Conjugates for prevention and treatment of viral infections and uses thereof

Abstract

The present invention relates to a conjugate for the prevention and treatment of viral infections and uses thereof, and more particularly, to a protein-anti-influenza compound conjugate having the formula I-A or formula I-B or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof, in which each symbol is as defined herein. The conjugate or the intermediate compound disclosed by the invention has remarkable anti-influenza virus activity, excellent in-vitro / in-vivo pharmacokinetic property and safety and high clinical application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to antiviral drugs against influenza (Flu) virus and antibodies or conjugates of their constant regions Fc. Specifically, it relates to compounds comprising antibodies or their constant regions Fc covalently coupled to virus surface protein inhibitor small molecules or inhibitor polypeptide drugs and intermediate compounds thereof, and to pharmaceutical compositions or drugs comprising these compounds, as well as their use for preventing and / or treating related viral infections. Background Art

[0002] Influenza virus causes nearly 3 million to 5 million cases of severe infection and about 500,000 deaths worldwide each year (Luliano et al., 2018, Lancet 391: 1285-1300). Although most healthy people will recover automatically within one to two weeks after being infected with the virus, influenza virus infection can develop into life-threatening severe infection and complications, such as pneumonia, for the elderly, those with chronic diseases and those with weakened immune systems.

[0003] Developing treatments to combat influenza viruses is an ongoing challenge for humanity. Although there are already approved anti-influenza drugs and preventive vaccines on the market, small molecule anti-influenza drugs usually need to be used within 48 hours of the onset of symptoms after infection to have clinical benefits. In addition, due to the high variability of influenza viruses, resistant strains of viruses to such commonly used drugs have been found. Therefore, there is a need to develop more effective and long-lasting therapies for the treatment and / or prevention of influenza viruses.

[0004] Influenza virus is a negative-strand, segmented RNA virus belonging to the family Orthomyxoviridae, including influenza virus A, B, and C. Among them, influenza virus A and B are the main infections in humans. Influenza virus infects respiratory epithelial cells. The first step of the process is the adsorption process of host cells mediated by the surface receptor binding protein of the virus (hemagglutinin protein, HA protein for influenza protein). Subsequently, under the action of hemagglutinin protein, the viral envelope and cell membrane fuse, and the influenza virus genome fragments and the viral RNA-dependent RNA polymerase complex are then released into the cell. The viral RNA-dependent RNA polymerase complex uses the genome fragments as a template to synthesize new progeny virus particles in the cell. The newly synthesized virus particles are released outside the cell by cell lysis or budding. For influenza virus, the complete release of progeny virus depends on the cleavage of sialic acid residues on the cell surface by neuraminidase NA. Neuraminidase inhibitors targeting influenza virus neuraminidase to reduce virus spread have been approved for clinical use, including oseltamivir (Tamiflu TM ), zanamivir (Relenza TM ) and peramivir (Rapivab TM ).

[0005] Patients who have received organ transplants or cancer patients cannot effectively eliminate viruses due to their suppressed immune systems. After being infected with influenza viruses, the virus can easily replicate in the body for a longer time, thereby increasing the possibility of producing drug-resistant strains. This has been found in clinical practice. At the same time, the problem of influenza drug resistance in ordinary patients is also a prominent medication challenge, so more effective new methods and therapeutic agents for treating influenza are needed. Summary of the invention

[0006] On the one hand, the present invention provides a conjugate, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, which comprises an anti-influenza virus small molecule compound conjugated to a protein (also referred to as a "protein-anti-influenza compound conjugate"), having significant anti-influenza virus biological activity.

[0007] In some embodiments, the present invention provides a conjugate having a structure of Formula IA or Formula IB, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof:

[0008]

[0009] in,

[0010] m is 1 or 2;

[0011] n is an integer from 1 to 20, preferably an integer of 1, 2, 3, 4, 5, 6, 7 or 8;

[0012] w is an integer from 0 to 8; for example, 0, 1, 2, 3, 4 or 5;

[0013] L is a linker;

[0014] E is selected from an antibody or antibody fragment, an Fc domain monomer, an Fc domain, an Fc binding peptide, albumin or an albumin-linked peptide;

[0015] R 1a , R 2a , R 3a , R 5a , R 6a , R 7a , R 9a and R 10a are each independently selected from hydrogen, deuterium, hydroxyl, -OC 1-6 Alkyl, hydroxyl C 1-6 Alkyl-, C 1-6 Alkyl-, C 3-8 Cycloalkyl-, C 3-8 Heterocycloalkyl-, C6-C 15 Aryl, 5-15 membered heteroaryl, halogen, cyano and amino; R 1a and R 2a Can form C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl;

[0016] or R 5a and R 6a Can form C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl;

[0017] or R 9a and R 10a Can form C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl, wherein the C 3-8 Cycloalkyl or C 3-8 The heterocycloalkyl group is optionally substituted with a substituent selected from halogen and hydroxy;

[0018] R 4a and R 8a Each independently selected from H, C 1-6 Alkyl, C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl;

[0019] x is 1 or 2;

[0020] y is an integer from 1 to 20, preferably an integer of 1, 2, 3, 4, 5, 6, 7 or 8;

[0021] z is an integer from 0 to 8; for example, 0, 1, 2, 3, 4 or 5;

[0022] q is 0, 1, 2, 3, or 4;

[0023] R 1’ , R 2’ , R 3’ , R 4’ and R 5’ are each independently selected from hydrogen, deuterium, hydroxyl, -OC 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl, C6-C 15 aryl, 5-15 membered heteroaryl, halogen, cyano and amino; or R 1’ and R 2’ Can form C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl; or two adjacent R 3’ Can form C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl, C6-C 15 Aryl or 5-15 membered heteroaryl.

[0024] It should be understood that in Formula IA and Formula IB, the brackets with subscripts n or y do not include (E) m The structural part covers the S atom and the structure below it.

[0025] In some embodiments, L is a linker having a structure of Formula I-3a, comprising L 1 and L 2a , L 1 The left and right ends of L are connected to the drug part (D). 2a The upper end of the formula IA and the -NR in the formula IB 8a connect,

[0026]

[0027] in,

[0028] L 1 Select from the following structures:

[0029]

[0030] in,

[0031] W is selected from O, S, NR b , CH 2- or does not exist;

[0032] R a and R b are each independently selected from H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl; preferably selected from H and methyl, more preferably methyl;

[0033] y1 and y2 are each independently 0, 1, 2, 3, 4, 5 or 6;

[0034] It should be understood that L 1 The bonds at the left and right ends of the structure are closely related to the drug (D 1 ) is connected to the oxygen atom of the middle amine group, and the bond marked by the wavy line on the middle amine group is connected to L 2a Connected;

[0035] Preferably L 1 Select from the following structures:

[0036]

[0037]

[0038] In some embodiments, wherein L 2a Having the structure of formula L2-1a to L2-7a:

[0039]

[0040]

[0041] in,

[0042] Z is NR, S and O;

[0043] The R groups are each independently selected from hydrogen, deuterium, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted 3-20 membered heterocycloalkyl, optionally substituted C6-C 15 aryl and optionally substituted 5-15 membered heteroaryl;

[0044] s and t are integers from 1 to 20; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and y is 0 or 1.

[0045] It should be understood that in formulas L2-1a to L2-7a, L 2aThe middle part of the wavy lines on both sides, (L 1 ) part does not belong to L 2a The components of L 1 Connected L 2a The end of the 2a Connection direction, L 2a The right end of -NR 8a connect.

[0046] In some embodiments, L 2a Selected from formula L2-1a, L2-5a and L2-7a,

[0047] Z is O,

[0048] R is H,

[0049] s and t are selected from integers of 3-10, such as 4, 5, 6, 7, or 8. Preferably, s and t are 4 or 8.

[0050] In some embodiments, the S atom on E is derived from a cysteine ​​(cys) residue or a disulfide bond on E;

[0051] Preferably, E is an antibody or an antibody fragment thereof, an Fc domain monomer, an Fc domain, an Fc binding peptide, albumin or an albumin-linked peptide, for example, the Fc domain monomer comprises or consists of an amino acid sequence described in any one of SEQ ID No.1-81 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0052] Preferably, it comprises or consists of an amino acid sequence selected from any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72.

[0053] In some embodiments, the conjugate has the structure of Formula IA.

[0054] In some embodiments, the conjugate has the structure of Formula IB.

[0055] In some embodiments, the structure between L and E is selected from the following structures:

[0056]

[0057] Among them, R 1a , R 2a , R 3a , R 5a , R 6a , R 7a , R 9a and R 10a are each independently selected from hydrogen, deuterium, -OC 1-6 Alkyl, hydroxyl C 1-6 Alkyl-, C 1-6 Alkyl- and C 3-8 Cycloalkyl-; or R 1a and R 2a Together they form C 3-8 Cycloalkyl;

[0058] or R 5a and R 6a Together they form C 3-8 Cycloalkyl;

[0059] or R 9a and R 10a Together they form C 3-8 Cycloalkyl, wherein the C 3-8 Cycloalkyl is optionally substituted with substituents selected from halogen and hydroxy;

[0060] R 4a and R 8a Each independently selected from H, C 1-6 Alkyl and C 3-8 Cycloalkyl;

[0061] w is 0, 1, or 2;

[0062] Preferably, wherein R 1a , R 3a , R 4a , R 5a , R 7a , R 9a and R 10a Each is hydrogen;

[0063] R 2a , R 6a and R8a are independently selected from hydrogen, C 1-6 Alkyl and C 3-8 cycloalkyl; and

[0064] w is 0;

[0065] More preferably, the structure of formula I-4a is selected from

[0066]

[0067] It should be understood that the S atom with a wavy line is connected to E, and the N atom with a wavy line is connected to L or L 2a In some embodiments, the conjugate is selected from compounds having formula C-1a to formula C-27a:

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] wherein m, n, x, y and E are as defined herein.

[0076] In some embodiments, the ratio of n to m is from 1 to 20, preferably from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0077] In some embodiments, the conjugate or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof has an average DAR value between 0.5 and 10.0, such as 2.0-4.5 or 4.8-8.5.

[0078] In some embodiments, E comprises an Fc domain monomer or an Fc domain comprising the Fc domain monomer, wherein the Fc domain monomer comprises the amino acid sequence of any one of SEQ ID No.1-81, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, or consists of the amino acid sequence.

[0079] In some embodiments, E is capable of recognizing viral surface antigens, such as CR6261, CR8020, MEDI8897, Palivizumab, SD38, etc.; or

[0080] The Fc domain monomer can be any immunoglobulin antibody subtype (e.g., IGHG1*01 (e.g., G1m(za)), IGHG1*07 (e.g., G1m(zax)), IGHG1*04 (e.g., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (e.g., G1m(fa)), IGHG2*01, IGHG2*02, IGHG2*06, IGHG3*01, IGHG3*04, IGHG3*05 , IGHG3*09,IGHG3*10,IGHG3*11,IGHG3*12,IGHG3*06,IGHG3*07,IGHG3*08,IGHG3*13,IGHG3*03,IGHG3*14,IGHG3*15,IGHG3*16,IGHG3*17,IGHG3*18,IGHG3*19,IGHG2*04,IGHG4*01,IGHG4*02,IGHG4*03) Fc domain monomer.

[0081] In some embodiments, the conjugate has the following structure:

[0082]

[0083] Wherein E is defined;

[0084] Preferably, wherein E is SEQ ID NO:67, SEQ ID NO:69 or SEQ ID NO:71;

[0085] and wherein m and n are as defined herein, m is preferably 1.

[0086] In some embodiments, The carbon to which the group (or the corresponding group) is attached (for example, in the case of Among the specific compounds of structure, it is also related to The carbon connected to the right nitrogen of the carbonyl group) is of R or S configuration, preferably of R configuration.

[0087] In some embodiments, the present invention provides a conjugate having a structure of Formula I-1, which comprises an anti-influenza virus small molecule compound conjugated to a protein (also referred to as a "protein-anti-influenza compound conjugate"), and has significant anti-influenza virus biological activity.

[0088]

[0089] Specifically, the protein-anti-influenza compound conjugate of the present invention has a linker (L 1 and L 2 ) is a small molecule (D) having anti-influenza virus activity connected to an antibody or an antibody fragment thereof, an Fc monomer, an Fc domain, an Fc connecting peptide, an albumin or an albumin connecting peptide (E) 1 The protein-anti-influenza compound conjugate of the present invention has significant anti-influenza virus activity, and also has excellent in vitro / in vivo pharmacokinetic properties and safety, a long half-life, and high clinical application prospects.

[0090] In some embodiments, the present invention provides a conjugate having formula I-1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof,

[0091]

[0092] in,

[0093] m is 1 or 2;

[0094] n is an integer selected from 1 to 20, preferably an integer from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and two n are the same;

[0095] D 1 With L 1 Covalently linked together, L 1 Through covalent bonds and L 2 Connect, L 2 Connected to -S- through a covalent bond; E is selected from an antibody or antibody fragment as defined above, an Fc domain monomer, an Fc domain, an Fc binding peptide, an albumin or

[0096] Albumin-linked peptide;

[0097] D 1 The small molecule drugs for anti-influenza virus are each independently selected from the anti-influenza virus drugs shown in formula D-1-1 to D-1-8.

[0098] Toxic compounds:

[0099]

[0100] R1 is selected from OH, NH2, -NH(=NH)NH2 and -NHC(=NH)NHR6;

[0101] R2 and R3 are each independently selected from H, OH, F, Cl and Br;

[0102] R4 is selected from -COOH, -P(=O)(OH)2 and -SO3H;

[0103] R5 is selected from -COC 1-6 Alkyl, -COC 1-6 Halogenated alkyl, -SO2C 1-6 Alkyl and -SO2C 1-6 Haloalkyl; preferably selected from -COCH3, -COCF3 and -SO2CH3;

[0104] X is selected from O or S;

[0105] R6 is selected from the following groups:

[0106]

[0107] Y is selected from the following groups, wherein "(connecting L 1 )" indicates that the group Y is connected to L 1 When N is drawn inside the ring, it indicates that N is an atom inside the ring:

[0108]

[0109] Wherein, the A ring is selected from C3-C 20 Cycloalkyl, substituted C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl, substituted C3-C 20 Cycloalkenyl, C6-C 15 Aryl, 3-20 membered heterocycloalkyl, substituted 3-20 membered heterocycloalkyl, substituted C6-C 15 Aryl and substituted 5-15 membered heteroaryl;

[0110] The R groups are each independently selected from H, deuterium, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted 3-20 membered heterocycloalkyl, optionally substituted C6-C 15 aryl and optionally substituted 5-15 membered heteroaryl;

[0111] q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0112] Preferably, D 1 The following anti-influenza virus small molecule drugs:

[0113]

[0114] L 1 To connect the drug (D 1 ) and L 2 The linker includes but is not limited to the following structures:

[0115] It should be understood that the bonds at the left and right ends of the following structure are 1 ) is connected to the oxygen atom of the middle amine group, and the bond marked by the wavy line on the middle amine group is connected to L 2 Connected;

[0116]

[0117] in,

[0118] W is selected from O, S, NR b , CH 2- or does not exist;

[0119] R a and R b are each independently selected from H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, preferably selected from H and methyl, more preferably methyl;

[0120] y1 and y2 are each independently 0, 1, 2, 3, 4, 5 or 6;

[0121] L 2 To connect L 1 and a linker of E having a structure of formula L2-1 to L2-14:

[0122]

[0123]

[0124]

[0125]

[0126] in,

[0127] Z is NR, S and O;

[0128] The R groups are each independently selected from hydrogen, deuterium, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted 3-20 membered heterocycloalkyl, optionally substituted C6-C 15 Aryl and optionally

[0129] 5-15 membered heteroaryl;

[0130] s and t are integers from 1 to 20; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0131] y is either 0 or 1.

[0132] It should be understood that in formulas L2-1 to L2-14, L2 is the portion between the wavy lines on both sides, (L 1 ) and (E)-S part does not belong to L 2 The components of L 1 L connected to E 2 to more clearly indicate the connection direction of L2, which is connected to the sulfur atom of E through a covalent bond.

[0133] It should be understood that "-S-" in Formula I-1 is formed by the reaction of the thiol group generated by the reduction of the disulfide bond in E with the linker compound.

[0134] In some embodiments,

[0135] L 1 Select from the following structures:

[0136]

[0137]

[0138] More preferably, L 1 for

[0139] In some embodiments, L 2 Selected from the formula L2-1, L2-5, L2-7, L2-8, L2-12 and L2-14, wherein Z is O,

[0140] R is H,

[0141] s and t are selected from integers of 3-10, preferably, s and t are 4 or 8.

[0142] In some embodiments, the conjugate having formula I-1 has the structure of formula I-1-1:

[0143]

[0144] Among them, E, L 1 , L 2 , m and n are as defined above.

[0145] In some embodiments, the conjugate is preferably selected from C-1 to C-12 conjugates:

[0146]

[0147]

[0148]

[0149]

[0150] wherein n and E are as defined above.

[0151] It should be understood that (E) in the above structures C-1 to C-12 means that the structural formula contains one E, which is equivalent to the case where m is 1 in the conjugate of formula I-1. The conjugates with similar structures herein can be understood in the same way.

[0152] In some embodiments, in C1 to C6, the carbon to which the NH2-CH2- on the left is attached (i.e., The carbon connected to the right nitrogen of the carbonyl group) is of R or S configuration, preferably of R configuration.

[0153] In some embodiments, in the structure of Formula I-1, the ratio of n to m is 1 to 20, preferably from 2 to 10, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0154] In some embodiments, m is 1 and n is an integer selected from 1-10.

[0155] In some embodiments, the conjugate, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotopically labeled thereof, has an average DAR between 0.5 and 10.0.

[0156] In some embodiments, E is an antibody or antibody fragment, an Fc domain monomer, an Fc domain, and / or an Fc binding peptide. In some embodiments, E comprises or consists of a sequence having at least 95% identity to an amino acid sequence described in any one of SEQ ID No. 1-81.

[0157] In one embodiment, in the compound having the conjugate including the structure of Formula I-1, the E single domain dimerizes to form the Fc domain.

[0158] In some embodiments, the antibody fragment is an antigen-binding fragment.

[0159] In some embodiments, the antibody or antibody fragment is a human, mouse, camelid, goat, sheep, rabbit, chicken, guinea pig, hamster, horse or rat antibody or antibody fragment.

[0160] In some embodiments, the antibody or antibody fragment is an IgG, IgA, IgD, IgE, or IgM antibody or antibody fragment.

[0161] In some embodiments, the antibody fragment comprises scFv, sdAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb or SMIP.

[0162] In some embodiments, the antibody or antibody fragment can recognize viral surface antigens, such as CR6261, CR8020, MEDI8897, Palivizumab, SD38, etc.

[0163] In some embodiments, the Fc domain monomer can be an antibody subtype from any immunoglobulin (e.g., IGHG1*01 (e.g., G1m(za)), IGHG1*07 (e.g., G1m(zax)), IGHG1*04 (e.g., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (e.g., G1m(fa)), IGHG2*01, IGHG2*02, IGHG2*06, IGHG3*01, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18, IGHG3*19, IGHG3*20, IGHG3*21, IGHG3*22, IGHG3*23, IGHG3*24, IGHG3*25, IGHG3*26, IGHG3*27, IGHG3*28, IGHG3*29, IGHG3*30, IGHG3*31, IGHG3*32, IGHG3*33, IGHG3*34, ​​IGHG3*35, IGHG3*36, IGHG3*37, IGHG3*38, IGHG3*39, IGHG3*31 05, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*13, IGHG3*03, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18, IGHG3*19, IGHG2*04, IGHG4*01, IGHG4*02, IGHG4*03) (such as Vidarsson et al., IgG subclasses and allotypes: from structure to effector function. Frontiers in Immunology. 5(520): 1-17 (2014)). The Fc domain monomer may contain one or more non-natural amino acid sequences as site-specific coupling sites for small molecule drugs. The Fc domain monomer may contain one or more site-specifically engineered solvent-exposed cysteine ​​or lysine residues to provide additional small molecule drug conjugation sites.

[0164] In some embodiments, L2 is connected to the S atom on E. The S atom on E is derived from a cysteine ​​(cys) residue on E or a disulfide bond.

[0165] In some embodiments, the Asn amino acid residue in E is replaced with an Ala amino acid residue to prevent conjugation at this site.

[0166] In some embodiments, E comprises additional Cys amino acid residues to increase the conjugation sites without affecting the spatial three-dimensional structure of the antibody protein.

[0167] In some embodiments, the end of E contains an additional amino acid sequence, such as a protein purification tag (e.g., a six-histidine tag), or a signal peptide sequence (e.g., the signal peptide sequence of human interleukin 2) or an MVRS (SEQ ID NO: 82) amino acid sequence or an ISAMVRS amino acid sequence (SEQ ID NO: 83)).

[0168] In some embodiments, the protein purification tag is located at the C-terminus of E. In some embodiments, the signal peptide sequence is located at the N-terminus of E.

[0169] In some embodiments, the protein purification tag is selected from a hexahistidine tag or a c-Myc tag. In some embodiments, the signal peptide is selected from a human IL-2 signal peptide sequence (e.g., MYRMQLLSCIALSLALVTNS (SEQ ID NO: 84)), a human serum albumin signal sequence (e.g., MKWVTFISLLFLFSSAYS (SEQ ID NO: 85)), a mouse heavy chain MIgG Vh signal sequence (e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 86)).

[0170] In some embodiments, the N-terminus of E further comprises a hinge region or a portion of a hinge region.

[0171] In some embodiments, E comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-81.

[0172] In some embodiments, E comprises or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1-81.

[0173] In some embodiments, E comprises or consists of the following amino acid sequence:

[0174] (i) is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-81; and

[0175] (ii) comprising the N-terminal addition sequence, mutation and / or C-terminal addition sequence described for each SEQ ID NO: in the "SEQ ID NO: 1-81 and description thereof" section of the present disclosure.

[0176] In some embodiments, E further comprises a linker. In one embodiment, a linker refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination. In one embodiment, the linker comprises the amino acid sequence (G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6 or 7. In one embodiment, the linker is GGGGS. In one embodiment, the linker comprises the amino acid sequence TS(G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In one embodiment, the linker comprises the amino acid sequence G(G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7.

[0177] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 1. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 1, and optionally, has an IL2 signal sequence at its N-terminus.

[0178] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 2. In some embodiments, E comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0179] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 3. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3, and optionally, an IL2 signal sequence at its N-terminus and an N-terminal MVRS amino acid sequence.

[0180] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 4. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 4, and optionally, having an MVRS amino acid sequence at its N-terminus.

[0181] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 5. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 5, and optionally, an IL2 signal sequence at its N-terminus and a six-histidine tag at its C-terminus.

