0.0307 mmol) and VX-787 (25 mg, 0.0626 mmol). After stirring at ambient temperature for 4 hours, THF was removed by concentration under vacuum. The remaining DMA solution was 15.5 g. The C18 Aq column was packed, and both solvents were eluted with 0-100% MeCN in H2O containing 0.05% HOAc. The fractions containing the pure product were combined and freeze-dried, resulting in compound 42f (7 mg, 16%). It was obtained as a white solid. MS(ESI, pos.): C 72 H 84 F2N 14 O 11 Calculated value of S: 1390.61; measured value 1391.45 (M+H).

[0233] General procedure for Fmoc removal: Compound 43a: 5% piperidine in DMF (1 mL) of compound 42a (1 mL) The reaction mixture was added to a 1 mL solution of 0 mg (0.01 mmol) of DMF and stirred for 45 minutes. Inject 15.5g of SCO into a C18 Aq column and dissolve in 5-95% MeCN:H2O (both containing 0.05% AcOH). The pure fractions were combined, frozen, and freeze-dried to obtain the title compound 43a (7 mg, 63 mg). %) was obtained as a fluffy, off-white solid. MS(ESI, pos.): C 47 H 55 F2N 11 O Calculated value for 6: 907.4; measured value: 908.4 (M+H).

[0234] Compound 43b: The general procedure for removing Fmoc was followed. From crude 42b', a fluffy off-ho Yield as a white solid = 78%. MS(ESI, pos.): C 42 H 51 F2N 11 Calculated value of O8: 875.4; actual value Measured value 876.4 (M+H).

[0235] Compound 43c: The general procedure for removing Fmoc was followed. A fluffy off-white compound was removed from 42c. As a solid of color, yield = 53%. MS (ESI, pos.): C 66 H 67 F2N 13 Calculated value of O8: 1139.5; measured value. The value is 1140.4 (M+H).

[0236] Compound 43d: General procedure for Fmoc removal was followed. A fluffy off-white compound was removed from 42d'. Yield as a light-colored solid = 59%. MS(ESI, pos.): C 43 H 53 F2N 11 Calculated value of O8: 889.4; measured value. The value is 890.4 (M+H).

[0237] Compound 43e: 10% piperidine in DMF (400 μL) compared to anhydrous DMF of Compound 42e (20 mg, 0.0182 mmol). It was added to (400 μL) of solution. After stirring at ambient temperature for 30 minutes, the reaction mixture was transferred to a 5.5 g C18 Aq column. The samples were packed and eluted in both solvents with 5-25% MeCN in H2O containing 0.1% TFA. The pure fractions were combined. When freeze-dried, the TFA salt of compound 43e (13 mg, 72%) was obtained as a white solid. MS(ESI, pos.): C 44 H 57 F2N 11 Calculated value of O6: 873.45; measured value: 874.40 (M+H).

[0238] Compound 43f: 10% piperidine / DMF solution (100 μL) with compound 42f (6.8 mg, 0.00489 mmol) in DMF ( It was added to 100 μL of solution. After stirring at ambient temperature for 1 hour, the solution was packed into a 5.5 g C18 Aq column. The compounds were then eluted in H2O containing 0.05% HOAc with 0-100% MeCN in both solvents. When the fractions are combined and freeze-dried, compound 43f (5.2 mg, 91%) is obtained as a white solid. MS(ESI, pos.): C 57 H 74 F2N 14 O9S calculated value: 1168.55; measured value: 1169.40 (M+H).

[0239] General procedure for incorporating Fmoc-N-amide-PEG8: Compound 44a: Compound 43a (7 mg, 0.008 mg) To a solution of (mol) DMF (1 mL), add Fmoc-N-amide-PEG8-NHS ester (7 mg, 0.009 mmol), then N, N-diisopropylethylamine (4 μL, 0.023 mmol) was added, and the reaction mixture was stirred for 2 hours. The resulting product was analyzed using a gradient elution method with 5-95% MeCN / H2O (both containing 0.05% AcOH) and 15.5g of C18 Aq. Purified using a Teledyne ISCO column. The pure fractions were combined, frozen, and freeze-dried. When this was done, the title compound 44a (5 mg, 42%) was obtained as a fluffy, off-white solid. MS(ESI, pos.): C 81 H 102 F2N 12 O 17 Calculated value: 1552.7; measured value: 1553.7 (M+H).

[0240] Compound 44b: The general procedure for incorporating Fmoc-N-amide-PEG8 was followed. As a fluffy, off-white solid, yield = 61%. MS(ESI, pos.): C 76 H 98 F2N 12 O 19 of Calculated value: 1520.7; Measured value: 1521.4 (M+H).

[0241] The general procedure for incorporating compound 44c: Fmoc-N-amide-PEG8 was followed. As a fluffy, off-white solid, yield = 83%. MS(ESI, pos.): C 97 H 114 F2N 14 O 17 of Calculated value: 1784.4; Measured value: 1785.6 (M+H).

[0242] Compound 44c': TFA (0.2 mL) was added to a solution of compound 44c (17 mg, 0.0073 mmol) in DCM (2 mL). After stirring for 3 hours, the reaction mixture was diluted with MeOH (3 mL), and volatile substances were removed under vacuum. Residue Dissolve in 1:1 MeCN:H2O (1.5 mL) and DMSO (0.2 mL), and filter on a Teledyne ISCO 15.5 g C18 Aq column. Inject into the solution and perform gradient elution of 5-95% MeCN:H2O (both containing 10 mmol ammonium acetate as a modifier). The fraction was eluted using ( ). The pure fractions were combined, frozen, and freeze-dried to obtain the title fraction. Compound 44c' (12 mg, 82%) was obtained as a fluffy, off-white solid. MS(ESI, po s.): C 78 H 100 F2N 14 O 17 Calculated value: 1542.7; measured value: 1543.8 (M+H).

