Conjugates and their use for the prevention and treatment of viral infections

Abstract

The present invention relates to conjugates and their use for the prevention and treatment of viral infections, and more specifically, to conjugates in which an anti-influenza compound represented by formula IA or IB, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, is conjugated to a protein. Each symbol is as defined herein. The conjugates or intermediate compounds of the present invention exhibit remarkable anti-influenza virus activity while possessing excellent in vitro / in vivo pharmacokinetic properties and safety, indicating high potential for clinical application. JPEG2026522131000283.jpg74143

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a conjugate of an antiviral agent against the influenza (Flu) virus and an antibody or its constant region Fc. Specifically, it relates to compounds in which a small molecule inhibitor or inhibitory peptide agent against the viral surface protein is covalently linked to an antibody or its constant region Fc, as well as pharmaceutical compositions or agents containing these compounds, and their use in the prevention and / or treatment of related viral infections. [Background technology]

[0002] Background of the Invention Influenza viruses cause approximately 3 to 5 million serious infections and around 500,000 deaths worldwide each year (Luliano et al., 2018, Lancet 391: 1285-1300). While most healthy individuals recover spontaneously within 1 to 2 weeks of infection, influenza virus infection can progress to life-threatening infections and complications such as pneumonia in the elderly, patients with chronic diseases, and immunocompromised individuals.

[0003] Developing treatments for influenza viruses remains a persistent challenge for humanity. While antiviral drugs and preventive vaccines are currently available, low-molecular-weight anti-influenza drugs typically require administration within 48 hours of symptom onset to achieve clinical efficacy. Furthermore, the high mutability of influenza viruses has led to the identification of drug-resistant strains. Therefore, there is a need for the development of more effective and sustainable treatments for the treatment and / or prevention of influenza virus infection.

[0004] Influenza viruses are a type of negative-chain segmented RNA virus belonging to the family Progressive RNA Viridae, and include influenza viruses A, B, and C. Of these, infection in humans is mainly caused by viruses A and B. Influenza viruses infect respiratory epithelial cells. In the first stage of this process, the virus is adsorbed to the host cell via a viral surface receptor-binding protein (hemagglutinin (HA protein) in the case of influenza viruses). Subsequently, the viral envelope fuses with the cell membrane under the action of hemagglutinin, and the influenza virus genome fragment and viral RNA-dependent RNA polymerase complex are released into the cell. The viral RNA-dependent RNA polymerase complex synthesizes new progeny virus particles within the cell using the genome fragment as a template. The newly synthesized virus particles are released extracellularly via cell lysis or budding. In influenza viruses, the complete release of progeny viruses depends on neuraminidase (NA), which cleaves sialic acid residues on the cell surface. Neuraminidase inhibitors, which target the neuraminidase of the influenza virus to suppress viral spread, include oseltamivir (Tamiflu®), zanamivir (Relenza®), and peramivir (Rapivab®), and are approved for clinical use.

[0005] Organ transplant recipients and cancer patients have suppressed immune systems and are therefore unable to effectively eliminate viruses. Influenza virus infections tend to allow the virus to replicate in the body for longer periods, increasing the likelihood of drug-resistant strains developing. These findings have already been observed clinically. Meanwhile, drug resistance in influenza viruses in the general population also presents a significant challenge in drug use. Therefore, more effective new methods and treatments for influenza are needed. [Overview of the project]

[0006] In one aspect, the present invention provides a conjugate, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound, in which an anti-influenza low molecular weight compound is conjugated to a protein (also called a conjugate of a protein and an anti-influenza compound), exhibiting significant anti-influenza virus activity. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1: MS detection spectrum of conjugate 1. [Figure 2] Figure 2: SEC detection chromatogram of conjugate 1. [Figure 3] Figure 3: Chromatogram of residual low molecule detection in conjugate 1. [Figure 4] Figure 4: MS detection spectrum of conjugate 2. [Figure 5] Figure 5: SEC detection chromatogram of conjugate 2. [Figure 6] Figure 6: Chromatogram of residual low molecule detection in conjugate 2. [Figure 7] Figure 7: MS detection results of product conjugate 1 in the screening experiment. [Figure 8] Figure 8: MS detection spectrum of conjugate 3. [Figure 9] Figure 9: SEC detection chromatogram of conjugate 3. [Figure 10] Figure 10: Chromatogram of detection of residual low molecular weight compounds in conjugate 3. [Figure 11] Figure 11: MS detection spectrum of conjugate 4. [Figure 12] Figure 12: SEC detection chromatogram of conjugate 4. [Figure 13] Figure 13: Detection chromatogram of residual low molecular weight compounds in conjugate 4. [Modes for carrying out the invention]