[0182] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 6. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 6, and optionally, a six-histidine tag at its C-terminus.

[0183] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 7. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 7, and optionally, an IL2 signal sequence and an MVRS amino acid sequence at the N-terminus and a six-histidine tag at the C-terminus.

[0184] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 8. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 8, and optionally, having an MVRS amino acid sequence at its N-terminus and a six-histidine tag at its C-terminus.

[0185] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 9. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 9, and optionally, an IL2 signal sequence and an MVRS amino acid sequence at its N-terminus, two additional cysteines (corresponding to the * positions of SEQ ID NO: 9) in the hinge region and a six-histidine tag at the C-terminus.

[0186] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 10. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 10, and optionally, an MVRS amino acid sequence at its N-terminus, two additional cysteines in the hinge region (corresponding to the * positions of SEQ ID NO: 10) and a six-histidine tag at the C-terminus.

[0187] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 11. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 11, and optionally, having an MVRS amino acid sequence at its N-terminus, and the hinge region contains two additional cysteines (corresponding to the * positions of SEQ ID NO: 11).

[0188] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 12. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 12, and optionally, having an IL2 signal sequence and an Asn to Ala substitution ((corresponding to the * position of SEQ ID NO: 12)) at its N-terminus and a six-histidine tag at its C-terminus.

[0189] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 13. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 13, and optionally, it contains an Asn to Ala substitution (corresponding to the * position of SEQ ID NO: 13) and has a six histidine tag at the C-terminus.

[0190] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 14. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 14, and optionally, an IL2 signal sequence and an MVRS amino acid sequence at its N-terminus, containing an Asn to Ala substitution (corresponding to the * position of SEQ ID NO: 14) and a six-histidine tag at its C-terminus.

[0191] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 15. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 15, and optionally, having an MVRS amino acid sequence at its N-terminus, containing an Asn to Ala substitution (corresponding to the * position of SEQ ID NO: 15) and a six-histidine tag at its C-terminus.

[0192] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 16. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 16, and optionally, having a human serum albumin signal sequence at its N-terminus and having an N-terminal ISAMVRS amino acid sequence.

[0193] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 17. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 17, and optionally, having a human serum albumin signal sequence at its N-terminus and having an N-terminal ISAMVRS amino acid sequence, containing a C-terminal G4S linker and a C-terminal c-Myc tag.

[0194] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 18. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 18, and optionally, having an ISAMVRS amino acid sequence at its N-terminus, a G4S linker at its C-terminus and a c-Myc tag at its C-terminus.

[0195] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 19. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 19, and optionally, having a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus, and containing a lysine to serine change (corresponding to the * position of SEQ ID NO: 19) to prevent coupling at this site.

[0196] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 20. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 20, and optionally, having an ISAMVRS amino acid sequence at its N-terminus and containing a lysine to serine (corresponding to the * position of SEQ ID NO: 20) change to prevent coupling at this site.

[0197] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 21. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 21, and optionally, having a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus, containing a lysine to serine change (corresponding to the * position of SEQ ID NO: 21) to prevent coupling at this site, and having a G4S linker and a c-Myc tag at its C-terminus.

[0198] In some embodiments, E includes the amino acid sequence in SEQ ID NO: 22. In some embodiments, E includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 22, and optionally, its N-terminus has an ISAMVRS amino acid sequence containing a lysine to serine change (corresponding to the * position of SEQ ID NO: 22) to prevent coupling at this site, and the C-terminus has a G4S linker and a c-Myc tag.

[0199] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 23. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 23, and optionally, having a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus, containing an Asn to Ala change (corresponding to the * position of SEQ ID NO: 23), and having a G4S linker and a c-Myc tag at its C-terminus.

[0200] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 24. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 24, and optionally, having an ISAMVRS amino acid sequence at its N-terminus, containing an Asn to Ala change (corresponding to the * position of SEQ ID NO: 24) and a G4S linker and a c-Myc tag at its C-terminus.

[0201] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 25. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 25, and optionally, having a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus, containing H310A and H435A changes to prevent FcRn binding, and having a G4S linker and a c-Myc tag at its C-terminus.

[0202] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 26. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 26, and optionally, its N-terminus has an ISAMVRS amino acid sequence containing H310A and H435A changes to prevent FcRn binding, and its C-terminus has a G4S linker and a c-Myc tag.

[0203] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 27. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 27, and optionally, a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus, and a G4S linker and a mutant (lysine to phenylalanine, corresponding to the bold position of SEQ ID NO: 27) and a c-Myc tag at its C-terminus.

[0204] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 28. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 28, and optionally, an ISAMVRS amino acid sequence at its N-terminus and a G4S linker and a mutant (lysine to phenylalanine, corresponding to the bold position of SEQ ID NO: 28) and a c-Myc tag at its C-terminus.

[0205] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 29. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 29, and optionally, having a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus, containing an Asn to Ala substitution and having a G4S linker and a mutant (lysine to phenylalanine, corresponding to the bold positions of SEQ ID NO: 29) and a c-Myc tag at its C-terminus.

[0206] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 30. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 30, and optionally, its N-terminus has an ISAMVRS amino acid sequence containing an Asn to Ala substitution (corresponding to the * position of SEQ ID NO: 30) and the C-terminus has a G4S linker and a mutant (lysine to phenylalanine, corresponding to the bold position of SEQ ID NO: 30) and a c-Myc tag.

[0207] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 31. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 31, and optionally, having a human serum albumin signal sequence at its N-terminus, having an allotype of G1m (fa), and having a G4S linker and a mutant (lysine to phenylalanine, corresponding to the bold position of SEQ ID NO: 31) and a c-Myc tag at its C-terminus.

[0208] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 32. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 32, and optionally, having a human serum albumin signal sequence at its N-terminus, and having the allotype G1m(fa).

[0209] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 33. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 33, and optionally, its N-terminus has an MVRS amino acid sequence and contains a YTE triple mutation.

[0210] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 34. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 34, and optionally, having a human serum albumin signal sequence at its N-terminus, containing the hinge region EPKSS amino acid sequence of mature human Fc-IgG1, and containing a cysteine ​​to serine change (corresponding to the # position of SEQ ID NO: 34) and the allotype G1m (fa).

[0211] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 35. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 35, and optionally, having a murine IgG signal sequence at its N-terminus, removing the EPKSSD amino acid sequence of the hinge region of mature human Fc-IgG, and having an allotype of G1m (fa).

[0212] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 36. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 36, and optionally, the hinge region EPKSSD amino acid sequence of mature human Fc-IgG is removed from its N-terminus, has an allotype of G1m (fa) and contains a YTE triple mutation.

[0213] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 37. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 37, and optionally, the hinge region EPKSSD amino acid sequence of mature human Fc-IgG is removed from its N-terminus, has a LS double mutation and has an allotype G1m (fa).

[0214] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 38. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 38, and optionally, having a human serum albumin signal sequence at its N-terminus, containing a YTE triple mutation, having an allotype of G1m (fa) and having a G4S linker and a c-Myc tag at its C-terminus.

[0215] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 39, and optionally, having at the position corresponding to X as set forth in SEQ ID NO: 39 the amino acid defined by SEQ ID NO: 39 at that position.

[0216] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 40, and optionally, having at the position corresponding to X as shown in SEQ ID NO: 40 the amino acid defined in SEQ ID NO: 40 at that position.

[0217] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 41, and optionally, having a YTE triple mutation and having an amino acid defined in SEQ ID NO: 41 at the position corresponding to X as shown in SEQ ID NO: 41.

[0218] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, E comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 42, and optionally, has a YTE triple mutation and has the allotype G1m(fa)

[0219] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, E comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 43, and optionally, has a YTE triple mutation and has the allotype G1m(f).

[0220] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 44, and optionally, having a LS double mutation and having an amino acid defined in SEQ ID NO: 44 at the position corresponding to X as shown in SEQ ID NO: 44.

[0221] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, E comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 45, and optionally, has a LS double mutation and has the allotype G1m(fa).

[0222] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, E comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 46, and optionally, has a LS double mutation and has the allotype G1m(f).

[0223] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 47. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 47, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 47), and having an amino acid defined by SEQ ID NO: 47 at the X position corresponding to SEQ ID NO: 47.

[0224] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 48. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 48, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 48), and containing the allotype G1m (fa).

[0225] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 49. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 49, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 49), and containing the allotype G1m(f).

[0226] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 50. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 50, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 50), having M428L and N434S mutations, and containing the allotype G1m (fa).

[0227] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 51. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 51, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 51), having M428L and N434S mutations, and containing allotype G1m(f).

[0228] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 52. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 52, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 52), having a YTE triple mutation, and containing the allotype G1m (fa).

[0229] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 53. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 53, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 53), having a YTE triple mutation, and containing the allotype G1m(f).

[0230] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 54. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 54, and optionally, having a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, having M428L, N434S mutations, having a G4S linker and a c-Myc tag at its C-terminus, and containing an allotype G1m(f).

[0231] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 55. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 55, and optionally, having a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, having M428L, N434S mutations, having a G4S linker and a c-Myc tag at its C-terminus, and containing an allotype G1m (fa).

[0232] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 56. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 56, and optionally, having a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, having a YTE triple mutation, having a G4S linker and a c-Myc tag at the C-terminus and containing an allotype G1m (f).

[0233] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 57. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 57, and optionally, having a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, having a YTE triple mutation, having a G4S linker and a c-Myc tag at the C-terminus and containing an allotype G1m (fa).

[0234] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 58. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 58, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position), a G4S linker and an IgA peptide tag at its C-terminus and containing an allotype G1m (fa).

[0235] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 59. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 59, and optionally, having a mouse heavy chain MIgG Vh signal sequence at its N-terminus, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 59), having M428L, N434S mutations, having a G4S linker and an IgA peptide tag at the C-terminus and containing an allotype G1m (fa).

[0236] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 60. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 60, and optionally, having an amino acid defined in SEQ ID NO: 60 at a position corresponding to Z or X as shown in SEQ ID NO: 60.

[0237] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 61. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 61, and optionally, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 61), and having at the X position corresponding to that shown in SEQ ID NO: 61 the amino acid defined in SEQ ID NO: 61 at that position.

[0238] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 62, and optionally, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 62), and having at the X position corresponding to SEQ ID NO: 62 the amino acid defined at that position by SEQ ID NO: 62.

[0239] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 63. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 63, and optionally, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 63) and having the allotype G1m(f).

[0240] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 64. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 64, and optionally, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 64) and having the allotype G1m(fa).

[0241] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 65. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 65, and optionally, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 65), M428L, N343S mutations, and having the allotype G1m(fa).

[0242] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 66. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 66, and optionally, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 66), M428L, N343S mutations, and having the allotype G1m(f).

[0243] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 67. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 67, and optionally, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 67) and a YTE triple mutation, and having the allotype G1m(fa).

[0244] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 68. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 68, and optionally, having a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 68) and a YTE triple mutation, and having the allotype G1m(f).

[0245] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 69. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 69, and optionally, has a YTE triple mutation and has the allotype G1m(fa).

[0246] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 70. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 70, and optionally, has a YTE triple mutation and has the allotype G1m(fa).

[0247] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 71, and optionally, it has M428L, N343S mutations, and has allotype G1m (fa).

[0248] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 72. In some embodiments, E comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 72.

[0249] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 73, and optionally, it has M428L, N343S mutations, and has allotype G1m(f).

[0250] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 74. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 74, and optionally, it has GAALIE, M428L, N343S mutations, and has the allotype G1m (fa).

[0251] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 75. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 75, and optionally, it has GAALIE, M428L, N343S mutations, and has allotype G1m(f).

[0252] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 76. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 76, and optionally, having a cysteine ​​to serine change (#), GAALIE, M428L, N343S mutation, and having the allotype G1m (fa).

[0253] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 77. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, and optionally, having a cysteine ​​to serine change (#), GAALIE, M428L, N343S mutation, and having the allotype G1m (f).

[0254] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 78. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 78, and optionally, has a cysteine ​​to serine change (#), a DHS triple mutation, and has the allotype G1m (fa).

[0255] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 79. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 79, and optionally, has a cysteine ​​to serine change (#), a DHS triple mutation, and has the allotype G1m (f).

[0256] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 80. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 80, and optionally, has a DHS triple mutation and has the allotype G1m(fa).

[0257] In some embodiments, E comprises the amino acid sequence in SEQ ID NO: 81. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 81, and optionally, has a DHS triple mutation and has the allotype G1m(f).

[0258] In any of the embodiments described herein, wherein E comprises an Fc domain monomer (e.g., having a sequence of any of SEQ ID NOs: 1-81) comprising a triple mutation corresponding to M252Y / S254T / T265E (YTE). One familiar with the art will understand that "corresponding to" an amino acid site that is a specific corresponding position obtained after sequence homology alignment using sequence analysis software (e.g., but not limited to DNA Star, Vector NTI, etc.). For example, any of SEQ ID NOs: 1-81 may comprise a mutation comprising YTE.

[0259] In any of the embodiments described herein, wherein E comprises an Fc domain monomer (e.g., having a sequence of any of SEQ ID NOs: 1-81), it comprises a double mutant corresponding to M428L / N434S (LS). One familiar with the art will understand that "corresponding to" an amino acid site that is a specific corresponding position obtained after sequence homology alignment using sequence analysis software (e.g., but not limited to DNA Star, Vector NTI, etc.). For example, any of SEQ ID NOs: 1-81 may comprise a mutation comprising LS.

[0260] In any of the embodiments described herein, wherein E comprises an Fc domain monomer (e.g., having a sequence of any of SEQ ID NOs: 1-81), it comprises a double mutant corresponding to N434H. One familiar with the art will understand that "corresponding to" an amino acid position that is a specific corresponding position obtained after sequence homology alignment using sequence analysis software (e.g., but not limited to DNA Star, Vector NTI, etc.). For example, any of SEQ ID NOs: 1-81 may comprise a mutation comprising N434H.

[0261] In any of the embodiments described herein, wherein E comprises an Fc domain monomer (e.g., having a sequence of any of SEQ ID NOs: 1-81), it comprises a double mutant corresponding to C220S. One familiar with the art will understand that "corresponding to" an amino acid site that is a specific corresponding position obtained after sequence homology alignment using sequence analysis software (e.g., but not limited to DNA Star, Vector NTI, etc.). For example, any of SEQ ID NOs: 1-81 may comprise a mutation comprising C220S.

[0262] In any of the embodiments described herein, wherein E comprises a fragment of an Fc domain monomer (e.g., a fragment of an Fc domain monomer from the sequence of any of SEQ ID NOs: 1-81), and is at least 25 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more), at least 50 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75 or more) or at least 75 (75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 or more) consecutive amino acids.

[0263] In one embodiment, the conjugate has the following structure:

[0264]

[0265]

[0266] wherein n is as defined above.

[0267] In one aspect, the present invention provides a linker-payload compound, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled compound thereof.

[0268] In some embodiments, the present invention provides a compound having a structure of Formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof,

[0269]

[0270] The symbols (such as L, R 2a , R 3a , R 5a , R 6a , R 7a , R 8a , R 9a , R 10a and w) as defined herein;

[0271]

[0272] The symbols (such as L, z, q, R 1’ , R 2’ , R 3’ , R 4’ and R 5’ ) as defined herein.

[0273] In some embodiments, the present invention provides a compound having a structure of Formula I-2, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof,

[0274]

[0275] Where L 1 and D 1 As defined in formula I-1 above;

[0276] L 3 Select from the following structures:

[0277]

[0278]

[0279]

[0280]

[0281] The variables such as Z, R, s, y, t are as above for L 2 It should be understood that in formulas L3-1 to L3-14, L 1 Part does not belong to L 3 The components of (L 1 ) is used to indicate the end and L 1 connect.

[0282] In some embodiments, the compound includes the compound of formula I-2 and preferably has the structure of formula C-inter-1 to formula C-inter-12:

[0283]

[0284]

[0285]

[0286] In some embodiments, The carbon to which the group is attached or The carbon to which the corresponding group is attached is of R or S configuration, preferably R configuration

[0287] In some embodiments, in C-inter-1 to C-inter-6, the carbon to which the left-side NH2-CH2- is attached is of R or S configuration, preferably of R configuration.

[0288] On the other hand, the present invention provides a pharmaceutical composition comprising a conjugate of Formula I-1, IA or IB, a compound of Formula I-2, I-5a or I-6a as described above, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists), and optionally a pharmaceutically acceptable excipient.

[0289] On the other hand, the present invention provides a drug combination comprising a conjugate of Formula I-1, IA or IB, a compound of Formula I-2, I-5a or I-6a as described above, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0290] On the other hand, the present invention provides a method for preventing or treating patients infected with a virus or patients who may be at risk of being infected with a virus, comprising the step of administering to the patient, for example by injection, an effective dose of any one of the conjugates of Formula I-1, IA or IB as described above, compounds of Formula I-2, I-5a or I-6a, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs or isotope labels thereof.

[0291] Use of the conjugate of formula I-1, IA or IB, the compound of formula I-2, I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof in the preparation of a medicament, wherein the medicament is used to prevent or treat viral infection in patients with viral infection or patients who may be at risk of viral infection.

[0292] In some embodiments, the viral infection is an infection caused by an influenza virus or a parainfluenza virus.

[0293] In some embodiments, the viral infection is an infection caused by influenza virus A, B, or C, or a parainfluenza virus.

[0294] In some embodiments, the patient who is infected with a virus or may be at risk of being infected with a virus may be a patient with a deficient immune system.

[0295] In some embodiments, the patient who is infected with a virus or may be at risk of being infected with a virus may be a patient who is receiving or is about to receive immunosuppressant therapy.

[0296] In some embodiments, the patient who is or may be at risk of being infected by a virus may be a patient diagnosed with a disease that causes immunosuppression.

[0297] In some embodiments, the patient diagnosed with a disease causing immunosuppression has cancer or acquired immunodeficiency syndrome.

[0298] In some embodiments, the patient diagnosed with a cancer disease causing immunosuppression has leukemia, lymphoma, humoral immunity deficiency, T-cell deficiency, complement deficiency, or multiple myeloma.

[0299] In some embodiments, the patient is a patient who is currently receiving or is about to receive a hematopoietic stem cell transplant.

[0300] In some embodiments, the patient is a patient who is currently receiving or is about to receive an organ transplant.

[0301] In some embodiments, the patient may be at risk for secondary infection.

[0302] A method for preventing the risk of secondary infection caused by influenza virus infection in a patient, by administering to the patient, for example by injection, an effective dose of any conjugate of Formula I-1, IA or IB as described above, a compound of Formula I-2, I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof.

[0303] In some embodiments, the secondary infection is a respiratory tract infection.

[0304] In some embodiments, the secondary infection is associated with pneumonia.

[0305] In some embodiments, the secondary infection is a bacterial, viral, or fungal infection.

[0306] In some embodiments, the bacterial infection is an infection caused by methicillin-resistant Staphylococcus aureus.

[0307] In some embodiments, the bacterial infection is an infection caused by Streptococcus pneumoniae.

[0308] In some embodiments, the conjugate of Formula I-1, IA or IB, the compound of Formula I-2, I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof can be administered by intramuscular injection, intravenous injection, intradermal injection, intraarterial injection, intraperitoneal injection, intralesional injection, intracranial injection, intraarticular injection, intrapleural injection, intratracheal injection, intraprostatic injection, intranasal injection, intravitreal injection, intravaginal injection, intrarectal injection, local, intratumor injection, intraperitoneal injection, subcutaneous injection, subconjunctival injection, intracapsular injection, mucosal injection, intrapericardial injection, intraumbilical injection, intraocular injection, oral, topical inhalation, injection or infusion.

[0309] In some embodiments, the conjugate of Formula I-1, IA or IB, the compound of Formula I-2, I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof is administered in combination with another therapeutic agent or used in combination to prepare a drug.

[0310] In some embodiments, the additional therapeutic agent is an antiviral drug.

[0311] In some embodiments, the antiviral drug is baloxavir, pimodivir, oseltamivir, zanamivir, peramivir, laninamivir, amantadine, MEDI8852, or rimantadine.

[0312] In some embodiments, the additional medication used by the patient is an antiviral vaccine.

[0313] In some embodiments, the antiviral drug and the conjugate of Formula I-1, IA or IB, the compound of Formula I-2, I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof are administered sequentially, for example, by injection, to the patient.

[0314] In some embodiments, the antiviral drug and the conjugate of Formula I-1, IA or IB, the compound of Formula I-2, I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof are administered to the patient simultaneously, for example, by injection.

[0315] Effects of the invention:

[0316] The conjugate of formula I-1, IA or IB, the compound of formula I-2, I-5a or I-6a or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof of the present invention has the following advantages:

[0317] It has a significant antiviral activity.

[0318] High activity against drug-resistant viral strains; and / or

[0319] It has excellent in vitro / in vivo pharmacokinetic properties and safety, such as a long half-life, a higher in vivo exposure, which can reduce the frequency of dosing and increase patient compliance, and has high prospects for clinical application.

[0320] Preparation of the conjugate of the present invention

[0321] In one aspect, the invention provides methods of making the conjugates described herein.

[0322] In some embodiments, the present invention provides a method for preparing a conjugate of Formula IA herein,

[0323]

[0324] The symbols (such as L, R 1a , R 2a , R 3a , R 4a , R 5a , R 6a , R 7a , R 8a , R 9a , R 10a , w, n, m and E) are as defined herein;

[0325] The method comprises the following steps:

[0326] The first step is to change (E)m Dissolved in a buffer, wherein E and m are as defined herein, add a reducing agent (such as TCEP, DTT) to convert (E) m The disulfide bond in is reduced to obtain a compound of formula II-1 containing a thiol residue,

[0327]

[0328] wherein E, m and n are as defined in the text;

[0329] In the second step, a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-5a is added to the compound of formula II-1,

[0330]

[0331] Among them, each symbol (such as L, R 2a , R 3a , R 5a , R 6a , R 7a , R 8a , R 9a , R 10a and w) as defined in Formula IA,

[0332] The Michael addition reaction is carried out at a pH of 5.5 to 7.0 (e.g., 5.8 to 6.8, 6.0 to 6.6) for 0.5 to 10 hours, preferably 0.5 to 2 hours, and then 3 to 7 molar equivalents of a buffer solution of the compound of formula I-5a are added in batches (e.g., 1 to 2 batches) and the reaction is continued for 0.5 to 10 hours (preferably 0.5 to 2 hours) to obtain a conjugate of formula I-3b.

