Conjugate for preventing and treating viral infection and use thereof

Abstract

The present invention relates to a conjugate for preventing and treating viral infection and a use thereof, and more specifically relates to a protein-anti-influenza compound conjugate of formula I-A or formula I-B or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope marker thereof, wherein the symbols are as defined herein. The conjugate or an intermediate compound has remarkable anti-influenza virus activity, has excellent in-vitro / in-vivo pharmacokinetic properties and safety, and has high clinical application prospect.

Description

Conjugates for preventing and treating viral infections and uses thereof Technical Field

[0001] The present invention relates to conjugates of antiviral drugs targeting influenza (Flu) viruses and antibodies or their constant regions (Fc regions). Specifically, the invention relates to compounds comprising antibodies or their constant regions (Fc regions) covalently coupled to small molecule or polypeptide inhibitors of viral surface proteins and intermediate compounds thereof, as well as pharmaceutical compositions or drugs containing these compounds, and 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 worldwide each year, with a death toll of around 500,000 (Luliano et al., 2018, Lancet 391:1285-1300). Although most healthy people recover within one to two weeks after being infected with the virus, influenza virus infection can develop into life-threatening severe infections and complications, such as pneumonia, for the elderly, those with chronic diseases, and those with weakened immune systems.

[0003] Developing treatments against influenza viruses is an ongoing challenge. While approved anti-influenza drugs and preventive vaccines are currently available, small molecule anti-influenza drugs typically require administration within 48 hours of symptom onset to achieve clinical benefit. Furthermore, due to the high variability of influenza viruses, drug-resistant strains have emerged to combat these commonly used drugs. Therefore, there is a need for more effective and long-lasting therapies for the treatment and / or prevention of influenza viruses.

[0004] Influenza viruses are a class of negative-stranded, segmented RNA viruses belonging to the Orthomyxoviridae family, encompassing influenza virus types A, B, and C. Influenza viruses A and B predominate in humans. Influenza viruses infect respiratory epithelial cells. The first step in this process is host cell attachment, mediated by viral surface receptor-binding proteins (hemagglutinin (HA) in the case of influenza). Subsequently, HA facilitates fusion of the viral envelope with the cell membrane, releasing the influenza virus genome fragments and the viral RNA-dependent RNA polymerase complex into the cell. The viral RNA-dependent RNA polymerase complex uses the genome fragments as templates to synthesize new progeny virus particles within the cell. These newly synthesized virus particles are released from the cell by cell lysis or budding. For influenza viruses, complete progeny virus release requires cleavage of sialic acid residues on the cell surface by neuraminidase (NA). Neuraminidase inhibitors targeting influenza virus neuraminidase to reduce viral spread have been approved for clinical use, including oseltamivir (Tamiflu TM ), zanamivir (Relenza TM ) and peramivir (Rapivab TM ).

[0005] Organ transplant recipients and cancer patients, whose immune systems are suppressed and unable to effectively clear viruses, are more susceptible to prolonged viral replication after influenza infection, increasing the likelihood of developing drug-resistant strains. This has been observed clinically. Meanwhile, influenza drug resistance in the general population also presents a significant medication challenge, necessitating the need for new, more effective methods and therapeutic agents for treating influenza.

[0006] Summary of the Invention

[0007] In one aspect, the present invention provides a conjugate, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof, comprising an anti-influenza virus small molecule compound conjugated to a protein (also referred to as a "protein-anti-influenza compound conjugate"), which has significant anti-influenza virus biological activity.

[0008] 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-labeled form thereof:

[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 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 together 3-8 Cycloalkyl or C 3-8 heterocycloalkyl;

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

[0017] or R 9a and R 10a Can form C together 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl, wherein the C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl 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 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 together 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 encompass (E) m The structural part covers the S atom and the structure below it.

[0025] In some embodiments, L is a linker having the 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 formula IB 8a connect,

[0026] in,

[0027] L 1 Selected from the following structures:

[0028] in,

[0029] W is selected from O, S, NR b 、CH 2-or does not exist;

[0030] 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;

[0031] y1 and y2 are each independently 0, 1, 2, 3, 4, 5 or 6;

[0032] It should be understood that L 1 The bonds at the left and right ends of the structure are connected to the drug (D 1 ) is connected to the oxygen atom of the middle amine group, and the wavy line on the middle amine group marks the bond with L 2a connected;

[0033] Preferably L 1 Selected from the following structures:

[0034] In some embodiments, wherein L 2a Having the structures of formula L2-1a to L2-7a:

[0035] in,

[0036] Z is NR, S and O;

[0037] Each R group is 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;

[0038] 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.

[0039] It should be understood that in formulas L2-1a to L2-7a, L 2a The middle part of the wavy lines on both sides, (L 1 ) part does not belong to L 2a The component that specifies the 1 Connected L 2a The end of the 2a Connection direction, L 2a The right end of -NR 8aconnect.

[0040] In some embodiments, L 2a Selected from formula L2-1a, L2-5a and L2-7a,

[0041] Z is O,

[0042] R is H,

[0043] 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.

[0044] In some embodiments, the S atom on E is derived from a cysteine ​​(cys) residue or a disulfide bond on E;

[0045] 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 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,

[0046] 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.

[0047] In some embodiments, the conjugate has the structure of Formula IA.

[0048] In some embodiments, the conjugate has the structure of Formula IB.

[0049] In some embodiments, the structure between L and E is selected from the following structures:

[0050] Among them, R 1a 、R 2a 、R 3a 、R 5a 、R 6a 、R 7a 、R 9a and R 10a 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;

[0051] or R 5a and R 6a Together they form C 3-8 Cycloalkyl;

[0052] or R 9a and R 10a Together they form C 3-8 Cycloalkyl, wherein the C 3-8 Cycloalkyl is optionally substituted with a substituent selected from halogen and hydroxy;

[0053] R 4a and R 8a Each independently selected from H, C 1-6 Alkyl and C 3-8 Cycloalkyl;

[0054] w is 0, 1, or 2;

[0055] Preferably, wherein R 1a 、R 3a 、R 4a 、R 5a 、R 7a 、R 9a and R 10a Each is hydrogen;

[0056] R 2a 、R 6a and R 8a are independently selected from hydrogen, C 1-6 Alkyl and C 3-8 cycloalkyl; and

[0057] w is 0;

[0058] More preferably, the structure of formula I-4a is selected from

[0059] 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 connect.

[0060] In some embodiments, the conjugate is selected from compounds having Formula C-1a to Formula C-27a:

[0061] wherein m, n, x, y and E are as defined herein.

[0062] 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.

[0063] In some embodiments, the conjugate or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopically labeled thereof has an average DAR value between 0.5 and 10.0, eg, 2.0-4.5 or 4.8-8.5.

[0064] In some embodiments, E comprises an Fc domain monomer or an Fc domain comprising said Fc domain monomer, wherein said 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 said amino acid sequence.

[0065] In some embodiments, E is capable of recognizing viral surface antigens, such as CR6261, CR8020, MEDI8897, Palivizumab, SD38, etc.; or

[0066] 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.

[0067] In some embodiments, the conjugate has the following structure:

[0068] Wherein E is defined;

[0069] Preferably, wherein E is SEQ ID NO: 67, SEQ ID NO: 69 or SEQ ID NO: 71;

[0070] and wherein m and n are as defined herein, m is preferably 1.

[0071] In some embodiments, The carbon to which the group (or its corresponding group) is attached (for example, in the case of Among the specific compounds of the structure, it is also The carbon to which the right nitrogen is connected) is in R or S configuration, preferably in R configuration.

[0072] 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.

[0073] Specifically, the protein-anti-influenza compound conjugate of the present invention has a 1 and L 2 ) small molecules (D) with anti-influenza virus activity linked to antibodies or antibody fragments thereof, Fc monomers, Fc domains, Fc connecting peptides, albumin or albumin connecting peptides (E) 1The protein-anti-influenza compound conjugate of the present invention has significant anti-influenza virus activity, excellent in vitro / in vivo pharmacokinetic properties and safety, a long half-life, and high clinical application prospects.

[0074] 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,

[0075] in,

[0076] m is 1 or 2;

[0077] 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;

[0078] 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, an Fc domain monomer, an Fc domain, an Fc binding peptide, albumin or an albumin-linked peptide as defined above;

[0079] D 1 are small molecule anti-influenza virus drugs, each independently selected from the compounds with anti-influenza virus activity represented by formulas D-1-1 to D-1-8:

[0080] R1 is selected from OH, NH2, -NH(=NH)NH2 and -NHC(=NH)NHR6;

[0081] R2 and R3 are each independently selected from H, OH, F, Cl and Br;

[0082] R4 is selected from -COOH, -P(=O)(OH)2 and -SO3H;

[0083] 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;

[0084] X is selected from O or S;

[0085] R6 is selected from the following groups:

[0086] 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:

[0087] 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;

[0088] Each R group is 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;

[0089] q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0090] Preferably, D 1 The following anti-influenza virus small molecule drugs:

[0091] L 1 To connect the drug (D 1 ) and L 2 The linker includes but is not limited to the following structures:

[0092] 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 wavy line on the middle amine group marks the bond with L 2 connected;

[0093] in,

[0094] W is selected from O, S, NR b 、CH 2- or does not exist;

[0095] R a and R b are each independently selected from H, optionally substituted C1-C 20Alkyl, optionally substituted C2-C 20 Alkenyl, preferably selected from H and methyl, more preferably methyl;

[0096] y1 and y2 are each independently 0, 1, 2, 3, 4, 5 or 6;

[0097] L 2 To connect L 1 and a linker of E having a structure of formula L2-1 to L2-14:

[0098] in,

[0099] Z is NR, S and O;

[0100] Each R group is 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;

[0101] s and t are integers from 1 to 20; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0102] y is either 0 or 1.

[0103] 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 components, which respectively indicate the 1 L connected to E 2 to the end of the group to more clearly indicate the connection direction of L2, which is connected to the sulfur atom of E through a covalent bond.

[0104] It should be understood that the "-S-" in Formula I-1 is formed by the reaction of the sulfhydryl group generated by the reduction of the disulfide bond in E with the linker compound.

[0105] In some embodiments,

[0106] L 1 Selected from the following structures:

[0107] More preferably, L 1 for

[0108] In some embodiments, L 2 is selected from the formula L2-1, L2-5, L2-7, L2-8, L2-12 and L2-14, wherein Z is O,

[0109] R is H,

[0110] s and t are selected from integers of 3-10, preferably, s and t are 4 or 8.

[0111] In some embodiments, the conjugate having formula I-1 has the structure of formula I-1-1:

[0112] Among them, E, L 1 、L 2 , m and n are as defined above.

[0113] In some embodiments, the conjugate is preferably selected from the group consisting of C-1 to C-12 conjugates:

[0114] wherein n and E are as defined above.

[0115] It should be understood that (E) in the above structures C-1 to C-12 indicates that the structural formula contains one E, which is equivalent to the case where m in the conjugate of formula I-1 is 1. The same understanding can be applied to conjugates with similar structures herein.

[0116] In some embodiments, in C1 to C6, the carbon to which the NH2-CH2- on the left is attached (i.e., The carbon to which the right nitrogen is connected) is in R or S configuration, preferably in R configuration.

[0117] In some embodiments, in the structure of Formula 1-1, the ratio of n to m is 1 to 20, preferably from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0118] In some embodiments, m is 1 and n is an integer selected from 1-10.

[0119] 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.

[0120] 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 of any one of SEQ ID No. 1-81.

[0121] In one embodiment, in the compound having the structure of the conjugate including formula I-1, the E single domain dimerizes to form the Fc domain.

[0122] In some embodiments, the antibody fragment is an antigen-binding fragment.

[0123] 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.

[0124] In some embodiments, the antibody or antibody fragment is an IgG, IgA, IgD, IgE, or IgM antibody or antibody fragment.

[0125] In some embodiments, the antibody fragment comprises scFv, sdAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, or SMIP.

[0126] In some embodiments, the antibody or antibody fragment can recognize viral surface antigens, such as CR6261, CR8020, MEDI8897, Palivizumab, SD38, etc.

[0127] In some embodiments, the Fc domain monomer can be an antibody from any immunoglobulin 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) (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 can contain one or more non-natural amino acid sequences as site-specific conjugation 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.

[0128] In some embodiments, L2 is connected to the S atom on E. The S atom on E is derived from a cysteine ​​(cys) residue or a disulfide bond on E.

[0129] In some embodiments, the Asn amino acid residue in E is replaced with an Ala amino acid residue to prevent conjugation at this site.

[0130] 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.

[0131] 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 MVRS (SEQ ID NO: 82) amino acid sequence or ISAMVRS amino acid sequence (SEQ ID NO: 83)).

[0132] 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.

[0133] 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)).

[0134] In some embodiments, the N-terminus of E further comprises a hinge region or a portion of a hinge region.

[0135] In some embodiments, E comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-81.

[0136] 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.

[0137] In some embodiments, E comprises or consists of the following amino acid sequence:

[0138] (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

[0139] (ii) comprising the N-terminal addition sequence, mutation and / or C-terminal addition sequence described for each SEQ ID NO: in the "Sequence SEQ ID NO: 1-81 and its description" section of this disclosure.

[0140] 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.

[0141] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 1. 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: 1, and optionally has an IL2 signal sequence at its N-terminus.

[0142] In some embodiments, E comprises the amino acid sequence of 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.

[0143] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 3. 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: 3, and optionally has an IL2 signal sequence at its N-terminus and an N-terminal MVRS amino acid sequence.

[0144] In some embodiments, E comprises the amino acid sequence of 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, has an MVRS amino acid sequence at its N-terminus.

[0145] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 5. 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: 5, and optionally, has an IL2 signal sequence at its N-terminus and a hexa-histidine tag at its C-terminus.

[0146] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 6. 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: 6, and optionally, has a hexa-histidine tag at its C-terminus.

[0147] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 7. 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: 7, and optionally, has an IL2 signal sequence and an MVRS amino acid sequence at its N-terminus and a hexa-histidine tag at its C-terminus.

[0148] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 8. 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: 8, and optionally, has an MVRS amino acid sequence at its N-terminus and a hexa-histidine tag at its C-terminus.

[0149] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 9. 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: 9, and optionally, has an IL2 signal sequence and an MVRS amino acid sequence at its N-terminus, two additional cysteines in the hinge region (corresponding to the * positions of SEQ ID NO: 9), and a hexa-histidine tag at its C-terminus.

[0150] In some embodiments, E comprises the amino acid sequence of 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 at the hinge region (corresponding to the * positions of SEQ ID NO: 10) and a hexa-histidine tag at its C-terminus.

[0151] In some embodiments, E comprises the amino acid sequence of 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 to the amino acid sequence of SEQ ID NO: 11, and optionally, an MVRS amino acid sequence at its N-terminus and two additional cysteines in the hinge region (corresponding to the * positions of SEQ ID NO: 11).

[0152] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 12. 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: 12, and optionally, has 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 hexa-histidine tag at its C-terminus.