[0243] Compound 44d was prepared using 43d according to a general procedure. Product 44d was then purified. However, it was used in the next step.

[0244] Compound 44e: iPr2NEt (15 μL, 0.0861 mmol) was mixed with compound 43e-TFA salt (20 mg, 0.0206 mmol) and Fm oc-N-amide-PEG8-NHS ester (20 mg, 0.0263 mmol) was added to a solution of anhydrous DMA (400 μL). After stirring at ambient temperature for 2 hours, the reaction mixture was packed into a 15.5 g C18 Aq column, and 0.1% TF was added to both solvents. A was eluted with 10-100% MeCN in H2O containing A. The pure fractions were combined and freeze-dried. Compound 44e (13 mg, 42%) was obtained as a white solid. MS (ESI, pos.): C 78 H 104 F2N 12 O 17 Calculated value: 1518.76; measured value: 1519.70 (M+H).

[0245] Compound 44f: Compound 43f (7 mg, 0.00599 mmol) in anhydrous DMF (100 μL) solution, with 10 in DMF (33 μL) %iPr2NEt solution, then Fmoc-N-amide-PEG8-NHS ester (6 mg, 0.0 mg) in anhydrous DMF (100 μL). (00789 mmol) was added. After 1 hour, LC-MS showed an incomplete reaction. Additional Fmoc-N- Add amide-PEG8-NHS ester (25 μL of 6% DMF solution) and stir the reaction mixture for a further 2 hours. The reaction was then terminated. 5.5g of 0-100% MeCN in H2O containing 0.05% HOAc was used as both solvents. Chromatographic purification on a C18 Aq ISCO column, followed by freezing of the pure fraction. Upon drying, compound 44f (9 mg, 82%) was obtained as a white solid. MS(ESI, pos.): C 91 H1 21 F2N 15 O 20 Calculated value of S: 1814.0; measured value: 1815.7 (M+H).

[0246] Compound 45a: Following the general procedure for Fmoc removal, the title compound 45a was fluffy. An off-white solid was obtained from 44a in 93% yield. MS(ESI, pos.): C 66 H 92 F2N 12 O 15 Calculated value: 1330.7; measured value: 1331.6 (M+H). [ka]

[0247] Compound 45b: Following the general procedure for Fmoc removal, the title compound 45b was fluffy. It was obtained from 44b in 69% yield as an off-white solid. MS(ESI, pos.): C 61 H88 F2N 12 O 17 Calculated value: 1298.6; measured value: 1299.5 (M+H). [ka]

[0248] Compound 45c: Following the general procedure for Fmoc removal, the title compound 45c was fluffy. It was obtained as an off-white solid from 44c' in 89% yield. MS(ESI, pos.): C 63 H 90 F2N 14 O1 Calculated value for 5: 1320.7; measured value: 1322.0 (M+H). [ka]

[0249] Compound 45d: Following the general procedure for Fmoc removal, the title compound 45d was fluffy. It was obtained as an off-white solid in unpurified form with a yield of 44 to 47% (over two steps). MS(ESI, p os.): C 62 H 90 F2N 12 O 17 Calculated value: 1312.7; measured value: 1313.6 (M+H). [ka]

[0250] Compound 45e: 10% piperidine in DMF (100 μL) of Compound 44e (16 mg, 0.0153 mmol) DMF (100 The reaction mixture was added to a μL solution. After stirring at ambient temperature for 30 minutes, the reaction mixture was packed into a 5.5g C18 Aq column. The compounds were then eluted in 5-100% MeCN in H2O containing 0.1% TFA in both solvents. The clean fractions were frozen. Upon drying, the TFA salt of compound 45e (7 mg, 47%) was obtained as a white solid. MS(ESI, p os.): C 63 H 94 F2N 12 O 15 Calculated value: 1296.69; measured value: 1297.60 (M+H). [ka]

[0251] Compound 45f: Triisopropyl in a CH2Cl2 (50 μL) suspension of Compound 44f (4 mg, 0.0022 mmol) Silane (5 μL) was added, followed by TFA (50 μL). After stirring at ambient temperature for 90 minutes, the reaction solution was prepared. The solution was concentrated in a vacuum. The residue was diluted with CH2Cl2 (200 μL) and concentrated. The product was diluted with CH2Cl2 (200 μL). The compound was further diluted three times with (part) and concentrated each time. The resulting yellow foamy substance (compound 44f') was then mixed with DMF (100 μL). The mixture was treated with 5% piperidine in ) and stirred at ambient temperature for 1 hour. After stirring, the reaction mixture was mixed with 5.5g of C18 Aq. The sample was packed into a container and eluted in both solvents with 0-100% MeCN in H2O containing 0.1% TFA. When fractions containing the compound are combined and freeze-dried, the TFA salt of compound 45f (1 mg, 47%) is white. It was obtained as a solid. MS(ESI, pos.): C 63 H 95 F2N 15 O 15 Calculated value: 1339.7; Measured value: 1340.5 (M+H). [ka]

[0252] (Example 2: Synthesis of anti-hemagglutinin non-cytotoxic antibody drug conjugate) The anti-hemagglutinin non-cytotoxic antibody drug conjugate was synthesized as follows.