[0008] In some embodiments, the present invention provides a conjugate represented by the structural formula of Formula I-A or Formula I-B, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof:

[0009]

Chemical formula

[0010]

Chemical formula

[0011] Wherein, m is 1 or 2; n is an integer from 1 to 20, preferably an integer of 1, 2, 3, 4, 5, 6, 7, or 8; w is an integer from 0 to 8; for example, 0, 1, 2, 3, 4, or 5; L is a linker; E is selected from an antibody or antibody fragment, Fc domain monomer, Fc domain, Fc-binding peptide, albumin, or albumin-binding peptide; R 1a 、R 2a 、R 3a 、R 5a 、R 6a 、R 7a 、R 9a and R 10a are each independently selected from H, deuterium, hydroxyl, -O-C 1-6 alkyl, hydroxyl C 1-6 alkyl group, C 1-6 alkyl group, C 3-8 cycloalkyl group, C 3-8 heterocycloalkyl group, C6-C 15 aryl, 5- to 15-membered heteroaryl, halogen, cyano, and amino; R 1a and R 2a may together form C 3-8 cycloalkyl or C 3-8 heterocycloalkyl; [[ID=5a and R 6a They are together C 3-8 Cycloalkyl or C 3-8 It can form heterocycloalkyl groups; or R 9a and R 10a They are together C 3-8 Cycloalkyl or C 3-8 A heterocycloalkyl can be formed. Here, the C 3-8 Cycloalkyl or C 3-8 Heterocycloalkyl groups can be optionally substituted with substituents selected from halogens and hydroxyls; R 4a and R 8a These are H and C, which are independent of each other. 1-6 Alkyl, C 3-8 Cycloalkyl or C 3-8 Selected from heterocycloalkyl groups; x is either 1 or 2; y is an integer between 1 and 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’ These are, independently, hydrogen, deuterium, hydroxyl, and -OC. 1-6 Alkyl, Hydroxyl C 1-6 Alkyl, C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl, C6-C 15 Selected from aryl, 5-15 member heteroaryl, halogen, cyano, and amino; R 1’ and R 2’ They are together C 3-8 Cycloalkyl or C 3-8 A heterocycloalkyl group may be formed. Alternatively, two adjacent R groups may be formed. 3’ C 3-8 Cycloalkyl, C3-8 Heterocycloalkyl, C6-C 15 They can form aryls or 5-15 member heteroaryls.

[0012] In formulas IA and IB, parentheses with subscripts n or y are (E) m It should be understood that this does not include the structural components, but includes the sulfur atom and the structure beneath it.

[0013] In some embodiments, L is a linker represented by the structure of formula I-3a, and L 1 and L 2a Includes L 1 The left and right ends are connected to the drug portion (D), L 2a The upper limit is -NR in equations IA and IB. 8a It connects to the network.

[0014] [ka]

[0015] Here, L 1 The following structures are selected:

[0016] [ka]

[0017] During the ceremony, W is O, S, NR b Selected from , CH2-, or lack; Each R a and R b These are, independently, H, and the optionally substituted C1-C 20 Alkyl and optionally substituted C2-C 20 Selected from alkenyls, preferably selected from H and methyl, and more preferably methyl; y1 and y2 are independently 0, 1, 2, 3, 4, 5, or 6;

[0018] L 1 The bonds at both ends of the structure are drug (D1 The bond connected to the oxygen atom of ) and indicated by the wavy line drawn on the intermediate amine group is L 2a It should be understood that it connects to [something].

[0019] Preferably, L 1 The following structures are selected:

[0020] [Table 1-1]

[0021] [Table 1-2]

[0022] In some embodiments, L 2a This can be represented by the structure of equations L2-1a to L2-7a:

[0023] [Table 2-1]

[0024] [Table 2-2]

[0025] During the ceremony, Z is selected from NR, S, and O; Each R group can independently be hydrogen, deuterium, or an optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted 3-20 member heterocycloalkyl, optionally substituted C6-C 15 Selected from aryls and optionally substituted 5- to 15-membered heteroaryls; s and t are integers from 1 to 20; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; y is 0 or 1.