[0153] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 13. 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: 13, and optionally, contains an Asn to Ala substitution (corresponding to the * position of SEQ ID NO: 13) and has a hexa-histidine tag at the C-terminus.

[0154] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 14. 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: 14, and optionally, has 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 hexa-histidine tag at its C-terminus.

[0155] In some embodiments, E comprises the amino acid sequence of 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, 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.

[0156] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 16. 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: 16, and optionally has a human serum albumin signal sequence at its N-terminus and an N-terminal ISAMVRS amino acid sequence.

[0157] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 17. 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: 17, and optionally, has a human serum albumin signal sequence at its N-terminus and an N-terminal ISAMVRS amino acid sequence, contains a C-terminal G4S linker and a C-terminal c-Myc tag.

[0158] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 18. 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: 18, and optionally, has 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.

[0159] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 19. 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: 19, and optionally has a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus, and contains a lysine to serine change (corresponding to the * position of SEQ ID NO: 19) to prevent conjugation at that site.

[0160] In some embodiments, E comprises the amino acid sequence of 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 to the amino acid sequence of SEQ ID NO: 20, and optionally, an ISAMVRS amino acid sequence at its N-terminus and containing a lysine to serine change (corresponding to the * position of SEQ ID NO: 20) to prevent coupling at this site.

[0161] In some embodiments, E comprises the amino acid sequence of 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, 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 a G4S linker and a c-Myc tag at its C-terminus.

[0162] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 22. 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: 22, and optionally, an ISAMVRS amino acid sequence at its N-terminus containing a lysine to serine change (corresponding to the * position of SEQ ID NO: 22) to prevent coupling at this site, and a G4S linker and a c-Myc tag at its C-terminus.

[0163] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 23. 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: 23, and optionally, has a human serum albumin signal sequence and an ISAMVRS amino acid sequence containing an Asn to Ala change (corresponding to the * position of SEQ ID NO: 23) at its N-terminus, and has a G4S linker and a c-Myc tag at its C-terminus.

[0164] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 24. 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: 24, and optionally, has 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.

[0165] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 25. 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: 25, and optionally, has a human serum albumin signal sequence and an ISAM VRS amino acid sequence at its N-terminus, contains H310A and H435A changes to prevent FcRn binding, and has a G4S linker and a c-Myc tag at its C-terminus.

[0166] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 26. 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: 26, and optionally, has an ISAMVRS amino acid sequence at its N-terminus containing H310A and H435A changes to prevent FcRn binding, and has a G4S linker and a c-Myc tag at its C-terminus.

[0167] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 27. 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: 27, and optionally, has a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus, and has a G4S linker and a mutant (lysine to phenylalanine, corresponding to the bold positions of SEQ ID NO: 27) and a c-Myc tag at its C-terminus.

[0168] In some embodiments, E comprises the amino acid sequence of 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 positions of SEQ ID NO: 28) and a c-Myc tag at its C-terminus.

[0169] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 29. 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: 29, and optionally, has a human serum albumin signal sequence and an ISAMVRS amino acid sequence at its N-terminus containing an Asn to Ala substitution and 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.

[0170] In some embodiments, E comprises the amino acid sequence of 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, an ISAMVRS amino acid sequence at its N-terminus containing an Asn to Ala substitution (corresponding to the * position of SEQ ID NO: 30) and a G4S linker and a mutant (lysine to phenylalanine, corresponding to the bold position of SEQ ID NO: 30) and a c-Myc tag at its C-terminus.

[0171] In some embodiments, E comprises the amino acid sequence of 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, a human serum albumin signal sequence having allotype G1m(fa) at its N-terminus, a G4S linker and a mutant (lysine to phenylalanine, corresponding to the bold positions of SEQ ID NO: 31) and a c-Myc tag at its C-terminus.

[0172] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 32. 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: 32, and optionally has a human serum albumin signal sequence at its N-terminus, and has the allotype G1m(fa).

[0173] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 33. 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: 33, and optionally, has an MVRS amino acid sequence at its N-terminus and contains a YTE triple mutation.

[0174] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 34. 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: 34, and optionally, has a human serum albumin signal sequence at its N-terminus, contains the hinge region EPKSS amino acid sequence of mature human Fc-IgG1, contains a cysteine ​​to serine change (corresponding to position # of SEQ ID NO: 34), and is allotype G1m(fa).

[0175] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 35. 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: 35, and optionally, has a murine IgG signal sequence at its N-terminus, the hinge region EPKSSD amino acid sequence of mature human Fc-IgG is removed, and has the allotype G1m(fa).

[0176] In some embodiments, E comprises the amino acid sequence of 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 N-terminus thereof is excluding the hinge region EPKSSD amino acid sequence of mature human Fc-IgG, has allotype G1m(fa) and contains a YTE triple mutation.

[0177] In some embodiments, E comprises the amino acid sequence of 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 N-terminus of the hinge region EPKSSD amino acid sequence of mature human Fc-IgG is deleted, has an LS double mutation and has the allotype G1m (fa).

[0178] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 38. 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: 38, and optionally, has a human serum albumin signal sequence at its N-terminus, contains a YTE triple mutation, has allotype G1m(fa) and has a G4S linker and a c-Myc tag at its C-terminus.

[0179] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 39. 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: 39, and optionally, has 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.

[0180] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 40. 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: 40, and optionally, has at the position corresponding to X in SEQ ID NO: 40 the amino acid defined in SEQ ID NO: 40 at that position.

[0181] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 41. 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: 41, and optionally, has a YTE triple mutation and has an amino acid defined in SEQ ID NO: 41 at the position corresponding to X in SEQ ID NO: 41.

[0182] 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)

[0183] 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).

[0184] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 44. 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: 44, and optionally, has an LS double mutation and has an amino acid defined in SEQ ID NO: 44 at the position corresponding to X as set forth in SEQ ID NO: 44.

[0185] 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 an LS double mutation and has the allotype G1m(fa).

[0186] 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 an LS double mutation and has the allotype G1m(f).

[0187] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 47. 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: 47, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus, has a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 47), and has an amino acid defined by SEQ ID NO: 47 at the position corresponding to the X position shown in SEQ ID NO: 47.

[0188] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 48. 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: 48, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus, has a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 48), and contains the allotype G1m(fa).

[0189] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 49. 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: 49, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus, has a cysteine ​​to serine change (at position # corresponding to SEQ ID NO: 49), and contains the allotype G1m(f).

[0190] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 50. 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: 50, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus, has a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 50), has M428L and N434S mutations, and contains the allotype G1m(fa).

[0191] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 51. 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: 51, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus, has a cysteine ​​to serine change (at position # corresponding to SEQ ID NO: 51), has M428L and N434S mutations, and contains allotype G1m(f).

[0192] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 52. 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: 52, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus, has a cysteine ​​to serine change (at positions corresponding to SEQ ID NO: 52), has a YTE triple mutation, and contains the allotype G1m(fa).

[0193] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 53. 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: 53, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus, has a cysteine ​​to serine change (at positions corresponding to SEQ ID NO: 53), has a YTE triple mutation, and contains allotype G1m(f).

[0194] In some embodiments, E comprises the amino acid sequence of 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, a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, M428L, N434S mutations, a G4S linker and a c-Myc tag at its C-terminus, and contains allotype G1m(f).

[0195] In some embodiments, E comprises the amino acid sequence of 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, a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, with M428L, N434S mutations, a G4S linker and a c-Myc tag at its C-terminus, and contains allotype G1m (fa).

[0196] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 56. 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: 56, and optionally, has a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, has a YTE triple mutation, has a G4S linker and a c-Myc tag at its C-terminus, and contains the allotype G1m(f).

[0197] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 57. 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: 57, and optionally, has a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, has a YTE triple mutation, has a G4S linker and a c-Myc tag at its C-terminus, and contains the allotype G1m(fa).

[0198] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 58. 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: 58, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus with a cysteine ​​to serine change (at the # position), a G4S linker and an IgA peptide tag at its C-terminus, and contains allotype G1m(fa).

[0199] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 59. 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: 59, and optionally, has a mouse heavy chain MIgG Vh signal sequence at its N-terminus, has a cysteine ​​to serine change (at position # corresponding to SEQ ID NO: 59), has M428L, N434S mutations, has a G4S linker and an IgA peptide tag at its C-terminus, and contains allotype G1m(fa).

[0200] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 60. 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: 60, and optionally, has an amino acid defined in SEQ ID NO: 60 at the position corresponding to Z or X as set forth in SEQ ID NO: 60.

[0201] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 61. 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: 61, and optionally, has a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 61) and has at the X position corresponding to SEQ ID NO: 61 the amino acid defined at that position by SEQ ID NO: 61.

[0202] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 62. 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: 62, and optionally, has a cysteine ​​to serine change (at the # position corresponding to SEQ ID NO: 62) and has an amino acid defined by SEQ ID NO: 62 at the position corresponding to the X position set forth in SEQ ID NO: 62 at that position.

[0203] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 63. 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: 63, and optionally, has a cysteine ​​to serine change (at position # corresponding to SEQ ID NO: 63) and has the allotype G1m(f).

[0204] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 64. 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: 64, and optionally, has a cysteine ​​to serine change (at position # corresponding to SEQ ID NO: 64) and has the allotype G1m(fa).

[0205] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 65. 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: 65, and optionally has a cysteine ​​to serine change (at position # corresponding to SEQ ID NO: 65), M428L, N343S mutations, and has the allotype G1m(fa).

[0206] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 66. 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: 66, and optionally has a cysteine ​​to serine change (at position # corresponding to SEQ ID NO: 66), M428L, N343S mutations, and has the allotype G1m(f).

[0207] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 67. 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: 67, and optionally has a cysteine ​​to serine change (at positions corresponding to SEQ ID NO: 67) and a YTE triple mutation, and has the allotype G1m(fa).

[0208] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 68. 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: 68, and optionally has a cysteine ​​to serine change (at positions corresponding to SEQ ID NO: 68) and a YTE triple mutation, and has the allotype G1m(f).

[0209] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 69. 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: 69, and optionally, has a YTE triple mutation and has the allotype G1m(fa).

[0210] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 70. 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: 70, and optionally, has a YTE triple mutation and has the allotype G1m(fa).

[0211] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 71. 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: 71, and optionally has M428L, N343S mutations, and has allotype G1m(fa).

[0212] In some embodiments, E comprises the amino acid sequence of 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.

[0213] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 73. 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: 73, and optionally has M428L, N343S mutations, and has allotype G1m(f).

[0214] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 74. 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: 74, and optionally has GAALIE, M428L, N343S mutations, and has the allotype G1m(fa).

[0215] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 75. 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: 75, and optionally has GAALIE, M428L, N343S mutations, and has allotype G1m(f).

[0216] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 76. 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: 76, and optionally has a cysteine ​​to serine change (#), GAALIE, M428L, N343S mutations, and has the allotype G1m (fa).

[0217] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 77. 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: 77, and optionally has a cysteine ​​to serine change (#), GAALIE, M428L, N343S mutations, and has the allotype G1m(f).

[0218] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 78. 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: 78, and optionally has a cysteine ​​to serine change (#), a DHS triple mutation, and has the allotype G1m(fa).

[0219] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 79. 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: 79, and optionally has a cysteine ​​to serine change (#), a DHS triple mutation, and has the allotype G1m(f).

[0220] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 80. 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: 80, and optionally, has a DHS triple mutation and has the allotype G1m(fa).

[0221] In some embodiments, E comprises the amino acid sequence of SEQ ID NO: 81. 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: 81, and optionally, has a DHS triple mutation and has the allotype G1m(f).

[0222] In any of the embodiments described herein, wherein E comprises an Fc domain monomer (e.g., having a sequence as set forth in any of SEQ ID NOs: 1-81), the Fc domain monomer comprises a triple mutation corresponding to M252Y / S254T / T265E (YTE). Those skilled in the art will understand that "corresponding to" refers to amino acid positions corresponding to specific positions obtained by sequence homology alignment using sequence analysis software (e.g., but not limited to, DNAStar, Vector NTI, etc.). For example, any of SEQ ID NOs: 1-81 may comprise a mutation comprising YTE.

[0223] In any of the embodiments described herein, wherein E comprises an Fc domain monomer (e.g., having a sequence as set forth in any of SEQ ID NOs: 1-81), the double mutant corresponding to M428L / N434S (LS) is included. Those skilled in the art will understand that "corresponding to" refers to amino acid positions corresponding to specific positions obtained by sequence homology alignment using sequence analysis software (e.g., but not limited to, DNAStar, Vector NTI, etc.). For example, any of SEQ ID NOs: 1-81 may comprise a mutation comprising LS.

[0224] In any of the embodiments described herein, wherein E comprises an Fc domain monomer (e.g., having a sequence as set forth in any of SEQ ID NOs: 1-81), comprising a double mutation corresponding to N434H. Those skilled in the art will understand that "corresponding to" refers to amino acid positions corresponding to specific positions obtained by 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.

[0225] In any of the embodiments described herein, wherein E comprises an Fc domain monomer (e.g., having a sequence as set forth in any of SEQ ID NOs: 1-81), the double mutant corresponding to C220S is included. Those skilled in the art will understand that "corresponding to" refers to amino acid positions corresponding to specific positions obtained by sequence homology alignment using sequence analysis software (e.g., but not limited to, DNAStar, Vector NTI, etc.). For example, any of SEQ ID NOs: 1-81 may comprise a mutation comprising C220S.

[0226] 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 or more), or at least 50 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 61 or more) of the Fc domain monomer (e.g., an Fc domain monomer from the sequence of any of SEQ ID NOs: 1-81). , 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.

[0227] In one embodiment, the conjugate has the following structure:

[0228] wherein n is as defined above.

[0229] 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.

[0230] 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 product thereof,

[0231] 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;

[0232] Among them, each symbol (such as L, z, q, R 1’ 、R 2’ 、R 3’ 、R 4’ and R 5’ ) as defined herein.

[0233] 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,

[0234] Among them L 1 and D 1 As defined in formula I-1 above;

[0235] L 3 Selected from the following structures:

[0236] Wherein each variable such as Z, R, s, y, t is as above for L 2 It should be understood that in formulas L3-1 to L3-14, L 1 Some do not belong to L 3 The components of (L 1 ) is used to indicate the end and L 1 connect.

[0237] In some embodiments, the compound includes the compound of formula I-2, preferably having the structure of formula C-inter-1 to formula C-inter-12:

[0238] In some embodiments, The carbon to which the group is attached or The carbon to which the corresponding group is connected is R or S configuration, preferably R configuration

[0239] In some embodiments, in C-inter-1 to C-inter-6, the carbon to which the left-side NH2-CH2- is attached is in R or S configuration, preferably in R configuration.

[0240] 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.

[0241] 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).

[0242] On the other hand, the present invention provides 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 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, compounds of Formula I-2, I-5a or I-6a as described above, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs or isotope labels thereof.

[0243] 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.

[0244] In some embodiments, the viral infection is an infection caused by influenza virus or parainfluenza virus.