[0253] The anti-hemagglutinin (anti-HA) monoclonal antibody mAb11729 was mutated to alter the heavy and light chains. The consensus LLQGA pentapeptide sequence was introduced to the C-terminus. This mutation resulted in Enzymatically conjugating antibodies to the heavy chain up to a maximum load of two (one on each heavy chain) is possible. This has become possible. Non-HA-binding mAbs containing the same consensus sequence at the C-terminus of the heavy chain (infectious diseases) An unbound isotype control (derived from an immunological antigen unrelated to the target antigen) was used.

[0254] Using PNGase F (NEB P0704L) at a concentration of 400 U / mg mAb in PBS pH 7.4, natural mAb 11729 and A The isotype control antibody was deglycosylated overnight at 37°C. Then, it was subjected to spin filtering (Amico Using n (30kDa cutoff), the reaction mixture was buffered to PBS pH 7.4. In order, the antibody is enzymatically conjugated at the Q295 site on the heavy chain up to a maximum load of 2. This became possible.

[0255] Second anti-hemagglutinin (N3H2) monoclonal antibody mAb5385 (J Virology 2014, vol 88, (as described in 7130-7144) is mutated, and the consensus is added to the C-terminus of the heavy chain. [ka] A pentapeptide sequence, or at the C-terminus of the light chain, [ka] This mutation was introduced. This mutation enzymatically conjugates antibodies up to a maximum load of 2. This has become possible. The same consensus sequence is contained at the C-terminus of the heavy chain or the C-terminus of the light chain. Non-HA-binding mAbs (derived from immunological antigens unrelated to infectious diseases) are paired with unbound isotypes. It was used as a light source.

[0256] An antibody having a conjugation site at either the C-terminus or the Q295 site of the heavy chain, The compounds were conjugated at 1 mg / mL in PBS pH 7.4. Compounds 6 or 11 (both containing VX-787) were used. A different linker is added in an excess of 10 to 40 times the molar concentration of the antibody, with 14 per 1 mg of antibody. The enzymatic reaction is initiated by adding a unit of bacterial transglutaminase (Zedira, T1001). The conjugates were incubated at 37°C for 16 hours. Purified using Pierce Protein A column (ThermoScientific, product number 20356). The gate is used for ESI-M to determine the payload:antibody ratio (DAR) using Waters Acquity UPLC. Analysis was performed using S. Separation by chromatography was performed using a 10-minute gradient (minutes of mobile phase B:percentage). Stages: 0:10%, 1:10%, 5:90%, 7:90%, 7.2:10%, 10:10%, C4 column (2.1×50mm) ACQUITY was achieved using UPLC BEH Protein C4 (1.7 μm, 300 A). Mobile phase A was 0.1% formic acid in water. The mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was set to 0.3 mL / min. The detector TOF scan is set from m / z 500 to 4500, and the main parameters are as listed. (Capillary voltage 3.0kV; sampling cone 80V; source offset 100V; so Spectrum (temperature 150°C; desolvation temperature 450°C; cone gas 0 L / hr; desolvation gas 800 L / hr). The data was deconvolved using the MaxEnt function in the MassLynx software. The molecular ions, when weighted according to their intensity, are listed in Tables 3 and 4. It is compatible with the spectroscopy. The actual mass spectrometry spectra are shown in Figures 1 and 2. Alternatively, anti To determine the loading of the payload onto the body, the conjugate is 1M potassium phosphate. TSK-NPR-butylHIC (hydrophobic interaction) using a linear gradient from pH 8.5 to water over 60 minutes. Chromatography was performed using an Agilent 1260 column. Payload load The ng corresponds to the species of conjugated and unconjugated antibodies. The peak area was determined by integration. The payload:antibody ratio is reported in Table 5. Exclusion HPLC confirmed that all conjugates were >92% monomers (Table 5). This procedure conjugates compound 11 via LLQGA pentapeptide at the C-terminus of the antibody heavy chain. mAb11729-VX-787 non-cytotoxic antibody-drug conjugate with gated mAb11729 (n cADC) ("11729-HC-Cterm-11"), compound 1 via LLQGA pentapeptide at the N-terminus of the antibody heavy chain. mAb11729-VX-787 ncADC ("11729-HC-Nterm-11") has a conjugated mAb11729. ), mA conjugated to compound 11 via LLQGA pentapeptide at the C-terminus of the antibody light chain mAb11729-VX-787 ncADC ("11729-LC-Cterm-11") containing b11729, antibody light chain N-terminus is LLQG mAb11729-VX has mAb11729 conjugated to compound 11 via A pentapeptide. -787 ncADC ("11729-LC-Nterm-11") has compound 11 conjugated at the Q295 site. mAb11729-VX-787 ncADC ("11729-Q295-11"), via LLQGA pentapeptide to the heavy chain C-terminal Isotype control antibody having compound 11 conjugated at the end ("Isotype control- Isotype control antibody having compound 11 conjugated at the Q295 site (HC-Cterm-11) The isotype ("Isotype Control-Q295-11") has compound 6 conjugated at the Q295 site. Ab11729-VX-787 ncADC ("11729-Q295-6"), via LLQGA pentapeptide at the heavy chain C-terminus Isotype control antibody having conjugated compound 6 ("Isotype control-HC-Ct An isotype control antibody ("A") having compound 6 conjugated at the Q295 site ("A") Isotype control (Q295-6), condylated at the C-terminus of mAb11729 heavy chain via LLQGA pentapeptide. mAb11729-baloxavir ncADC ("11729-HC-Cterm-15") having compound 15 that has been modified. , and an isotype control antibody having compound 15 conjugated at the heavy chain C-terminus ("A The isotype control "HC-Cterm-15" was produced.