[0333]

[0334] Among them, each symbol (such as L, R 2a , R 3a R 5a , R 6a , R 7a , R 8a , R 9a , R 10a , w, n, m and E) are as defined in Formula IA;

[0335] The third step is to adjust the pH of the solution containing the conjugate of formula I-3b to 7-8 (e.g., 7.1-7.9, 7.3-7.7), and perform a hydrolysis reaction to obtain the final conjugate of formula IA;

[0336] or,

[0337] A method for preparing the conjugate of formula IB described herein,

[0338]

[0339] Among them, each symbol (such as L, z, q, x, y, R 1’ , R 2’ , R 3’ , R 4’ , R 5’ , R 8a and E) as defined herein;

[0340] The method comprises the following steps:

[0341] The first step is to change (E) m Dissolved in a buffer, wherein E and m are as defined herein, add a reducing agent (such as TCEP, DTT) to convert (E) m The disulfide bond in is reduced to obtain a compound of formula II-1 containing a thiol residue,

[0342]

[0343] wherein E, m and n are as defined in the text;

[0344] In the second step, a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-6a is added to the compound of formula II-1,

[0345]

[0346] Among them, each symbol (such as L, z, q, R 1’ , R 2’ , R 3’ , R 4’ , R 5’ and R 8a ) is as defined in Formula IB,

[0347] The Michael addition reaction is carried out at a pH of 5.5 to 8 (eg, 5.8 to 7.6, 6.0 to 7.5) to obtain an IB conjugate.

[0348] In some embodiments, the present invention provides two methods for preparing conjugates of I-1.

[0349] Method 1 includes the following steps:

[0350]

[0351] in, Corresponding to L 3 , where (L 1 ) indicates L 2 'With L 1 Connect, L 1 Not part of the above structure;

[0352] in, Corresponding to L 2 As explained above, the structure between the two wavy lines corresponds to L 2 .

[0353] Among them, variables such as n, m, E, D 1 , L 1 , L 2 , L 3 As defined above, preferably, wherein L 3 Selected from formula L3-1 to L3-7, L 2 Selected from formula L2-1 to L2-7;

[0354] The first step is to change (E) m Dissolve in buffer and add reducing agent (such as TCEP, DTT) to (E) m Reducing the disulfide bonds in to obtain a compound of formula II-1 containing a thiol residue;

[0355] The second step is to add a buffer containing 6 to 14 molar equivalents of the compound of formula I-2a to the compound of formula II-1, and carry out Michael addition reaction at pH 5.5 to 7.0 (e.g., 5.8 to 6.8, 6.0 to 6.6) for 0.5 to 10 hours, preferably 0.5 to 2 hours, and then add 3 to 7 molar equivalents of the compound of formula I-2a in portions (e.g., 1 to 2 times) to continue the reaction for 0.5 to 10 hours, preferably 0.5 to 2 hours, to obtain a conjugate of formula I-3;

[0356] In the third step, the pH of the solution containing the I-3 conjugate is adjusted to 7-8 (eg, 7.1-7.9, 7.3-7.7), and a hydrolysis reaction is performed to obtain the final conjugate of formula I-1a.

[0357] In some embodiments, the buffer has a pH of 5.0-7.5, eg, 5.5-7.0, 6.0-6.5, 5.5-6.5.

[0358] In some embodiments, the buffer is selected from the group consisting of phosphate buffer, citrate buffer, acetate buffer, and histidine buffer.

[0359] In some embodiments, the reaction in the step is carried out at 10-30°C, such as 15-28°C, 20-25°C, such as 22°C.

[0360] In some embodiments, the reducing agent and (E) m The molar ratio is 30:1-1:1, such as 25:1-2:1, 20:1-5:1, 15:1-8:1, such as 10:1.

[0361] In some embodiments, the compound of formula II-1 used in the second step is present in the reaction solution after the first step reaction is completed.

[0362] In some embodiments, the solution comprising the conjugate of Formula 1-3 in the third step is a solution of the conjugate of Formula 1-3 in a buffer (eg, a buffer as described above).

[0363] In some embodiments, the solution containing the conjugate of formula I-3 in the third step is the reaction solution after the reaction in the second step is completed.

[0364] The inventors unexpectedly found that in the second step, adding the raw material (compound of formula I-2a) in batches can make the DAR value of the final product more uniform, and obtain a product with improved quality. In addition, in the second step, adjusting the pH to acidic conditions, such as 5.5-7.0, 5.8-6.8, 6.0-6.6, also helps to improve the uniformity of the DAR value of the final product.

[0365] Method 2 includes the following steps:

[0366]

[0367] in, Corresponding to L 3 ,

[0368] in, Corresponding to L 2 ,

[0369] Among them, n, m, E, D 1 , L 1 , L 2 , L 3 As defined above, preferably, wherein L 3 Selected from formula L3-8 to L3-14, L 2 Selected from formula L2-8 to L2-14;

[0370] In the first step, (E) m Dissolve in buffer and add reducing agent (such as TCEP, DTT) to (E) m Reducing the disulfide bonds in to obtain a compound of formula II-1 containing a thiol residue;

[0371] In the second step, a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-2b is added to the compound of formula II-1, and Michael addition reaction is carried out at pH 5.5 to 8 (eg, 5.8 to 7.6, 6.0 to 7.5) to obtain the I-1b conjugate.

[0372] In some embodiments, the buffer, reaction temperature, reducing agent and (E)m The molar ratio is as defined in Method 1.

[0373] In some embodiments, the compound of formula II-1 used in the second step is present in the reaction solution after the first step reaction is completed.

[0374] Pharmaceutical composition

[0375] In some embodiments, the present invention provides a pharmaceutical composition comprising a conjugate of formula I-1, IA or IB, a conjugate or compound of a compound of formula I-2, I-5a or I-6a or a subformula thereof, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof. For the sake of simplicity, a conjugate of formula I-1, IA or IB, a conjugate or compound of a compound of formula I-2, I-5a or I-6a or a subformula thereof, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof may be referred to as a "compound of the present invention". In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition comprises a compound of the present invention, and a combination of one or more other therapeutic agents.

[0376] As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0377] For the use of pharmaceutically acceptable excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0378] The pharmaceutical compositions of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0379] Medicaments containing the compounds of the present invention can be prepared by mixing the compounds of the present invention having the desired degree of purity with one or more optional pharmaceutically acceptable excipients, preferably in the form of a lyophilized preparation or an aqueous solution.

[0380] definition:

[0381] The following are definitions of the terms involved in the present invention. In addition, those skilled in the art can also understand the following terms in combination with the prior art. For undefined terms, they have the same meaning as the common understanding of those skilled in the art in the field to which the present invention belongs.

[0382] The term "inhibit neuraminidase activity" as used herein refers to an IC of less than or equal to 1000 nM. 50 For example, the neuraminidase inhibition assay described in the examples herein is used. In some embodiments, the neuraminidase activity is inhibited with an IC of less than or equal to 100 nM or less than or equal to 10 nM. 50 It refers to a compound that inhibits neuraminidase activity.

[0383] As used herein, the term "inhibit viral growth" refers to an EC of less than or equal to 1000 nM. 50 For example, the activity of inhibiting viral growth is less than or equal to 100 nM or less than or equal to 10 nM EC. 50 Represents compounds that have the activity of inhibiting viral growth.

[0384] The term "Fc domain monomer" as used herein refers to a polypeptide chain or a functional fragment thereof (e.g., a fragment capable of forming a dimer with another Fc domain monomer and binding to an Fc receptor) having the following characteristics, wherein the polypeptide chain comprises a second and a third antibody constant region (CH2 and CH3) and optionally a fourth antibody constant region, and in some cases, the Fc domain monomer further comprises at least one hinge region or a portion of a hinge region. The Fc domain monomer may be any immunoglobulin antibody type, including IgG, IgE, IgM, IgA, IgD. In addition, the Fc domain monomer may be any IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). For example, in a natural antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or comprises the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise specified herein, the amino acid numbering in the IgG or Fc domain monomer is based on the EC numbering system for antibodies (this system is also called Kabat EU, or EU numbering system, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Services, National Institute of Health, Bethesda, MD 1991). The Fc domain monomer can be of any species origin, such as synthetic, human, mouse, rat, llama, etc. The Fc domain monomer can contain one or more non-natural amino acid sequences as site-specific conjugation sites for small molecule drugs. The Fc domain monomer can contain one or more solvent-exposed cysteine ​​or lysine residues that have been site-specifically modified to provide additional small molecule drug conjugation sites.

[0385] The term "Fc domain" as used herein refers to a dimer formed by the interaction between two Fc domain monomers through the hinge region, and / or CH2, and / or CH3 antibody constant regions. In some embodiments, there are one or more disulfide bonds between the monomers of the dimer. The Fc domain can be of any species origin, such as synthetic, human, mouse, rat, llama, etc. The Fc domain can contain one or more non-natural amino acid sequences as site-specific coupling sites for small molecule drugs. The Fc domain can contain one or more solvent-exposed cysteine ​​or lysine residues that have been subjected to site-specific modification to provide additional small molecule drug coupling sites.

[0386] As used herein, the term "covalently linked" refers to two parts of a conjugate being linked to each other by a covalent bond formed between two atoms in the two parts of the conjugate.

[0387] The term "Fc binding peptide" as used herein refers to a polypeptide consisting of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 40, 10 to 30, or 10 to 20) consecutive amino acid sequences, and has affinity for an Fc domain (e.g., any Fc domain described herein) and has the function of binding to the Fc domain. The Fc binding peptide can be derived from any species, such as synthetic, human, mouse, rat, llama, etc. The Fc binding peptide can contain one or more non-natural amino acid sequences as site-specific coupling sites for small molecule drugs. The Fc binding peptide can contain one or more solvent-exposed cysteine ​​or lysine residues that have been site-specifically modified to provide additional small molecule drug coupling sites.

[0388] The term "solvent exposure" as used herein refers to an amino acid residue that has solvent molecules surrounding the protein or polypeptide. The solvent-exposed amino acid molecules may be naturally occurring or artificially modified (including natural amino acids and non-natural amino acids). In some embodiments, the site modification does not affect the spatial three-dimensional structure of the protein.

[0389] As used herein, the term "% identity" refers to the percentage of amino acid residues in a protein or polypeptide sequence that are identical to those of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent identity (i.e., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment for the purpose of sequence alignment percent identity may be achieved in a variety of ways within the skill of the art, such as, but not limited to, publicly available computer software such as BLAST, ALIGN, MegAlign (DNA Star) software, etc.

[0390] As used herein, the term "treat" or "treat" refers to therapeutic treatment of an individual after infection with a virus. In some embodiments, therapeutic treatment can slow the progression of the viral infection, alleviate symptoms of the individual, and / or eliminate the viral infection.

[0391] The term "effective amount" or "effective dose" refers to an amount or dose of an antibody or fragment or composition or combination of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration in single or multiple doses to the patient.

[0392] Individually or in combination (e.g. C1-C20 The term "C1-C 20 The term "alkyl" refers to a saturated straight or branched chain alkyl group containing 1 to 20, for example 1 to 15, 1 to 10 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), especially 1 to 6 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, n-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3,-dimethyl-2-butyl, etc. Preferably, "C 1-20 The "alkyl group" is any one of methyl, ethyl, isopropyl and tert-butyl.

[0393] The term "C2-C 20 "Alkenyl" means a straight or branched alkyl group as defined above, but containing 1 or more, e.g. 1 to 10, e.g. 1, 2, 3, 4 or 5 double bonds, but does not include alkynyl. Exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, isobutenyl, n-pentenyl, 2-pentenyl, 3-pentenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 3-methyl-1-butenyl, 2-methyl-1-butenyl, n-hexenyl, 2-hexenyl, 3-hexenyl, 2-methyl-2-pentenyl, and the like.

[0394] Individually or in combination (e.g. C3-C 20 The term "C 3-20 "Cycloalkyl" means a saturated cycloalkyl group having 3-20, for example 3-15, 3-10 (e.g. 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), especially 3-6 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. In particular, "C 3-7 "Cycloalkyl" refers to cyclopropyl, cyclopentyl, cyclohexyl, etc.

[0395] The term "C3-C 20 "Cycloalkenyl" means a cycloalkyl group having 3-20, such as 3-15, 3-10 (e.g. 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), especially 3-6 carbon atoms, which contains 1 or more, such as 1-10, such as 1, 2, 3, 4 or 5 double bonds, but does not contain an alkynyl group and is not an aromatic group. Exemplary cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, etc. Particularly "C 3-7"Cycloalkenyl" means cyclopropenyl, cyclopentenyl, cyclohexenyl and the like.

[0396] The term "halogen", alone or in combination, denotes fluorine, chlorine, bromine or iodine, in particular fluorine, chlorine or bromine.

[0397] The term "haloalkyl", alone or in combination, refers to an alkyl group as defined above substituted by one or more (eg 1, 2, 3, 4 or 5) halogens.

[0398] The term "amino" alone or in combination means a primary amino group (-NH2), a secondary amino group (-NH-) or a tertiary amino group.

[0399] The term "carbonyl", also known as "-C(=O)-", refers to a divalent group consisting of only one carbon atom and one oxygen atom, the carbon atom and the oxygen atom are connected by a double bond, and the carbon atom in its own structure is also connected to the other two fragments by single bonds.

[0400] The term "heterocycloalkyl", also known as "heterocyclic group", refers to a saturated or partially unsaturated (containing 1 or 2 double bonds) non-aromatic cyclic group composed of carbon atoms and heteroatoms such as nitrogen, oxygen or sulfur. This cyclic group can be a monocyclic or bicyclic group. In the present invention, the heterocycloalkyl can be a 3-20-membered heterocycloalkyl, such as a 3-15-membered, 3-10-membered, 3-7-membered, 3-6-membered, 5-7-membered, 4-6-membered heterocycloalkyl. The number of carbon atoms in the heterocycloalkyl can be 2-16, such as 2-11, and the number of heteroatoms can be 1 or more, preferably 1, 2, 3 or 4. The nitrogen, carbon or sulfur atoms in the heterocycloalkyl can be optionally oxidized. The hydrogen atoms on the "heterocycloalkyl" are independently and optionally substituted by one or more substituents described in the present invention. The "heterocycloalkyl" can be linked to the parent molecule through any ring atom on the ring. The terms "3-6 membered heterocycloalkyl" and "3-7 membered heterocycloalkyl" refer to saturated or partially unsaturated monocyclic or polycyclic heterocycloalkyl groups containing 3-6 and 3-7 ring members, respectively, selected from carbon atoms and heteroatoms or heteroatom groups, wherein the heteroatoms or heteroatom groups are selected from N, O, S(O), m (wherein m is any integer from 0 to 2); for example, aziridinyl, azetidinyl, oxetanyl, tetrahydropyrrolyl, oxopyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, morpholinyl, piperazinyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxidothiomorpholinyl, etc.

[0401] The term "aryl" refers to any stable 6-15 membered, for example 6-10 membered, monocyclic or bicyclic aromatic carbocyclic hydrocarbon group, including phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindanyl or biphenyl, etc. The hydrogen atoms on the "aryl" group are independently optionally substituted with one or more substituents described in the present invention.

[0402] The term "heteroaryl" or "heterocyclic aromatic group" refers to an aromatic ring group formed by replacing the carbon atoms in the ring with at least one heteroatom selected from sulfur, oxygen or nitrogen, and the aromatic ring group can be a 5-15-membered ring, such as a 5-7-membered or 5-6-membered monocyclic or 7-12-membered bicyclic group, including but not limited to a 5-, 6-, 7-, 8-, 9- or 12-membered heteroaryl group. In the present invention, the number of heteroatoms in the heteroaryl group is preferably 1, 2, 3 or 4. Exemplary heteroaryl groups are selected from, for example, thienyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, pyridin-2(1H)-onyl, pyridin-4(1H)-onyl, pyrrolyl, pyrazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, imidazolyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, benzothienyl, indolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, quinolyl, isoquinolyl, quinazolinyl, etc. The hydrogen atoms on the “heteroaryl” group are independently optionally substituted with one or more substituents described herein.

[0403] The term "C 6-15 "Aryl" means an aromatic group having 6 to 15 carbon atoms, wherein "aryl" is as defined above.

[0404] The term "5-15 membered heteroaryl" refers to an aromatic hetero group having 5 to 15 ring atoms, wherein "heteroaryl" is as defined above.

[0405] The term "cyano" refers to the group -CN.

[0406] The term "carboxy" refers to the group -COOH.

[0407] The term "hydroxy" refers to the group -OH.

[0408] The term "substituted" refers to having one or more substituents such as 1-25, 1-20, 1-10 or 1-5, for example 1, 2, 3, 4, 5 substituents. Substituents include, but are not limited to, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, alkaryl, acyl, heteroaryl, heteroalkyl, heterocycloalkyl, heteroalkenyl, heteroalkynyl, heteroalkaryl, halogen, oxo, cyano, nitro, amino, alkylamino, hydroxy, alkoxy, alkanoyl, carbonyl, carbamoyl, guanidinyl, ureido, amidinyl, and combinations of the above groups or moieties. Substituents include, but are not limited to, F, Cl, methyl, phenyl, benzyl, OR, NR2, SR, SOR, S02R, OCOR, NRCOR, NRCONR2, NRCOOR, OCONR2, RCO, COOR, alkyl-OOCR, S03R, CONR2, SO2NR2, NRSO2NR2, CN, CF3, OCF3, SiR3, and NO2, wherein each R is independently H, alkyl, alkenyl, aryl, heteroalkyl, heteroalkenyl, or heteroaryl, and wherein two optional substituents on the same or adjacent atoms may join to form a fused, optionally substituted, aromatic or non-aromatic, saturated or unsaturated ring containing 3-8 members, or two optional substituents on the same atom may join to form an optionally substituted, aromatic or non-aromatic, saturated or unsaturated ring containing 3-8 members.

[0409] The term "optionally" means that the following event or situation etc. may occur / exist or may not occur / exist. For example, "optionally substituted" means that the group involved may be substituted or not substituted.

[0410] The term "comprising" also encompasses the case where the component consists of or consists essentially of the features or elements mentioned.

[0411] The term "stereoisomer" includes all isomeric forms, including enantiomers, diastereomers and geometric isomers (cis-trans isomers). Therefore, the single stereochemical isomers of the compounds designed in the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or cis-trans isomers) all fall within the scope of the present invention.

[0412] The term "pharmaceutically acceptable salt" means that the compounds of the present invention exist in the form of their pharmaceutical salts, including acid addition salts and base addition salts. In the present invention, pharmaceutically acceptable non-toxic acid addition salts refer to salts formed between the compounds of the present invention and organic or inorganic acids, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed between the compounds of the present invention and organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, such as ammonium salts or N-containing organic bases formed by reacting with N-containing groups. + (C 1-6 Alkyl)4 salt.

[0413] The term "solvate" refers to an association formed by one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0414] The term "hydrate" refers to an association formed between water and a compound of the present invention.

[0415] The term "prodrug" refers to a chemical derivative of the compound of the present invention, which is converted into a compound represented by the general formula I-1 or I-2 through a chemical reaction in vivo.

[0416] The term "isotope-labeled substance" refers to an isotope-labeled substance in which one or more, for example, 1, 2, 3, 4 or 5 hydrogen atoms in the general formula I-1 and formula I-2 are replaced by deuterium atoms, a carbon atom is replaced by one or more, for example, 1 to 3 carbon 14 atoms ( 14 C) The isotope label obtained by substitution.

[0417] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) a compound of the present invention (including a conjugate of formula I-1, IA or IB, a compound of formula I-2, I-5a or I-6a or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof), and (ii) other therapeutic agents) are administered to a patient in sequence as separate entities, without specific time restrictions or at the same or different time intervals, wherein such administration provides two or more active agents at effective levels of prevention or treatment in the patient. In some embodiments, the compounds of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those when they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. It is preferred that the dosage and / or time interval of two or more active agents be selected so that the combined use of each part can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component may be in a separate preparation form, and the preparation forms may be the same or different.

[0418] The term "drug:antibody ratio" or "DAR" refers to a small molecule drug moiety (D) conjugated to an E moiety (e.g., an antibody, an Fc domain, or albumin) as described herein. 1 and D 2 ) to the E moiety. In some embodiments described herein, DAR is represented by n:m in Formula I-1 and is 1 to 20, preferably from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10. DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the small molecule drug moiety (DAR) conjugated to the E moiety (e.g., antibody, Fc domain or albumin) described herein in the product measured by a detection method (e.g., by MS method) 1 and D 2) to the overall ratio of the E portion, this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugate of the invention is between 0.5 and 10.0, such as 1.0-8.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, or a range having two of these values ​​as endpoints.

[0419] In the text, when variables such as n, y, etc. are or are selected from a range of integers, it means that all integers in the range are considered in the present invention, just as if they were listed one by one in the text. For example, when n is described as an integer of 1-8, it is equivalent to describing that n is an integer of 1, 2, 3, 4, 5, 6, 7 or 8 in the text.