[0245] In some embodiments, the viral infection is an infection caused by influenza virus A, B, or C, or a parainfluenza virus.

[0246] In some embodiments, the patient who is infected with a virus or who may be at risk of being infected with a virus may be a patient with a deficient immune system.

[0247] 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 immunosuppressive therapy.

[0248] 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.

[0249] In some embodiments, the patient diagnosed with a disease causing immunosuppression has cancer or acquired immunodeficiency syndrome.

[0250] In some embodiments, the patient diagnosed with a cancer disorder that causes immunosuppression has leukemia, lymphoma, humoral immunity deficiency, T-cell deficiency, complement deficiency, or multiple myeloma.

[0251] In some embodiments, the patient is a patient who is currently receiving or is about to receive a hematopoietic stem cell transplant.

[0252] In some embodiments, the patient is a patient who is currently receiving or is about to receive an organ transplant.

[0253] In some embodiments, the patient may be at risk for a secondary infection.

[0254] 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 of the conjugates of Formula I-1, IA or IB, compounds of Formula I-2, I-5a or I-6a as described above, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs or isotope labels thereof.

[0255] In some embodiments, the secondary infection is a respiratory tract infection.

[0256] In some embodiments, the secondary infection is associated with pneumonia.

[0257] In some embodiments, the secondary infection is a bacterial, viral, or fungal infection.

[0258] In some embodiments, the bacterial infection is an infection caused by methicillin-resistant Staphylococcus aureus.

[0259] In some embodiments, the bacterial infection is an infection caused by Streptococcus pneumoniae.

[0260] 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 injection, topical inhalation, injection or infusion.

[0261] 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 isotopically labeled thereof is administered in combination with another therapeutic agent or used in combination to prepare a medicament.

[0262] In some embodiments, the additional therapeutic agent is an antiviral drug.

[0263] In some embodiments, the antiviral drug is baloxavir, pimodivir, oseltamivir, zanamivir, peramivir, laninamivir, amantadine, MEDI8852, or rimantadine.

[0264] In some embodiments, the additional medication being used by the patient is an antiviral vaccine.

[0265] 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 thereof are administered sequentially, for example, by injection, to the patient.

[0266] 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 thereof are administered to the patient simultaneously, for example, by injection.

[0267] Effects of the invention:

[0268] The conjugates of Formula I-1, IA or IB, compounds of Formula I-2, I-5a or I-6a, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs or isotope-labeled products thereof of the present invention have the following advantages:

[0269] It has significant antiviral activity.

[0270] High activity against drug-resistant viral strains; and / or

[0271] It has excellent in vitro / in vivo pharmacokinetic properties and safety, such as a long half-life and a higher in vivo exposure, which can reduce the frequency of dosing and increase patient compliance, and has high prospects for clinical application.

[0272] Preparation of the conjugate of the present invention

[0273] In one aspect, the invention provides methods of preparing the conjugates described herein.

[0274] In some embodiments, the present invention provides a method for preparing a conjugate of Formula IA herein,

[0275] 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;

[0276] The method comprises the following steps:

[0277] The first step is to change (E) m Dissolved in a buffer solution, where E and m are as defined above, add a reducing agent (e.g., TCEP, DTT) to reduce (E) m The disulfide bond in is reduced to obtain a compound of formula II-1 containing a sulfhydryl residue,

[0278] Wherein E, m and n are as defined in the text;

[0279] 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.

[0280] 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) are as defined in formula IA,

[0281] A 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, followed by adding 3 to 7 molar equivalents of a buffer solution of the compound of formula I-5a in batches (e.g., 1 to 2 batches), and continuing the reaction for 0.5 to 10 hours (preferably 0.5 to 2 hours) to obtain a conjugate of formula I-3b.

[0282] 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;

[0283] 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;

[0284] or,

[0285] A method for preparing the conjugate of formula IB,

[0286] 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;

[0287] The method comprises the following steps:

[0288] The first step is to change (E) m Dissolved in a buffer solution, wherein E and m are as defined herein, add a reducing agent (such as TCEP, DTT) to (E) m The disulfide bond in is reduced to obtain a compound of formula II-1 containing a sulfhydryl residue,

[0289] Wherein E, m and n are as defined in the text;

[0290] 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.

[0291] 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,

[0292] The 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 an IB conjugate.

[0293] In some embodiments, the present invention provides two methods for preparing conjugates of I-1.

[0294] Method 1 includes the following steps:

[0295] in, Corresponding to L 3 , where (L 1 ) indicates L 2 'With L 1 Connect, L 1 Not part of the above structure;

[0296] in, Corresponding to L 2 As explained above, the structure between the two wavy lines corresponds to L 2 .

[0297] 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 formulas L2-1 to L2-7;

[0298] The first step is to change (E) m Dissolve in buffer, add reducing agent (such as TCEP, DTT) to (E) m Reducing the disulfide bond in to obtain a compound of formula II-1 containing a sulfhydryl residue;

[0299] In the second step, a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-2a is added to the compound of formula II-1, and a Michael addition reaction is carried out 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. Then, 3 to 7 molar equivalents of the compound of formula I-2a are added in portions (e.g., 1 to 2 times) and the reaction is continued for 0.5 to 10 hours, preferably 0.5 to 2 hours, to obtain a conjugate of formula I-3.

[0300] 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.

[0301] 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.

[0302] In some embodiments, the buffer is selected from the group consisting of phosphate buffer, citrate buffer, acetate buffer, and histidine buffer.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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).

[0307] In some embodiments, the solution containing the conjugate of formula I-3 in the third step is the reaction solution after the second step reaction is completed.

[0308] The inventors unexpectedly discovered that adding the starting material (the compound of Formula I-2a) in batches during the second step can make the DAR value of the final product more uniform, resulting in a product of improved quality. Furthermore, adjusting the pH to acidic conditions, such as 5.5-7.0, 5.8-6.8, or 6.0-6.6, during the second step also helps improve the uniformity of the DAR value of the final product.

[0309] Method 2 includes the following steps

[0310] in, Corresponding to L 3 ,

[0311] in, Corresponding to L 2 ,

[0312] 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 formulas L2-8 to L2-14;

[0313] Among them, the first step is to convert (E) m Dissolve in buffer, add reducing agent (such as TCEP, DTT) to (E) m Reducing the disulfide bond in to obtain a compound of formula II-1 containing a sulfhydryl residue;

[0314] 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 a Michael addition reaction is carried out at pH 5.5 to 8 (e.g., 5.8 to 7.6, 6.0 to 7.5) to obtain the I-1b conjugate.

[0315] In some embodiments, the buffer, reaction temperature, reducing agent and (E) m The molar ratio is as defined in Method 1.

[0316] 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.

[0317] Pharmaceutical composition

[0318] 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 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 simplicity, a conjugate of Formula I-1, IA or IB, a conjugate or 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 invention." In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition comprises a compound of the invention and a combination of one or more other therapeutic agents.

[0319] 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.

[0320] For the use of pharmaceutically acceptable excipients and their uses, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0321] The pharmaceutical compositions of the present invention can be in a variety of 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.

[0322] 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.

[0323] definition:

[0324] The following are definitions of terms used 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 commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0325] The term "inhibit neuraminidase activity" as used herein refers to an IC of less than or equal to 1000 nM. 50 For example, the inhibition of neuraminidase activity is determined according to the neuraminidase inhibition assay described in the Examples herein. In some embodiments, the inhibition of neuraminidase activity is less than or equal to 100 nM or less than or equal to 10 nM IC 50 It refers to a compound that inhibits neuraminidase activity.

[0326] As used herein, the term "inhibit viral growth" refers to an EC value less than or equal to 1000 nM. 50 For example, the activity of inhibiting influenza virus-mediated cytopathic effect is determined according to the influenza virus-mediated cytopathic effect assay described in the Examples herein. In some embodiments, the activity of inhibiting viral growth is less than or equal to 100 nM or less than or equal to 10 nM EC. 50 Compounds that inhibit viral growth.

[0327] As used herein, the term "Fc domain monomer" refers to a polypeptide chain or 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 third antibody constant region (CH2 and CH3) and optionally a fourth antibody constant region. 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 can be of any immunoglobulin antibody type, including IgG, IgE, IgM, IgA, and IgD. In addition, the Fc domain monomer can be of any IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). For example, in a natural antibody, the 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 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 known as the Kabat EU, or EU numbering system, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. 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 specifically engineered to provide additional small molecule drug conjugation sites.

[0328] 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 conjugation 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 conjugation sites.

[0329] 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.

[0330] The term "Fc-binding peptide" as used herein refers to a polypeptide consisting of a continuous amino acid sequence 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), 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 conjugation sites for small molecule drugs. The Fc-binding peptide 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 conjugation sites.

[0331] As used herein, the term "solvent exposed" refers to amino acid residues that are surrounded by solvent molecules surrounding the protein or polypeptide. The solvent-exposed amino acid molecules can be naturally occurring or artificially modified (including natural amino acids and unnatural amino acids). In some embodiments, the site modification does not affect the spatial three-dimensional structure of the protein.

[0332] 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 in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and nonhomologous sequences can be disregarded for comparison purposes). Alignment for the purpose of percent identity in sequence comparisons can 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, and MegAlign (DNA Star).

[0333] 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 the individual's symptoms, and / or eliminate the viral infection.

[0334] 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.

[0335] Individually or in combination (e.g. C1-C20 The term "C1-C 20 "Alkyl" means 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), in particular 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.

[0336] The term "C2-C 20 "Alkenyl" means a straight or branched chain alkyl group as defined above, but which contains one or more, e.g., 1 to 10, e.g., 1, 2, 3, 4, or 5, double bonds, but which 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.

[0337] 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. Particularly "C 3-7 "Cycloalkyl" includes cyclopropyl, cyclopentyl, cyclohexyl, etc.

[0338] The term "C3-C 20 "Cycloalkenyl" means a cycloalkyl group having 3-20, for example 3-15, 3-10 (for example 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), in particular 3-6 carbon atoms, which contains 1 or more, for example 1-10, for example 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 and the like. Particularly "C 3-7"Cycloalkenyl" includes cyclopropenyl, cyclopentenyl, cyclohexenyl and the like.

[0339] The term "halogen", alone or in combination, signifies fluorine, chlorine, bromine or iodine, in particular fluorine, chlorine or bromine.

[0340] 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.

[0341] The term "amino", alone or in combination, means a primary amino group (-NH2), a secondary amino group (-NH-) or a tertiary amino group.

[0342] 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 being connected by a double bond, and the carbon atom in the structure being further connected to two other fragments by single bonds.

[0343] The term "heterocycloalkyl", also known as "heterocyclyl", 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, or 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 optionally substituted with one or more substituents described herein. 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.

[0344] 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 herein.

[0345] The term "heteroaryl" or "heterocyclic aromatic group" refers to an aromatic ring group in which a carbon atom in the ring is replaced by at least one heteroatom selected from sulfur, oxygen, or nitrogen. This aromatic ring group can be a 5-15 membered ring, such as a 5-7 membered or 5-6 membered monocyclic ring or a 7-12 membered bicyclic ring, including but not limited to 5-, 6-, 7-, 8-, 9-, or 12-membered heteroaryl groups. 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, pyrimidinyl, 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.

[0346] The term "C 6-15 "Aryl" means an aromatic group having 6 to 15 carbon atoms, wherein "aryl" is as defined above.

[0347] The term "5-15 membered heteroaryl" refers to an aromatic hetero group having 5 to 15 ring atoms, wherein "heteroaryl" is as defined above.

[0348] The term "cyano" refers to the group -CN.

[0349] The term "carboxy" refers to the group -COOH.

[0350] The term "hydroxy" refers to the group -OH.

[0351] The term "substituted" refers to a group having one or more substituents, such as 1-25, 1-20, 1-10, or 1-5, for example, 1, 2, 3, 4, or 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, guanidino, urea, amidino, and combinations of the foregoing 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, NRSON2NR2, 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.

[0352] The term "optionally" means that the following event or situation can occur / exist or not occur / exist. For example, "optionally substituted" means that the group involved can be substituted or not substituted.

[0353] The term "comprising" also encompasses the case where the component consists of or consists essentially of the referenced features or elements.

[0354] The term "stereoisomer" encompasses all isomeric forms, including enantiomers, diastereomers, and geometric isomers (cis-trans isomers). Therefore, single stereochemical isomers of the compounds contemplated herein, or mixtures thereof, of enantiomers, diastereomers, or geometric isomers (or cis-trans isomers), are within the scope of the present invention.

[0355] 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 by the compounds of the present invention with 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 by the compounds of the present invention with 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 salts formed by reacting with organic bases containing N groups. + (C 1- 6 alkyl) 4 salt.

[0356] The term "solvate" refers to an association formed between one or more solvent molecules and a 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.

[0357] The term "hydrate" refers to an association complex formed between water and a compound of the present invention.

[0358] The term "prodrug" refers to a chemical derivative of the compound of the present invention, which is converted into the compound represented by the general formula I-1 or I-2 through a chemical reaction in vivo.

[0359] 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, or a carbon atom is replaced by one or more, for example, 1 to 3 carbon 14 atoms ( 14 C) The isotope label obtained by replacement.

[0360] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and 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 simultaneously, without specific time restrictions, or at the same or different time intervals, in sequence as separate entities, wherein such administration provides two or more active agents at a preventive or therapeutically effective level in the patient's body. In some embodiments, the compound of the present invention and other therapeutic agents used in the drug combination are administered at a level no greater than 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. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts 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 formulation, and the formulations may be the same or different.

[0361] The term "drug:antibody ratio" or "DAR" refers to the small molecule drug moiety (D) conjugated to an E moiety (e.g., an antibody, 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 a population of molecules in the product, i.e., the small molecule drug moiety (DAR) conjugated to the E moiety described herein (e.g., an antibody, Fc domain, or albumin) in the product as measured by a detection method (e.g., by MS) 1 and D 2) to the overall ratio of moieties E, 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 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, or any range having two of these values ​​as endpoints.

[0362] As used herein, when a variable such as n, y, or the like is or is selected from a range of integers, it is intended that all integers within that range are contemplated by the present invention, as if they were listed individually herein. For example, when n is described as an integer from 1 to 8, it is intended that n be described herein as an integer of 1, 2, 3, 4, 5, 6, 7, or 8.