[0257] Native mAb11729 and isotype control antibody in 50 mM HEPES, 150 mM NaCl, pH 7.5 (1-10 mg / mL) was treated with 1 mM dithiothreitol at 37°C for 30 minutes. Gel filtration (G-25, pH 4.5 Sodium acetate) followed by maleimide linker payload derivatin compounds in DMSO (10 mg / ml) Add 18 (1.2 equivalents / SH group) to the reduced antibody, and adjust the pH of the mixture to 7.0 with 1M HEPES (pH 7.4). The conjugate was then size-exclusion chromatography-analyzed to remove the conjugate from PBS containing 5% glycerol. The sample was purified using [method / tool ​​name] and then sterile filtered. Protein concentration and payload-to-antibody ratio were measured using UV spectroscopy. Determined by analysis. Size exclusion HPLC determined that all conjugates used were >95%. It was confirmed to be monomeric. All conjugated antibodies were linked to the linker payload. The loading values ​​were analyzed by mass spectrometry. The payload-to-antibody ratio is reported in Table 6. It has been reported.

[0258] Tables 3, 4, and 5 contain loading data (ESI-MS DAR) for specific conjugates. Hmm. Compounds 11 and 15, which are conjugated with transglutaminase, bring the theoretical value to 2. Closer DAR values ​​were obtained. Table 6 includes a list of ncADC purity and DAR values. Table 3. Intensity-weighted average payload row in 11729-Q295-11 and isotype control-Q295-11. Summary of Ding [Table 3] Table 4. Intensity-weighted average payload in 11729-HC-Cterm-11 and isotype control-HC-Cterm-11 Download summary [Table 4] Table 5. Intensity-weighted average payload in 11729-HC-Cterm-15 and isotype control-HC-Cterm-15 Download summary [Table 5] Table 6. Purity (by SEC) and DAR of compound conjugates 6, 11, and 15. [Table 6]

[0259] (Example 3: Anti-HA infected cell binding ELISA and drug antiviral efficacy assay) MDCK London cells were placed in a 96-well plate with 1% sodium pyruvate (Life Technolog ies), 0.21% low IgG BSA solution (Sigma Aldrich), and 0.5% gentamicin (Life Technolog Seed 40,000 cells / well in 50 μL of infection medium (DMEM (Life Technologies)) containing ies) The cells were incubated in 5% CO2 at 37°C for 4 hours. Then, 50 μL was placed on a plate. Infect with the H1N1 A / Puerto Rico / 08 / 1934 influenza virus at a MOI of 1.2 and lightly strike. First, the plates were rehydrated in 5% CO2 at 37°C for 20 hours. After that, the plates were phosphate-buffered saline (PBS), Wash once with Life Technologies, then add 200 μL of 4% paraformaldehyde (PFA, Alfa) in PBS. The plates were fixed with Aesar and incubated at room temperature for 15 minutes. The plates were washed three times with PBS and 300 μL of water was used. The StartingBlock was blocked for 1 hour at room temperature with blocking buffer (ThermoFisher). The control antibodies were 11729-HC-Cterm-11, 11729-HC-Nterm-11, 11729-LC-Cterm-11, and 11729 -Tested against LC-Nterm-11. The control antibody used was unconjugated mA. b11729, unconjugated isotype control antibody, isotype control-HC-Cter m-6, isotype control - LC-Cterm-6, isotype control - HC-Cterm-45a, isotype control Reference -LC-Cterm-45a, Isotype control -HC-Cterm-34, Isotype control -LC-Cterm-34, A The isotype controls were HC-Cterm-45d and HC-Cterm-11.

[0260] Dilute the antibody in StartingBlock blocking buffer to a starting concentration of 100 μg / mL, and each 6.1 × 10 -3 The concentration was gradually reduced in a 1:4 ratio until the final concentration of μg / mL was reached. After incubating the plate, Remove the StartingBlock blocking buffer and add the diluted antibody to the cells in 75 μL / well. It was added to the plate. The plate was incubated at room temperature for 1 hour. After incubation, pre Wash the tweed with a washing buffer (imidazole-buffered saline and Milli-Q water diluted to 1x). Wash three times with en(registered trademark) 20; KPL) and then in StartingBlock Blocking Buffer at a ratio of 1:2000. Diluted 75 μL / well secondary antibody (HRP-conjugated donkey anti-human IgG; Jackson Imm) (unoResearch) was layered. This secondary solution was incubated on the plate at room temperature for 1 hour. Then, the plate was washed three times with washing buffer, and then 75 μL / well of ELISA P was added. A 1:1 preparation of ico chemiluminescent substrate was added to the plate. The plate was then examined for luminescence using a Molecular Device. I read it immediately with the Spectramax i3x plate reader.