[0420] Sequence SEQ ID NO: 1-81 and its description

[0421] SEQ ID NO: 1: Murine Fc-IgG2a with IL2 signal sequence (bold) at the N-terminus (bold)

[0422]

[0423] SEQ ID NO: 2: Mature murine Fc-IgG2a

[0424] PRGPTIKPC(!)PPC(!)KCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL PAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0425] SEQ ID NO: 3: Human Fc-IgG1 with IL2 signal sequence at the N-terminus (bold) and MVRS amino acid sequence added at the N-terminus (underlined)

[0426]

[0427] SEQ ID NO: 4: Mature human Fc-IgG1 with MVRS amino acid sequence (underlined) added to the N-terminus MVRS DKTHTC(!)PPC(!)PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0428] SEQ ID NO: 5: Murine Fc-IgG2a with IL2 signal sequence (bold) added to the N-terminus and six histidine tags (italics) added to the C-terminus

[0429]

[0430] SEQ ID NO:6: Mature murine Fc-IgG2a with six histidine tags (italics) added to the C-terminus

[0431]

[0432] SEQ ID NO:7: Human Fc-IgG1 with IL2 signal sequence (bold) and MVRS amino acid sequence (underlined) added to the N-terminus and a six-histidine tag (italics) added to the C-terminus

[0433]

[0434] SEQ ID NO: 8: Human mature Fc-IgG1 with MVRS amino acid sequence (underlined) added to the N-terminus and six histidine tags (italics) added to the C-terminus

[0435]

[0436] SEQ ID NO:9: Human Fc-IgG1 with IL2 signal sequence (bold) and MVRS amino acid sequence (underlined) added to the N-terminus, two additional cysteines (*) in the hinge region, and a six-histidine tag (italics) added to the C-terminus

[0437]

[0438] SEQ ID NO: 10: Mature human Fc-IgG1 with MVRS amino acid sequence added to the N-terminus (underlined), two additional cysteines (*) in the hinge region, and a six-histidine tag (italics) added to the C-terminus

[0439]

[0440] SEQ ID NO: 11: Mature human Fc-IgG1 with MVRS amino acid sequence added to the N-terminus (underlined) and two additional cysteines (*) in the hinge region

[0441] MVRS DKTHTC(!)PPC(!)PPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQMQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0442] SEQ ID NO: 12: Murine Fc-IgG2a with IL2 signal sequence (bold) and Asn to Ala substitution (*) added to the N-terminus and a hexa-histidine tag (italics) added to the C-terminus

[0443]

[0444] SEQ ID NO: 13: Murine Fc-IgG2a containing an Asn to Ala substitution (*) and a hexa-histidine tag (italics) added to the C-terminus

[0445]

[0446] SEQ ID NO: 14: Human Fc-IgG1 with IL2 signal sequence (bold) and MVRS amino acid sequence (underlined) added to the N-terminus, containing an Asn to Ala substitution (*), and a hexa-histidine tag (italics) added to the C-terminus

[0447]

[0448] SEQ ID NO: 15: Human Fc-IgG1 with MVRS amino acid sequence added to the N-terminus (underlined), containing an Asn to Ala substitution (*) and a hexa-histidine tag added to the C-terminus (italics)

[0449] MVRS DKTHTC(!)PPC(!)PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 16: Human Fc-IgG1 with human serum albumin signal sequence (bold) added to the N-terminus and with N-terminal ISAMVRS amino acid sequence (underlined) added

[0450]

[0451] SEQ ID NO: 17: Human Fc-IgG1 with human serum albumin signal sequence (bold) added to the N-terminus and with N-terminal ISAMVRS amino acid sequence (underlined), containing C-terminal G4S linker (italics) and C-terminal c-Myc tag (underlined, italics)

[0452]

[0453]

[0454] SEQ ID NO: 18: Mature human Fc-IgG1 with ISAMVRS amino acid sequence (underlined) added to the N-terminus, a G4S linker (italics) added to the C-terminus, and a c-Myc tag (underlined, italics) added to the C-terminus

[0455]

[0456] SEQ ID NO: 19: Human Fc-IgG1 with human serum albumin signal sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus and containing a lysine to serine change (*) to prevent coupling at this site

[0457]

[0458] SEQ ID NO:20: Mature human Fc-IgG1 with the ISAMVRS amino acid sequence (underlined) added to the N-terminus and containing a lysine to serine change (*) to prevent coupling at this site

[0459] ISAMVRS DKTHTC(!)PPC(!)PAPELLGGPSVFLFPPKPS*DTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0460] SEQ ID NO:21: Human Fc-IgG1 with human serum albumin signal sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing a lysine to serine change (*) to prevent coupling at this site, and a G4S linker (italics) and c-Myc tag (underlined, italics) added to the C-terminus

[0461]

[0462] SEQ ID NO:22: Mature human Fc-IgG1 with the ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing a lysine to serine change (*) to prevent coupling at this site, and a G4S linker (italics) and c-Myc tag (underlined, italics) added to the C-terminus

[0463]

[0464] SEQ ID NO:23: Human Fc-IgG1 with human serum albumin signal sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing an Asn to Ala change (*), and a G4S linker (italics) and c-Myc tag (underlined, italics) added to the C-terminus

[0465]

[0466]

[0467] SEQ ID NO:24: Mature human Fc-IgG1 with ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing an Asn to Ala change (*), and a G4S linker (italics) and c-Myc tag (underlined, italics) added to the C-terminus

[0468]

[0469] SEQ ID NO:25: Human Fc-IgG1 with human serum albumin signal sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing H310A (*) and H435A (*) changes to prevent FcRn binding, and G4S linker (italics) and c-Myc tag (underlined, italics) added to the C-terminus

[0470]

[0471] SEQ ID NO:26: Human Fc-IgG1 with ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing H310A (*) and H435A (*) changes to prevent FcRn binding, and G4S linker (italics) and c-Myc tag (underlined, italics) added to the C-terminus

[0472]

[0473] SEQ ID NO:27: Human Fc-IgG1 with human serum albumin signal sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, and a G4S linker (italics) and mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italics) added to the C-terminus

[0474]

[0475] SEQ ID NO:28: Mature human Fc-IgG1 with ISAMVRS amino acid sequence (underlined) added to the N-terminus and a G4S linker (italics) and a mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italics) added to the C-terminus

[0476]

[0477] SEQ ID NO:29: Human Fc-IgG1 with human serum albumin signal sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing an Asn to Ala substitution (*) and a G4S linker (italics) and mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italics) added to the C-terminus

[0478]

[0479] SEQ ID NO:30: Mature human Fc-IgG1 with ISAMVRS amino acid sequence added to the N-terminus (underlined), containing an Asn to Ala substitution (*) and a G4S linker (italics) and a mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italics) added to the C-terminus

[0480]

[0481] SEQ ID NO:31: Human Fc-IgG1 with human serum albumin signal sequence (bold) added to the N-terminus, allotype G1m(fa) (bold italic), and G4S linker (italic) and mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italic) added to the C-terminus

[0482]

[0483] SEQ ID NO:32: Human Fc-IgG1 with human serum albumin signal sequence (bold) added to the N-terminus and allotype G1m(fa) (bold italics)

[0484]

[0485] SEQ ID NO:33: Mature human Fc-IgG1 with MVRS amino acid sequence added to the N-terminus (underlined) and containing a YTE triple mutation (bold, underlined)

[0486]

[0487] SEQ ID NO:34: Human Fc-IgG1 with human serum albumin signal sequence (bold) added to the N-terminus, containing the EPKSS amino acid sequence of the hinge region of mature human Fc-IgG1 (underlined), and containing a cysteine ​​to serine change (#) and allotype G1m (fa) (bold italics)

[0488]

[0489] SEQ ID NO:35: Human Fc-IgG1 with a murine IgG signal sequence added to the N-terminus (bold), the EPKSSD amino acid sequence of the hinge region of mature human Fc-IgG removed, and allotype G1m(fa) (bold italics)

[0490]

[0491] SEQ ID NO:36: N-terminally removed hinge region EPKSSD amino acid sequence of mature human Fc-IgG, having allotype G1m(fa) (bold italics) and containing the YTE triple mutation (bold, underlined)

[0492]

[0493] SEQ ID NO:37: N-terminally removed EPKSSD amino acid sequence of a mature human Fc-IgG with double LS mutation (bold, underlined) and allotype G1m(fa) (bold italic)

[0494]

[0495] SEQ ID NO:38: Human Fc-IgG1 with human serum albumin signal sequence added to the N-terminus (bold), containing a YTE triple mutation (bold, underlined), having allotype G1m(fa) (bold italic), and with a G4S linker (italics) and c-Myc tag (underlined) added to the C-terminus

[0496]

[0497] SEQ ID NO:39: Mature human Fc-IgG1, wherein X1 is Met or Trp, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser DKTHTC(!)PPC(!)PAPELLGGPSVFLFPPKPKDTLX1IX2RX3PEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRX4EX5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX6HEALHX7HYTQKSLSLSPG

[0498] SEQ ID NO:40: Mature human Fc-IgG1, wherein X4 is Asp or Glu, and X5 is Leu or Met DKTHTC(!)PPC(!)PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRX4EX5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0499] SEQ ID NO:41: Mature human Fc-IgG1 with a YTE triple mutation (bold, underlined), and wherein X4 is Asp or Glu, and X5 is Leu or Met

[0500]

[0501]

[0502] SEQ ID NO:42: Mature human Fc-IgG1 with a YTE triple mutation (bold, underlined) and allotype G1m(fa) (bold italics)

[0503]

[0504] SEQ ID NO:43: Mature human Fc-IgG1 with a YTE triple mutation (bold, underlined) and allotype G1m(f) (bold italics)

[0505]

[0506] SEQ ID NO:44: Mature human Fc-IgG1 with LS double mutation (bold, underlined), and wherein X4 is Asp or Glu, and X5 is Leu or Met

[0507]

[0508] SEQ ID NO:45: Mature human Fc-IgG1 with LS double mutation (bold, underlined) and allotype G1m(fa) (bold italics)

[0509]

[0510] SEQ ID NO:46: Mature human Fc-IgG1 with LS double mutation (bold, underlined) and allotype G1m(f) (bold italics)

[0511]

[0512] SEQ ID NO:47: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) added to the N-terminus, with a cysteine ​​to serine change (#), wherein X1 is Met or Trp, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0513]

[0514] SEQ ID NO:48: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), with a cysteine ​​to serine change (#), and containing allotype G1m(fa) (bold italics)

[0515]

[0516] SEQ ID NO:49: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), with a cysteine ​​to serine change (#), and containing allotype G1m(f) (bold italics)

[0517]

[0518] SEQ ID NO:50: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), with a cysteine ​​to serine change (#), with M428L and N434S mutations (bold, underlined), and containing allotype G1m(fa) (bold italics)

[0519]

[0520] SEQ ID NO:51: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), with a cysteine ​​to serine change (#), with M428L and N434S mutations (bold, underlined), and containing allotype G1m(f) (bold italics)

[0521]

[0522] SEQ ID NO:52: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), with a cysteine ​​to serine change (#), with a YTE triple mutation (bold, underlined), and containing allotype G1m(fa) (bold italics)

[0523]

[0524] SEQ ID NO:53: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), with a cysteine ​​to serine change (#), with a YTE triple mutation (bold, underlined), and containing allotype G1m(f) (bold italics)

[0525]

[0526] SEQ ID NO:54: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) and ISAMVRS amino acid sequence (italic) added to the N-terminus, with M428L, N434S mutations (bold, underlined), G4S linker (italics) and c-Myc tag (underlined) added to the C-terminus, and containing allotype G1m(f) (bold italics)

[0527]

[0528] SEQ ID NO:55: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) and ISAMVRS amino acid sequence (italic) added to the N-terminus, with M428L, N434S mutations (bold, underlined), G4S linker (italics) and c-Myc tag (underlined) added to the C-terminus, and containing allotype G1m(fa) (bold italics)

[0529]

[0530] SEQ ID NO:56: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) and ISAMVRS amino acid sequence (italic) added to the N-terminus, with a YTE triple mutation (bold, underlined), a G4S linker (italics) and a c-Myc tag (underlined) added to the C-terminus, and containing allotype G1m(f) (bold italics)

[0531]

[0532] SEQ ID NO:57: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) and ISAMVRS amino acid sequence (italic) added to the N-terminus, with a YTE triple mutation (bold, underlined), a G4S linker (italics) and a c-Myc tag (underlined) added to the C-terminus, and containing allotype G1m(fa) (bold italics)

[0533]

[0534] SEQ ID NO:58: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) added to the N-terminus, with a cysteine ​​to serine change (#), a G4S linker (italics) and an IgA peptide tag (underlined) added to the C-terminus, and containing allotype G1m(fa) (bold italics)

[0535]

[0536] SEQ ID NO:59: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), with cysteine ​​to serine change (#), with M428L, N434S mutations (bold, underlined), with G4S linker (italics) and IgA peptide tag (underlined) added to the C-terminus and containing allotype G1m(fa) (bold italics)

[0537]

[0538] SEQ ID NO:60: Mature human Fc-IgG1, Z1 is Cys or Ser, and wherein X1 is Met or Trp, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or SerNVNHKPSNTKVDKKVEPKSZ1DKTHTC(!)PPC(!)PAPELLGGPSVFLFPPKPKDTLX1IX2RX3P EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX4EX5TKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX6HEALHX7HYTQKSLSLSPGK

[0539] SEQ ID NO:61: mature human Fc-IgG1, cysteine ​​to serine change (#), and wherein X1 is Met or Trp, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, and X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser

[0540] NVNHKPSNTKVDKKVEPKSS(#)DKTHTC(!)PPC(!)PAPELLGGPSVFLFPPKPKDTLX1IX2RX3PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX4EX5TKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX6HEALHX7HYTQKSLSLSPGK

[0541] SEQ ID NO:62: Mature human Fc-IgG1, cysteine ​​to serine change (#), and wherein X4 is Asp or Glu, and X5 is Leu or Met

[0542] NVNHKPSNTKVDKKVEPKSS(#)DKTHTC(!)PPC(!)PAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX4EX5TKNQVSLTCLVKGFYPSD ​​IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[0543] SEQ ID NO:63: Mature human Fc-IgG1, cysteine ​​to serine change (#), allotype G1m(f) (bold italics)

[0544]

[0545] SEQ ID NO:64: Mature human Fc-IgG1, cysteine ​​to serine change (#), allotype G1m (fa) (bold italics)

[0546]

[0547] SEQ ID NO:65: Mature human Fc-IgG1, cysteine ​​to serine change (#), M428L, N343S mutations (bold, underlined), allotype G1m (fa) (bold italics)

[0548]

[0549]

[0550] SEQ ID NO:66: Mature human Fc-IgG1, cysteine ​​to serine change (#), M428L, N343S mutations (bold, underlined), allotype G1m(f) (bold italics)

[0551]

[0552] SEQ ID NO:67: Mature human Fc-IgG1, cysteine ​​to serine change (#), YTE triple mutation (bold, underlined), allotype G1m (fa) (bold italics)

[0553]

[0554] SEQ ID NO:68: Mature human Fc-IgG1, cysteine ​​to serine change (#), YTE triple mutation (bold, underlined), allotype G1m(f) (bold italics)

[0555]

[0556] SEQ ID NO: 69: Mature human Fc-IgG1, YTE triple mutation (bold, underlined), allotype G1m (fa) (bold italics)

[0557]

[0558] SEQ ID NO:70: Mature human Fc-IgG1, glycine to alanine change (#), alanine to leucine change ($), isoleucine to glutamate change (*), YTE triple mutation (bold, underlined), allotype G1m (fa) (bold italics)

[0559]

[0560] SEQ ID NO:71: Mature human Fc-IgG1, M428L, N343S mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0561]

[0562] SEQ ID NO:72: (wild type)

[0563] NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV

[0564] VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

[0565] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES

[0566] NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

[0567] SPGK

[0568] SEQ ID NO:73: Mature human Fc-IgG1, M428L, N343S mutation (bold, underlined), allotype G1m(f) (bold italics)

[0569]

[0570] SEQ ID NO:74: Mature human Fc-IgG1, GAALIE, M428L, N343S mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0571]

[0572] SEQ ID NO:75: Mature human Fc-IgG1, GAALIE, M428L, N343S mutation (bold, underlined), allotype G1m(f) (bold italics)

[0573]

[0574] SEQ ID NO:76: Mature human Fc-IgG1, cysteine ​​to serine change (#), GAALIE, M428L, N343S mutations (bold, underlined), allotype G1m (fa) (bold italics)

[0575]

[0576]

[0577] SEQ ID NO:77: Mature human Fc-IgG1, cysteine ​​to serine change (#), GAALIE, M428L, N343S mutations (bold, underlined), allotype G1m(f) (bold italics)

[0578]

[0579] SEQ ID NO:78: Mature human Fc-IgG1, cysteine ​​to serine change (#), DHS triple mutation (bold, underlined), allotype G1m (fa) (bold italics)

[0580]

[0581] SEQ ID NO:79: Mature human Fc-IgG1, cysteine ​​to serine change (#), DHS triple mutation (bold, underlined), allotype G1m(f) (bold italics)

[0582]

[0583] SEQ ID NO:80: Mature human Fc-IgG1, DHS triple mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0584]

[0585] SEQ ID NO:81: Mature human Fc-IgG1, DHS triple mutation (bold, underlined), allotype G1m(f) (bold italics)

[0586]

[0587]

[0588] The C (!) in SEQ ID NO: 1 to 81 can be connected to L 2 of cysteine. BRIEF DESCRIPTION OF THE DRAWINGS

[0589] Figure 1 : MS detection image of conjugate 1.

[0590] Figure 2 : SEC detection diagram of conjugate 1.

[0591] Figure 3 : Residual small molecule detection diagram of conjugate 1.

[0592] Figure 4 : MS detection image of conjugate 2.

[0593] Figure 5 : SEC detection diagram of conjugate 2.

[0594] Figure 6 : Residual small molecule detection diagram of conjugate 2.

[0595] Figure 7 : MS detection results of the screening test product conjugate 1.

[0596] Figure 8 : MS detection image of conjugate 3.

[0597] Fig. 9 : SEC detection diagram of conjugate 3.

[0598] Fig.10 : Residual small molecule detection diagram of conjugate 3.

[0599] Fig.11 : MS detection image of conjugate 4.

[0600] Fig.12 : SEC detection diagram of conjugate 4.

[0601] Fig.13 : Residual small molecule detection diagram of conjugate 4. DETAILED DESCRIPTION

[0602] The above-mentioned and other aspects and embodiments of the present invention are exemplified in the following examples.Any or all of the features discussed above and throughout this application can be combined in various embodiments of the present invention.The following examples further illustrate the present invention, however, it should be understood that the embodiments are described in an illustrative and non-limiting manner, and that those skilled in the art can make multiple modifications.

[0603] Example 1: Preparation of Fc part

[0604] The protein amino acid sequence (SEQ ID No: 1-68) was reverse translated and the corresponding nucleotide sequence was synthesized. The nucleotide sequence was end-bound with appropriate restriction sites (XbaI+SalI) and then cloned into the pWX4.1 expression vector (WuXi Biologics, Shanghai). Each constructed vector either carried the signal peptide sequence of human interleukin-2 or the signal peptide sequence of human serum albumin. The pWX4.1 plasmid was transfected into Escherichia coli Top10 strain (Life Technologies) to amplify the DNA and try The HiPURE Plasmid Filter Maxiprep Kit (Life Technologies) was used for purification. TM 293 Transfection Kit (Life Technologies) was used to transfect the plasmid into CHO cells (ATCC). The transfected cells were cultured for 7 days, then centrifuged and filtered, the supernatant was collected, and purified with MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA) to obtain purified h-IgG Fc. The purified samples were subjected to 4-12% Bis Tris SDS-PAGE electrophoresis, 1-2 μg was loaded, and then stained with Coomassie Brilliant Blue. Each sample was simultaneously treated with reduction (R) and non-reduction (NR).

[0605] Example 2: Preparation of Fc part

[0606] The protein amino acid sequence (SEQ ID No: 69-81) was reverse translated and the corresponding nucleotide sequence was synthesized, and the sequence was homologously recombined into the pCDNA3.4 vector. Each constructed vector carries the signal peptide sequence of human interleukin 10. The constructed plasmid was electrotransfected into CHO cells, and the transfected cells were cultured for seven days, then centrifuged and precipitated, and the supernatant was filtered and purified using a protein A affinity chromatography column to obtain purified h-IgG Fc. The purified samples were subjected to Bis Tris SDS-PAGE electrophoresis and then stained with Coomassie Brilliant Blue. Each sample was simultaneously subjected to reduction (R) and non-reduction treatment (NR).

[0607] Example 3: General Synthesis Method of h-IgG1 Fc-SH

[0608]

[0609] 50 mM PB, pH 6.5 buffer, antibody ((E) m ) solution (30 mg) and 10 mM reducing agent (TCEP) solution (reducing agent / (E) m The molar equivalent ratio is 10), control (E) m The reaction concentration was 5 mg / mL, and the reaction temperature was controlled at 22°C in a metal bath for 2 hours. wherein E, m and n are as defined herein.

[0610] Example 4: General method 1 for preparing conjugates:

[0611]

[0612] The variables are as defined in the text.

[0613] Add 50mM PB, pH 6.5 buffer and formula I-2a compound solution (I-2 / II-1=7-12) to the reaction solution containing II-1, place in a metal bath and control the reaction temperature to 22°C for 1 hour. After the reaction time is reached, add formula I-2a compound (I-2 / II-1=3.5-6) ​​to the reaction solution, place in a metal bath and control the reaction temperature to 22°C for 0.5 hour. After the reaction time is reached, add formula I-2a compound (I-2 / II-1=3.5-6) ​​to the reaction solution again, place in a metal bath and control the reaction temperature to 22°C for 1.0 hour to obtain I-3.

[0614] The reaction solution was adjusted to pH = 7.5 with 0.5 M NaOH, and continued to be placed in a metal bath and the reaction temperature was controlled at 22° C. for 2 hours until I-3 was completely hydrolyzed to I-1a. The reaction solution was concentrated by ultrafiltration and centrifugation, and the purified target conjugated compound was prepared after ultrafiltration and liquid exchange, and the average DAR (drug / antibody ratio) of the conjugate was analyzed by MS.

[0615] Example 5: Synthesis of drug intermediate (INT-DRUG)

[0616]

[0617] Step 1. Synthesis of INT-DRUG-2

[0618]

[0619] Add INT-DRUG-1 (3.0 g, 6.57 mmol) to the reaction bottle, then add tetrahydrofuran (30 mL), triphenylphosphine (1.9 g, 7.23 mmol), and stir the reaction at 30 ° C for 2 hours. After the reaction is completed, add lithium hydroxide monohydrate (28 mg) and water (1.5 mL), and continue stirring at 30 ° C for 16 hours. After the reaction is completed by TLC monitoring, add N, N'-di-Boc-1H-1-guanidinopyrazole (2.1 g, 6.91 mmol) and stir at 30 ° C for 48 hours. After the reaction is completed, concentrate the reaction solution and separate and purify it by silica gel column chromatography (ethyl acetate / petroleum ether = 1: 1). Purification gives INT-DRUG-2 (2.2 g) as a colorless oil. MS: [M+1] + found673.40 Step 2, Synthesis of INT-DRUG-3

[0620]

[0621] Add INT-DRUG-2 (2.19 g, 3.25 mmol) to the reaction flask, and protect with nitrogen, then add a solution of dry sodium methoxide (0.65 mmol) in methanol (8 mL), stir at room temperature for 10 minutes, and quench the reaction with HCl (0.4 N, 1,4-dioxane solution, 2.5 mL). Concentrate the reaction solution, separate and purify by silica gel column chromatography (ethyl acetate / petroleum ether, 70% to 100%), and purify to obtain INT-DRUG-3 (1.15 g) as a white solid. MS: [M+1] + found 547.33.

[0622] Step 3. Synthesis of INT-DRUG-4

[0623]

[0624] Add INT-DRUG-3 (386.9 mg, 0.71 mmol), acetonitrile (10 mL), CDI (160.8 mg, 0.99 mmol), and DMAP (302.7 mg, 2.47 mmol) to the reaction flask. Stir the reaction at room temperature for 16 hours. After the reaction, concentrate the reaction solution and purify and separate it by silica gel column chromatography (ethyl acetate / petroleum ether, 0% to 45%) to obtain INT-DRUG-4 (242.8 mg) as a white solid. MS: [M+1] + Found 573.35.