[0026] In equations L2-1a to L2-7a, L 2a (L) represents the part between the two wavy lines; 1 ) The substructure is L 2a It is not a component of L 2a L 1 To more clearly indicate the direction of bonding to L, 1 L that binds 2a This indicates the end of L. 2a The rightmost part is -NR 8a It is connected to.

[0027] In some embodiments, L 2a This is selected from equations L2-1a, L2-5a, and L2-7a. Z is O, R is H, s and t are selected from integers between 3 and 10 (e.g., 4, 5, 6, 7, or 8); preferably, s and t are 4 or 8.

[0028] In some embodiments, the sulfur atom on E originates from a cysteine ​​(cys) residue or a disulfide bond in E.

[0029] 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-binding peptide. For example, the Fc domain monomer contains or consists of any of the amino acid sequences described in SEQ ID NOs: 1 to 81, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any of these sequences.

[0030] Preferably, it comprises or consists of an amino acid sequence shown in 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.

[0031] In some embodiments, the conjugate has a structure of Formula I-A.

[0032] In some embodiments, the conjugate has a structure of Formula I-B.

[0033] In some embodiments, the structure between L and E is selected from the following structures:

[0034]

Chemical formula

[0035] wherein R 1a , R 2a , R 3a , R 5a , R 6a , R 7a , R 9a and R 10a are each independently selected from hydrogen, deuterium, -O-C 1-6 alkyl, hydroxyl, C 1-6 alkyl, C 1-6 alkyl, and C 3-8 cycloalkyl. Alternatively, R 1a and R 2a may together form a C 3-8 cycloalkyl group; Alternatively, R 5a and R 6a may together form a C 3-8 cycloalkyl; Alternatively, R 9a and R 10a They are together C 3-8 A cycloalkyl group may be formed, and the C 3-8 The cycloalkyl group may optionally be substituted with substituents selected from halogens and hydroxyls; R 4a and R 8a These are H and C, which are independent of each other. 1-6 Alkyl and C 3-8 Cycloalkyl から Selected; w is 0, 1, or 2; Preferably, R 1a , R 3a , R 4a , R 5a , R 7a , R 9a , and R 10a Each of them is independently a hydrogen atom; R 2a , R 6a and R 8a Each of them is independently of hydrogen and C 1-6 Alkyl and C 3-8 Selected from cycloalkyl groups; w is 0; More preferably, the structure of formula I-4a is selected from the following:

[0036] [ka]

[0037] S atoms indicated by a dashed line become E, and N atoms indicated by a dashed line become L or L 2a It should be understood that they are connected.

[0038] In some embodiments, the conjugate is selected from compounds represented by formulas C-1a to C-27a:

[0039] [Table 3-1]

[0040] [Table 3-2]

[0041] [Table 3-3]

[0042] [Table 3-4]

[0043] [Table 3-5]

[0044] [Table 3-6]

[0045] [Table 3-7]

[0046] [Table 3-8]

[0047] In the formula, m, n, x, y, and E are as defined herein.

[0048] In some embodiments, the ratio of n to m is in the range of 1 to 20, preferably in the range of 2 to 10, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0049] In some embodiments, the conjugate or pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound has an average DAR value between 0.5 and 10.0, for example, 2.0–4.5 or 4.8–8.5.

[0050] In some embodiments, E comprises an Fc domain monomer or an Fc domain containing the Fc domain monomer, wherein the Fc domain monomer is composed of any of the amino acid sequences described in SEQ ID NOs: 1 to 81, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto, or the aforementioned amino acid sequence.

[0051] In some embodiments, E can recognize viral surface antigens such as CR6261, CR8020, MEDI8897, palivizumab, SD38; or

[0052] Fc domain monomers can be used with any antibody subtype of immunoglobulin (e.g., IGGH1*01 (e.g., G1m(za)), IGGH1*07 (e.g., G1m(zax)), IGGH1*04 (e.g., G1m(zav)), IGGH1*03 (G1m(f)), IGGH1*08 (e.g., G1m(fa)), IGGH2*01, IGGH2*02, IGGH2*06, IGGH3*01, IGGH3*04, IGGH3 *05, IGGH3*09, IGGH3*10, IGGH3*11, IGGH3*12, IGGH3*06, IGGH3*07, IGGH3*08, IGGH3*13, IGGH3*03, IGGH3*14, IGGH3*15, IGGH3*16, IGGH3*17, IGGH3*18, IGGH3*19, IGGH2*04, IGGH4*01, IGGH4*02, IGGH4*03) may be Fc domain monomers.