[0261] 11729-HC-Cterm-11, 11729-LC-Cterm-11, and 11729-LC-Nterm-11 are shown in Table 7 and Figure 3. As described above, it binds to influenza A-infected cells at concentrations below nanomolar, and Heterospecific binding was observed. In particular, this specific binding was observed with the unconjugated mAb11729 antibody. It is within the same range, and the addition of the payload does not have an excessively harmful effect on binding. This demonstrated that the linker-payload to the N-terminus of the heavy chain is as shown in Figure 11. The denaturation leads to a decrease in binding to influenza A / H1N1 / PR8-infected cells. However, due to the conjugation of the linker-payload to the C-terminus of the heavy chain, influenza Binding to Enza A / H1N1 / PR8-infected cells was retained. Table 7. Anti-HA binding ELISA for influenza A-infected cells [Table 7]

[0262] To test its antiviral efficacy, we suppress the infection of cells by the influenza virus. The ability of 11729-HC-Cterm-11 was assayed. MDCK London cells were subjected to a 96-well assay. 100 μL of growth medium in the plate (1% sodium pyruvate, 10% fetal bovine serum, and 0.5 Cells were seeded at 20,000 cells / well in DMEM containing % gentamicin. Cells were incubated at 37°C and 5°C. The antibodies were incubated in %CO2 for 18 hours. The following day, all antibodies were triple-pressed to a starting concentration of 500 μg / mL. Syn infection medium (1% sodium pyruvate, 0.21% low IgG BSA solution, 1 mg / mL TPCK treated trip Dilute with DMEM containing 0.5% gentamicin, and 1.143 × 10 -1Final concentration of μg / mL The dose was gradually reduced in a 1:3 ratio until it reached a certain degree. This was H1, which was modified to express GFP in the cells it infected. N1 A / Puerto Rico / 08 / 1934 Influenza Virus ("H1N1 A / Puerto Rico / 08 / 1934-GF") Dilute P) to MOI 1 in trypsin infection medium (Life Technologies), and use the diluted antibody or AD Mixed with C in a 1:1 ratio. Removed the growth medium from the seeded 96-well plate and used with virus-antibody or The virus-ADC mixture was added to the cells at a rate of 100 μL per well. The plate was gently tapped and then cooled to 37°C. Then, the plates were returned to 5% CO2 for 20 hours. After that, the plates were washed once with PBS and then treated with 4% PFA in 50 μL of PBS. The specimens were fixed and incubated at room temperature for 15 minutes. The plate was washed twice with PBS and then topped with 50 μL of PBS. The plates were layered. The GFP signal was analyzed using an ImmunoSpot analyzer (Cellular Technology I immediately recognized it as "Limited."

[0263] As shown in Table 8 and Figure 4, phosphorus is present at the C-terminus of the heavy and light chains, and at the N-terminus of the light chain. Car-payload 11 conjugation enables antiviral action of antibody-drug conjugates. Activity was 3 to 163 times enhanced against influenza A / H1N1 / Pr8 virus compared to the parental antibody. As shown in Table 9 and Figure 6, the linker-payload 11 conjugates to the C-terminus of the heavy chain. Through pharmacoagulation, the antiviral activity of antibody-drug conjugates is enhanced in influenza A / H1N. The antibody level against the 1 / Cal09 virus increased tenfold compared to the parental antibody level, as shown in Table 8 and Figure 7. Conjugation of the linker-payload 6 to the C-terminus of the heavy chain enables antibody-drug conjugation. The antiviral activity of jugate was 51 times greater than that of the parent antibody against influenza A / H1N1 / PR8. However, due to the conjugation of 6 to the C-terminus of the light chain, the antiviral activity is greater than that of the parent antibody. It did not increase either. As shown in Table 8 and Figure 8, linker pay to the C-terminus of the heavy chain Due to the conjugation of load, the antiviral activity of the antibody-drug conjugate is In the case of 11729-HC-Cterm-34, the antibody level against influenza A / H1N1 / PR8 virus is 7 times higher than that of the parent antibody. In the case of 1129-HC-Cterm-45a, the increase was 35-fold, but the linker payload to the C-terminus of the light chain Conjugation did not increase antiviral activity compared to the parent antibody. (Table 9 and Figure 9) As shown, the linker-payload conjugate to the C-terminus of the light and heavy chains According to the study, the antiviral activity of antibody-drug conjugates is related to influenza A / H3N2 / HK The antibody levels against the 68X31 virus increased 3 to 10 times compared to the parental antibody levels, as shown in Table 8 and Figure 10. Furthermore, the conjugation of the linker-payload to the C-terminus of the heavy chain allows for antibody-drug conjugation. The antiviral activity of jugate was 71 times greater than that of the parent antibody against influenza A / H1N1 / PR8. That's impressive. Table 8. Antiviral activity of antibodies against influenza A / H1N1 / PR8 virus [Table 8] Table 9. Antiviral activity of antibodies against influenza A / H1N1 / CalO9 virus [Table 9] Table 10. Antiviral activity of antibodies against influenza A / H3N2 / HK68x31 virus [Table 10]

[0264] (Example 4: Anti-influenza HA mAb11729 antibody-drug condyloma in monkey or IgG-depleted human plasma) (In vitro plasma stability of the gate) 11729-HC-Cterm-11 was incubated in vitro with plasma from various species, and DAR I evaluated it.

[0265] ncADC samples were placed in pooled fresh cynomolgus monkey plasma (BioReclamation, Lot CYN260056- CYN260057) or IgG-depleted human plasma (BioIVT, lot#BRH1097869) is mixed with an Eppendorf tube ( In Eppendorf (Cat# 022363514), the solution was added independently up to a final concentration of 50 μg / mL, and then in a water bath... The samples were incubated at 37°C for 0 to 72 hours. Samples were removed at 0, 24, 48, and 72 hours. Then, it was frozen and stored at -80°C until analysis.