[0625] Step 4, Synthesis of INT-DRUG

[0626]

[0627] INT-DRUG-4 (403 mg, 0.7 mmol), DMAP (1.37 g, 11.2 mmol), pyridine (60 mL), p-nitrophenyl chloroformate (2.11 g, 10.5 mmol) were added to the reaction bottle, and the mixture was stirred at 40°C for 4 h after nitrogen replacement. After LCMS detection showed a conversion of >90%, water (20 mL) was added to quench the reaction (not to be left for a long time), and the mixture was separated by reverse column (acetonitrile / water 0% to 90%), and lyophilized to obtain INT-DRUG (173 mg) as a white solid. MS: [M+1] + Found 738.33.

[0628] Example 6: Synthesis of Intermediate A (inter-A)

[0629]

[0630] Step 1: Synthesis of intermediate 2

[0631]

[0632] Compound 1 (150 g, 1.43 mol) and triethylamine (289 g, 2.85 mol) were added to a reaction flask, and tetrahydrofuran (1 L) was added, and the mixture was stirred at 0 ° C after nitrogen replacement. CbzCl (243 g, 1.43 mol) was dissolved in tetrahydrofuran (500 mL) and then dropped into the reaction solution. The reaction solution was stirred at room temperature overnight. After the reaction was completed, water (3 L) was added to quench the reaction, and then extracted with ethyl acetate (300 mL x 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain a yellow oily compound 2 (255 g).

[0633] Step 2: Synthesis of Intermediate 3

[0634]

[0635] Compound 2 (245 g, 1.02 mol) and carbon tetrabromide (509 g, 1.54 mol) were added to a reaction flask, and then dichloromethane (2 L) was added. After nitrogen replacement, the mixture was stirred at 0 ° C. Triphenylphosphine (403 g, 1.54 mol) was dissolved in dichloromethane (500 mL) and then dropped into the reaction solution. The reaction solution was stirred at room temperature for 1.5 h. After the reaction was completed, the crude product was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain a yellow oily compound 3 (200 g, crude product).

[0636] Step 3: Synthesis of intermediate 4

[0637]

[0638] Compound 3 (200 g, crude product) was added to a reaction flask, and N, N-dimethylformamide (2 L) was added. Bianamine (28.3 g, 264 mmol) and sodium carbonate (73.0 g, 528 mmol) were added to the reaction solution. The reaction solution was stirred at 60 ° C overnight. After the reaction was completed, the temperature was lowered to room temperature, water (3 L) was added, and then extracted with ethyl acetate (300 mL x 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain a yellow oily compound 4 (45 g).

[0639] Step 4-5: Synthesis of intermediate 6

[0640]

[0641] Compound 4 (43.0 g, 78.2 mmol) and tetrahydrofuran (450 mL) were added to the reaction flask, and the mixture was stirred at 0 ° C after nitrogen replacement. LiAlH4 (23.7 g, 62.6 mmol) was added to the reaction solution. The reaction solution was stirred at room temperature for 1.5 h, and then heated to reflux and continued to stir for 6 h. After the reaction was completed, the temperature was lowered to 0 ° C, and sodium sulfate decahydrate (90 g) was added to quench the reaction. The reaction solution was stirred at room temperature for 1 h. Triethylamine (31.7 g, 31.3 mmol) and Boc anhydride (68.3 g, 31.3 mmol) were added to the reaction solution and stirred at room temperature overnight. After the reaction was completed, water (2 L) was added, and then extracted with ethyl acetate (300 mL x2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 30 / 1) to obtain a yellow oily compound 6 (20 g). MS: [M+1] + found 510.2.

[0642] Step 6: Synthesis of inter A

[0643]

[0644] Compound 6 (19 g, 3.73 mmol), palladium carbon (2 g) and methanol (400 mL) were added to the reaction flask under nitrogen atmosphere. The gas was replaced with hydrogen several times. The reaction solution was stirred at room temperature overnight. After the reaction, the reaction solution was filtered, the filter cake was rinsed with methanol (50 mL), and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 15 / 1) to obtain yellow oily intermediate A (14 g). MS: [M+1] + Found 420.2.

[0645] Example 7: Synthesis of Intermediate B

[0646] Step 1: Synthesis of intermediate 9

[0647]

[0648] At zero degrees, 8 (8.4 g, 54 mmol) was slowly added to a saturated sodium bicarbonate aqueous solution (110 ml) of 7 (5.5 g, 27 mmol), and the mixture was stirred at room temperature for 18 hours. After the reaction was completed, the pH value of the reaction solution was adjusted to weak acidity with a saturated potassium bisulfate aqueous solution. After the acid adjustment was completed, the solution was extracted with DCM and the extract was dried by spin drying, purified with a reverse column (water / acetonitrile), and freeze-dried to obtain the product 9 (470 mg). MS [M+1] +: 285.1

[0649] Step 2: Synthesis of Inter B

[0650]

[0651] At zero degrees, EDCI (201 mg, 1.05 mmol) was added to a DCM (20 ml) solution of 9 (150 mg, 0.53 mmol) and 10 (149 mg, 0.79 mmol), and the reaction solution was stirred at room temperature for 4 hours. After the reaction was completed, the solvent was removed by rotary evaporation and purified by silica gel column chromatography (PE: EA = 3: 1) to obtain a white solid Inter B (110 mg). MS [M + 1] +: 451.32

[0652] Example 8: Synthesis of Inter C

[0653]

[0654] 11 (1 g, 4.33 mmol), 10 (796 mg, 4.33 mmol), 12 (892 mg, 4.33 mmol) were dissolved in DMF (40 mL) and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was filtered and the filtrate was concentrated to obtain a crude product, which was then purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain Inter C (1.34 g) as a colorless oil.

[0655] Example 9: Synthesis of Linker 1

[0656]

[0657] Step 1: Synthesis of L1-2

[0658]

[0659] Dissolve chlorosulfonyl isocyanate (400 mg, 2.83 mmol) in dichloromethane (10 mL), protect with nitrogen, and stir at 0°C. Add a solution of bromoethanol (353 mg, 2.83 mmol) in dichloromethane (5 mL) dropwise, stir at 0°C for 1 hour, then add a solution of L1-1 (2.83 mmol) in dichloromethane (5 mL) dropwise, and stir at 0°C for 1 hour. After the reaction is complete, add dichloromethane (10 mL), then wash with dilute hydrochloric acid (1M, 20 mL), dry the organic phase over anhydrous sodium sulfate, remove the solvent by rotary evaporation, and purify by silica gel column chromatography (PE:EA=1:1) to obtain a yellow oily crude product L1-2 (800 mg).

[0660] Step 2: Synthesis of L1-3

[0661]

[0662] The crude product L1-2 (800 mg) was dissolved in dichloromethane (10 mL), TEA (2 mL) was added, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, dichloromethane (10 mL) was added, and the mixture was washed with dilute hydrochloric acid (1M, 20 mL). The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography (PE:EA=1:3) to obtain a yellow oil L1-3 (670 mg).

[0663] Step 3: Synthesis of L1-4

[0664]

[0665] L1-3 (300 mg, 0.79 mmol), Inter-A (330 mg, 0.79 mmol) and TEA (160 mg, 1.60 mmol) were dissolved in acetonitrile (10 mL) and stirred at 80°C for 10 hours. After the reaction was completed, the solvent was removed by rotary evaporation and purified by reverse phase column chromatography (acetonitrile / water) to obtain a yellow oil L1-4 (190 mg). MS [M+1] + :713.43

[0666] Step 4: Synthesis of Linker 1

[0667]

[0668] L1-4 (107 mg, 0.15 mmol) was placed in a hydrochloric acid 1,4-dioxane solution (5 ml) and stirred at room temperature for 1 hour. After the reaction was completed, the crude product Linker 1 was concentrated and directly subjected to the next step of reaction. MS [M+1] + :513.51.

[0669] Example 10: Synthesis of Linker 2

[0670]

[0671] Step 1: Synthesis of L2-2

[0672]

[0673] L2-1 (464 mg, 1.57 mmol), Inter A (600 mg, 4.06 mmol), potassium carbonate (395 mg, 2.86 mmol), and acetonitrile (10 mL) were added to a reaction flask and stirred at 85°C for 18 hours. After the reaction, the reaction solution was concentrated and the crude product was purified by reverse column to obtain L2-2 (320 mg) as a colorless oil. MS [M+1] + :634.31

[0674] Step 2: Synthesis of Linker 2

[0675]

[0676] L2-2 (320 mg, 0.51 mmol) was dissolved in dichloromethane (10 mL), and HCl (4 mL, 4 M in 1,4-dioxane) was added, and stirred at room temperature for 4 hours. After the reaction was completed, the solution was concentrated to obtain Linker 2 (219 mg) as a colorless liquid. MS [M+1] + :434.53 Example 11: Synthesis of Linker3

[0677]

[0678] Step 1: Synthesis of L3-2

[0679]

[0680] L3-1 (1 g, 2.7 mmol) and NaH (216 mg, 5.4 mmol) were added to a reaction flask, and tetrahydrofuran (20 mL) was added, and the mixture was replaced with nitrogen and stirred at 25 ° C for 3 h. After the reaction was completed, water (5 mL) was added to quench the reaction, and the crude product was concentrated and purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain yellow oily L3-2 (400 mg).

[0681] Step 2: Synthesis of L3-3

[0682]

[0683] L3-2 (1.7 g, 4.16 mmol) and DMAP (1.01 g, 8.32 mmol) were added to a reaction flask, and then dichloromethane (20 mL) was added. After nitrogen replacement, the mixture was stirred at 25 ° C. Tos-Cl (1.18 g, 6.24 mmol) and TEA (840 mg, 8.32 mmol) were added to the above system. The reaction solution was stirred at room temperature for 15 h. After the reaction was completed, it was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain yellow oily L3-3 (1.1 g, crude product).

[0684] Step 3: Synthesis of L3-4

[0685]

[0686] Dissolve L3-3 (440 mg, 0.78 mmol) in acetonitrile (10 mL) and start stirring. Add Inter A (326 mg, 0.78 mmol) and DIPEA (201 mg, 1.56 mmol) to the reaction solution. Stir at 80°C for 10 hours. After the reaction is completed, remove the solvent by rotary evaporation and purify by reverse phase column chromatography (acetonitrile / water) to obtain a yellow oil L3-4 (310 mg).

[0687] Step 4: Synthesis of Linker 3

[0688]

[0689] Compound 4 (247 mg, 0.3 mmol) was dissolved in dichloromethane (10 mL), added to a hydrochloric acid 1,4-dioxane solution (3 ml) and stirred at room temperature for 1 hour. After the reaction was completed, the mixture was concentrated to obtain Linker 3 (182 mg) as a colorless oil. MS [M+1] +:610.52 Example 12: Synthesis of C-Inter-1

[0690]

[0691] Step 1: Synthesis of C-Inter-1-1

[0692]

[0693] Linker 1 (0.2 mmol) was dissolved in DCM (2 ml) and DIEA (0.6 g, 4.5 mmol) was added. INT-DRUG (280 mg, 0.4 mmol) was dissolved in DCM (2 ml) and then added dropwise to the mixture. The mixture was stirred at room temperature for 16 hours, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase column chromatography (acetonitrile / water) and freeze-dried to obtain a white solid C-Inter-1-1 (240 mg). MS [M / 2+1] + :855.5

[0694] Step 2: Synthesis of C-Inter-1-2

[0695]

[0696] C-Inter-1-1 (240 mg, 0.146 mmol) was dissolved in methanol (5 ml) and lithium hydroxide aqueous solution (0.55 mmol, 1 ml) was added. After stirring at room temperature for 1 hour, acetic acid (0.3 mL) was added. After low temperature concentration, the mixture was purified by reverse phase column chromatography (acetonitrile / water) to obtain C-Inter-1-2 (213 mg, 0.131 mmol) as a white solid. MS [M / 2+1] + :815.4

[0697] Step 3: Synthesis of C-Inter-1-3

[0698]

[0699] At zero degrees, add THPTA (45.5 mg, 0.105 mmol), sodium ascorbate (51.8 mg, 0.262 mmol) and CuSO4 (51.8 mg, 0.262 mmol) in clear water (2 ml) to a solution of C-Inter-1-2 (213 mg, 0.131 mmol) and azide-tetraethylene glycol-ammonia (137 mg, 0.523 mmol) in MeCN (2 ml), protect with nitrogen, stir the reaction solution at room temperature for 1 hour, after the reaction is completed, purify by high pressure preparative liquid chromatography to obtain C-Inter-1-3 (187 mg). MS [M / 2+1] + :946.4

[0700] Step 4: Synthesis of C-Inter-1-4

[0701]

[0702] At zero degrees, add Inter B (77.3 mg, 0.171 mmol) and DIEA (57.56 mg, 0.445 mmol) to a DMF (3 ml) solution of C-Inter-1-3 (270 mg, 0.143 mmol), stir the reaction solution at room temperature for 1 hour, add 1.5 ml of trifluoroacetic acid to the reaction solution after the reaction is complete, and then freeze-dry to obtain a crude product C-Inter-1-4. MS [M / 2+1] + :1079.5

[0703] Step 5: Synthesis of C-Inter-1

[0704]

[0705] The crude product of C-Inter-1-4 was dissolved in a 10% trifluoroacetic acid solution (30 ml), stirred at room temperature for 15 minutes, and then dried by spin drying. C-Inter-1 (85 mg) was purified by high pressure preparative liquid chromatography. MS [M / 2+1] + :829.4

[0706] Example 13: Synthesis of C-Inter-3

[0707]

[0708] Step 1: Synthesis of C-Inter-3-1

[0709]

[0710] Linker 2 (219 mg, 0.5 mmol) and DIEA (396 mg, 3 mmol) were dissolved in dichloromethane (10 mL), and INT-DRUG (885 mg, 1.2 mmol) was dissolved in dichloromethane (5 mL), and then added dropwise to the reaction solution, and stirred at room temperature for 16 hours. After the reaction, the reaction solution was concentrated and purified by reverse phase column to obtain C-Inter-3-1 (730 mg) as a white solid. MS [M / 2+1] + :815.8

[0711] Step 2: C-Inter-3-2

[0712]

[0713] C-Inter-3-1 (730 mg, 0.45 mmol) was dissolved in methanol (10 mL), and lithium hydroxide monohydrate (112.7 mg, 2.69 mmol) was dissolved in water (3 mL) and added dropwise to the reaction solution, and stirred at room temperature for 4 hours. After the reaction was completed, acetic acid (1 mL) was added, the reaction solution was concentrated, and C-Inter-3-2 (290 mg) was purified by reverse column to obtain a white solid. MS [M / 2+1] + :775.8Step 3: C-Inter-3-3

[0714]

[0715] C-Inter-3-2 (290 mg), azide-tetraethylene glycol-amine (196 mg, 0.75 mmol) were dissolved in acetonitrile, THPTA (65.1 mg, 0.15 mmol), sodium ascorbate (74 mg, 0.374 mmol), copper sulfate (14.9 mg, 0.093 mmol) were dissolved in water (5 mL) and added dropwise to the reaction solution. After nitrogen replacement, the mixture was stirred at room temperature for 2 hours. Reverse column purification gave C-Inter-3-3 (123 mg) as a white solid. MS [M / 2+1] + :907.0

[0716] Step 4: Synthesis of C-Inter-3-4

[0717]

[0718] Dissolve C-Inter-3-3 (123 mg) and intermediate B (36.6 mg, 0.081 mmol) in DMF (7 mL), and add DIEA (27.36 mg). Stir at room temperature for 2 hours. After the reaction is completed, freeze-dry to obtain the crude product of C-Inter-3-4. MS [M+1] + :1040.0Step 5: Synthesis of C-Inter-3

[0719]

[0720] C-Inter-3-4 (crude), TFA (10 mL), and water (0.5 mL) were added to a reaction flask, stirred at room temperature for 30 minutes, concentrated at low temperature to remove the solvent, and the crude product was purified by high pressure preparative liquid chromatography to obtain C-Inter-3 (65 mg). MS [M / 2+1] + :789.8 Example 14: Synthesis of C-Inter-6

[0721]

[0722] Step 1: Synthesis of C-Inter-6-1

[0723]

[0724] Linker 3 (182 mg, 0.3 mmol) was dissolved in DCM (5 ml) and TEA (60.6 mg, 0.6 mmol) was added. INT-DRUG (220 mg, 0.3 mmol) was dissolved in DCM (3 ml) and then added dropwise to the mixture and stirred at room temperature for 16 hours. The mixture was then concentrated under reduced pressure to obtain a crude product, which was purified by reverse phase column chromatography (acetonitrile / water) and freeze-dried to obtain a white solid C-Inter-6-1 (397 mg). MS [M / 2+1] + :903.9

[0725] Step 2: Synthesis of C-Inter-6-2

[0726]

[0727] C-Inter-6-1 (440 mg, 0.24 mmol) was dissolved in methanol (6 ml) and lithium hydroxide aqueous solution (0.48 mmol, 1 ml) was added. After stirring at room temperature for 1 hour, acetic acid (0.2 mL) was added. After low temperature concentration, the mixture was purified by reverse phase column chromatography (acetonitrile / water) to obtain C-Inter-6-2 (310 mg) as a white solid. MS [M / 2+1] + :863.9

[0728] Step 3: Synthesis of C-Inter-6-3

[0729]

[0730] At zero degrees, add THPTA (62.6 mg, 0.14 mmol), sodium ascorbate (71.28 mg, 0.36 mmol) and CuSO4 (57.2 mg, 0.36 mmol) in clear water (2 ml) to a solution of C-Inter-6-2 (310 mg, 0.18 mmol) and azide-octaethylene glycol-ammonia (188 mg, 0.72 mmol) in MeCN (2 ml), protect with nitrogen, stir the reaction solution at room temperature for 1 hour, after the reaction is completed, purify by high pressure preparative liquid chromatography to obtain C-Inter-6-3 (176 mg). MS [M / 2+1] + :1083.0

[0731] Step 4: Synthesis of C-Inter-6-4

[0732]

[0733] At zero degrees, add Inter B (43.04 mg, 0.09 mmol) and DIEA (30.96 mg, 0.24 mmol) to a DMF (3 ml) solution of C-Inter-6-3 (176 mg, 0.08 mmol), stir the reaction solution at room temperature for 1 hour, add 1.5 ml of trifluoroacetic acid to the reaction solution after the reaction is complete, and then freeze-dry to obtain C-Inter-6-4. MS [M / 2+1] + :1216.1

[0734] Step 5: Synthesis of C-Inter-6

[0735]

[0736] Dissolve C-Inter-6-4 in 5% trifluoroacetic acid solution (10 ml), stir at room temperature for 15 minutes, spin dry, and purify by high pressure preparative liquid chromatography to obtain the target C-Inter-6 (25 mg). MS [M / 2+1] + :967.4

[0737] Example 15: Synthesis of C-Inter-9

[0738]

[0739] Step 1: Synthesis of C-Inter-9-1

[0740]

[0741] C-Inter-3-3 (155 mg), Inter-C (40.7 mg), and DIEA (34.89 mg) were dissolved in DMF and stirred at room temperature for 2 hours. After the reaction was completed, the crude product of C-Inter-9-1 was obtained by freeze-drying. MS [M / 2+1] + :1013.5

[0742] Step 2: Synthesis of C-Inter-9

[0743]

[0744] The crude product of C-Inter-9-1, TFA (10 mL), and water (0.5 mL) were added to the reaction flask and stirred at room temperature for 30 minutes. After the reaction was completed, the solvent was removed by low temperature concentration and purified by high pressure preparative liquid chromatography to obtain C-Inter-9 (26 mg). MS [M / 2+1] +: 813.5

[0745] Example 16: Expression and purification of hIgG1-Fc

[0746] 1. Experimental Materials

[0747] name Manufacturer Part Number ExpiCHO-S cells Thermo A29127 <![CDATA[ExpiFectamine TM CHO Transfection Kit]]> Thermo A29130 <![CDATA[OptiPRO TM SFM Reduced Serum Medium]]> Thermo 12309019

[0748] 2. Expression of hIgG1-Fc

[0749] 1) One day before transfection (day -1), dilute the cells to a final density of 3-4×10 6 viable cells / mL and allowed the cells to grow overnight.

[0750] 2) On the day of transfection, the cell density should be 5-6×10 6 Cell viability must be >95%.

[0751] 3) Transfect 0.8 μg of pcDNA3.1 plasmid expressing hIgG1-Fc per ml of cells and dilute 0.8 μg into 40 μL OptiPRO TM Add to SFM medium (4°C) and mix gently.

[0752] 4) Use 37 μL of OptiPRO TM Dilute 3.2 μL ExpiFectamine in SFM medium (4°C) TM CHO, mix gently.

[0753] 5) Dilute ExpiFectamine TM Add CHO reagent to the diluted DNA, mix gently, and incubate at room temperature for 1-5 minutes.

[0754] 6) Slowly add the above complex to each shake flask.

[0755] 7) Place the cells in a shaker at 37°C and 8% CO2 and set the rotation speed to 125 rpm.

[0756] 8) After incubating the cells for 20 hours, mix 10 μL ExpiCHO Transfection Enhancer and 400 μL ExpiCHO Feed per mL of cells and add the two to each culture flask.

[0757] 9) After culturing for eight days, harvest the cell supernatant.

[0758] 3. Purification of hIgG1-Fc

[0759] 1) Collection of supernatant

[0760] Transfer the transfected Fc protein fermentation liquid to a 50ml centrifuge tube labeled with the sequence and name, and centrifuge at 10000rpm / min for 5min. Pour the supernatant after centrifugation into a new 50ml centrifuge tube labeled with the name until the same Fc fermentation liquid has been centrifuged and the supernatant has been collected.

[0761] 2) Preprocessing

[0762] The gravity column is soaked in 0.5M NaOH solution. Add the Pro A filler solution to the empty gravity column. After the solution in the gravity column is dripped, add 0.1M NaOH solution to the column. After the solution in the gravity column is dripped, add pure water to the column.

[0763] 3) Balanced loading

[0764] Use 5-10 column volumes of Pro A equilibration buffer to equilibrate the Pro A column. Add the supernatant to be purified after centrifugation to the Pro A column and collect the supernatant that flows through into a clean 250 ml shake bottle. After all the supernatant has passed through the column, add 5-10 column volumes of Pro-A equilibration buffer until all the equilibration buffer flows out at the bottom.

[0765] 4) Protein elution

[0766] Seal the bottom of the washed column with a clean plug. Add 2-3 column volumes of Pro-A / G elution buffer to the column that has been washed and sealed at the bottom. Use a pipette to fully suspend the filler in the column with elution buffer and let it stand for 5 minutes. Prepare for elution, prepare a new collection tube and add a certain proportion of Tris neutralizing solution to neutralize and prevent protein denaturation, open the plug and collect the liquid into the collection tube. Measure the protein concentration and freeze it in a -80℃ refrigerator.

[0767] 4. Characterization of hIgG1 Fc

[0768] According to the sequence information of Fc described herein, the target Fc was obtained according to the expression and purification steps of this example.