[0363] Sequence SEQ ID NO: 1-81 and its description

[0364] SEQ ID NO: 1: Murine Fc-IgG2a with IL2 signal sequence (bold) at the N-terminus (bold)

[0365] SEQ ID NO: 2: Mature murine Fc-IgG2a

[0366] SEQ ID NO: 3: Human Fc-IgG1 with an IL2 signal sequence at the N-terminus (bold) and an additional N-terminal MVRS amino acid sequence (underlined)

[0367] SEQ ID NO: 4: Mature human Fc-IgG1 with MVRS amino acid sequence (underlined) added to the N-terminus

[0368] SEQ ID NO: 5: Murine Fc-IgG2a with an IL2 signal sequence (bold) added to the N-terminus and a six-histidine tag (italics) added to the C-terminus

[0369] SEQ ID NO: 6: Mature murine Fc-IgG2a with a six-histidine tag (italics) added to the C-terminus

[0370] SEQ ID NO: 7: Human Fc-IgG1 with an IL2 signal sequence (bold) and MVRS amino acid sequence (underlined) added to the N-terminus and a hexa-histidine tag (italics) added to the C-terminus

[0371] SEQ ID NO: 8: Human mature Fc-IgG1 with MVRS amino acid sequence (underlined) added to the N-terminus and a hexa-histidine tag (italics) added to the C-terminus

[0372] SEQ ID NO:9: Human Fc-IgG1 with the IL2 signal sequence (bold) and MVRS amino acid sequence (underlined) added to the N-terminus, two additional cysteines (*) in the hinge region, and a hexa-histidine tag (italics) added to the C-terminus

[0373] SEQ ID NO: 10: Mature human Fc-IgG1 with the MVRS amino acid sequence (underlined) added to the N-terminus, two additional cysteines (*) in the hinge region, and a hexa-histidine tag (italics) added to the C-terminus

[0374] 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

[0375] SEQ ID NO: 12: Murine Fc-IgG2a with the 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

[0376] SEQ ID NO: 13: Murine Fc-IgG2a containing an Asn to Ala substitution (*) and a hexa-histidine tag (italics) added to the C-terminus

[0377] SEQ ID NO: 14: Human Fc-IgG1 with the 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

[0378] SEQ ID NO: 15: Human Fc-IgG1 with 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

[0379] SEQ ID NO: 16: Human Fc-IgG1 with human serum albumin signal sequence (bold) added to the N-terminus and ISAMVRS amino acid sequence (underlined) added to the N-terminus

[0380] SEQ ID NO: 17: Human Fc-IgG1 with human serum albumin signal sequence (bold) added to the N-terminus and the N-terminal ISAMVRS amino acid sequence (underlined), containing a C-terminal G4S linker (italics) and a C-terminal c-Myc tag (underlined, italics)

[0381] SEQ ID NO: 18: Mature human Fc-IgG1 with the 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

[0382] 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

[0383] 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 conjugation at this site

[0384] SEQ ID NO:21: Human Fc-IgG1 with the 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

[0385] 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

[0386] SEQ ID NO:23: Human Fc-IgG1 with the 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

[0387] SEQ ID NO:24: Mature human Fc-IgG1 with the 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

[0388] SEQ ID NO:25: Human Fc-IgG1 with the 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 with a G4S linker (italics) and c-Myc tag (underlined, italics) added to the C-terminus

[0389] SEQ ID NO:26: Human Fc-IgG1 with the ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing H310A (*) and H435A (*) changes to prevent FcRn binding, and a G4S linker (italics) and c-Myc tag (underlined, italics) added to the C-terminus

[0390] 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

[0391] SEQ ID NO:28: Mature human Fc-IgG1 with the 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

[0392] SEQ ID NO:29: Human Fc-IgG1 with the human serum albumin signal sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing an Asn to Ala substitution (*), and with a G4S linker (italics) and a mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italics) added to the C-terminus

[0393] SEQ ID NO:30: Mature human Fc-IgG1 with the ISAMVRS amino acid sequence (underlined) added to the N-terminus, 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

[0394] 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

[0395] 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)

[0396] 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)

[0397] SEQ ID NO:34: Human Fc-IgG1 with a 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), a cysteine ​​to serine change (#), and allotype G1m(fa) (bold italics)

[0398] SEQ ID NO:35: Human Fc-IgG1 with a murine IgG signal sequence (bold) added to the N-terminus, the EPKSSD amino acid sequence of the hinge region of mature human Fc-IgG removed, and allotype G1m(fa) (bold italics)

[0399] SEQ ID NO:36: Amino acid sequence of mature human Fc-IgG with the hinge region EPKSSD removed at the N-terminus, having allotype G1m(fa) (bold italics) and containing the YTE triple mutation (bold, underlined).

[0400] SEQ ID NO:37: Amino acid sequence of mature human Fc-IgG with the hinge region EPKSSD removed at the N-terminus, having the LS double mutation (bold, underlined) and allotype G1m(fa) (bold italics).

[0401] SEQ ID NO:38: Human Fc-IgG1 with human serum albumin signal sequence (bold) added to the N-terminus, containing a YTE triple mutation (bold, underlined), having the allotype G1m(fa) (bold italic), and with a G4S linker (italics) and c-Myc tag (underlined) added to the C-terminus

[0402] 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

[0403] SEQ ID NO: 40: Mature human Fc-IgG1, wherein X4 is Asp or Glu, and X5 is Leu or Met

[0404] SEQ ID NO:41: Mature human Fc-IgG1 with a YTE triple mutation (bold, underlined), wherein X4 is Asp or Glu, and X5 is Leu or Met

[0405] SEQ ID NO:42: Mature human Fc-IgG1 with a YTE triple mutation (bold, underlined) and allotype G1m(fa) (bold italics)

[0406] SEQ ID NO:43: Mature human Fc-IgG1 with a YTE triple mutation (bold, underlined) and allotype G1m(f) (bold italics)

[0407] SEQ ID NO: 44: Mature human Fc-IgG1 with LS double mutation (bold, underlined), wherein X4 is Asp or Glu, and X5 is Leu or Met

[0408] SEQ ID NO:45: Mature human Fc-IgG1 with LS double mutation (bold, underlined) and allotype G1m(fa) (bold italics)

[0409] SEQ ID NO:46: Mature human Fc-IgG1 with LS double mutation (bold, underlined) and allotype G1m(f) (bold italics)

[0410] SEQ ID NO:47: Mature human Fc-IgG1 with a 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

[0411] SEQ ID NO:48: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), a cysteine ​​to serine change (#), and containing allotype G1m(fa) (bold italics)

[0412] SEQ ID NO:49: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), a cysteine ​​to serine change (#), and containing allotype G1m(f) (bold italics)

[0413] 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)

[0414] 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)

[0415] SEQ ID NO:52: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), a cysteine ​​to serine change (#), a YTE triple mutation (bold, underlined), and allotype G1m(fa) (bold italics)

[0416] SEQ ID NO:53: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence added to the N-terminus (bold), a cysteine ​​to serine change (#), a YTE triple mutation (bold, underlined), and allotype G1m(f) (bold italics)

[0417] SEQ ID NO: 54: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) and ISAMVRS amino acid sequence (italics) added to the N-terminus, 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)

[0418] SEQ ID NO:55: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) and ISAMVRS amino acid sequence (italics) added to the N-terminus, 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)

[0419] SEQ ID NO:56: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) and ISAMVRS amino acid sequence (italics) added to the N-terminus, 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)

[0420] SEQ ID NO:57: Mature human Fc-IgG1 with mouse heavy chain MIgG Vh signal sequence (bold) and ISAMVRS amino acid sequence (italics) added to the N-terminus, a YTE triple mutation (bold, underlined), a G4S linker (italics) and a c-Myc tag (underlined) added to the C-terminus, and containing the allotype G1m(fa) (bold italics)

[0421] SEQ ID NO:58: Mature human Fc-IgG1 with a mouse heavy chain MIgG Vh signal sequence (bold) added to the N-terminus, a cysteine ​​to serine change (#), a G4S linker (italics) and an IgA peptide tag (underlined) added to the C-terminus, and containing the allotype G1m(fa) (bold italics)

[0422] SEQ ID NO:59: 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, N434S mutations (bold, underlined), with a G4S linker (italics) and IgA peptide tag (underlined) added to the C-terminus, and containing allotype G1m(fa) (bold italics)

[0423] 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 Ser

[0424] 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

[0425] 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

[0426] SEQ ID NO:63: Mature human Fc-IgG1, cysteine ​​to serine change (#), allotype G1m(f) (bold italics)

[0427] SEQ ID NO:64: Mature human Fc-IgG1, cysteine ​​to serine change (#), allotype G1m(fa) (bold italics)

[0428] SEQ ID NO:65: Mature human Fc-IgG1, cysteine ​​to serine change (#), M428L, N343S mutations (bold, underlined), allotype G1m(fa) (bold italics)

[0429] SEQ ID NO:66: Mature human Fc-IgG1, cysteine ​​to serine change (#), M428L, N343S mutations (bold, underlined), allotype G1m(f) (bold italics)

[0430] SEQ ID NO:67: Mature human Fc-IgG1, cysteine ​​to serine change (#), YTE triple mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0431] SEQ ID NO:68: Mature human Fc-IgG1, cysteine ​​to serine change (#), YTE triple mutation (bold, underlined), allotype G1m(f) (bold italics)

[0432] SEQ ID NO: 69: Mature human Fc-IgG1, YTE triple mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0433] 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)

[0434] SEQ ID NO:71: Mature human Fc-IgG1, M428L, N343S mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0435] SEQ ID NO:72: (wild type)

[0436] SEQ ID NO:73: Mature human Fc-IgG1, M428L, N343S mutation (bold, underlined), allotype G1m(f) (bold italics)

[0437] SEQ ID NO:74: Mature human Fc-IgG1, GAALIE, M428L, N343S mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0438] SEQ ID NO:75: Mature human Fc-IgG1, GAALIE, M428L, N343S mutation (bold, underlined), allotype G1m(f) (bold italics)

[0439] SEQ ID NO:76: Mature human Fc-IgG1, cysteine ​​to serine change (#), GAALIE, M428L, N343S mutations (bold, underlined), allotype G1m(fa) (bold italics)

[0440] SEQ ID NO:77: Mature human Fc-IgG1, cysteine ​​to serine change (#), GAALIE, M428L, N343S mutations (bold, underlined), allotype G1m(f) (bold italics)

[0441] SEQ ID NO:78: Mature human Fc-IgG1, cysteine ​​to serine change (#), DHS triple mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0442] SEQ ID NO:79: Mature human Fc-IgG1, cysteine ​​to serine change (#), DHS triple mutation (bold, underlined), allotype G1m(f) (bold italics)

[0443] SEQ ID NO:80: Mature human Fc-IgG1, DHS triple mutation (bold, underlined), allotype G1m(fa) (bold italics)

[0444] SEQ ID NO:81: Mature human Fc-IgG1, DHS triple mutation (bold, underlined), allotype G1m(f) (bold italics)

[0445] The C (!) in SEQ ID NO: 1 to 81 is a C that can be connected to L 2 of cysteine. BRIEF DESCRIPTION OF THE DRAWINGS

[0446] Figure 1: MS detection of conjugate 1.

[0447] Figure 2: SEC detection of conjugate 1.

[0448] Figure 3: Residual small molecule detection diagram of conjugate 1.

[0449] Figure 4: MS detection of conjugate 2.

[0450] Figure 5: SEC detection of conjugate 2.

[0451] Figure 6: Residual small molecule detection diagram of conjugate 2.

[0452] Figure 7: MS detection results of the screening test product conjugate 1.

[0453] Figure 8: MS detection graph of conjugate 3.

[0454] Figure 9: SEC detection chart of conjugate 3.

[0455] Figure 10: Residual small molecule detection diagram of conjugate 3.

[0456] Figure 11: MS detection graph of conjugate 4.

[0457] Figure 12: SEC detection chart of conjugate 4.

[0458] Figure 13: Residual small molecule detection diagram of conjugate 4. DETAILED DESCRIPTION

[0459] The above 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 may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art.

[0460] Example 1: Preparation of Fc part

[0461] 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-to-end with appropriate restriction sites (XbaI + SalI) and then cloned into the pWX4.1 expression vector (WuXi Biologics Co., Ltd., Shanghai). Each constructed vector 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 it out. The HiPURE Plasmid Filter Maxiprep Kit (Life Technologies) was then used for purification. TM The plasmid was transfected into CHO cells (ATCC) using the 293 Transfection Kit (Life Technologies). The transfected cells were cultured for 7 days, then centrifuged and filtered. The supernatant was collected and purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA) to obtain purified h-IgG Fc. The purified sample was subjected to 4-12% Bis Tris SDS-PAGE electrophoresis, with 1-2 μg loaded and then stained with Coomassie Brilliant Blue. Each sample was treated with both reducing (R) and non-reducing (NR).

[0462] Example 2: Preparation of Fc part

[0463] The protein amino acid sequence (SEQ ID Nos: 69-81) was reverse-translated and the corresponding nucleotide sequence was synthesized. This sequence was then homologously recombined into the pCDNA3.4 vector. Each constructed vector carries the signal peptide sequence of human interleukin-10. The constructed plasmids were electroporated into CHO cells. The transfected cells were cultured for seven days, then precipitated by centrifugation. The supernatant was collected by filtration and purified using a protein A affinity column to obtain purified h-IgG Fc. The purified samples were subjected to Bis Tris SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue. Each sample was treated with both reducing (R) and non-reducing (NR) treatments.

[0464] Example 3: General Synthesis Method of h-IgG1 Fc-SH

[0465] 50mM PB, pH 6.5 buffer, antibody ((E) m ) solution (30 mg) and 10 mM reducing agent (TCEP) solution (reducing agent / (E) mThe 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.

[0466] Example 4: General method 1 for preparing conjugates:

[0467] The variables are as defined in the text.

[0468] To the reaction solution containing II-1, 50 mM PB, pH 6.5 buffer and a solution of the compound of formula I-2a (I-2 / II-1 = 7 to 12) were added, and the reaction temperature was controlled at 22°C in a metal bath for 1 hour. After the reaction time was reached, the compound of formula I-2a (I-2 / II-1 = 3.5 to 6) was added to the reaction solution, and the reaction temperature was controlled at 22°C in a metal bath for a further 0.5 hour. After the reaction time was reached, the compound of formula I-2a (I-2 / II-1 = 3.5 to 6) was added to the reaction solution again, and the reaction temperature was controlled at 22°C in a metal bath for a further 1.0 hour to obtain I-3.

[0469] The reaction solution was adjusted to pH 7.5 with 0.5 M NaOH and continued in a metal bath at 22°C for 2 hours, until I-3 was completely hydrolyzed to I-1a. The reaction solution was concentrated by ultrafiltration and centrifugation. After ultrafiltration and liquid exchange, the purified target conjugate was prepared. The average DAR (drug / antibody ratio) of the conjugate was analyzed by MS.