[0053] In some embodiments, the conjugate has the following structure:

[0054] [Table 4]

[0055] In the formula, E is as defined herein; Preferably, E is sequence number 67, sequence number 69, or sequence number 71; m and n are as defined herein, where m is preferably 1.

[0056] In some embodiments,

[0057] [ka]

[0058] The carbon to which the group (or the corresponding group) is bonded (for example,

[0059] [ka]

[0060] In certain compounds containing a specific structure, it is,

[0061] [ka]

[0062] The carbon atom bonded to the nitrogen atom on the right side of the molecule is in an R or S configuration, preferably an R configuration.

[0063] In some embodiments, the present invention provides a conjugate represented by the structure of formula I-1, which is a small anti-influenza compound conjugated to a protein (also called a "protein-anti-influenza compound conjugate") that exhibits significant anti-influenza virus activity.

[0064] [ka]

[0065] Specifically, the protein and anti-influenza compound conjugate of the present invention has a low molecular weight compound (D1) having anti-influenza virus activity, and the low molecular weight compound is a linker (L 1 and L 2The protein is linked to an antibody or an antibody fragment, an Fc monomer, an Fc domain, an FC-binding peptide, albumin, or an albumin-binding peptide (E) via a pharmacokinetic linkage. The protein-anti-influenza compound complex of the present invention exhibits remarkable anti-influenza virus activity, as well as excellent in vitro / in vivo pharmacokinetic properties and safety, a long half-life, and good clinical applicability.

[0066] In some embodiments, the present invention provides a conjugate of formula I-1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

[0067] [ka]

[0068] During the ceremony, m is either 1 or 2; n is an integer from 1 to 20, preferably an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and both values ​​of n are the same; D 1 L 1 L is connected by a covalent bond. 1 L is formed via a covalent bond. 2 It is connected to L 2 E is linked to an S-bond via a covalent bond; E is selected from the antibody or antibody fragment defined above, an Fc domain monomer, an Fc domain, an Fc-binding peptide, albumin, or an albumin-binding peptide; D 1 These are small molecule anti-influenza virus agents, each independently selected from compounds having anti-influenza virus activity represented by formulas D-1-1 to D-1-8:

[0069] [ka]

[0070] R1 is selected from OH, NH2, -NH(=NH)NH2, and -NHC(=NH)NHR6; R2 and R3 are each independently selected from H, OH, F, Cl, and Br; R4 、 Selected from -COOH, -P(=O)(OH)2, and -SO3H; R5 is -COC 1-6 Alkyl, -COC 1-6 Haloalkyl, -SO2C 1-6 Alkyl and -SO2C 1-6 Selected from haloalkyl groups, preferably from -COCH3, -COCF3, and -SO2CH3; X is selected from either O or S; R6 is selected from the following bases:

[0071] [ka]

[0072] Y is selected from the following group, where "(L 1 (to be connected to) means that base Y is L 1 This indicates that it is linked to the end of the ring, and if N is depicted within the ring, it indicates that N is an atom within the ring:

[0073] [ka]

[0074] In the formula, ring A is C3-C 20 Cycloalkyl, substituted C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl, substituted C3-C 20 Cycloalkenyl, C6-C 15 Aryl, 3-20 member heterocycloalkyl, substituted 3-20 member heterocycloalkyl, substituted C6-C 15 Selected from aryls and substituted 5- to 15-membered heteroaryls; Each R group can independently be H, deuterium, or optionally substituted C1-C20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted 3-20 member heterocycloalkyl, optionally substituted C6-C 15 Selected from aryls and optionally substituted 5- to 15-membered heteroaryls; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Preferably, D 1 These are the following small molecule anti-influenza virus drugs:

[0075] [ka]

[0076] L 1 is a drug (D 1 ) and L 2 A linker that connects and includes, but is not limited to, the following structures.