[0769]

[0770] Example 17: Expression and purification of hIgG1-Fc

[0771] hIgG1-Fc (SEQ ID NO: 69) was prepared and expressed as described in Example 16.

[0772] 2. Purification of hIgG1-Fc

[0773] Experimental method: Protein A affinity chromatography column purification

[0774] 1) Balance chromatography column: 1xPBS, flow rate 1ml / min, 20ml

[0775] 2) Sample loading: flow rate 1ml / min

[0776] 3) Washing: 1xPBS, flow rate 1ml / min, 20ml

[0777] 4) Elution: Sodium acetate buffer (PH3.2), 1 ml / min, collect in separate tubes, about 500 ul per tube. Collect 10 tubes in total, and read the absorbance at 280 nm using a NanoDrop instrument.

[0778] 5) Dialysis: Pipette high-concentration protein into a dialysis bag and dialyze it in a beaker containing 1X PBS.

[0779] 3. Characterization of hIgG1 Fc

[0780] According to the sequence information of Fc described herein, the target Fc was obtained according to the expression and purification steps of this example.

[0781]

[0782] Example 18: Synthesis and Characterization of Conjugate 1 1. Experimental Materials and Methods

[0783] 1.1 Instruments / Equipment

[0784] Serial number Instrument / Equipment Name Device Model Equipment No. 1 pH Meter S470K RD-080 2 Centrifuge Sorvall SI 16R BP-0317 3 Oscillating metal bath SD2-100 BP-0379 4 Nanodrop lite NanoDrop One BP-0318 5 Clean bench SW-CJ-1D QC-085 Clean bench NA QC-438 Test tube thermostat NA QC-324

[0785] 1.2 Reagents / test solutions

[0786] Serial number Reagent / Test Solution Name Reagent / test solution batch number / article number Remark 1 50 mM PB, pH 6.5 buffer SO-014-087 NA 2 10 mM His-HCl, pH 5.5 buffer SO-014-088 NA 3 10mM TCEP solution SO-014-089 NA 4 10mg / mL C-Inter-1 aqueous solution SO-014-090 NA 5 Endotoxin Standards 150601-202292 Manufacturer: China Inspection Institute, 90EU / piece 6 Limulus test kit 21061034 Manufacturer Xiamen horseshoe crab reagent, 0.06EU / mL 7 Endotoxin test water 2008130 Manufacturer: Zhanjiang Andos, <0.003EU / mL 8 Tris buffer 2109240 Manufacturer: Zhanjiang Andos, <0.03EU / mL

[0787] 1.3 Experimental methods

[0788]

[0789] 1.3.1 Ultrafiltration

[0790] 1) Ultrafiltration centrifuge tube pretreatment

[0791] Take two 15mL 10KDa ultrafiltration centrifuge tubes, add 0.1M NaOH solution, and centrifuge at 4000G for 10

[0792] min, repeat 3 times.

[0793] Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4000G for 10 min, and repeat twice.

[0794] Add 50 mM PB, pH 6.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4000G for 10 min. Repeat until the pH of the permeate is about 6.5.

[0795] 2) Antibody ultrafiltration and fluid exchange

[0796] Add Fc (SEQ ID NO: 67) antibody to the treated ultrafiltration centrifuge tube and use 50mM PB, pH 6.5

[0797] The volume of the solution in the ultrafiltration tube was supplemented with buffer to 12 mL, and the solution was centrifuged at 4000 G for 10 min. This was repeated 4 times, and samples were taken to detect the antibody concentration after the solution was replaced.

[0798] 3) Antibody concentration detection

[0799] The Protein A280 method in NanoDrop One was used for detection, and the sample category was selected as "Other

[0800] protein E 1%, the extinction coefficient E1% L / gm-cm was set to 13.5, and the baseline correction wavelength was 340 nm.

[0801] Use 50 mM PB, pH 6.5 buffer as blank, draw 2.5 μL sample for detection, and take the average of three test results as the sample concentration.

[0802] 1.3.2 Reduction reaction

[0803] 50 mM PB, pH 6.5 buffer, Fc (SEQ ID NO: 67) antibody solution (30

[0804] mg) and 10 mM TCEP solution (TCEP / mAb molar equivalent ratio was 10), and the antibody reaction concentration was controlled to be 5

[0805] mg / mL, placed in a metal bath and controlled at 22°C for 2 hours.

[0806] 1.3.3 Coupling reaction

[0807] After the reduction reaction reached 2 hours, 50 mM PBS, pH 6.5 buffer and 10 mg / mL

[0808] C-Inter-1 solution (C-Inter / Fc molar equivalent ratio is 7) was placed in a metal bath and the reaction temperature was controlled to be 22° C. for 1 hour.

[0809] After the reaction time was reached, 10 mg / mL C-Inter-1 with a molar equivalent ratio of C-Inter-1 / Fc of 3.5 was added to the reaction solution.

[0810] The Inter-1 solution was placed in a metal bath and the reaction temperature was controlled at 22°C and the reaction was continued for 0.5 hour.

[0811] After the reaction time was reached, 10% C-Inter-1 / mAb was added to the reaction solution again with a molar equivalent ratio of 3.5.

[0812] mg / mL C-Inter-1 solution was placed in a metal bath and the reaction temperature was controlled at 22°C and the reaction was continued for 1.0 hour.

[0813] 1.3.4 Hydrolysis reaction

[0814] 0.5 M NaOH solution was added to the reaction solution to adjust the pH to about 7.5, and the solution was placed in a metal bath and the reaction temperature was controlled at 22° C. to continue the reaction for 2 hours to obtain conjugate 1.

[0815] 1.3.5 Ultrafiltration II

[0816] 1) Ultrafiltration centrifuge tube pretreatment

[0817] Take two 15mL 10KDa ultrafiltration centrifuge tubes, add 0.1M NaOH solution, and centrifuge at 4000G for 10

[0818] min, repeat 3 times.

[0819] Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4000G for 10 min, and repeat twice.

[0820] Add 10 mM His-HCl, pH 5.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4000G for 10 min. Repeat until the pH of the permeate is about 5.6.

[0821] 2) Antibody ultrafiltration and fluid exchange

[0822] Add conjugate 1 to the treated ultrafiltration centrifuge tube, use 10mM His-HCl, pH5.5 buffer to replenish the volume of the solution in the ultrafiltration tube to 12mL, centrifuge at 4000G for 10min, repeat 6 times, filter the ultrafiltered solution using a 0.22μm PES filter membrane, and detect the antibody concentration after the liquid change.

[0823] 3) Antibody concentration detection

[0824] The Protein A280 method in NanoDrop One was used for detection, and the sample category was selected as "Other

[0825] protein E 1%, the extinction coefficient E1% L / gm-cm was set to 13.5, and the baseline correction wavelength was 340 nm.

[0826] Use 10 mM His-HCl, pH 5.5 buffer as blank, draw 2.5 μL sample for detection, and take the average of three test results as the sample concentration.

[0827] According to the concentration and volume of the test sample, use 10mM His-HCl, pH5.5 buffer to dilute the sample concentration to about 10mg / mL, and re-test the diluted sample concentration.

[0828] 2. Experimental results and analysis

[0829] 2.1 LC-MS DAR value detection

[0830] The DAR value of the sample was detected by LC-MS. The relevant method conditions are shown in the following table:

[0831] LC method conditions

[0832]

[0833] MS method conditions

[0834] name Parameter settings instrument SCIEX 5600 Scan range 900-5000 polarity Positive Atomizing gas (GS1) 50 Auxiliary gas (GS2) 50 Curtain Air (CUR) 25 Ion transfer tube temperature (TEM) 450 Spray voltage (ISVF) 5500 Declustering Potential (DP) 250 Collision Energy (CE) 10 Cumulative time (AT) 1s

[0835] The MS-DAR value test results of the samples are shown in the table below. The MS test results show that the MS-DAR value of the conjugate 1 sample is 4.0. After the sample is treated with sugar removal and reduction, the peak is a single-chain peak. C0, C1, C2, and C3 are the molecular weights of 0, 1, 2, and 3 small molecules, respectively.

[0836]

[0837] Sample MS detection spectrum see Figure 1 :

[0838] 2.2SEC-HPLC purity test

[0839] The sample was tested for purity by SEC-HPLC. The relevant method conditions are shown in the table below:

[0840]

[0841] The small molecule residue test results of the samples are shown in Figure 2 , SEC test results showed that the purity of conjugate 1 was 98.21%.

[0842] 2.3 RP-HPLC small molecule residue detection

[0843] The samples were tested for residual free drug levels (mol / mol, free drug / bound drug) by RP-HPLC. Two standards (2% & 5%) containing free drug and Fc were prepared first. The free drug percentage was determined by comparing the EIC signal peak area with the residual free drug in the conjugate sample. The relevant method conditions are shown in the table below:

[0844]

[0845]

[0846] Preparation of standards:

[0847] C Fc(SEQ ID NO：67) =1mg / mL, C Fc(SEQ ID NO：69) =1 mg / mL; the DAR values ​​of the samples were 4.0 and 6.0 respectively;

[0849] MW Fc(SEQ ID NO:67) =55414 g / mol, MW Fc(SEQ ID NO:69) =56000 g / mol, MW C-Inter-1 =1656

[0850] g / mol;

[0851] M mAb =1 / MW mAb , M Total drug =DAR / MW mAb ;

[0852] 2% standard moles = DAR × M mAb ×2%;

[0853] Number of moles of 5% standard = DAR × M mAb ×5%;

[0854] The weight of small molecules added to 100 μL of 2% standard is 2% m C-Inter-1 = Number of moles of 2% standard × 100 μL ×

[0855] MW C-Inter-1 ;

[0856] The weight of small molecules added to 100 μL of 5% standard is 5% m C-Inter-1 = Number of moles of 5% standard × 100 μL ×

[0857] MW C-Inter-1 ;

[0858] The calculated small molecule weight was added to 100 μL of 1 mg / mL antibody solution to obtain a standard.

[0859] The small molecule residue test results of the samples are shown in Figure 3 , where AKSZ-2627-0.01mg / mL sample is the AKSZ2627 small molecule sample, AKSZ-2627-2% and AKSZ-2627-5% are samples with 2% and 5% mol / mol small molecules added to the corresponding Fc, and C060-7607B-230424 is the conjugate 1 sample. The RP test results show that the two samples have a RP-

[0860] No small molecule peaks were detected in HPLC. Compared with antibody samples with 2% mol / mol and 5% mol / mol small molecules added, the sample residues were less than 2% mol / mol.

[0861] 2.4 Endotoxin Residue Detection

[0862] The endotoxin residues in the samples were tested using the following steps:

[0863] 1) Take one endotoxin working standard, add 1 mL of BET water, vortex for 15 minutes, and gradually dilute to 4λ

[0864] (E 4λ )、2λ(E 2λ )spare.

[0865] 2) Preparation of test solution: Take the sample solution and dilute it gradually to 80 times (S 80 ) and 160 times

[0866] (S 160 )spare.

[0867] 3) Preparation of each solution

[0868] Test group A: S 160 Solution;

[0869] Test sample positive control group B: one times the amount of S 80 The solution was added to one amount of E 4λ Solution, mix well, prepare

[0870] S 160 E 2λ ;

[0871] Positive control group C:E 2λ Solution;

[0872] Negative control group D: BET water.

[0873] 4) Reconstitution of horseshoe crab reagent: Take horseshoe crab reagent and add 0.1 mL of BET water to each vial for reconstitution.

[0874] 5) After the horseshoe crab reagent is reconstituted, take 0.1 mL of each of the four solutions prepared in step 3) and add them to the horseshoe crab reagent, with two parallel horseshoe crab reagents for each group.

[0875] 6) Incubation: Place the mixed ampoule into a test tube thermostat preheated to 37°C and incubate for 60 minutes.

[0876] Test results:

[0877] The concentration of conjugate 1 was 10.13 mg / mL, and the residual endotoxin in the sample was less than 1 EU / mg.

[0878] Example 19: Synthesis and characterization of conjugate 2

[0879] 1. Experimental Materials and Methods

[0880] 1.1 Instruments / equipment are the same as in Example 18

[0881] 1.2 Reagents / test solution Same as Example 18

[0882] 1.3 Experimental methods

[0883]

[0884] 1.3.1 Ultrafiltration

[0885] 1) Ultrafiltration centrifuge tube pretreatment

[0886] Take two 15mL 10KDa ultrafiltration centrifuge tubes, add 0.1M NaOH solution, and centrifuge at 4000G for 10

[0887] min, repeat 3 times.

[0888] Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4000G for 10 min, and repeat twice.

[0889] Add 50 mM PB, pH 6.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4000G for 10 min. Repeat until the pH of the permeate is about 6.5.

[0890] 2) Antibody ultrafiltration and fluid exchange

[0891] Add Fc (SEQ ID NO: 69) antibody to the treated ultrafiltration centrifuge tube, use 50mM PB, pH6.5 buffer to replenish the solution volume in the ultrafiltration tube to 12mL, centrifuge at 4000G for 10min, repeat 4 times, and take samples to detect the antibody concentration after changing the solution.

[0892] 3) Antibody concentration detection

[0893] The Protein A280 method in NanoDrop One was used for detection, and the sample category was selected as "Other

[0894] protein E 1%", the extinction coefficient E1% L / gm-cm was set to 13.44, and the baseline correction wavelength was 340nm.

[0895] Use 50 mM PB, pH 6.5 buffer as blank, draw 2.5 μL sample for detection, and take the average of three test results as the sample concentration.

[0896] 1.3.2 Reduction reaction

[0897] 50 mM PBS, pH 6.5 buffer, Fc (SEQ ID NO: 69) antibody solution (30 mg) and 10 mM TCEP solution (TCEP / Fc molar equivalent ratio is 10) were added to the centrifuge tube in sequence to control the antibody reaction concentration to 5

[0898] mg / mL, placed in a metal bath and controlled at 22°C for 2 hours.

[0899] 1.3.3 Coupling Reaction After the reduction reaction reaches 2 hours, add 50mM PBS, pH 6.5 buffer and 10mg / mL C-Inter-1 solution (C-Inter-1 / Fc molar equivalent ratio is 12) to the reaction solution to control the final concentration of the reaction to 2mg / mL. Place the reaction solution in a metal bath and control the reaction temperature to 22°C for 1 hour.

[0900] After the reaction time was reached, 10 mg / mL C-Inter-1 solution with a C-Inter-1 / Fc molar equivalent ratio of 6.0 was added to the reaction solution, and the solution was placed in a metal bath and the reaction temperature was controlled to be 22°C and the reaction was continued for 0.5 hour.

[0901] After the reaction time was reached, 10 mg / mL C-Inter-1 solution with a C-Inter-1 / Fc molar equivalent ratio of 6.0 was added to the reaction solution again, and the reaction solution was placed in a metal bath and the reaction temperature was controlled to be 22° C. and the reaction was continued for 1.0 hour.

[0902] 1.3.4 Hydrolysis reaction: Add 0.5 M NaOH solution to the reaction solution, adjust the pH to about 7.5, place in a metal bath and control the reaction temperature to 22° C. to continue the reaction for 2 hours to obtain conjugate 2.

[0903] 1.3.5 Ultrafiltration II

[0904] 1) Ultrafiltration centrifuge tube pretreatment

[0905] Take two 15mL 10KDa ultrafiltration centrifuge tubes, add 0.1M NaOH solution, and centrifuge at 4000G for 10

[0906] min, repeat 3 times.

[0907] Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4000G for 10 min, and repeat twice.

[0908] Add 10 mM His-HCl, pH 5.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4000G for 10 min. Repeat until the pH of the permeate is about 5.6.

[0909] 2) Antibody ultrafiltration and fluid exchange

[0910] Add conjugate 2 to the treated ultrafiltration centrifuge tube, use 10mM His-HCl, pH5.5 buffer to replenish the volume of the solution in the ultrafiltration tube to 12mL, centrifuge at 4000G for 10min, repeat 6 times, filter the ultrafiltered solution using a 0.22μm PES filter membrane, and detect the antibody concentration after changing the solution.

[0911] 3) Antibody concentration detection

[0912] The Protein A280 method in NanoDrop One was used for detection, and the sample category was selected as "Other

[0913] protein E 1%", the extinction coefficient E1% L / gm-cm was set to 13.44, and the baseline correction wavelength was 340nm.

[0914] Use 10 mM His-HCl, pH 5.5 buffer as blank, draw 2.5 μL sample for detection, and take the average of three test results as the sample concentration.

[0915] According to the concentration and volume of the test sample, use 10mM His-HCl, pH5.5 buffer to dilute the sample concentration to about 10mg / mL, and re-test the diluted sample concentration.

[0916] 2. Experimental results and analysis

[0917] 2.1 LC-MS DAR value detection

[0918] The method was the same as Example 18, and the MS-DAR value of the conjugate 2 sample was 6.0.

[0919] Sample MS detection spectrum see Figure 4 After the sample was treated with sugar removal and reduction, the peak was a single-chain peak. C0, C1, C2,

[0920] C3 is the molecular weight obtained by adding 0, 1, 2, and 3 small molecules respectively.

[0921]

[0922] 2.2SEC-HPLC purity test

[0923] The method is the same as in Example 18, and the SEC test results are shown in Figure 5 , the purity of conjugate 2 was 94.47%.

[0924] 2.3 RP-HPLC small molecule residue detection

[0925] The method is the same as Example 18, and the small molecule residue is <2%. Figure 6 shown.

[0926] 2.4 Endotoxin Residue Detection

[0927] The method was the same as in Example 18, the concentration of conjugate 2 was 10.26 mg / mL, and the endotoxin residue in the sample was less than 1

[0928] EU / mg.

[0929] Example 20: Preparation of Conjugate 3

[0930] 1. Materials and Methods

[0931] 1) Experimental instruments

[0932] Device Name factory Model / Specification Equipment No. Oscillating metal bath Titan Technology SD2-100 BP-0379 pH Meter Mettler Toledo S470K RD-080 Electronic balance Mettler Toledo XSR205DU / A RD-079 Electronic balance Mettler Toledo Me802E BP-0308 Vortex mixer IKA IKA MS3 Basic BP-113 Nanodrop lite Thermo NanoDrop One BP-0318 Medical low temperature refrigerator Haier DW-25L262 BP-291 Ultra-low temperature refrigerator Thermo 902-ULTS BP-290

[0933] 2) Reagents / Solutions

[0934] name batch number 50 mM PB, pH 6.5 buffer SO-007-096 10 mM His-HCl, pH 5.5 buffer SO-038-032 10mM TCEP solution SO-038-037 10mg / mL C-Inter-3 aqueous solution S0-883-098(N)

[0935] 2. Experimental Methods

[0936]

[0937] 1) Ultrafiltration

[0938] (1) Antibody ultrafiltration and fluid exchange

[0939] Add Fc (SEQ ID NO: 71) to the ultrafiltration centrifuge tube and use 50mM PB, pH 6.5 buffer to replenish the solution in the ultrafiltration tube.

[0940] The volume was increased to 12 mL, and the mixture was centrifuged at 4500 G for 10 min. This was repeated 4 times, and samples were taken to detect the antibody concentration.

[0941] (2) Antibody concentration detection

[0942] The Protein A280 method was used in NanoDrop One, and the sample category was selected as "Otherprotein E 1%".

[0943] The extinction coefficient E1% L / gm-cm was set to 13.44 (7607H), and the baseline correction wavelength was 340 nm. 50 mM Pb, pH 6.5 buffer was used as a blank, 2.5 μL of sample was drawn for detection, and the average of three test results was taken as the sample concentration.

[0944] 2) Reduction reaction

[0945] 50 mM PB, pH 6.5 buffer, the corresponding antibody solution (28 mg) and 10 mM TCEP solution (TCEP / mAb equivalent ratio is 20) were added to the centrifuge tube in sequence, the antibody reaction concentration was controlled to be 5 mg / mL, and the reaction temperature was controlled to be 25° C. in a metal bath for 2 hours. Equivalents herein refer to molar equivalents unless otherwise specified or contradictory to the context.

[0946] 3) Coupling reaction

[0947] 1. After the reduction reaction reaches 2 hours, add 10 mg / mL C-Inter-3 solution (Drug / mAb

[0948] 2. The equivalent ratio is 10), placed in a metal bath and controlled to react at 25°C for 1 hour, and 10 mg / mL C-Inter-3 aqueous solution (AKSZ2665 aqueous solution) (Drug / mAb equivalent ratio is 10) is added repeatedly every 1 hour until the total reaction equivalent is 50 (add 0.5 M NaOH to adjust the solution pH to about 6.5). After the reaction is completed, adjust the pH to 7.0-7.5 with 0.5 M NaOH, and continue to react at 25°C in the metal bath for 2 hours.

[0949] 4) Ultrafiltration

[0950] (1) Antibody ultrafiltration and fluid exchange

[0951] 3. Add the conjugate sample to the treated ultrafiltration centrifuge tube, use 10mM His-HCl, pH5.5 buffer to replenish the volume of the solution in the ultrafiltration tube to 12mL, centrifuge at 4500G for 10min, repeat 12 times, filter the ultrafiltered solution with a 0.22μm PES filter membrane, and detect the antibody concentration after the liquid change.

[0952] (2) Antibody concentration detection

[0953] The Protein A280 method in NanoDrop One was used for detection, and the sample category was selected as "Otherprotein E

[0954] 1%”, the extinction coefficient E1%L / gm-cm was set to 13.44, and the baseline correction wavelength was 340nm.

[0955] Use 10 mM His-HCl, pH 5.5 buffer as blank, draw 2.5 μL sample for detection, and take the average of three test results as the sample concentration.

[0956] 5) Experimental results and analysis

[0957] (1) LC-MS DAR value detection

[0958] The DAR value of the sample was detected by LC-MS. The relevant method conditions are shown in the following table:

[0959] LC method conditions

[0960]

[0961]

[0962] MS method conditions

[0963] name Parameter settings instrument SCIEX 5600 Scan range 900-5000 polarity Positive Atomizing gas (GS1) 50 Auxiliary gas (GS2) 50 Curtain Air (CUR) 25 Ion transfer tube temperature (TEM) 450 Spray voltage (ISVF) 5500 Declustering Potential (DP) 250 Collision Energy (CE) 10 Cumulative time (AT) 1s

[0964] The MS-DAR value test results of the samples are shown in the table (figure) below. The MS test results show that the MS-DAR value of the C060-7607H-2665-240125 (conjugate 3) sample is 5.9.