[0266] For DAR analysis, a DynaMag-2 magnetic particle processor (Life Technologies, Cat#12321D) is used. By using this method, ncADCs were purified from plasma samples by affinity capture. First, Bio Chinified anti-human kappa antibody (reagent from Regeneron) is used with streptavidin paramagnetic beads (Invitr The sample was fixed to ogen (Cat#605602). Each plasma sample containing ncADC was heated at 1850 rpm with 1 mg of bead. Mix the ingredients in a ThermoMixer C unit (Eppendorf, catalog number 2231000574) at room temperature for 2 hours. Combined. Then, the beads were added to 600 μL of 50 mM Tris-HCl pH 7.5 buffer (1 M Tris-HCl pH 7.5). Wash three times with 5 buffer (diluted from Invitrogen, Cat#15567-027), then 600 Washed once with 10% acetonitrile (VWR Chemicals, Cat#BDH83640.100E) in μL of water. After purification, the beads were left at room temperature for 15 minutes with 1% formic acid in 50 μL of 25:75 acetonitrile:water (v / v). ncADC was eluted by incubation. 2 μL of 0.5 M TCEP (Sigma, Cat 6465) was added. By adding 47-10X1ML to each sample (19.2 mM TCEP in the final solution), the eluted samples were obtained. The material was further reduced and incubated in a ThermoMixer C unit at 50°C for 30 minutes.

[0267] The reduced ncADC sample was subjected to a 1.7 μm BEH300 connected to a Synapt G2-Si mass spectrometer (Waters). The sample was injected into a C4 column (Waters Corporation, Cat# 186007567). The flow rate was 8 μL / min (mobile phase A). (Phase B: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile) and liquid chromatography - The gradient is set to a 10-minute gradient, and the ncADC dissolves between 2.1 and 6.5 minutes, corresponding to 26-40% of mobile phase B. I took it out.

[0268] MaxEnt1 software (Waters Corporation) with the following parameters: Mass range: 20~60kD a, m / z range: 700 Da~4000 Da; division: 1.0 Da / channel; half-width: 1.0 Da; minimum intensity ratio: 33%; The acquired spectrum was deconvolved using a maximum iteration value of 15. .

[0269] After 72 hours of incubation with cynomolgus monkey or IgG-free human plasma, ncADCs No significant linker-payload loss was observed (Figure 5). Sequence List [ka] TIFF2026076189000107.tif243170TIFF2026076189000108.tif244170TIFF2026076189000109.tif242170TIFF2026076189000110.tif243170TIFF2026076189000111.tif246170TIFF2026076189000112.tif242170TIFF2026076189000113.tif246170TIFF2026076189000114.tif247170TIFF2026076189000115.tif248170TIFF2026076189000116.tif243170TIFF2026076189000117.tif247170TIFF2026076189000118.tif246170TIFF2026076189000119.tif244170TIFF2026076189000120.tif248170TIFF2026076189000121.tif248170TIFF2026076189000122.tif244170TIFF2026076189000123.tif249170TIFF2026076189000124.tif237170TIFF2026076189000125.tif247170TIFF2026076189000126.tif244170TIFF2026076189000127.tif247170TIFF2026076189000128.tif247170TIFF2026076189000129.tif246170TIFF2026076189000130.tif244170TIFF2026076189000131.tif248170TIFF2026076189000132.tif243170TIFF2026076189000133.tif249170TIFF2026076189000134.tif247170TIFF2026076189000135.tif244170TIFF2026076189000136.tif248170TIFF2026076189000137.tif247170TIFF2026076189000138.tif249170TIFF2026076189000139.tif246170TIFF2026076189000140.tif247170TIFF2026076189000141.tif243170TIFF2026076189000142.tif247170TIFF2026076189000143.tif240170TIFF2026076189000144.tif241170TIFF2026076189000145.tif246170TIFF2026076189000146.tif247170TIFF2026076189000147.tif248170TIFF2026076189000148.tif241170TIFF2026076189000149.tif244170TIFF2026076189000150.tif247170TIFF2026076189000151.tif243170TIFF2026076189000152.tif247170TIFF2026076189000153.tif247170TIFF2026076189000154.tif248170TIFF2026076189000155.tif243170TIFF2026076189000156.tif248170TIFF2026076189000157.tif239170TIFF2026076189000158.tif241170TIFF2026076189000159.tif247170TIFF2026076189000160.tif247170TIFF2026076189000161.tif247170TIFF2026076189000162.tif248170TIFF2026076189000163.tif243170TIFF2026076189000164.tif248170TIFF2026076189000165.tif243170TIFF2026076189000166.tif247170TIFF2026076189000167.tif247170TIFF2026076189000168.tif248170TIFF2026076189000169.tif247170TIFF2026076189000170.tif247170TIFF2026076189000171.tif246170TIFF2026076189000172.tif243170TIFF2026076189000173.tif243170TIFF2026076189000174.tif246170TIFF2026076189000175.tif239170TIFF2026076189000176.tif175170.

Claims

1. Anti-influenza antibodies or antigen-binding fragments conjugated to antiviral compounds Antibody-drug conjugates containing these substances.

2. The anti-influenza antibody or its antigen-binding fragment undergoes antiviral activation via a linker. The antibody-drug conjugate according to claim 1, which is conjugated in a compound.

3. The antibody-drug conjugate according to claim 1 having the following structure 【Chemistry 1】 (Here, Ab is an anti-influenza antibody or its antigen-binding fragment; L is the linker; P is an antiviral compound; and k is an integer between 1 and 30.

4. The antibody-drug conjugate according to claim 3, wherein P is an influenza inhibitor.

5. The antibody-drug conjugate according to claim 3, wherein P is a polymerase inhibitor.

6. The antibody-drug conjugate according to claim 3, wherein P is VX-787, a derivative thereof, or a residue thereof. .

7. The antibody-drug compound according to claim 3, wherein P is baloxavir, a derivative thereof, or a residue thereof. Jugate.