[0077] In the following structural formula, the bonds at both ends are drug (D 1 The bond is linked to the oxygen atom of ), and the bond shown by the wavy line drawn on the intermediate amine group is L 2 It should be understood that it is linked to;

[0078] [ka]

[0079] During the ceremony, W is O, S, NR b Selected from , CH2-, or lack; Each R a and R b These are, independently, H, and the optionally substituted C1-C 20 Alkyl and optionally substituted C2-C 20 Selected from alkenyls, preferably selected from H and methyl, and more preferably methyl; y1 and y2 are independently 0, 1, 2, 3, 4, 5, or 6;

[0080] L 2 L 1 This is a linker that connects to E, and has the structure of equations L2-1 to L2-14:

[0081] [Table 5-1]

[0082] [Table 5-2]

[0083] [Table 5-3]

[0084] [Table 5-4]

[0085] [Table 5-5]

[0086] During the ceremony, Z is selected from NR, S, and O; Each R group can independently be hydrogen, deuterium, or an optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted 3-20 member heterocycloalkyl, optionally substituted C6-C 15 Selected from aryls and optionally substituted 5- to 15-membered heteroaryls; s and t are integers from 1 to 20; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; y is either 0 or 1.

[0087] In Formulas L2-1 to L2-14, L2 refers to the portion between two wavy lines, and the substructure (L 1 ) and (E)-S should be understood not to be components of L 2 . These are, respectively, for the purpose of more clearly indicating the linking direction of L 2 , and show both ends of L 1 and L 2 linked to L 2 and E. L 2 is linked to the sulfur atom of E via a covalent bond.

[0088] It should be understood that "-S-" in Formula I-1 is formed by the reaction of a sulfhydryl group generated by the reduction of a disulfide bond in E with a linker compound.

[0089] In some embodiments, L 1 is selected from the following structures:

[0090] [Table 6-1]

[0091] [Table 6-2]

[0092] More preferably, L 1 is as follows.

[0093] [Chemical formula]

[0094] In some embodiments, L 2 is selected from Formulas L2-1, L2-5, L2-7, L2-8, L2-12, and L2-14, where Z is O, R is H, s and t are selected from integers from 3 to 10, preferably s and t are 4 or 8.

[0095] In some embodiments, the conjugate of Formula I-1 is represented by the structure of Formula I-1-1:

[0096]

Chemical Formula

[0097] Wherein, E, L 1 , L 2 , m, and n are as defined above.

[0098] In some embodiments, the conjugate is preferably selected from conjugates C-1 to C-12:

[0099]

Table 7-1

[0100]

Table 7-2

[0101]

Table 7-3

[0102] Wherein, n and E are defined as above.

[0103] In the above structures C-1 to C-12, (E) indicates that there is one E in the structure, which corresponds to the situation where m is 1 in the conjugate of Formula I-1. The same understanding can be obtained for conjugates having similar structures in this specification.

[0104] In some embodiments, in C-1 to C-6, the carbon to which the left NH2-CH2- is attached (i.e.,

[0105] [Chemical]

[0106] The carbon atom bonded to the nitrogen on the right side of (is in the R or S configuration, preferably the R configuration.

[0107] In some embodiments, in the structure of Formula I-1, the ratio of n to m ranges from 1 to 20, preferably from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0108] In some embodiments, m is 1 and n is selected from integers from 1 to 10.

[0109] In some embodiments, the conjugate or pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound has an average DAR ranging from 0.5 to 10.0.

[0110] In some embodiments, E is an antibody or antibody fragment, Fc domain monomer, Fc domain, and / or Fc-binding peptide. In some embodiments, E comprises, or consists of, a sequence having at least 95% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-81.

[0111] In one embodiment, in a conjugate comprising a compound having the structure of Formula I-1, the E monomer domain is dimerized to form an Fc domain.

[0112] In some embodiments, the antibody fragment is an antigen-binding fragment.

[0113] In some embodiments, the antibody or antibody fragment is an antibody or antibody fragment derived from human, mouse, camel, goat, sheep, rabbit, chicken, guinea pig, hamster, horse, or rat.

[0114] In some embodiments, the antibody or antibody fragment is an IgG, IgA, IgD, IgE, or IgM type antibody or antibody fragment.

[0115] In some embodiments, the antibody fragment comprises scFv, sdAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, or SMIP.

[0116] In some embodiments, the antibody or antibody fragment can recognize viral surface antigens such as CR6261, CR8020, MEDI8897, palivizumab, and SD38.