[0470] Example 5: Synthesis of drug intermediate (INT-DRUG)

[0471] Step 1, Synthesis of INT-DRUG-2

[0472] INT-DRUG-1 (3.0 g, 6.57 mmol) was added to a reaction flask, followed by tetrahydrofuran (30 mL) and triphenylphosphine (1.9 g, 7.23 mmol). The reaction was stirred at 30°C for 2 hours. After the reaction, lithium hydroxide monohydrate (28 mg) and water (1.5 mL) were added, and stirring was continued at 30°C for 16 hours. After TLC monitoring of the reaction, N,N'-di-Boc-1H-1-guanidinopyrazole (2.1 g, 6.91 mmol) was added, and the mixture was stirred at 30°C for 48 hours. After the reaction, the reaction mixture was concentrated and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:1). INT-DRUG-2 (2.2 g) was obtained as a colorless oil. MS: [M+1]+ found 673.40

[0473] Step 2, Synthesis of INT-DRUG-3

[0474] INT-DRUG-2 (2.19 g, 3.25 mmol) was added to a reaction flask under nitrogen protection. A solution of dry sodium methoxide (0.65 mmol) in methanol (8 mL) was then added and stirred at room temperature for 10 minutes. The reaction was quenched with HCl (0.4 N, 1,4-dioxane solution, 2.5 mL). The reaction solution was concentrated and purified by silica gel column chromatography (ethyl acetate / petroleum ether, 70% to 100%) to afford INT-DRUG-3 (1.15 g) as a white solid. MS: [M+1] + found 547.33.

[0475] Step 3, Synthesis of INT-DRUG-4

[0476] 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) were added to a reaction flask. The reaction was stirred at room temperature for 16 hours. After completion of the reaction, the reaction solution was concentrated and purified 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.

[0477] Step 4, Synthesis of INT-DRUG

[0478] INT-DRUG-4 (403 mg, 0.7 mmol), DMAP (1.37 g, 11.2 mmol), pyridine (60 mL), and p-nitrophenyl chloroformate (2.11 g, 10.5 mmol) were added to a reaction flask. After nitrogen replacement, the mixture was stirred at 40°C for 4 h. After LCMS detection, the reaction was >90% conversion. Water (20 mL) was added to quench the reaction (do not store for an extended period). The reaction was separated by reverse column chromatography (acetonitrile / water 0% to 90%) and lyophilized to obtain INT-DRUG (173 mg) as a white solid. MS: [M+1] + found 738.33.

[0479] Example 6: Synthesis of Intermediate A (inter-A)

[0480] Step 1: Synthesis of Intermediate 2

[0481] Compound 1 (150 g, 1.43 mol) and triethylamine (289 g, 2.85 mol) were added to a reaction flask, followed by tetrahydrofuran (1 L), nitrogen replacement, and stirring at 0 ° C. CbzCl (243 g, 1.43 mol) was dissolved in tetrahydrofuran (500 mL) and added dropwise to 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 give a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to give compound 2 (255 g) as a yellow oil.

[0482] Step 2: Synthesis of Intermediate 3

[0483] Compound 2 (245 g, 1.02 mol) and carbon tetrabromide (509 g, 1.54 mol) were added to a reaction flask, followed by dichloromethane (2 L). After nitrogen replacement, the mixture was stirred at 0°C. Triphenylphosphine (403 g, 1.54 mol) was dissolved in dichloromethane (500 mL) and added dropwise to 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 compound 3 (200 g, crude product) as a yellow oil.

[0484] Step 3: Synthesis of Intermediate 4

[0485] 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 give a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to give compound 4 (45 g) as a yellow oil.

[0486] Step 4-5: Synthesis of intermediate 6

[0487] Compound 4 (43.0 g, 78.2 mmol) and tetrahydrofuran (450 mL) were added to a reaction flask, replaced with nitrogen, and stirred at 0°C. 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, then heated to reflux and stirred 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 the mixture was 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, 30 / 1) to obtain compound 6 (20 g) as a yellow oil. MS: [M+1] + found 510.2.

[0488] Step 6: Synthesis of inter A

[0489] Compound 6 (19 g, 3.73 mmol), palladium carbon (2 g) and methanol (400 mL) were added to a reaction flask under a nitrogen atmosphere. The atmosphere was replaced with hydrogen several times. The reaction solution was stirred at room temperature overnight. After the reaction was completed, 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 intermediate A (14 g) as a yellow oil. MS: [M+1] + found 420.2.

[0490] Example 7: Synthesis of Intermediate B

[0491] Step 1: Synthesis of intermediate 9

[0492] At zero degrees Celsius, 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, the pH of the reaction solution was adjusted to a weakly acidic state with a saturated potassium bisulfate aqueous solution. After acidification, the solution was extracted with DCM and the extract was dried by rotary evaporation. The product 9 (470 mg) was purified by reverse column chromatography (water / acetonitrile) and lyophilized to obtain the product. MS [M+1] +: 285.1

[0493] Step 2: Synthesis of Inter B

[0494] To a solution of compounds 9 (150 mg, 0.53 mmol) and 10 (149 mg, 0.79 mmol) in DCM (20 ml) was added EDCI (201 mg, 1.05 mmol) at zero degrees Celsius. The reaction mixture was stirred at room temperature for 4 hours. After completion of the reaction, the solvent was removed by rotary evaporation and purified by silica gel column chromatography (PE:EA=3:1) to obtain Inter B (110 mg) as a white solid. MS [M+1]+: 451.32

[0495] Example 8: Synthesis of Inter C

[0496] Compounds 11 (1 g, 4.33 mmol), 10 (796 mg, 4.33 mmol), and 12 (892 mg, 4.33 mmol) were dissolved in DMF (40 mL) and stirred at room temperature for 2 hours. After the reaction, 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.

[0497] Example 9: Synthesis of Linker 1

[0498] Step 1: Synthesis of L1-2

[0499] Dissolve chlorosulfonyl isocyanate (400 mg, 2.83 mmol) in dichloromethane (10 mL) under nitrogen and stir at 0°C. Add a solution of bromoethanol (353 mg, 2.83 mmol) in dichloromethane (5 mL) dropwise, and 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 completion of the reaction, add dichloromethane (10 mL), and wash with dilute hydrochloric acid (1 M, 20 mL). The organic phase is dried over anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and the mixture is purified by silica gel column chromatography (PE:EA = 1:1) to afford crude L1-2 (800 mg) as a yellow oil.

[0500] Step 2: Synthesis of L1-3

[0501] The crude product L1-2 (800 mg) was dissolved in dichloromethane (10 mL), and TEA (2 mL) was added. The mixture was stirred at room temperature for 16 hours. After completion of the reaction, dichloromethane (10 mL) was added, and the mixture was washed with dilute hydrochloric acid (1 M, 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).

[0502] Step 3: Synthesis of L1-4

[0503] 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 complete, the solvent was removed by rotary evaporation and the mixture was purified by reverse phase column chromatography (acetonitrile / water) to obtain a yellow oil L1-4 (190 mg). MS [M+1] + :713.43

[0504] Step 4: Synthesis of Linker 1

[0505] L1-4 (107 mg, 0.15 mmol) was placed in a 1,4-dioxane hydrochloride 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 used for the next reaction. MS [M+1] + :513.51.

[0506] Example 10: Synthesis of Linker 2

[0507] Step 1: Synthesis of L2-2

[0508] 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-phase column chromatography to obtain L2-2 (320 mg) as a colorless oil. MS [M+1] + :634.31

[0509] Step 2: Synthesis of Linker 2

[0510] Dissolve L2-2 (320 mg, 0.51 mmol) in dichloromethane (10 mL), add HCl (4 mL, 4 M in 1,4-dioxane), and stir at room temperature for 4 hours. After the reaction, concentrate to obtain Linker 2 (219 mg) as a colorless liquid. MS [M+1] + :434.53

[0511] Example 11: Synthesis of Linker3

[0512] Step 1: Synthesis of L3-2

[0513] L3-1 (1 g, 2.7 mmol) and NaH (216 mg, 5.4 mmol) were added to a reaction flask, followed by tetrahydrofuran (20 mL). After nitrogen replacement, the mixture was stirred at 25°C for 3 h. After completion of the reaction, water (5 mL) was added to quench the reaction and the mixture was concentrated to obtain a crude product, which was then purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to afford L3-2 (400 mg) as a yellow oil.

[0514] Step 2: Synthesis of L3-3

[0515] L3-2 (1.7 g, 4.16 mmol) and DMAP (1.01 g, 8.32 mmol) were added to a reaction flask, followed by dichloromethane (20 mL). 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, the crude product was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain L3-3 (1.1 g, crude product) as a yellow oil.

[0516] Step 3: Synthesis of L3-4

[0517] Dissolve L3-3 (440 mg, 0.78 mmol) in acetonitrile (10 mL) and stir. Add Inter A (326 mg, 0.78 mmol) and DIPEA (201 mg, 1.56 mmol) to the reaction mixture. Stir at 80°C for 10 hours. After the reaction is complete, remove the solvent by rotary evaporation and purify by reverse phase column chromatography (acetonitrile / water) to obtain a yellow oil L3-4 (310 mg).

[0518] Step 4: Synthesis of Linker 3

[0519] Compound 4 (247 mg, 0.3 mmol) was dissolved in dichloromethane (10 mL), added to a 1,4-dioxane hydrochloride solution (3 ml), and stirred at room temperature for 1 hour. After the reaction, the mixture was concentrated to obtain Linker 3 (182 mg) as a colorless oil. MS [M+1] + :610.52

[0520] Example 12: Synthesis of C-Inter-1

[0521] Step 1: Synthesis of C-Inter-1-1

[0522] 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 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 lyophilized to obtain C-Inter-1-1 (240 mg) as a white solid. MS [M / 2+1] + :855.5

[0523] Step 2: Synthesis of C-Inter-1-2

[0524] Dissolve C-Inter-1-1 (240 mg, 0.146 mmol) in methanol (5 ml) and add aqueous lithium hydroxide solution (0.55 mmol, 1 ml). After stirring at room temperature for 1 hour, add acetic acid (0.3 mL). Concentrate at low temperature and purify 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

[0525] Step 3: Synthesis of C-Inter-1-3

[0526] 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) was added 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) at zero degrees Celsius. The reaction mixture was stirred at room temperature for 1 hour under nitrogen protection. After completion of the reaction, C-Inter-1-3 (187 mg) was purified by high pressure preparative liquid chromatography. MS [M / 2+1] + :946.4

[0527] Step 4: Synthesis of C-Inter-1-4

[0528] At zero degrees Celsius, to a solution of C-Inter-1-3 (270 mg, 0.143 mmol) in DMF (3 ml) were added Inter B (77.3 mg, 0.171 mmol) and DIEA (57.56 mg, 0.445 mmol). The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, 1.5 ml of trifluoroacetic acid was added to the reaction mixture, and then lyophilized to obtain crude C-Inter-1-4. MS [M / 2+1] + :1079.5

[0529] Step 5: Synthesis of C-Inter-1

[0530] The crude product of C-Inter-1-4 was dissolved in 10% trifluoroacetic acid solution (30 ml), stirred at room temperature for 15 minutes, and then dried by spin drying. The product was purified by high pressure preparative liquid chromatography to obtain C-Inter-1 (85 mg). MS [M / 2+1] + :829.4

[0531] Example 13: Synthesis of C-Inter-3

[0532] C-Inter-3

[0533] Step 1: Synthesis of C-Inter-3-1

[0534] Linker 2 (219 mg, 0.5 mmol) and DIEA (396 mg, 3 mmol) were dissolved in dichloromethane (10 mL). INT-DRUG (885 mg, 1.2 mmol) was then dissolved in dichloromethane (5 mL) and added dropwise to the reaction mixture. The mixture was stirred at room temperature for 16 hours. After the reaction, the reaction mixture 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

[0535] Step 2: C-Inter-3-2

[0536] 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 mixture. The mixture was stirred at room temperature for 4 hours. After the reaction, acetic acid (1 mL) was added, the reaction mixture was concentrated, and purified using a reverse-phase column chromatography column to obtain C-Inter-3-2 (290 mg) as a white solid. MS [M / 2+1] + :775.8

[0537] Step 3: C-Inter-3-3

[0538] C-Inter-3-2 (290 mg) and 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), and copper sulfate (14.9 mg, 0.093 mmol) were dissolved in water (5 mL) and added dropwise to the reaction mixture. After nitrogen substitution, the mixture was stirred at room temperature for 2 hours. Reverse-phase column purification afforded C-Inter-3-3 (123 mg) as a white solid. MS [M / 2+1] + :907.0

[0539] Step 4: Synthesis of C-Inter-3-4

[0540] 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 complete, freeze-dry to obtain the crude product of C-Inter-3-4. MS [M+1] + :1040.0

[0541] Step 5: Synthesis of C-Inter-3

[0542] C-Inter-3-4 (crude product), TFA (10 mL), and water (0.5 mL) were added to a reaction flask, stirred at room temperature for 30 minutes, and concentrated at low temperature to remove the solvent. The crude product was purified by high pressure preparative liquid chromatography to obtain C-Inter-3 (65 mg). MS [M / 2+1] + :789.8

[0543] Example 14: Synthesis of C-Inter-6

[0544] Step 1: Synthesis of C-Inter-6-1

[0545] 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 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 lyophilized to obtain C-Inter-6-1 (397 mg) as a white solid. MS [M / 2+1]+ :903.9

[0546] Step 2: Synthesis of C-Inter-6-2

[0547] Dissolve C-Inter-6-1 (440 mg, 0.24 mmol) in methanol (6 ml) and add aqueous lithium hydroxide solution (0.48 mmol, 1 ml). Stir at room temperature for 1 hour, then add acetic acid (0.2 mL). Concentrate at low temperature and purify 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

[0548] Step 3: Synthesis of C-Inter-6-3

[0549] 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) was added 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) at zero degrees Celsius. The reaction mixture was stirred at room temperature for 1 hour under nitrogen protection. After completion of the reaction, the product was purified by high pressure preparative liquid chromatography to give C-Inter-6-3 (176 mg). MS [M / 2+1] + :1083.0

[0550] Step 4: Synthesis of C-Inter-6-4

[0551] At zero degrees Celsius, Inter B (43.04 mg, 0.09 mmol) and DIEA (30.96 mg, 0.24 mmol) were added to a DMF (3 ml) solution of C-Inter-6-3 (176 mg, 0.08 mmol). The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, 1.5 ml of trifluoroacetic acid was added to the reaction mixture, and then lyophilized to obtain C-Inter-6-4. MS [M / 2+1] + :1216.1

[0552] Step 5: Synthesis of C-Inter-6

[0553] Dissolve C-Inter-6-4 in 5% trifluoroacetic acid solution (10 ml), stir at room temperature for 15 minutes, then 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

[0554] Example 15: Synthesis of C-Inter-9

[0555] C-Inter-9

[0556] Step 1: Synthesis of C-Inter-9-1

[0557] 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, the crude product of C-Inter-9-1 was obtained by lyophilization. MS [M / 2+1] + :1013.5

[0558] Step 2: Synthesis of C-Inter-9

[0559] The crude product of C-Inter-9-1, TFA (10 mL), and water (0.5 mL) were added to a reaction flask and stirred at room temperature for 30 minutes. After the reaction, 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

[0560] Example 16: Expression and purification of hIgG1-Fc

[0561] 1. Experimental Materials

[0562] 2. Expression of hIgG1-Fc

[0563] 1) One day before transfection (day -1), dilute the cells to a final density of 3-4×10 6 viable cells / mL and allowed to grow overnight.