[0965]

[0966] Remark:

[0967] Load distribution calculation formula: drug load distribution (%) = (single-chain peak area with drug loading of n / sum of two single-chain peak areas) × 100 (%);

[0968] Where n is the drug loading, and one single chain is the heavy chain constant domain 2 and heavy chain constant domain 3 and 1 / 2 hinge region (CH2+CH3+1 / 2Hi);

[0969] DAR value calculation formula: Single-chain weighted peak area = (drug load n × drug load distribution %)

[0970] Drug to antibody ratio = 2 × ∑ (single chain weighted peak area)

[0971] Sample MS detection spectrum see Figure 8 :

[0972] (2) SEC-HPLC purity test

[0973] The sample was tested for purity by SEC-HPLC. The relevant method conditions are shown in the table below:

[0974]

[0975]

[0976] The purity test results of the samples are shown in Fig. 9 , SEC test results showed that the purity of C060-7607H-2665-240125 (conjugate 3) was 97.50%.

[0977] (3) RP-HPLC small molecule residue detection

[0978] The samples were tested for residual free drug levels (mol / mol, free drug / bound drug) by RP-HPLC.

[0979] Two standards of free drug and Fc (2% & 5%) were used to compare the EIC with the residual free drug in the conjugate sample.

[0980] The signal peak area is used to determine the percentage of free drug. The relevant method conditions are shown in the table below:

[0981]

[0982] Preparation of standards:

[0983] C 7607H =1 mg / mL; the sample DAR value is 5.9;

[0984] MW 7607H =56000 g / mol, MW drug =1578.63 g / mol;

[0985] M mAb =1 / MW mAb , M Totaldrug =DAR / MW mAb ;

[0986] 2% standard moles = DAR × M mAb ×2%;

[0987] Number of moles of 5% standard = DAR × M mAb ×5%;

[0988] The weight of small molecules added to 100 μL of 2% standard is 2% m drug = Number of moles of 2% standard × 100 μL × MW drug ;

[0989] The weight of small molecules added to 100 μL of 5% standard is 5% m drug = Number of moles of 5% standard × 100 μL × MW drug ;

[0990] The calculated small molecule weight was added to 100 μL of 1 mg / mL antibody solution to obtain a standard.

[0991] The small molecule residue test results of the samples are shown in Fig.10 ,The RP test results showed that: compared with the antibody samples with 2% mol / mol and 5% mol / mol small molecules added, the sample residue in RP-HPLC was less than 2% mol / mol.

[0992] Example 21: Preparation of Conjugate 4

[0993] 1. Experimental Overview

[0994] The experiment used the reducing agent TCEP to open the disulfide bonds between antibody chains, and then used -SH to react with the maleimide on the Linker-payload to produce a coupling product with the target DAR6. The DAR value of the coupling product was detected by LC-MS, the purity of the coupling product was detected by SEC-HPLC, and the small molecule residues of the coupling product were detected by RP-HPLC.

[0995]

[0996] 2. Summary of experimental results

[0997]

[0998] 3. Experimental Materials and Methods

[0999] 3.1 Instruments / Equipment

[1000] Serial number Instrument / Equipment Name Device Model Equipment No. 1 pH Meter S470K RD-080 2 Centrifuge Sorvall SI 16R BP-0317 3 Oscillating metal bath SD2-100 BP-0379 4 Nanodrop lite NanoDrop One BP-0318 5 Clean bench SW-CJ-1D QC-085

[1001] 3.2 Reagents / test solutions

[1002] Serial number Reagent / Test Solution Name Reagent / test solution batch number Remark 1 50 mM PB, pH 6.5 buffer SO-007-076 NA 2 10 mM His-HCl, pH 5.5 buffer SO-007-077 NA 3 10mM TCEP solution SO-014-089 NA 4 10mg / mL C-Inter-1 aqueous solution SO-007-045 NA

[1003] 3.3 Experimental methods

[1004] 3.3.1 Ultrafiltration

[1005] 1) Ultrafiltration centrifuge tube pretreatment

[1006] Take a 15mL 10KDa ultrafiltration centrifuge tube, add 0.1M NaOH solution, centrifuge at 4500G for 10min, repeat 3 times. Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4500G for 10min, repeat 2 times. Add 50

[1007] mM PB, pH 6.5 buffer was added to an ultrafiltration centrifuge tube and centrifuged at 4500G for 10 min. This was repeated until the pH of the permeate was

[1008] Around 6.5.

[1009] 2) Antibody ultrafiltration and fluid exchange

[1010] Add 7607H antibody (SEQ ID NO: 71) to the treated ultrafiltration centrifuge tube, use 50mM PB, pH6.5 buffer to replenish the solution volume in the ultrafiltration tube to 12mL, centrifuge at 4500G for 10min, repeat 4 times, filter with 0.22μm filter membrane, and take samples to detect antibody concentration.

[1011] 3) Antibody concentration detection

[1012] The Protein A280 method in NanoDrop One was used for detection, and the sample category was selected as "Otherprotein E

[1013] 1%”, the extinction coefficient E1% L / gm-cm was set to 13.44 (7607H), and the baseline correction wavelength was 340nm. Using 50

[1014] mM PB, pH 6.5 buffer was used as blank, 2.5 μL of sample was drawn for detection, and the average of three test results was taken as the sample concentration.

[1015] 3.3.2 Reduction reaction

[1016] Add 50mM PB, pH 6.5 buffer, the corresponding antibody solution (45mg) and 10mM

[1017] TCEP solution (TCEP / mAb equivalent ratio is 12), the antibody reaction concentration is controlled to be 5 mg / mL, and the reaction temperature is controlled to be 22° C. in a metal bath for 2 hours.

[1018] 3.3.3 Coupling reaction and hydrolysis reaction

[1019] 4. After the reduction reaction reached 2 hours, 50mM PB, pH 6.5 buffer and small molecules were added to the reaction solution.

[1020] 10mg / mL C-Inter-1 aqueous solution (AKSZ2627 aqueous solution) with a drug / mAb equivalent ratio of 10

[1021] The equivalent ratio was 10), the final concentration of the reaction was controlled to be 2 mg / mL, and the reaction temperature was controlled to be 22°C in a metal bath.

[1022] After the reaction time is reached, a 10 mg / mL C-Inter-1 aqueous solution (AKSZ2627 solution) with a Drug / mAb equivalent ratio of 5 is added to the 7607H antibody reaction solution, and the reaction is continued for 1 hour at a controlled reaction temperature of 22°C in a metal bath. After the reaction is completed, the pH is adjusted to 7.0-7.5 with 0.5 M NaOH, and the reaction is continued for 2 hours at 22°C in a metal bath.

[1023] 3.3.4 Ultrafiltration II

[1024] 1) Ultrafiltration centrifuge tube pretreatment

[1025] 5. Take a 15mL 10KDa ultrafiltration centrifuge tube, add 0.1M NaOH solution, centrifuge at 4500G for 10min, repeat 3 times. Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4500G for 10min, repeat 2 times. Add 10mM His-HCl, pH5.5 buffer to the ultrafiltration centrifuge tube, centrifuge at 4500G for 10min, repeat until the pH of the permeate is about 5.5.

[1026] 2) Antibody ultrafiltration and fluid exchange

[1027] 6. Add the coupled sample to the treated ultrafiltration centrifuge tube, use 10mM His-HCl, pH5.5 buffer to replenish the volume of the solution in the ultrafiltration tube to 12mL, centrifuge at 4500G for 10min, repeat 8 times, filter the ultrafiltration solution with a 0.22μm PES filter membrane, and detect the antibody concentration after the liquid change.

[1028] 3) Antibody concentration detection

[1029] 7. Use the Protein A280 method in NanoDrop One for detection, select the sample category as "Otherprotein E 1%, set the extinction coefficient E1% L / gm-cm to 13.44, and the baseline correction wavelength to 340nm. Use 10

[1030] mM His-HCl, pH5.5 buffer was used as blank, 2.5μL sample was drawn for detection, and the average of three test results was taken as the sample concentration. According to the test sample concentration and sample volume, the sample concentration was diluted to about 10mg / mL with 10mM His-HCl, pH5.5 buffer, and the diluted sample concentration was re-detected.

[1031] 4. Experimental results and analysis

[1032] 4.1 LC-MS DAR value detection

[1033] The DAR value of the sample was detected by LC-MS. The relevant method conditions are shown in the following table:

[1034] LC method conditions

[1035]

[1036] MS method conditions

[1037] name Parameter settings instrument SCIEX 5600 Scan range 900-5000 polarity Positive Atomizing gas (GS1) 50 Auxiliary gas (GS2) 50 Curtain Air (CUR) 25 Ion transfer tube temperature (TEM) 450 Spray voltage (ISVF) 5500 Declustering Potential (DP) 250 Collision Energy (CE) 10 Cumulative time (AT) 1s

[1038] The MS-DAR value test results of the samples are shown in the table (figure) below. The MS test results show that the MS-DAR value of the conjugate 4 (internal number C060-7607H-231008) sample is 5.9.

[1039]

[1040] Remark:

[1041] The formula for calculating the load distribution is: drug load distribution (%) = (peak area of ​​the single chain with drug loading n / sum of the peak areas of the two single chains) × 100 (%);

[1042] Where n is the drug loading, and one single chain is the heavy chain constant domain 2 and heavy chain constant domain 3 and 1 / 2 hinge region (CH2+CH3+1 / 2Hi);

[1043] DAR value calculation formula: single chain weighted peak area = (drug loading n × drug loading distribution %)

[1044] Drug-antibody ratio = 2 × ∑ (single-chain weighted peak area)

[1045] Sample MS detection spectrum see Fig.11 .

[1046] 4.2SEC-HPLC purity test

[1047] The sample was tested for purity by SEC-HPLC. The relevant method conditions are shown in the table below:

[1048]

[1049] The purity test results of the samples are shown in Fig.12 SEC test results showed that the purity of conjugate 4 (C060-7607H-231008) was 98.57%.

[1050] 4.3 RP-HPLC small molecule residue detection

[1051] The samples were tested for residual free drug levels (mol / mol, free drug / bound drug) by RP-HPLC. Two standards (2% & 5%) containing free drug and Fc were prepared first. The free drug percentage was determined by comparing the EIC signal peak area with the residual free drug in the conjugate sample. The relevant method conditions are shown in the table below:

[1052]

[1053]

[1054] Preparation of standards:

[1055] C 7607H =1 mg / mL; the sample DAR value is 5.9;

[1056] MW 7607H =56000 g / mol, MW drug =1950.79 g / mol;

[1057] M mAb =1 / MW mAb , M Total drug =DAR / MW mAb ;

[1058] 2% standard moles = DAR × M mAb ×2%;

[1059] Number of moles of 5% standard = DAR × M mAb ×5%;

[1060] The weight of small molecules added to 100 μL of 2% standard is 2% m drug = Number of moles of 2% standard × 100 μL × MW drug ;

[1061] The weight of small molecules added to 100 μL of 5% standard is 5% m drug = Number of moles of 5% standard × 100 μL × MW drug ;

[1062] The calculated small molecule weight was added to 100 μL of 1 mg / mL antibody solution to obtain a standard.

[1063] The small molecule residue test results of the samples are shown in Fig.13 The RP test results showed that no small molecule peak was detected in the sample in RP-HPLC. Compared with the antibody samples with 2% mol / mol and 5% mol / mol small molecules added, the sample residue (C060-7607H-231008) was less than 2% mol / mol.

[1064] 4.4 Endotoxin Residue Detection

[1065] Test results:

[1066] According to the comparison with 1EU / mg positive control, the endotoxin residues of the samples were all less than 1EU / mg.

[1067] Example 22: In vitro anti-influenza virus activity of the conjugate (influenza virus H1N1-mediated cytopathic inhibition assay)

[1068] 1. Experimental Methods

[1069] A cytopathicity-based in vitro anti-IAV / IBV activity assay using the MDCK cell line was used to quantify the IAV / IBV-induced cytopathicity of the test compounds.

[1070] 2. Experimental Materials

[1071] Material source batch number H1N1 (A / PR / 8 / 34) virus ATCC VR1469 MDCK cells ATCC CCL-34 96-well plate Corning 3917 Fetal Bovine Serum(FBS) Hyclone SH30406.05 100x Pen-Strep solution (P / S) Hyclone SV30010 DMEM Hyclone SH30243.01 TPCK treated trypsin Sigma T1426 Cell-titer Glo Promega G7571 Zanamivir Adamas P1695633

[1072] 3. Experimental Procedure

[1073] 1) MDCK cells were digested and diluted to 2×10 5 / mL, 50 μL per well was inoculated into a 96-well plate and cultured in an incubator overnight.

[1074] 2) First dilute the compound to 2 μM, then dilute it three-fold from 2 μM, for a total of 8 dilutions. Add 5 μL of the diluted compound to each well and place it in the incubator for 1 hour.

[1075] 3) Dilute influenza virus to 2222 pfu / mL with DMEM medium containing 2% FBS, 1% P / S and 4 μg / mL TPCK treated trypsin, and add 45 μL of virus to each well. At the same time, set up a control without adding compound but adding virus and a cell control without adding compound and virus. Place in an incubator and culture for 4 days.

[1076] 4) Add 50 μL of Cell-titer Glo to each well and detect chemiluminescence using an enzyme reader.

[1077] 4. Data analysis: Calculate the virus inhibition rate of the compound.

[1078] Inhibition rate = (value of the sample measured - average value of the virus control group) / (average value of the cell control group - average value of the virus control group) * 100%

[1079] The calculated inhibition rate was used to fit the inhibition curve using the log (inhibitor) vs. response--Variable slope (four parameters) in the Nonlinear regression (curvefit) function of GraphPad Prism 8 software to calculate the EC50 value.

[1080] Reference molecule A and reference molecule B were prepared according to the specific operations of Example 156 (Conjugate 45b) and Example 188 (Conjugate 46) in patent WO2021046549A1, respectively.

[1081] The purity of reference molecule A was 99.2% (SEC-HPLC) and the DAR value was 4.26.

[1082] The purity of reference molecule B was 98.8% (SEC-HPLC) and the DAR value was 5.87.

[1083] Summary of results:

[1084] Compound DAR value <![CDATA[EC 50 (nM)]]> Conjugate 1 4 0.068 Conjugate 2 6 0.038 Conjugate 3 5.9 0.060 Conjugate 4 5.9 0.060 Reference molecule A 4.26 0.60 Reference molecule B 5.87 0.40

[1085] The data show that the tested conjugate molecules have excellent in vitro anti-H1N1 virus activity. Compared with reference molecules A and B, the activity of conjugate 1 is increased by 8.8 times and 5.9 times, respectively. The activity of conjugate 2 is increased by 15.8 times and 10.5 times, respectively. The activity of conjugates 3 and 4 is increased by 10.0 times and 6.7 times, respectively.

[1086] The higher in vitro antiviral activity indicates that the conjugate molecule of the present invention has more excellent antiviral activity in animals and clinical humans.

[1087] Example 23: Conjugate 1 Coupling Condition Screening Experiment

[1088] Except for the different coupling conditions, the experimental materials, experimental methods and detection methods are the same as those in Example 18.

[1089] The coupling reaction conditions for adding C-Inter-1 at one time are as follows: after the reduction reaction reaches 2 hours, 50mM PBS, pH 6.5 buffer and 10mg / mL C-Inter-1 solution (C-Inter-1 / Fc molar equivalent ratio is 7) are added to the reaction solution at one time to control the final concentration of the reaction to be 1mg / mL. The reaction is placed in a metal bath and the reaction temperature is controlled at 22°C for 2 hours.

[1090] The DAR values ​​detected by LC-MS are as follows

[1091]

[1092] The MS test results are shown in Figure 7 .

[1093] Example 24: Pharmacokinetic study of conjugate 1 in mice

[1094] 1. Experimental information

[1095] Male CD-1 (ICR) mice were used for pharmacokinetic studies. Blood samples were analyzed by ELISA analysis. The specific experimental information and design are shown in the following table.

[1096] Test compound Conjugate 1 Dosage Intravenous (IV) Dosage (mg / kg) 5.0 solvent 1 mg / mL dissolved in 10 mM histidine solution (pH 5.5) Analytical methods ELISA Blood collection point design 0，0.083，1，3，5，24，72，168h Number of animals 15

[1097] 2. Key experimental materials

[1098] category name Batch number / article number supplier Capture Agent Conjugate capture agent 40017-VNAHC Sino Biological HRP anti-human-IgG-HRP 151886 Jackson Wash buffer 0.1% PBST WB-20220314-GKS WuXi AppTec Coating buffer CBS CB-20220314-GKS WuXi AppTec Blocking buffer (BSA) 5% BSA in 0.1% PBST BB-20220316-GKS WuXi AppTec Assay Buffer 1% BSA in 0.1% PBST AB-20220314-SWZ WuXi AppTec TMB TMB Substrate Kit 10537557 <![CDATA[SeraCare TM ]]> stop solution <![CDATA[2N H2SO4]]> SS-20220219-YJC WuXi AppTec

[1099] 3. Detection steps

[1100] Method description: The concentration of the conjugate in CD-1 mouse plasma was determined by ELISA. The lower limit of quantification (LLOQ) of the conjugate was

[1101] The upper limit of quantification (ULOQ) is 3000 ng / mL. The specific steps are as follows:

[1102] 1) Blood was collected at the set time points, EDTA-K2 was used as an anticoagulant, and corresponding test samples were prepared.

[1103] 2) Add 100 μL of coating solution to a 96-well microplate, seal, and incubate overnight at 2-8°C before use.

[1104] 3) Wash the plate 5 times with washing buffer (300 μL / well).

[1105] 4) Block the plate by adding 300 μL of blocking buffer to each well. Seal and incubate at room temperature for 2 hours without shaking.

[1106] 5) Repeat step 2.

[1107] 6) Add 100 μL of standard solution and test sample to each well, seal and incubate at room temperature with shaking at 450 RPM for 2 hours ± 10 minutes.

[1108] 7) Repeat step 2.

[1109] 8) Add 100 μL of HRP solution to each well, seal and incubate at room temperature with shaking at 450 RPM for 1 hour ± 10 minutes.

[1110] 9) Repeat step 2.

[1111] 10) Add 100 μL TMB to each well and incubate at room temperature for 5-20 minutes.

[1112] 11) Add 100 μL of stop solution to each well.

[1113] 12) Read the plate within 30 minutes using a SpectraMax M5e / M5 / Plus 384 microplate reader at 450 nm and 630 nm.

[1114] 13) Convert the reading of the standard solution into the corresponding concentration of the conjugate.

[1115] 14) Data processing was performed using Watson LIMS V7.6 or SoftMax Pro GxP 7.0.3 and Microsoft Excel 2007 or higher.

[1116] 4. Results Summary

[1117]

[1118] From the mouse pharmacokinetic data, it can be seen that conjugate 1 has a long half-life of 165 hours. At the same time, conjugate 1 has a good in vivo exposure. The above data indicate that conjugate 1 has a long half-life and high exposure in clinical practice, and may have excellent antiviral activity.

[1119] Example 25: Pharmacokinetic study of conjugate 2 in mice

[1120] The pharmacokinetic study of conjugate 2 was carried out according to the method of Example 22, and the results are summarized as follows:

[1121]

[1122]

[1123] From the mouse pharmacokinetic data, it can be seen that conjugate 2 has a long half-life of 140 hours. At the same time, conjugate 1 has a good in vivo exposure. The above data indicate that conjugate 1 has a long half-life and high exposure in clinical practice, and may have excellent antiviral activity.

[1124] Example 26: Pharmacokinetic study of conjugate 4 in mice

[1125] 1. Experimental information

[1126] Male CD-1 (ICR) mice were used for pharmacokinetic studies. Blood samples were analyzed by ELISA analysis. The specific experimental information and design are shown in the following table.

[1127]

[1128] 2. ELISA method to test the conjugate drug concentration (ADC concentration) in animal plasma

[1129] 1) Key Reagents

[1130]

[1131] 2) Key experimental parameters

[1132]

[1133] 3) Experimental process

[1134] (1) Add 100 μL of coating solution (Solarbio) to a 96-well microplate, seal it, and incubate it at 2-8°C overnight before use.

[1135] (2) Wash the plate three times with wash buffer (300 μL / well).

[1136] (3) Block the plate by adding 300 μL of blocking solution to each well. Seal and incubate at room temperature for 2 hours without shaking.

[1137] (4) Repeat step (2).

[1138] (5) Add 100 μL of standard / control and sample to each well, seal and incubate at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes.

[1139] (6) Repeat step 5.2.

[1140] (7) Add 100 μL of HRP solution to each well, seal and incubate at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes.

[1141] (8) Repeat step 5.2.

[1142] (9) Add 100 μL of TMB to each well and incubate at room temperature for 5-20 minutes.

[1143] (10) Add 100 μL 2N H2SO4 to each well.

[1144] (11) Read the absorbance at 450 nm using a plate reader within 30 minutes.

[1145] 3. ELISA method to test the conjugate drug concentration in animal plasma (Total drug concentration)

[1146] 1) Key Reagents

[1147]

[1148] 2) Key experimental parameters

[1149]

[1150] 3) Experimental process

[1151] (1) Add 100 μL of coating solution to a 96-well microplate, seal, and incubate overnight at 2-8°C before use.

[1152] (2) Wash the plate 5 times with wash buffer (300 μL / well).

[1153] (3) Block the plate by adding 300 μL of blocking solution to each well. Seal and incubate at room temperature for 2 hours without shaking.

[1154] (4) Repeat step (2).

[1155] (5) Add 100 μL of standard / control and sample to each well, seal and incubate at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes.

[1156] (6) Repeat step 5.2.

[1157] (7) Add 100 μL of HRP solution to each well. Seal and incubate at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes.

[1158] (8) Repeat step 5.2.

[1159] (9) Add 100 μL of TMB to each well and incubate at room temperature for 5-20 minutes.

[1160] (10) Add 100 μL 2N H2SO4 to each well.

[1161] (11) Read the absorbance at 450 nm and 630 nm using a plate reader within 30 minutes.

[1162] 4. Experimental Results

[1163]

[1164]

[1165]

[1166] From the pharmacokinetic data of mice, it can be seen that conjugate 4 has a long half-life, and the three administration methods (intravenous administration IV, subcutaneous injection SC, intraperitoneal injection IM) have reached more than 190 hours. At the same time, conjugate 4 has a good in vivo exposure. The above data indicate that conjugate 4 has a long half-life and high exposure in clinical practice, and may have excellent antiviral activity.

[1167] Example 27: Pharmacokinetic study of conjugate 4 in rats

[1168] 1. Experimental information

[1169] Male SD rats were used for pharmacokinetic study. Blood samples were analyzed by ELISA analysis method. The specific experimental information and design are shown in the following table.

[1170]

[1171] 2. ELISA method to test the concentration of conjugate drugs in animal plasma

[1172] Referring to the conjugate test method in animal plasma in Example 26, the drug concentrations of ADC and Total in rat plasma were tested.