8. The antibody-drug combination according to claim 3, wherein Ab is an anti-hemagglutinin antibody or an antigen-binding fragment thereof. Jugate.

9. The antibody-drug conjugate according to claim 8, wherein P is an influenza inhibitor.

10. The antibody-drug conjugate according to claim 8, wherein P is a polymerase inhibitor.

11. The antibody-drug conjugate according to claim 8, wherein P is VX-787, a derivative thereof, or a residue thereof. .

12. The antibody-drug compound according to claim 8, wherein P is baloxavir, a derivative thereof, or a residue thereof. Jugate.

13. Compounds having the following structure 【Chemistry 2】 (Here, L is the linker; BA is a binder; and k is an integer between 1 and 30. 【Request Item 14】 【Chemistry 3】 【change】 【change】 Selected from the group consisting of, Here, BA is an antibody or its antigen-binding fragment; and k is an integer between 1 and 30. The compound according to claim 13.

15. The compound according to claim 13, wherein BA is an antibody or an antigen-binding fragment thereof.

16. Ab or BA contains at least one glutamine residue used in conjugation. A lance glutaminase-modified antibody or an antigen-binding fragment thereof, any one of claims 1 to 15 The antibody-drug conjugate or compound described in the section.

17. Ab or BA contains at least two glutamine residues used in conjugation. A lance glutaminase-modified antibody or an antigen-binding fragment thereof, any one of claims 1 to 16 The antibody-drug conjugate or compound described in the section.

18. Ab or BA contains at least three glutamine residues used in conjugation. A lance glutaminase-modified antibody or an antigen-binding fragment thereof, any one of claims 1 to 17 The antibody-drug conjugate or compound described in the section.

19. Ab or BA contains at least four glutamine residues that can be used for conjugation. A lance glutaminase-modified antibody or an antigen-binding fragment thereof, any one of claims 1 to 18 The antibody-drug conjugate or compound described in the section.

20. Ab or BA is a transglutaminase-modified antibody or its antigen-binding fragment, where, The denjugation is at two Q295 residues in the EU numbering system; and k is 2. The antibody-drug conjugate or compound according to claim 19.

21. Ab or BA is a transglutaminase-modified antibody containing an antibody heavy chain or its antigen-binding fragment. Here, the conjugation is at the C-terminus of the heavy chain, and k is 2. or the antibody-drug conjugate or compound according to claim 19.

22. The antibody-drug according to claim 21, wherein the conjugation is mediated by glutamine. Conjugate or compound.

23. The conjugation is mediated by glutamine in the LLQGA sequence at the C-terminus of the antibody heavy chain. The antibody-drug conjugate or compound according to claim 21.

24. The conjugation is at two Q295 residues and two N297Q residues; and k is 4 The antibody-drug conjugate or compound according to claim 19.

25. The antibody-drug conjugate according to any one of claims 1 to 24, wherein Ab or BA is mAb11729. A compound.

26. An antibody-drug conjugate or compound according to any one of claims 1 to 25, and a pharmaceutical product. A pharmaceutical composition comprising an acceptable excipient, carrier, or diluent.

27. The pharmaceutical composition is selected from the group consisting of oral, intravenous, intraperitoneal, inhalation, and intranasal administration. A pharmaceutical composition according to claim 26, formulated for a selected dose.

28. For the treatment, prevention, mitigation, or inhibition of infection-related diseases, disorders, or illnesses in the target population. A method for applying an antibody-drug conjugate according to any one of claims 1 to 27 to the target. The method comprising administering an effective amount of a compound or pharmaceutical composition.

29. The method according to claim 28, wherein the infection is a viral infection.

30. The method according to claim 29, wherein the infection is an influenza virus infection.

31. The method according to claim 29, wherein the infection is an influenza A virus infection.

32. The method according to claim 29, wherein the infection is caused by influenza B virus.

33. The aforementioned infections are influenza A virus infection and influenza B virus infection. The method according to claim 29.

34. The side effects of the compound when administered to the subject are conjugated to the comparison subject. Any one of claims 28 to 33, which is reduced compared to the administration of an antiviral compound that is not present. The method described in item 1.

35. Methods for treating, preventing, mitigating, or inhibiting influenza infection in a subject. The subject is an antibody-drug conjugate, compound, or according to any one of claims 1 to 34. The method comprising administering an effective amount of a pharmaceutical composition.

36. The aforementioned influenza infection is caused by an influenza A virus infection. The method according to claim 35.

37. The aforementioned influenza infection is caused by influenza A group 1 virus. The method according to claim 35.

38. The aforementioned influenza infection is caused by the influenza A H1 virus. The method described in item 35.

39. The aforementioned influenza infection was caused by influenza group 2 virus type A. The method according to claim 35.

40. The aforementioned influenza infection is caused by the influenza A H3 virus. The method described in item 35.

41. The aforementioned influenza infection is caused by an unknown or unidentified influenza virus. The method according to claim 35.

42. The aforementioned influenza infection is caused by an influenza B virus infection. The method according to claim 35.

43. The aforementioned influenza infection is a type A influenza virus infection and a type B influenza virus infection. The method according to claim 35, caused by Rus infection.

44. The antibody-drug conjugate, compound, or pharmaceutical composition is used in combination with an adjunct therapeutic agent. The method according to any one of claims 28 to 43, which is administered.