[0564] 2) On the day of transfection, the cell density should be 5-6×10 6 Cell viability must be >95%.

[0565] 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.

[0566] 4) Use 37 μL of OptiPRO TM Dilute 3.2 μL ExpiFectamine in SFM medium (4°C) TM CHO, mix gently.

[0567] 5) Dilute ExpiFectamine TM Add CHO reagent to the diluted DNA, mix gently, and incubate at room temperature for 1-5 minutes.

[0568] 6) Slowly add the above complex to each shake flask.

[0569] 7) Place the cells in a shaker at 37°C and 8% CO2, and set the rotation speed to 125 rpm.

[0570] 8) After incubating the cells for 20 hours, mix 10 μL of ExpiCHO Transfection Enhancer and 400 μL of ExpiCHO Feed per mL of cells, and add the mixture to each culture flask.

[0571] 9) After culturing for eight days, harvest the cell supernatant.

[0572] 3. Purification of hIgG1-Fc

[0573] 1) Collection of supernatant

[0574] Transfer the transfected Fc protein fermentation broth to a labeled 50ml centrifuge tube. Centrifuge at 10,000 rpm for 5 minutes. Pour the supernatant into a new labeled 50ml centrifuge tube. Continue centrifuging and collecting the supernatant until all the Fc protein fermentation broths have been centrifuged and collected.

[0575] 2) Preprocessing

[0576] Soak the gravity column in 0.5M NaOH solution. Add the Pro A filler solution to the empty gravity column. Once the solution in the gravity column has been drained, add 0.1M NaOH solution to the column. Once the solution in the gravity column has been drained, add pure water to the column.

[0577] 3) Balanced loading

[0578] Equilibrate the Pro A column with 5-10 column volumes of Pro A equilibration buffer. Add the supernatant from the centrifugation to the Pro A column and collect the flow-through into a clean 250 ml shake flask. After all the supernatant has passed through the column, add 5-10 column volumes of Pro A equilibration buffer until all the equilibration buffer has flowed out of the bottom.

[0579] 4) Protein elution

[0580] Seal the bottom of the washed column with a clean stopper. Add 2-3 column volumes of Pro-A / G elution buffer to the washed and sealed column. Use a pipette to thoroughly suspend the filler in the elution buffer and let it rest for 5 minutes. To prepare for elution, prepare a new collection tube and add a certain ratio of Tris neutralizing solution to prevent protein denaturation. Remove the stopper and collect the liquid into the collection tube. Measure the protein concentration and freeze at -80°C.

[0581] 4. Characterization of hIgG1 Fc

[0582] Based on the sequence information of Fc described herein, the target Fc was obtained according to the expression and purification steps of this example.

[0583] Example 17: Expression and purification of hIgG1-Fc

[0584] hIgG1-Fc (SEQ ID NO: 69) was prepared and expressed as described in Example 16.

[0585] 2. Purification of hIgG1-Fc

[0586] Experimental method: Protein A affinity chromatography column purification

[0587] 1) Equilibration column: 1xPBS, flow rate 1ml / min, 20ml

[0588] 2) Sample loading: flow rate 1 ml / min

[0589] 3) Wash: 1xPBS, flow rate 1ml / min, 20ml

[0590] 4) Elution: Sodium acetate buffer (pH 3.2) at 1 ml / min. Collect approximately 500 μl of fluid in 10 tubes. Read the absorbance at 280 nm using a NanoDrop instrument.

[0591] 5) Dialysis: Pipette the high-concentration protein into a dialysis bag and dialyze it in a beaker containing 1X PBS.

[0592] 3. Characterization of hIgG1 Fc

[0593] Based on the sequence information of Fc described herein, the target Fc was obtained according to the expression and purification steps of this example.

[0594] Example 18: Synthesis and characterization of conjugate 1

[0595] 1. Materials and Methods

[0596] 1.1 Instruments / Equipment

[0597] 1.2 Reagents / test solutions

[0598] 1.3 Experimental methods

[0599] 1.3.1 Ultrafiltration

[0600] 1) Ultrafiltration centrifuge tube pretreatment

[0601] Take two 15 mL 10 KDa ultrafiltration centrifuge tubes, add 0.1 M NaOH solution, centrifuge at 4000G for 10 min, and repeat 3 times.

[0602] Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4000G for 10 minutes, and repeat twice.

[0603] Add 50 mM PB, pH 6.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4000G for 10 minutes. Repeat until the pH of the permeate is about 6.5.

[0604] 2) Antibody ultrafiltration and fluid exchange

[0605] Add Fc (SEQ ID NO: 67) antibody to the treated ultrafiltration centrifuge tube, add 50 mM PB, pH 6.5 buffer to the ultrafiltration tube to 12 mL, centrifuge at 4000 G for 10 min, repeat 4 times, and take samples to detect the antibody concentration after the solution is replaced.

[0606] 3) Antibody concentration detection

[0607] The Protein A280 method in NanoDrop One was used for detection. The sample category was selected as "Other protein E 1%", the extinction coefficient E1% L / gm-cm was set to 13.5, and the baseline correction wavelength was 340 nm.

[0608] Use 50 mM PB, pH 6.5 buffer as a blank, draw 2.5 μL of sample for detection, and take the average value of three detection results as the sample concentration.

[0609] 1.3.2 Reduction reaction

[0610] 50 mM PB, pH 6.5 buffer, Fc (SEQ ID NO: 67) antibody solution (30 mg), and 10 mM TCEP solution (TCEP / mAb molar equivalent ratio of 10) were added to the centrifuge tube in sequence to control the antibody reaction concentration to 5 mg / mL. The tube was placed in a metal bath and the reaction temperature was controlled at 22°C for 2 hours.

[0611] 1.3.3 Coupling reaction

[0612] After the reduction reaction reaches 2 hours, 50mM PBS, pH 6.5 buffer and 10mg / mL C-Inter-1 solution (C-Inter / Fc molar equivalent ratio is 7) are added to the reaction solution to control the final reaction concentration to 2mg / mL. Place it in a metal bath and control the reaction temperature to 22°C for 1 hour.

[0613] After the reaction time was reached, a 10 mg / mL C-Inter-1 solution with a C-Inter-1 / Fc molar equivalent ratio of 3.5 was added to the reaction solution, and the reaction solution was placed in a metal bath and the reaction temperature was controlled at 22°C for another 0.5 hour.

[0614] After the reaction time was up, 10 mg / mL C-Inter-1 solution with a C-Inter-1 / mAb molar equivalent ratio of 3.5 was added to the reaction solution again, and the reaction 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.

[0615] 1.3.4 Hydrolysis reaction

[0616] 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.

[0617] 1.3.5 Ultrafiltration II

[0618] 1) Ultrafiltration centrifuge tube pretreatment

[0619] Take two 15 mL 10 KDa ultrafiltration centrifuge tubes, add 0.1 M NaOH solution, centrifuge at 4000G for 10 min, and repeat 3 times.

[0620] Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4000G for 10 minutes, and repeat twice.

[0621] Add 10 mM His-HCl, pH 5.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4000G for 10 minutes. Repeat until the pH of the permeate is about 5.6.

[0622] 2) Antibody ultrafiltration and fluid exchange

[0623] Add conjugate 1 to the treated ultrafiltration centrifuge tube, use 10mM His-HCl, pH 5.5 buffer to replenish the volume of the solution in the ultrafiltration tube to 12mL, centrifuge at 4000G for 10min, repeat 6 times, filter the ultrafiltration solution using a 0.22μm PES filter membrane, and detect the antibody concentration after the liquid change.

[0624] 3) Antibody concentration detection

[0625] The Protein A280 method in NanoDrop One was used for detection. The sample category was selected as "Other protein E 1%", the extinction coefficient E1% L / gm-cm was set to 13.5, and the baseline correction wavelength was 340 nm.

[0626] Use 10 mM His-HCl, pH 5.5 buffer as a blank, draw 2.5 μL of sample for detection, and take the average of three detection results as the sample concentration.

[0627] According to the test sample concentration and sample volume, use 10mM His-HCl, pH5.5 buffer to dilute the sample concentration to about 10mg / mL, and re-test the diluted sample concentration.

[0628] 2. Experimental Results and Analysis

[0629] 2.1 LC-MS DAR value detection

[0630] The DAR values ​​of the samples were detected by LC-MS. The relevant method conditions are shown in the table below:

[0631] LC method conditions

[0632] MS method conditions

[0633] The MS-DAR values ​​for the samples are shown in the table below. The MS results indicate that the MS-DAR value for conjugate 1 is 4.0. After sugar cleavage and reduction, the sample exhibits a single-stranded peak. C0, C1, C2, and C3 represent the molecular weights of 0, 1, 2, and 3 small molecules, respectively.

[0634] The MS detection spectrum of the sample is shown in Figure 1:

[0635] 2.2 SEC-HPLC purity test

[0636] The sample was tested for purity by SEC-HPLC. The relevant method conditions are shown in the table below:

[0637] The small molecule residue detection results of the sample are shown in FIG2 . The SEC detection results show that the purity of conjugate 1 is 98.21%.

[0638] 2.3 RP-HPLC small molecule residue detection

[0639] The samples were tested for residual free drug levels (mol / mol, free drug / bound drug) by RP-HPLC. Two standards containing free drug and Fc (2% & 5%) were prepared. 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:

[0640] Preparation of standards:

[0641] 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;

[0642] MW Fc(SEQ ID NO:67) =55414 g / mol, MW Fc(SEQ ID NO:69) =56000g / mol, MW C-Inter-1 =1656 g / mol;

[0643] M mAb =1 / MW mAb , M Total drug =DAR / MW mAb ;

[0644] Number of moles of 2% standard = DAR × M mAb ×2%;

[0645] Number of moles of 5% standard = DAR × M mAb ×5%;

[0646] 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 × MW C-Inter-1 ;

[0647] 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 × MW C-Inter-1 ;

[0648] The calculated small molecule weight was added to 100 μL of 1 mg / mL antibody solution to obtain a standard.

[0649] The small molecule residue detection results of the samples are shown in Figure 3, where the AKSZ-2627-0.01 mg / 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 detection results show that no small molecule peaks were detected in both samples by RP-HPLC. Compared with the antibody samples with 2% mol / mol and 5% mol / mol small molecules added, the sample residues were less than 2% mol / mol.

[0650] 2.4 Endotoxin Residue Detection

[0651] Endotoxin residues in samples were tested using the following steps:

[0652] 1) Take one endotoxin working standard, add 1 mL of BET water, vortex for 15 minutes, and gradually dilute to 4λ (E 4λ )、2λ(E 2λ )spare.

[0653] 2) Preparation of test solution: dilute the sample solution to 80 times (S 80 ) and 160 times (S 160 )spare.

[0654] 3) Preparation of solutions for each group

[0655] Test group A: S 160 solution;

[0656] Test sample positive control group B: one times the amount of S 80 The solution was added to twice the amount of E 4λ Solution, mix well, prepare S 160 E 2λ ;

[0657] Positive control group C:E 2λ solution;

[0658] Negative control group D: BET water.

[0659] 4) Reconstitution of the limulus amebocyte lysate: Take each vial of limulus amebocyte lysate and add 0.1 mL of BET water to reconstitute the limulus amebocyte lysate.

[0660] 5) After the limulus amebocyte lysate is reconstituted, 0.1 mL of each of the four solutions prepared in step 3) is added to the limulus amebocyte lysate, with two parallel tubes of limulus amebocyte lysate for each group.

[0661] 6) Incubation: Place the mixed ampoule into a test tube thermostat preheated to 37°C and incubate for 60 minutes.

[0662] Test results:

[0663] The concentration of conjugate 1 was 10.13 mg / mL, and the residual endotoxin in the sample was less than 1 EU / mg.

[0664] Example 19: Synthesis and characterization of conjugate 2

[0665] 1. Materials and Methods

[0666] 1.1 Instruments / Equipment

[0667] Same as Example 18

[0668] 1.2 Reagents / test solutions

[0669] Same as Example 18

[0670] 1.3 Experimental methods

[0671] 1.3.1 Ultrafiltration

[0672] 1) Ultrafiltration centrifuge tube pretreatment

[0673] Take two 15 mL 10 KDa ultrafiltration centrifuge tubes, add 0.1 M NaOH solution, centrifuge at 4000G for 10 min, and repeat 3 times.

[0674] Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4000G for 10 minutes, and repeat twice.

[0675] Add 50 mM PB, pH 6.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4000G for 10 minutes. Repeat until the pH of the permeate is about 6.5.

[0676] 2) Antibody ultrafiltration and fluid exchange

[0677] Add Fc (SEQ ID NO: 69) antibody to the treated ultrafiltration centrifuge tube, add 50 mM PB, pH 6.5 buffer to the ultrafiltration tube to 12 mL, centrifuge at 4000 G for 10 min, repeat 4 times, and take samples to detect the antibody concentration after the solution is replaced.

[0678] 3) Antibody concentration detection

[0679] The Protein A280 method in NanoDrop One was used for detection. The sample category was selected as "Other protein E 1%", the extinction coefficient E1% L / gm-cm was set to 13.44, and the baseline correction wavelength was 340 nm.

[0680] Use 50 mM PB, pH 6.5 buffer as a blank, draw 2.5 μL of sample for detection, and take the average value of three detection results as the sample concentration.

[0681] 1.3.2 Reduction reaction

[0682] 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 of 10) were added to the centrifuge tube in sequence to control the antibody reaction concentration to 5 mg / mL. The tube was placed in a metal bath and the reaction temperature was controlled at 22°C for 2 hours.

[0683] 1.3.3 Coupling reaction

[0684] 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 reaction concentration to 2mg / mL. Place it in a metal bath and control the reaction temperature to 22°C for 1 hour.

[0685] After the reaction time was reached, a 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 reaction solution was placed in a metal bath and the reaction temperature was controlled at 22°C for another 0.5 hour.

[0686] After the reaction time was up, 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 at 22°C and the reaction was continued for 1.0 hour.

[0687] 1.3.4 Hydrolysis reaction

[0688] 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 2.

[0689] 1.3.5 Ultrafiltration II

[0690] 1) Ultrafiltration centrifuge tube pretreatment

[0691] Take two 15 mL 10 KDa ultrafiltration centrifuge tubes, add 0.1 M NaOH solution, centrifuge at 4000G for 10 min, and repeat 3 times.

[0692] Add ultrapure water to the ultrafiltration centrifuge tube, centrifuge at 4000G for 10 minutes, and repeat twice.

[0693] Add 10 mM His-HCl, pH 5.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4000G for 10 minutes. Repeat until the pH of the permeate is about 5.6.

[0694] 2) Antibody ultrafiltration and fluid exchange

[0695] Add conjugate 2 to the treated ultrafiltration centrifuge tube, use 10mM His-HCl, pH 5.5 buffer to replenish the volume of the solution in the ultrafiltration tube to 12mL, centrifuge at 4000G for 10min, repeat 6 times, filter the ultrafiltration solution using a 0.22μm PES filter membrane, and detect the antibody concentration after the liquid change.