[1173] 3. Experimental results

[1174]

[1175]

[1176]

[1177] From the pharmacokinetic data of rats, it can be seen that conjugate 4 has a long half-life of more than 190 hours. At the same time, conjugate 4 has a good in vivo exposure. The above data indicate that conjugate 4 has a long half-life and a high exposure in clinical practice, and may have excellent antiviral activity.

[1178] Example 28: Evaluation of the in vivo antiviral efficacy of the test substance (conjugate 4) using an influenza virus mouse infection model 1. Experimental materials and methods

[1179] 1) Animals: 6-7 weeks old, 18-20g, specific pathogen-free female BALB / c mice, purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., arrived at the animal room of the Biological Department of Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd. on November 03, 2023, and were kept in independent ventilated cages. The laboratory animal quality certificate number is 20220009014323. The breeding and use of mice were in accordance with the experimental protocol approved by WuXi AppTec IACUC-QD (Animal Use Protocol Number: ID01-QD031-2022v1.0). After at least 3 days of environmental adaptation, qualified animals were taken for experiments.

[1180] 2) Solvent and test substance

[1181] 10 mM histidine buffer (pH = 5.5). Taking the preparation of 1 L solution as an example, 0.372 g histidine and 1.593 g histidine hydrochloride were added to 1 L ddH2O respectively, and vortexed and stirred evenly for later use.

[1182] Test substance information:

[1183]

[1184] 3) Viruses

[1185] Influenza A virus, A / California / 07 / 2009 (H1N1), the original strain was purchased from ATCC, catalog number: VR1843. The strain used was amplified by WuXi AppTec Biological Department, batch number: 20201228YNR. Titer: 7.0E+06PFU / mL.

[1186] 4) Reagents and instruments

[1187] Main reagents

[1188]

[1189]

[1190] Main instruments

[1191] Instrument Name brand model Analytical Balance Mettler Toledo XSE205DU Electronic balance Changzhou Keyuan YH-2000 Biohazard Class II Safety Cabinets Sujing Antai BSC-1604IIA2 Centrifuge eppendorf 5427R ELISA reader Molecular Devices SpectraMax iD3 Microplate mixer Thermo Fisher 88882006

[1192] 2. Experimental methods and procedures

[1193] 1) The day of the first administration was defined as Day 0 of the experiment, the day before was Day -1, the day after was Day 1, and so on;

[1194] 2) Virus inoculation: On day 28, mice were deeply anesthetized by intraperitoneal injection of Zotai 50 (30 mg / kg) / Xylazine hydrochloride (6 mg / kg) (injection volume 6 mL / kg), and the virus was inoculated by intranasal drops. The inoculation amount for each mouse was 500 PFU, and the inoculation volume was 50 μL.

[1195] 3) Grouping: According to the experimental design, the animals were randomly divided into 11 groups, with 10 mice in each group, and the weight of the animals was adjusted to ensure that there was no statistical difference in the average weight of the animals between the groups.

[1196] 4) Administration: Except for the healthy control group (Group 1), the mice in the other groups were treated with the vehicle or the test substance, respectively. The administration method was subcutaneous injection, and the administration time was 28 days before the virus inoculation, and the administration was a single dose.

[1197] In vivo experimental design—dosing regimen

[1198]

[1199] * Reference conjugate: prepared according to the synthesis and characterization of conjugate 9 in Example 3 of patent WO2023 / 125806A1.

[1200] 5) Sample collection: Blood samples were collected from 3 mice in each group of groups 4-9 at 48, 336, 672, 885 or 1008 hours after administration. The collected whole blood samples were incubated at 4°C for 8 hours and centrifuged at 3000g, 4°C for 10 minutes to separate the serum. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected from groups 4 and 7 at 1008 hours after administration. All samples were stored in a -80°C refrigerator.

[1201] 6) Health monitoring: During the experiment, the mice were weighed three times a week from day 0 to day 27 and once a day from day 28 to day 42 to observe their condition and record their weight.

[1202] 7) Median survival time: the survival time corresponding to the cumulative survival rate of 50%.

[1203] 8) Humane endpoint: According to the IACUC protocol, any mouse that loses more than 35% (inclusive) of its initial body weight during the experiment, or / and shows symptoms of dying, will be euthanized and recorded as a dead animal in the results. In actual experimental operations, the above standards will be followed.

[1204] 3. Sample Analysis

[1205] ELISA experiment to detect the concentration of the test substance in the sample: 1) Use the coating solution to dilute the ADC capture antibody at a ratio of 1:400 to coat a 96-well plate, 100 μL per well, and coat overnight at 4°C; 2) Discard the liquid in the ELISA plate and wash the plate 3 times with PBST (300 μL / well); 3) Add 200 μL of PBST containing 5% BSA to each well and block at room temperature for 2 hours; 4) Discard the liquid in the ELISA plate and wash the plate 3 times with PBST (300 μL / well); 5) Add 100 μL of the sample to be tested to each well and incubate at room temperature for 2 hours; 6) Discard the liquid in the ELISA plate and wash the plate 3 times with PBST (300 μL / well); 7) Add 100 μL of HRP (see Example 24) (1:20000 dilution) to each well and incubate the microplate at room temperature for 40 minutes; 8) Discard the liquid in the ELISA plate and wash the plate 3 times with PBST (300 μL / well); 9) Add 100 μL of TMB color substrate, color development at room temperature in the dark for 5-10 minutes, when the solution is more obvious blue, add 100 μL stop solution to each well to stop color development; 10) Detect absorbance with 450 / 620nm dual wavelength microplate reader, and fit the standard curve using the four-parameter method. Use the above method to determine the content of the test substance in the sample.

[1206] 4. Data Analysis

[1207] The data were statistically analyzed using Prism, and the survival status of mice was analyzed using Log-rank (Mantel-cox) test.

[1208] 5. Results

[1209] The in vivo efficacy of the test compound in the influenza A virus mouse infection model was evaluated by mouse body weight changes and survival rate; the pharmacokinetic characteristics of the test compound were analyzed by detecting the concentration of the test compound in serum, lung tissue and lung epithelial cell lining fluid; and the tolerability of the test compound in mice was evaluated by monitoring animal body weight.

[1210] 1) The test substance protects the body weight of mice in the model

[1211] Healthy group: During the experiment, the weight of the healthy control group remained stable and no significant decrease was observed;

[1212] Vehicle group: The body weight of mice began to decrease significantly from the 31st day, and then continued to decrease until death or euthanasia, with an average maximum decrease of 37.77% (euthanasia was performed);

[1213] hIgG1-Fc(LS) (2.5 mg / kg) group: The body weight of mice began to decrease significantly from the 31st day of the experiment, with the average maximum decrease of 36.08%. The body weight of surviving mice began to recover from the 39th day;

[1214] Conjugate 4 (2.5 mg / kg) group: The body weight of mice began to decrease significantly from the 31st day of the experiment, with the average maximum decrease of 16.83%. The body weight of surviving mice began to recover to normal levels from the 36th day;

[1215] Conjugate 4 (0.5 mg / kg) group: The body weight of mice began to decrease significantly from the 31st day of the experiment, with the average maximum decrease of 34.58%. The body weight of surviving mice began to recover from the 38th day;

[1216] Conjugate 4 (0.1 mg / kg) group: The body weight of mice began to decrease significantly from the 31st day, and then continued to decrease until death or euthanasia, with an average maximum decrease of 33.00%;

[1217] Reference conjugate (0.5 mg / kg) group: The body weight of mice began to decrease significantly from the 31st day, and then continued to decrease until death or euthanasia, with an average maximum decrease of 37.03% (euthanasia was performed);

[1218] 2) The test substance protects the survival of mice in the model

[1219] After virus inoculation, the survival status of mice in each group is summarized as follows:

[1220] Healthy group: No mice died, the final animal survival rate was 100%, and the median survival time was >42 days;

[1221] Vehicle group: Mice began to die on the 35th day and all died on the 37th day, with a final survival rate of 0% and a median survival period of 36 days;

[1222] hIgG-Fc (LS) (2.5 mg / kg) group: mice began to die from the 36th day, the final survival rate was 10%, and the median survival time was 36.5 days;

[1223] Conjugate 4 (2.5 mg / kg) group: mice began to die on the 38th day, the final survival rate was 90%, and the median survival period was >42 days;

[1224] Conjugate 4 (0.5 mg / kg) group: mice began to die from the 36th day, the final survival rate was 50%, and the median survival time was 39.5 days;

[1225] Conjugate 4 (0.1 mg / kg) group: Mice began to die on the 34th day and all died on the 37th day. The final survival rate was 0% and the median survival period was 36 days.

[1226] Reference conjugate (0.5 mg / kg) group: Mice began to die from the 36th day and all died on the 38th day, with a final survival rate of 0% and a median survival period of 37 days;

[1227]

[1228] Statistical method: Log-rank (Mantel-Cox) test; #, compared with the vehicle group; NS, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001, ***, P<0.001.

[1229] From the analysis of the results, it can be seen that the conjugate 4 obtained by the thiol-directed conjugation technology described in this patent showed excellent antiviral activity in the mouse in vivo model. Under high-dose (2.5 mg / kg) conditions, the survival rate of mice reached 90%. Under conditions compared with the reference conjugate, at a dose of 0.5 mg / kg, the survival rate of the conjugate mice reached 50%, and the survival rate of the reference conjugate molecule mice reached 0%. It can be seen that conjugate 4 shows better antiviral activity, indicating better clinical effects in clinical influenza prevention and / or treatment.

[1230] The present invention has been illustrated by various specific embodiments. However, it will be understood by those skilled in the art that the present invention is not limited to the specific embodiments, and that those skilled in the art can make various changes or modifications within the scope of the present invention, and that the various technical features mentioned in various places in this specification can be combined with each other without departing from the spirit and scope of the present invention. Such changes and modifications are all within the scope of the present invention.

Claims

1. A conjugate having a structure of Formula IA or Formula IB, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: in, m is 1 or 2; n is an integer from 1 to 20, preferably an integer of 1, 2, 3, 4, 5, 6, 7 or 8; w is an integer from 0 to 8; for example, 0, 1, 2, 3, 4 or 5; L is a linker; E is selected from an antibody or antibody fragment, an Fc domain monomer, an Fc domain, an Fc binding peptide, albumin or an albumin-linked peptide; R 1a , R 2a , R 3a , R 5a , R 6a , R 7a ,,R 9a and R 10a are each independently selected from hydrogen, deuterium, hydroxyl, -OC 1-6 Alkyl, hydroxyl C 1-6 Alkyl-, C 1-6 Alkyl-, C 3-8 Cycloalkyl-, C 3-8 Heterocycloalkyl-, C6-C 15 Aryl, 5-15 membered heteroaryl, halogen, cyano and amino; R 1a and R 2a Can form C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl; or R 5a and R 6a Can form C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl; or R 9a and R 10a Can form C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl, wherein the C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl is optionally substituted with substituents selected from halogen and hydroxy; R 4a and R 8a Each independently selected from H, C 1-6 Alkyl, C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl; x is 1 or 2; y is an integer from 1 to 20, preferably an integer of 1, 2, 3, 4, 5, 6, 7 or 8; z is an integer from 0 to 8; for example, 0, 1, 2, 3, 4 or 5; q is 0, 1, 2, 3, or 4; R 1’ , R 2’ , R 3’ , R 4’ and R 5’ are each independently selected from hydrogen, deuterium, hydroxyl, -OC 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl, C6-C 15 aryl, 5-15 membered heteroaryl, halogen, cyano and amino; or R 1’ and R 2’ Can form C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl; or two adjacent R 3’ Can form C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl, C6-C 15 Aryl or 5-15 membered heteroaryl.

2. The conjugate according to claim 1 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: L is a linker having a structure of formula I-3a, comprising L 1 and L 2a , L 1 The left and right ends of L are connected to the drug part. 2a The upper end of -NR 8a connect, in, L 1 Select from the following structures: in, W is selected from O, S, NR b , CH 2- or does not exist; R a and R b are each independently selected from H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl; preferably selected from H and methyl, more preferably methyl; y1 and y2 are each independently 0, 1, 2, 3, 4, 5 or 6; Preferably, L 1 Select from the following structures:

3. The conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: L 2a Having the structure of formula L2-1a to L2-7a: in, Z is NR, S and O; The R groups are each independently selected from hydrogen, deuterium, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted 3-20 membered heterocycloalkyl, optionally substituted C6-C 15 aryl and optionally substituted 5-15 membered heteroaryl; s and t are integers from 1 to 20; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and y is either 0 or 1.

4. The conjugate according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: L 2a Selected from formula L2-1a, L2-5a and L2-7a, Z is O, R is H, s and t are selected from integers of 3-10, preferably, s and t are 4 or 8.

5. The conjugate according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, in, The S atom on E comes from the cysteine ​​(cys) residue or disulfide bond on E; Preferably, E is an antibody or an antibody fragment thereof, an Fc domain monomer, an Fc domain, an Fc binding peptide, albumin or an albumin-linked peptide, for example, the Fc domain monomer comprises or consists of an amino acid sequence described in any one of SEQ ID No.1-81 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, Preferably, it comprises or consists of an amino acid sequence selected from any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:

72.

6. The conjugate according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: The conjugate has the structure of Formula IA.

7. The conjugate according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: The conjugate has the structure of Formula IB.

8. The conjugate according to claim 6 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: The structure between L and E is selected from the following structures: Among them, R 1a , R 2a , R 3a , R 5a , R 6a , R 7a , R 9a and R 10a are each independently selected from hydrogen, deuterium, -OC 1-6 Alkyl, hydroxyl C 1-6 Alkyl-, C 1-6 Alkyl- and C 3-8 Cycloalkyl-; or R 1a and R 2a Together they form C 3-8 Cycloalkyl; or R 5a and R 6a Together they form C 3-8 Cycloalkyl; or R 9a and R 10a Together they form C 3-8 Cycloalkyl, wherein the C 3-8 Cycloalkyl is optionally substituted with substituents selected from halogen and hydroxy; R 4a and R 8a Each independently selected from H, C 1-6 Alkyl and C 3-8 cycloalkyl; and w is 0, 1, or 2; Preferably, wherein R 1a , R 3a , R 4a , R 5a , R 7a , R 9a and R 10a Each is hydrogen; R 2a , R 6a and R 8a are independently selected from hydrogen, C 1-6 Alkyl and C 3-8 cycloalkyl; and w is 0; More preferably, the structure of formula I-4a is selected from 9. The conjugate according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, characterized in that: The conjugate is selected from compounds having formula C-1a to formula C-27a: wherein m, n, x, y and E are as defined in any one of claims 1-5.

10. The conjugate according to any one of claims 1 to 9 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: The ratio of n to m is from 1 to 20, preferably from 2 to 10, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10.

11. The conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof, having an average DAR value between 0.5 and 10.0, such as 2.0-4.5 or 4.8-8.

5.

12. The conjugate according to any one of claims 1 to 11 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: E comprises an Fc domain monomer or an Fc domain comprising the Fc domain monomer, wherein the Fc domain monomer comprises the amino acid sequence of any one of SEQ ID No.1-81, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, or consists of the amino acid sequence.

13. The conjugate according to any one of claims 1 to 12 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: E can recognize viral surface antigens, such as CR6261, CR8020, MEDI8897, Palivizumab, SD38, etc.; or The Fc domain monomer can be any immunoglobulin antibody subtype (e.g., IGHG1*01 (e.g., G1m(za)), IGHG1*07 (e.g., G1m(zax)), IGHG1*04 (e.g., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (e.g., G1m(fa)), IGHG2*01, IGHG2*02, IGHG2*06, IGHG3*01, IGHG3*04, IGHG3*05 , IGHG3*09,IGHG3*10,IGHG3*11,IGHG3*12,IGHG3*06,IGHG3*07,IGHG3*08,IGHG3*13,IGHG3*03,IGHG3*14,IGHG3*15,IGHG3*16,IGHG3*17,IGHG3*18,IGHG3*19,IGHG2*04,IGHG4*01,IGHG4*02,IGHG4*03) Fc domain monomer.

14. The conjugate according to any one of claims 1 to 13 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: The conjugate has the following structure: Wherein E is as defined in any one of claims 1 to 13; Preferably, wherein E is SEQ ID NO:67, SEQ ID NO:69 or SEQ ID NO:71; And wherein m and n are as defined in claim 1, m is preferably 1.

15. The conjugate according to any one of claims 1 to 14 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein The carbon to which the group is attached or The carbon to which the corresponding group is attached is of R or S configuration, preferably R configuration.

16. A compound having the structure of formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein each symbol is as defined in one of claims 1 to 15; wherein each symbol is as defined in one of claims 1 to 15; 17. The compound according to claim 16 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, wherein: The compounds have structures of formula C-Inter-1 to formula C-Inter-12:

18. A process for preparing a conjugate of formula IA as claimed in any one of claims 1 to 15, wherein each symbol is as defined in one of claims 1 to 15; The method comprises the following steps: The first step is to change (E) m dissolved in a buffer, wherein E and m are as defined in any one of claims 1 to 15, and a reducing agent (such as TCEP, DTT) is added to convert (E) m The disulfide bond in is reduced to obtain a compound of formula II-1 containing a thiol residue, wherein E, m and n are as defined in any one of claims 1 to 15; In the second step, a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-5a is added to the compound of formula II-1, Wherein, each symbol is as defined in Formula IA, The Michael addition reaction is carried out at a pH of 5.5 to 7.0 (e.g., 5.8 to 6.8, 6.0 to 6.6) for 0.5 to 10 hours, preferably 0.5 to 2 hours, and then 3 to 7 molar equivalents of a buffer solution of the compound of formula I-5a are added in batches (e.g., 1 to 2 batches) and the reaction is continued for 0.5 to 10 hours (preferably 0.5 to 2 hours) to obtain a conjugate of formula I-3b. Wherein, each symbol is as defined in Formula IA; The third step is to adjust the pH of the solution containing the conjugate of formula I-3b to 7-8 (e.g., 7.1-7.9, 7.3-7.7), and perform a hydrolysis reaction to obtain the final conjugate of formula IA; or, A method for preparing a conjugate of formula IB according to any one of claims 1 to 15, Wherein, each symbol is defined in any one of claims 1 to 15; The method comprises the following steps: The first step is to change (E) m dissolved in a buffer, wherein E and m are as defined in any one of claims 1 to 15, and a reducing agent (such as TCEP, DTT) is added to convert (E) m The disulfide bond in is reduced to obtain a compound of formula II-1 containing a thiol residue, wherein E, m and n are as defined in any one of claims 1 to 15; In the second step, a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-6a is added to the compound of formula II-1, Wherein, each symbol is as defined in the formula IB, The Michael addition reaction is carried out at a pH of 5.5 to 8 (eg, 5.8 to 7.6, 6.0 to 7.5) to obtain an IB conjugate.

19. A pharmaceutical composition comprising a conjugate of formula IA or formula IB as described in any one of claims 1 to 15, or a compound of formula I-5a or I-6a as described in claim 16 or 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists), and optionally a pharmaceutically acceptable excipient.

20. A method for preventing or treating a patient infected with a virus or a patient who may be at risk of being infected by a virus, comprising administering to the patient, for example by injection, an effective dose of any one of the conjugates of formula IA or IB as described in any one of claims 1 to 15, or a compound of formula I-5a or I-6a as described in claim 16 or 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof.

21. Use of a conjugate of formula IA or formula IB as claimed in any one of claims 1 to 15, or a compound of formula I-5a or I-6a as claimed in claim 16 or 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled product thereof in the preparation of a medicament, wherein the medicament is used to prevent or treat viral infection in a patient with a viral infection or a patient who may be at risk of viral infection; Preferably, the viral infection is an infection caused by influenza virus or parainfluenza virus; Preferably, the viral infection is an infection caused by influenza virus A, B or C, or parainfluenza virus; Preferably, the patient who is infected by a virus or may be at risk of being infected by a virus may be a patient with a defective immune system; Preferably, the patient who is infected with a virus or may be at risk of being infected with a virus may be a patient who is receiving or is about to receive immunosuppressant therapy; preferably, the patient who is infected with a virus or may be at risk of being infected with a virus may be a patient diagnosed with a disease that causes immunosuppression; Preferably, the patient diagnosed with a disease causing immunosuppression suffers from cancer or acquired immunodeficiency syndrome; Preferably, the patient diagnosed with a disease causing immunosuppression suffers from a cancer selected from leukemia, lymphoma, humoral immunity deficiency, T-cell deficiency, complement deficiency or multiple myeloma; Preferably, the patient is a patient who is currently receiving or is about to receive a hematopoietic stem cell transplant; Preferably, the patient is a patient who is currently receiving or is about to receive an organ transplant; and / or Preferably, the patient may be at risk of secondary infection; 22. A method for preventing the risk of secondary infection caused by influenza virus infection in a patient, by administering, for example, injecting, into the patient an effective dose of any conjugate of formula IA or formula IB as described in any one of claims 1 to 15, or a compound of formula I-5a or I-6a as described in claim 16 or 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof, Preferably, the secondary infection is a respiratory tract infection; Preferably, the secondary infection is related to pneumonia; Preferably, the secondary infection is a bacterial, viral, or fungal infection; Preferably, the bacterial infection is an infection caused by methicillin-resistant Staphylococcus aureus; Preferably, the bacterial infection is an infection caused by Streptococcus pneumoniae; Preferably, the conjugate of Formula IA or Formula IB, or the compound of Formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof can be administered by intramuscular injection, intravenous injection, intradermal injection, intraarterial injection, intraperitoneal injection, intralesional injection, intracranial injection, intraarticular injection, intrapleural injection, intratracheal injection, intraprostatic injection, intranasal injection, intravitreal injection, intravaginal injection, intrarectal injection, local, intratumor injection, intraperitoneal injection, subcutaneous injection, subconjunctival injection, intracapsular injection, mucosal injection, intrapericardial injection, intraumbilical injection, intraocular injection, oral administration, topical inhalation, injection or infusion; Preferably, the conjugate of Formula IA or Formula IB, the compound of Formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof is administered in combination with another therapeutic drug or used in combination to prepare a drug; Preferably, the additional therapeutic agent is an antiviral drug; Preferably, the antiviral drug is baloxavir, pimodivir, oseltamivir, zanamivir, peramivir, laninamivir, amantadine, MEDI8852 or rimantadine; Preferably, the other drug used by the patient is an antiviral vaccine; Preferably, the antiviral drug and the conjugate of Formula IA or Formula IB, the compound of Formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof are sequentially administered, for example, by injection to the patient; and / or Preferably, the antiviral drug and the conjugate of Formula IA or Formula IB, the compound of Formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof are administered to the patient simultaneously, for example, by injection.

23. A drug combination comprising a conjugate of formula IA or formula IB as described in any one of claims 1-15, or a compound of formula I-5a or I-6a as described in claim 16 or 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope label thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

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