45. The aforementioned auxiliary therapeutic agents include antiviral drugs, anti-inflammatory drugs, and other drugs used to treat influenza infection. Antibodies that specifically bind to influenza HA, influenza vaccines, nutritional supplements, and The method according to claim 44, selected from the group consisting of: and palliative therapy.

46. The aforementioned anti-inflammatory drug is selected from the group consisting of corticosteroids and non-steroidal anti-inflammatory drugs. The method according to claim 45.

47. The method according to claim 46, wherein the nutritional supplement is an antioxidant.

48. The aforementioned adjuvant therapeutic agent, via various routes of administration, delivers the antibody-drug conjugate and compound The method according to any one of claims 44 to 47, or administered as a pharmaceutical composition.

49. The method according to any one of claims 44 to 48, wherein the auxiliary therapeutic agent is administered orally.

50. The method according to claim 45, wherein the antiviral agent is oseltamivir.

51. The oseltamivir is used in the antibody-drug conjugate, compound, or pharmaceutical composition. The method according to claim 50, administered before administration.

52. The oseltamivir is used in the antibody-drug conjugate, compound, or pharmaceutical composition. The method according to claim 50, administered simultaneously with the administration of the drug.

53. The oseltamivir is used in the antibody-drug conjugate, compound, or pharmaceutical composition. The method according to claim 50, administered after administration.

54. The claim states that the antiviral agent is an anti-influenza A drug or an anti-influenza B drug. The method described in section 45.

55. The aforementioned anti-influenza A drug or the aforementioned anti-influenza B drug is an antibody or its antigen-binding portion The method according to claim 54, which is a minute.

56. The antibody specifically binds to influenza A HA or influenza B HA, according to the claim. The method described in 55.

57. The antibody-drug conjugate, compound, or pharmaceutical composition is delivered subcutaneously, intravenously, intradermally, or intramuscularly. The method according to any one of claims 28 to 56, wherein the administration is intramuscular, intranasal, or orally.

58. The following structure 【Chemistry 4】 (Here, L is a linker; and RG is the reactive part. A linker-antiviral compound having a pharmaceutically acceptable salt thereof.

59. The aforementioned linker-payload is 【Transformation 5】 (Here, SP 1 and SP 2 If present, it is a spacer base; RG is the portion that reacts with the antibody or its antigen-binding fragment; Each AA is an amino acid; and n is an integer between 1 and 10. The linker-antiviral compound according to claim 58, or a pharmaceutically acceptable salt thereof. 【Request Item 60】 【Transformation 6】 A linker-antiviral compound according to claim 59, selected from the group consisting of the following.

61. comprising an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is as described in claim 58. An antibody-drug conjugate, which is conjugated to the compound shown.

62. The conjugated compound is 【Transformation 7】 (Here, L is the linker.) An antibody-drug conjugate according to claim 61, selected from the following.

63. A method for preparing an antibody-drug conjugate, wherein the binder is the linker described in claim 58. The method comprising contacting with an antiviral compound.

64. Any of claims 1 to 63, wherein Ab or BA is an anti-influenza A antibody or an antigen-binding fragment thereof Any antibody-drug conjugate or compound described in item one.

65. Claim 1, wherein Ab or BA is an anti-influenza A group 1 antibody or its antigen-binding fragment. An antibody-drug conjugate or compound as described in any one of items ~64.

66. Any of claims 1 to 65, Ab or BA is an anti-influenza H1 antibody or its antigen-binding fragment. Any antibody-drug conjugate or compound described in item one.

67. Claim 1, wherein Ab or BA is an anti-influenza A group 2 antibody or its antigen-binding fragment. An antibody-drug conjugate or compound as described in any one of items ~66.

68. Ab or BA is an anti-influenza H3 antibody or an antigen-binding fragment thereof, according to any of claims 1 to 67. Any antibody-drug conjugate or compound described in item one.

69. Any of claims 1 to 68, wherein Ab or BA is an anti-influenza B antibody or its antigen-binding fragment. Any antibody-drug conjugate or compound described in item one.

70. Anti-influenza antibodies conjugated to antiviral compounds via a linker or The antigen-binding fragment comprises, where the antibody-drug conjugate is a polymerase base Sex protein 2 (PB2) (VX-787), polymerase acid protein (PA) (baloxavir marboxy (Lu), and / or bind to polymerase basic protein 1 (PB1), and / or these An antibody-drug conjugate according to any one of claims 1 to 69 that inhibits

71. The antibody-drug conjugate or compound comprises an antibody heavy chain, and the C-terminus of the antibody heavy chain The antibody-drug conjugate according to any one of claims 1 to 70, further comprising a pentapeptide. Or a compound.

72. The antibody-drug conjugate according to claim 71, comprising the pentapeptide amino acid sequence LLQGA. A compound or compound.

73. The antibody or its antigen-binding fragment further comprises the amino acid sequence shown in SEQ ID NO:

26. ; and any of claims 1 to 72, comprising HCVR further comprising the amino acid sequence shown in SEQ ID NO:

18. The antibody-drug conjugate described above.

74. The antibody or its antigen-binding fragment is antibody mAb11729, according to any one of claims 1 to 73. Antibody-drug conjugate.

75. The aforementioned antibody (a) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 20; (b) HCDR2 containing the amino acid sequence shown in SEQ ID NO: 22; (c) HCDR3 containing the amino acid sequence shown in SEQ ID NO: 24; (d) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28; (e) LCDR2 containing the amino acid sequence shown in SEQ ID NO: 30; and (f) LCDR3 containing the amino acid sequence shown in Sequence ID No. 32 An antibody-drug conjugate according to any one of claims 1 to 74, comprising:

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