[0696] 3) Antibody concentration detection

[0697] The Protein A280 method in NanoDrop One was used for detection. The sample category was selected as "Other protein E 1%", the extinction coefficient E1% L / gm-cm was set to 13.44, and the baseline correction wavelength was 340 nm.

[0698] Use 10 mM His-HCl, pH 5.5 buffer as a blank, draw 2.5 μL of sample for detection, and take the average of three detection results as the sample concentration.

[0699] According to the test sample concentration and sample volume, use 10mM His-HCl, pH5.5 buffer to dilute the sample concentration to about 10mg / mL, and re-test the diluted sample concentration.

[0700] 2. Experimental Results and Analysis

[0701] 2.1 LC-MS DAR value detection

[0702] The method was the same as that of Example 18, and the MS-DAR value of the conjugate 2 sample was 6.0.

[0703] The MS spectrum of the sample is shown in Figure 4. After sugar cleavage and reduction, the sample exhibits a single-strand peak. C0, C1, C2, and C3 represent the molecular weights obtained by attaching 0, 1, 2, and 3 small molecules, respectively.

[0704] 2.2 SEC-HPLC purity test

[0705] The method was the same as that of Example 18. The SEC test results are shown in FIG5 . The purity of conjugate 2 was 94.47%.

[0706] 2.3 RP-HPLC small molecule residue detection

[0707] The method was the same as Example 18, and the small molecule residue was <2%, as shown in FIG6 .

[0708] 2.4 Endotoxin Residue Detection

[0709] The method was the same as that of Example 18, except that the concentration of conjugate 2 was 10.26 mg / mL, and the residual endotoxin in the sample was less than 1 EU / mg.

[0710] Example 20: Preparation of Conjugate 3

[0711] 1. Materials and Methods

[0712] 1) Experimental instruments

[0713] 2) Reagents / Solutions

[0714] 2. Experimental Methods

[0715] 1) Ultrafiltration

[0716] (1) Antibody ultrafiltration and fluid exchange

[0717] Fc (SEQ ID NO: 71) was added to an ultrafiltration centrifuge tube, and the volume of the solution in the ultrafiltration tube was supplemented to 12 mL with 50 mM PB, pH 6.5 buffer. The tube was centrifuged at 4500 G for 10 min, repeated 4 times, and samples were taken to detect the antibody concentration.

[0718] (2) Antibody concentration detection

[0719] Detection was performed using the Protein A280 method in NanoDrop One, selecting "Other protein E 1%" as the sample type, setting the extinction coefficient E1% L / gm-cm to 13.44 (7607H), and the baseline correction wavelength to 340 nm. A 2.5 μL sample was aspirated and tested using 50 mM PB, pH 6.5 buffer as a blank. The sample concentration was calculated as the average of three test results.

[0720] 2) Reduction reaction

[0721] 50 mM PB, pH 6.5 buffer, the corresponding antibody solution (28 mg), and 10 mM TCEP solution (TCEP / mAb equivalent ratio of 20) were added sequentially to a centrifuge tube to control the antibody reaction concentration to 5 mg / mL. The tube was placed in a metal bath and reacted at 25°C for 2 hours. Equivalents herein refer to molar equivalents unless otherwise specified or inconsistent with the context.

[0722] 3) Coupling reaction

[0723] 1. After the reduction reaction reaches 2 hours, add 10mg / mL C-Inter-3 solution (Drug / mAb

[0724] 2. Place the reaction mixture in a metal bath at 25°C for 1 hour. Repeat the addition of a 10 mg / mL C-Inter-3 aqueous solution (AKSZ2665 aqueous solution) (drug / mAb equivalent ratio of 10) every hour until the total reaction equivalent reaches 50 (add 0.5 M NaOH to adjust the solution pH to approximately 6.5). After the reaction, adjust the pH to 7.0-7.5 with 0.5 M NaOH and continue the reaction in the metal bath at 25°C for 2 hours.

[0725] 4) Ultrafiltration

[0726] (1) Antibody ultrafiltration and fluid exchange

[0727] 3. Add the conjugate sample to the treated ultrafiltration centrifuge tube, use 10mM His-HCl, pH 5.5 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 using a 0.22μm PES filter membrane, and detect the antibody concentration after the liquid change.

[0728] (2) Antibody concentration detection

[0729] The Protein A280 method in NanoDrop One was used for detection. The sample category was selected as "Other protein E 1%", the extinction coefficient E1% L / gm-cm was set to 13.44, and the baseline correction wavelength was 340 nm.

[0730] Use 10 mM His-HCl, pH 5.5 buffer as a blank, draw 2.5 μL of sample for detection, and take the average of three detection results as the sample concentration.

[0731] 5) Experimental results and analysis

[0732] (1) LC-MS DAR value detection

[0733] The DAR values ​​of the samples were detected by LC-MS. The relevant method conditions are shown in the table below:

[0734] LC method conditions

[0735] MS method conditions

[0736] 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.

[0737] Remark:

[0738] The formula for calculating drug loading distribution is: drug loading distribution (%) = (peak area of ​​single chain with drug loading of n / sum of peak areas of two single chains) × 100 (%);

[0739] Where n is the drug loading, and one single chain is the heavy chain constant domain 2, heavy chain constant domain 3, and 1 / 2 hinge region (CH2+CH3+1 / 2Hi);

[0740] DAR value calculation formula: Single chain weighted peak area = (drug load n × drug load distribution %)

[0741] Drug to antibody ratio = 2 × ∑ (single chain weighted peak area)

[0742] The MS detection spectrum of the sample is shown in Figure 8:

[0743] (2) SEC-HPLC purity test

[0744] The sample was tested for purity by SEC-HPLC. The relevant method conditions are shown in the table below:

[0745] The purity test results of the samples are shown in FIG9 . The SEC test results show that the purity of C060-7607H-2665-240125 (conjugate 3) is 97.50%.

[0746] (3) RP-HPLC small molecule residue detection

[0747] The samples were tested for residual free drug levels (mol / mol, free drug / bound drug) by RP-HPLC. Two standards containing free drug and Fc (2% & 5%) were prepared. 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:

[0748] Preparation of standards:

[0749] C 7607H =1 mg / mL; the sample DAR value is 5.9;

[0750] MW 7607H =56000g / mol, MW drug =1578.63 g / mol;

[0751] M mAb =1 / MW mAb , M Total drug =DAR / MW mAb ;

[0752] Number of moles of 2% standard = DAR × M mAb ×2%;

[0753] Number of moles of 5% standard = DAR × M mAb ×5%;

[0754] 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 ;

[0755] 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 ;

[0756] The calculated small molecule weight was added to 100 μL of 1 mg / mL antibody solution to obtain a standard.

[0757] The small molecule residue detection results of the samples are shown in FIG10 . The RP detection results show that the sample residue in the RP-HPLC is less than 2% mol / mol compared with the antibody samples added with 2% mol / mol and 5% mol / mol small molecules.

[0758] Example 21: Preparation of Conjugate 4

[0759] 1. Experimental Overview

[0760] The experiment used the reducing agent TCEP to break the disulfide bonds between antibody chains. A Michael addition reaction then occurred between -SH and the maleimide group on the linker-payload, yielding the conjugated product targeting DAR6. The DAR value of the conjugated product was determined by LC-MS, its purity by SEC-HPLC, and its residual small molecules by RP-HPLC.

[0761] 2. Summary of experimental results

[0762] 3. Experimental Materials and Methods

[0763] 3.1 Instruments / Equipment

[0764] 3.2 Reagents / test solutions

[0765] 3.3 Experimental methods

[0766] 3.3.1 Ultrafiltration

[0767] 1) Ultrafiltration centrifuge tube pretreatment

[0768] Take a 15mL 10kDa ultrafiltration centrifuge tube, add 0.1M NaOH solution, and centrifuge at 4500g for 10 minutes. Repeat three times. Add ultrapure water to the ultrafiltration centrifuge tube and centrifuge at 4500g for 10 minutes. Repeat two times. Add 50mM PB, pH 6.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4500g for 10 minutes. Repeat until the permeate pH is approximately 6.5.

[0769] 2) Antibody ultrafiltration and fluid exchange

[0770] Add 7607H antibody (SEQ ID NO: 71) to the treated ultrafiltration centrifuge tube, add 50 mM PB, pH 6.5 buffer to the ultrafiltration tube to 12 mL, centrifuge at 4500 G for 10 min, repeat 4 times, filter through a 0.22 μm filter membrane, and sample to detect the antibody concentration.

[0771] 3) Antibody concentration detection

[0772] Detection was performed using the Protein A280 method in NanoDrop One, selecting "Other protein E 1%" as the sample type, setting the extinction coefficient E1% L / gm-cm to 13.44 (7607H), and the baseline correction wavelength to 340 nm. A 2.5 μL sample was aspirated and tested using 50 mM PB, pH 6.5 buffer as a blank. The sample concentration was calculated as the average of three test results.

[0773] 3.3.2 Reduction reaction

[0774] 50 mM PB, pH 6.5 buffer, the corresponding antibody solution (45 mg), and 10 mM TCEP solution (TCEP / mAb equivalent ratio of 12) were added to the centrifuge tube in sequence to control the antibody reaction concentration to 5 mg / mL. The tube was placed in a metal bath and the reaction temperature was controlled at 22°C for 2 hours.

[0775] 3.3.3 Coupling reaction and hydrolysis reaction

[0776] 4. After the reduction reaction reached 2 hours, 50 mM PB, pH 6.5 buffer and a 10 mg / mL C-Inter-1 aqueous solution (AKSZ2627 aqueous solution) with a small molecule / mAb equivalent ratio of 10 were added to the reaction solution to achieve a final concentration of 2 mg / mL. The reaction was then allowed to react at 22°C in a metal bath for 1 hour. After the reaction time expired, a 10 mg / mL C-Inter-1 aqueous solution (AKSZ2627 solution) with a drug / mAb equivalent ratio of 5 was added to the 7607H antibody reaction solution. The reaction was then continued at 22°C in a metal bath for 1 hour. After the reaction was complete, the pH was adjusted to 7.0-7.5 with 0.5 M NaOH and the reaction was continued at 22°C in the metal bath for 2 hours.

[0777] 3.3.4 Ultrafiltration II

[0778] 1) Ultrafiltration centrifuge tube pretreatment

[0779] 5. Take a 15mL 10kDa ultrafiltration centrifuge tube, add 0.1M NaOH solution, and centrifuge at 4500g for 10 minutes. Repeat three times. Add ultrapure water to the ultrafiltration centrifuge tube and centrifuge at 4500g for 10 minutes. Repeat two times. Add 10mM His-HCl, pH 5.5 buffer to the ultrafiltration centrifuge tube and centrifuge at 4500g for 10 minutes. Repeat until the permeate pH is approximately 5.5.

[0780] 2) Antibody ultrafiltration and fluid exchange

[0781] 6. Add the coupled sample to the treated ultrafiltration centrifuge tube, use 10mM His-HCl, pH 5.5 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 using a 0.22μm PES filter membrane, and detect the antibody concentration after the solution change.

[0782] 3) Antibody concentration detection

[0783] 7. Perform the assay using the Protein A280 method in NanoDrop One, selecting "Other protein E 1%" as the sample type, setting the extinction coefficient E1% L / gm-cm to 13.44, and the baseline correction wavelength to 340 nm. Using 10 mM His-HCl, pH 5.5 buffer as a blank, aspirate 2.5 μL of sample for assay, and average the three assay results as the sample concentration. Based on the assay sample concentration and volume, dilute the sample to approximately 10 mg / mL with 10 mM His-HCl, pH 5.5 buffer and re-assay the diluted sample.

[0784] 4. Experimental Results and Analysis

[0785] 4.1 LC-MS DAR value detection

[0786] The DAR values ​​of the samples were detected by LC-MS. The relevant method conditions are shown in the table below:

[0787] LC method conditions

[0788] MS method conditions

[0789] 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.

[0790] Remark:

[0791] The formula for calculating drug loading distribution is: drug loading distribution (%) = (peak area of ​​drug-loaded single chain n / sum of the peak areas of two single chains) × 100 (%);

[0792] Where n is the drug loading, and one single chain is the heavy chain constant domain 2, heavy chain constant domain 3, and 1 / 2 hinge region (CH2+CH3+1 / 2Hi);

[0793] DAR value calculation formula: Single chain weighted peak area = (drug loading n × drug loading distribution %)

[0794] Drug-antibody ratio = 2 × ∑ (single-chain weighted peak area)

[0795] The MS detection spectrum of the sample is shown in Figure 11.

[0796] 4.2 SEC-HPLC purity test

[0797] The sample was tested for purity by SEC-HPLC. The relevant method conditions are shown in the table below:

[0798] The purity test results of the samples are shown in FIG12 . The SEC test results show that the purity of conjugate 4 (C060-7607H-231008) is 98.57%.

[0799] 4.3 RP-HPLC small molecule residue detection

[0800] The samples were tested for residual free drug levels (mol / mol, free drug / bound drug) by RP-HPLC. Two standards containing free drug and Fc (2% & 5%) were prepared. 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:

[0801] Preparation of standards:

[0802] C 7607H =1 mg / mL; the sample DAR value is 5.9;

[0803] MW 7607H =56000g / mol, MW drug =1950.79 g / mol;

[0804] M mAb =1 / MW mAb , M Total drug =DAR / MW mAb ;

[0805] Number of moles of 2% standard = DAR × M mAb ×2%;

[0806] Number of moles of 5% standard = DAR × M mAb ×5%;

[0807] 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 ;

[0808] 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 ;

[0809] The calculated small molecule weight was added to 100 μL of 1 mg / mL antibody solution to obtain a standard.

[0810] The small molecule residue detection results of the sample are shown in Figure 13. The RP detection results show 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.

[0811] 4.4 Endotoxin Residue Detection

[0812] Test results:

[0813] Compared with the 1EU / mg positive control, the endotoxin residues in the samples were all less than 1EU / mg.

[0814] Example 22: In vitro anti-influenza virus activity of the conjugate (influenza virus H1N1-mediated cytopathic inhibition assay)

[0815] 1. Experimental Methods

[0816] An in vitro cytopathic activity assay based on IAV / IBV activity was used using the MDCK cell line to quantify the IAV / IBV-induced cytopathic effect of the test compounds.

[0817] 2. Experimental Materials

[0818] 3. Experimental Procedure

[0819] 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.

[0820] 2) First, dilute the compound to 2 μM, then perform three-fold dilutions from 2 μM for a total of 8 dilutions. Add 5 μL of the diluted compound to each well and place in the incubator for 1 hour.

[0821] 3) Dilute influenza virus to 2222 pfu / mL in DMEM medium supplemented with 2% FBS, 1% P / S, and 4 μg / mL TPCK-treated trypsin. Add 45 μL of virus to each well. Simultaneously, establish a control with virus added but no compound, as well as a control with cells without compound or virus. Incubate in an incubator for 4 days.

[0822] 4) Add 50 μL of Cell-titer Glo to each well and detect chemiluminescence using a microplate reader.

[0823] 4. Data analysis: Calculate the viral inhibition rate of the compound.

[0824] Inhibition rate = (value of the sample tested - average value of the virus control group) / (average value of the cell control group - average value of the virus control group) * 100%

[0825] The calculated inhibition rate was fitted to the inhibition curve using the log (inhibitor) vs. response--Variable slope (four parameters) in the Nonlinear regression (curve fit) function of GraphPad Prism 8 software to calculate the EC50 value.

[0826] 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.

[0827] The purity of reference molecule A was 99.2% (SEC-HPLC) and the DAR value was 4.26.

[0828] The purity of reference molecule B was 98.8% (SEC-HPLC) and the DAR value was 5.87.

[0829] Summary of results:

[0830] The data demonstrates that the tested conjugates exhibited excellent in vitro anti-H1N1 activity. Compared to reference molecules A and B, conjugate 1 exhibited 8.8-fold and 5.9-fold increases in activity, respectively. Conjugate 2 exhibited 15.8-fold and 10.5-fold increases, respectively. Conjugates 3 and 4 exhibited 10.0-fold and 6.7-fold increases, respectively.

[0831] 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.

[0832] Example 23: Conjugate 1 Coupling Condition Screening Experiment

[0833] Except for the different coupling conditions, the experimental materials, experimental methods and detection methods are the same as those in Example 18.

[0834] 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.

[0835] The DAR values ​​detected by LC-MS are as follows

[0836] The MS detection results are shown in Figure 7.

[0837] Example 24: Pharmacokinetic Study of Conjugate 1 in Mice

[0838] 1. Experimental Information

[0839] Pharmacokinetic studies were conducted in male CD-1 (ICR) mice. Blood samples were analyzed using ELISA. Detailed experimental information and design are provided in the table below.

[0840] 2. Key experimental materials

[0841] 3. Detection steps

[0842] Method: The concentration of the conjugate in CD-1 mouse plasma was determined using an ELISA. The lower limit of quantification (LLOQ) of the conjugate was 50.0 ng / mL, and the upper limit of quantification (ULOQ) was 3000 ng / mL. The specific steps are as follows:

[0843] 1) Blood was collected at the set time points, and EDTA-K2 was used as an anticoagulant to prepare the corresponding test samples.

[0844] 2) Add 100 μL of coating solution to a 96-well microplate, seal, and incubate overnight at 2-8°C before use.

[0845] 3) Wash the plate 5 times with wash buffer (300 μL / well).

[0846] 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.

[0847] 5) Repeat step 2.

[0848] 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.

[0849] 7) Repeat step 2.

[0850] 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.

[0851] 9) Repeat step 2.

[0852] 10) Add 100 μL of TMB to each well and incubate at room temperature for 5-20 minutes.

[0853] 11) Add 100 μL of stop solution to each well.

[0854] 12) Read the plate within 30 minutes using a SpectraMax M5e / M5 / Plus 384 microplate reader at wavelengths between 450 nm and 630 nm.

[0855] 13) Convert the reading of the standard solution into the corresponding concentration of the conjugate.

[0856] 14) Data processing was performed using Watson LIMS V7.6 or SoftMax Pro GxP 7.0.3 and Microsoft Excel 2007 or higher.

[0857] 4. Results Summary

[0858] Mouse pharmacokinetic data indicate that Conjugate 1 has a long half-life of 165 hours and exhibits excellent in vivo exposure. These data suggest that Conjugate 1, with its long half-life and high exposure in clinical practice, may possess excellent antiviral activity.

[0859] Example 25: Pharmacokinetic study of conjugate 2 in mice

[0860] Pharmacokinetic studies were performed on Conjugate 2 according to the method of Example 22. The results are summarized as follows:

[0861] Mouse pharmacokinetic data show that Conjugate 2 has a long half-life of 140 hours. Conjugate 1 also exhibits excellent in vivo exposure. These data suggest that Conjugate 1, with its long half-life and high exposure in clinical practice, may possess excellent antiviral activity.

[0862] Example 26: Pharmacokinetic Study of Conjugate 4 in Mice

[0863] 1. Experimental Information

[0864] Pharmacokinetic studies were conducted in male CD-1 (ICR) mice. Blood samples were analyzed using ELISA. Detailed experimental information and design are provided in the table below.

[0865] 2. ELISA method to test the conjugate drug concentration (ADC concentration) in animal plasma

[0866] 1) Key reagents

[0867] 2) Key experimental parameters

[0868] 3) Experimental process

[0869] (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.

[0870] (2) Wash the plate three times with wash buffer (300 μL / well).

[0871] (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.

[0872] (4) Repeat step (2).

[0873] (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.

[0874] (6) Repeat step 5.2.

[0875] (7) Add 100 μL of HRP solution to each well, seal the well, and incubate at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes.

[0876] (8) Repeat step 5.2.

[0877] (9) Add 100 μL of TMB to each well and incubate at room temperature for 5-20 minutes.

[0878] (10) Add 100 μL of 2N H2SO4 to each well.

[0879] (11) Read the absorbance at 450 nm using a plate reader within 30 minutes.

[0880] 3. ELISA method to test the conjugate drug concentration in animal plasma (Total drug concentration)

[0881] 1) Key reagents

[0882] 2) Key experimental parameters

[0883] 3) Experimental process

[0884] (1) Add 100 μL of coating solution to a 96-well microplate, seal, and incubate overnight at 2-8°C before use.

[0885] (2) Wash the plate 5 times with wash buffer (300 μL / well).

[0886] (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.

[0887] (4) Repeat step (2).

[0888] (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.

[0889] (6) Repeat step 5.2.

[0890] (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.

[0891] (8) Repeat step 5.2.

[0892] (9) Add 100 μL of TMB to each well and incubate at room temperature for 5-20 minutes.

[0893] (10) Add 100 μL of 2N H2SO4 to each well.

[0894] (11) Read the absorbance at 450 nm and 630 nm using a plate reader within 30 minutes.

[0895] 4. Experimental Results

[0896] Mouse pharmacokinetic data demonstrate that conjugate 4 exhibits a long half-life, exceeding 190 hours across all three administration routes (intravenous, subcutaneous, and intraperitoneal). Furthermore, conjugate 4 exhibits excellent in vivo exposure. These data suggest that conjugate 4, with its long half-life and high exposure in clinical practice, may possess excellent antiviral activity.

[0897] Example 27: Pharmacokinetic study of conjugate 4 in rats

[0898] 1. Experimental Information

[0899] Pharmacokinetic studies were conducted in male Sprague-Dawley rats. Blood samples were analyzed using ELISA. Detailed experimental information and design are shown in the table below.

[0900] 2. ELISA method to test the concentration of conjugate drugs in animal plasma

[0901] Referring to the conjugate test method for animal plasma in Example 26, the drug concentrations of ADC and Total in rat plasma were tested.

[0902] 3. Experimental Results

[0903] Pharmacokinetic data in rats show that Conjugate 4 has a long half-life of over 190 hours. Conjugate 4 also exhibits excellent in vivo exposure. These data suggest that Conjugate 4, with its long half-life and high exposure in clinical practice, may possess excellent antiviral activity.

[0904] Example 28: Evaluation of the in vivo antiviral efficacy of the test substance (Conjugate 4) using an influenza virus mouse infection model

[0905] 1. Materials and Methods

[0906] 1) Animals: Female BALB / c mice, 6-7 weeks old, weighing 18-20 g, were purchased from Shanghai Jihui Laboratory Animal Husbandry Co., Ltd. and housed in individually ventilated cages at the Animal Room of the Biological Department of Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd. on November 3, 2023. Laboratory Animal Quality Certificate Number: 20220009014323. Mice were housed and used according to the WuXi AppTec IACUC-QD-approved experimental protocol (Animal Use Protocol Number: ID01-QD031-2022 v1.0). After at least 3 days of acclimatization, qualified animals were used in experiments.

[0907] 2) Solvent and test substance

[0908] 10 mM histidine buffer (pH = 5.5). Taking the preparation of 1 L of solution as an example, add 0.372 g of histidine and 1.593 g of histidine hydrochloride to 1 L of ddH2O, vortex and stir evenly before use.

[0909] Test substance information:

[0910] 3) Viruses

[0911] Influenza A virus, A / California / 07 / 2009 (H1N1), purchased from ATCC, catalog number VR1843. The strain used was amplified by WuXi AppTec's Biological Department, batch number 20201228YNR. Titer: 7.0E+06 PFU / mL.

[0912] 4) Reagents and instruments

[0913] Main reagents

[0914] Main instruments

[0915] 2. Experimental Methods and Procedures

[0916] 1) The day of the first administration was defined as day 0 of the experiment, the day before that was day -1, the day after that was day 1, and so on;

[0917] 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 intranasally. The inoculum dose for each mouse was 500 PFU in a 50 μL inoculation volume.

[0918] 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.

[0919] 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 a single dose 28 days before virus inoculation.

[0920] In vivo experimental design—dosing regimen

[0921] * Reference conjugate: prepared according to the synthesis and characterization of conjugate 9 in Example 3 of patent WO2023 / 125806A1.

[0922] 5) Sample Collection: Blood was collected from three mice in each group for serum collection at 48, 336, 672, 885, or 1008 hours after dosing. Whole blood samples were incubated at 4°C for 8 hours and then centrifuged at 3000g for 10 minutes at 4°C to separate serum. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected from groups 4 and 7 at 1008 hours after dosing. All samples were stored at -80°C.

[0923] 6) Health monitoring: During the experiment, 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.

[0924] 7) Median survival time: the survival time corresponding to the cumulative survival rate of 50%.

[0925] 8) Humane Endpoints: According to IACUC protocols, any mouse that loses more than 35% of its initial body weight during the experiment, or / and exhibits signs of moribundity, will be euthanized and recorded as dead in the results. In actual experimental procedures, these standards will be followed.

[0926] 3. Sample Analysis

[0927] ELISA assay to detect the concentration of the test substance in the sample: 1) Coat a 96-well plate with ADC capture antibody diluted 1:400 with coating solution, 100 μL per well, and coat overnight at 4°C; 2) Discard the liquid in the ELISA plate and wash the plate three 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 three times with PBST (300 μL / well); 5) Add 100 μL of the test sample to each well and incubate at room temperature for 2 hours; 6) Discard the liquid in the ELISA plate and wash the plate three times with PBST (300 μL / well); 7) Add 100 μL of HRP (see Example 24) (1:20,000 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 three times with PBST (300 μL / well); 9) Add 100 μL of Develop the solution with TMB chromogenic substrate at room temperature in the dark for 5-10 minutes. Once the solution turns a distinct blue, add 100 μL of stop solution to each well to terminate the color development. 10) Measure the absorbance at 450 / 620 nm using a microplate reader and fit the standard curve using the four-parameter method. Determine the test substance content in the sample using this method.

[0928] 4. Data Analysis

[0929] The data were statistically analyzed using Prism, and the survival status of mice was analyzed using Log-rank (Mantel-cox) test.

[0930] 5. Results

[0931] The in vivo efficacy of the test compounds in the influenza A virus mouse infection model was evaluated by changes in mouse body weight and survival rate; the pharmacokinetic characteristics of the test compounds were analyzed by detecting the concentrations of the test compounds in serum, lung tissue and lung epithelial cell lining fluid; and the tolerability of the test compounds in mice was assessed by monitoring animal body weight.

[0932] 1) The test substance protects the body weight of mice in the model

[0933] Healthy group: During the experiment, the weight of the healthy control group remained stable and no significant decrease was observed;

[0934] Vehicle group: The body weight of mice began to decrease significantly from day 31 and continued to decrease until death or euthanasia, with an average maximum decrease of 37.77% (euthanasia was performed);

[0935] hIgG1-Fc(LS) (2.5 mg / kg) group: The body weight of mice decreased significantly starting from day 31 of the experiment, with the maximum average decrease of 36.08%. The body weight of surviving mice began to recover from day 39.

[0936] Conjugate 4 (2.5 mg / kg) group: The body weight of mice began to decrease significantly from day 31 of the experiment, with the maximum average decrease of 16.83%. The body weight of surviving mice began to recover to normal levels from day 36.

[0937] Conjugate 4 (0.5 mg / kg) group: The body weight of mice began to decrease significantly from day 31 of the experiment, with an average maximum decrease of 34.58%. The body weight of surviving mice began to recover from day 38.

[0938] Conjugate 4 (0.1 mg / kg) group: The body weight of mice began to decrease significantly from day 31 and continued to decrease until death or euthanasia, with an average maximum decrease of 33.00%;

[0939] Reference conjugate (0.5 mg / kg) group: The body weight of mice began to decrease significantly from day 31 and continued to decrease until death or euthanasia, with an average maximum decrease of 37.03% (euthanasia was performed);

[0940] 2) The test substance protects the survival of mice in the model

[0941] After virus inoculation, the survival status of mice in each group is summarized as follows:

[0942] Healthy group: No mice died, the final animal survival rate was 100%, and the median survival time was >42 days;

[0943] Vehicle group: Mice began to die on day 35 and all died on day 37, with a final survival rate of 0% and a median survival time of 36 days;

[0944] hIgG-Fc(LS) (2.5 mg / kg) group: mice began to die from day 36, with a final survival rate of 10% and a median survival time of 36.5 days;

[0945] Conjugate 4 (2.5 mg / kg) group: mice began to die on day 38, with a final survival rate of 90% and a median survival time of >42 days;

[0946] Conjugate 4 (0.5 mg / kg) group: mice began to die from day 36, with a final survival rate of 50% and a median survival time of 39.5 days;

[0947] Conjugate 4 (0.1 mg / kg) group: Mice began to die on day 34 and all died on day 37, with a final survival rate of 0% and a median survival time of 36 days.

[0948] Reference conjugate (0.5 mg / kg) group: Mice began to die on day 36 and all died on day 38, with a final survival rate of 0% and a median survival time of 37 days.

[0949] 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.

[0950] From the analysis of the results, it can be seen that conjugate 4 obtained by the thiol-directed conjugation technology described in this patent showed excellent antiviral activity in a mouse model. Under high-dose conditions (2.5 mg / kg), 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 conjugate mice reached 50%, while the survival rate of mice containing the reference conjugate molecule reached 0%. It can be seen that conjugate 4 shows better antiviral activity, indicating better clinical effects in the prevention and / or treatment of clinical influenza.

[0951] The present invention has been illustrated through various specific embodiments. However, those skilled in the art will appreciate that the present invention is not limited to these specific embodiments. Those skilled in the art may make various modifications and variations within the scope of the present invention, and the various technical features described throughout this specification may be combined without departing from the spirit and scope of the present invention. Such modifications and variations are 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 an 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 , CH2- or absent; 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 A 5-15 membered heteroaryl group; 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 label 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 the disease causing immunosuppression has cancer or acquired immunodeficiency syndrome. disease; 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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