[0117] In some embodiments, the Fc domain monomer is any subtype of immunoglobulin (e.g., IGGH1*01 (e.g., G1m(za)), IGGH1*07 (e.g., G1m(zax)), IGGH1*04 (e.g., G1m(zav)), IGGH1*03 (G1m(f)), IGGH1*08 (e.g., G1m(fa)), IGGH2*01, IGGH2*02, IGGH2*06, IGGH3*01, IGGH3*0 These may be Fc domain monomers derived from 4, IGG3*05, IGG3*09, IGG3*10, IGG3*11, IGG3*12, IGG3*06, IGG3*07, IGG3*08, IGG3*13, IGG3*03, IGG3*14, IGG3*15, IGG3*16, IGG3*17, IGG3*18, IGG3*19, IGG2*04, IGG4*01, IGG4*02, IGG4*03 (e.g., Vidarsson et al., IgG subclasses and allotypes: from structure to effector function. Frontiers in Immunology. 5(520): 1-17 (2014)). The Fc domain monomer may contain one or more non-natural amino acid sequences that function as site-specific binding sites for small molecules. The Fc domain monomer may contain one or more solvent-exposed cysteine ​​or lysine residues that are site-specifically designed, thereby providing additional sites for small molecule binding.

[0118] In some embodiments, L2 is linked to an S atom on E, and the S atom on E originates from a cysteine ​​(Cys) residue or disulfide bond within E.

[0119] In some embodiments, the asparagine (Asn) amino acid residue in E is replaced with an alanine (Ala) amino acid residue to prevent binding at this site.

[0120] In some embodiments, E includes additional Cys amino acid residues to increase the number of binding sites without affecting the spatial three-dimensional structure of the antibody protein.

[0121] In some embodiments, the E terminus includes an additional amino acid sequence such as a protein purification tag (e.g., a 6-histidine tag), a signal peptide sequence (e.g., a signal peptide sequence of human interleukin-2), an MVRS (SEQ ID NO: 82) amino acid sequence, or an ISAMVRS amino acid sequence (SEQ ID NO: 83).

[0122] 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.

[0123] In some embodiments, the protein purification tag is selected from a 6-histidine 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)), or a mouse heavy chain IgG Vh signal sequence (e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 86)).

[0124] In some embodiments, the N-end of E includes a hinge region or a portion of a hinge region.

[0125] In some embodiments, E comprises or is one of the amino acid sequences of SEQ ID NOs: 1 to 81.

[0126] In some embodiments, E includes or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 81.

[0127] In some embodiments, E comprises or consists of the following amino acid sequence: (i) Having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one amino acid sequence of sequence numbers 1 to 81; (ii) Includes N-terminal append sequences, mutants, and / or C-terminal append sequences described for each sequence number as described in the “Sequences of Sequence Numbers 1-81 and Their Descriptions” section of this Specified Specification.

[0128] In some embodiments, E further comprises a connector. In one embodiment, the connector refers to a short amino acid sequence consisting of amino acids, using glycine (G) and / or serine (S) and / or threonine (T) residues individually or in combination. In one embodiment, the connector comprises an amino acid sequence (G4S)n, where n is an integer of 1 or more. For example, n is an integer of 1, 2, 3, 4, 5, 6, or 7. In one embodiment, the connector is GGGGS. In one embodiment, the connector comprises an amino acid sequence TS(G4S)n, where n is an integer of 1 or more. For example, n is an integer of 2, 3, 4, 5, 6, or 7. In another embodiment, the connector comprises an amino acid sequence G(G4S)n, where n is an integer of 1 or more, for example, n is an integer of 2, 3, 4, 5, 6, or 7.

[0129] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 1. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 1, and optionally having an IL2 signal sequence at its N-terminus.

[0130] In some embodiments, E includes the amino acid sequence described in SEQ ID NO: 2. In some embodiments, E includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 2.

[0131] In some embodiments, E includes the amino acid sequence described in SEQ ID NO: 3. In some embodiments, E includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 3, and optionally having an IL2 signal sequence and an N-terminal MVRS amino acid sequence at its N-terminus.

[0132] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 4. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 4, and optionally having an MVRS amino acid sequence at its N-terminus.

[0133] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 5. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 5, and optionally having an IL2 signal sequence at its N-terminus and a 6-histidine tag at its N-terminus.

[0134] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 6. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 6, and optionally having a 6-histidine tag at its C-terminus.

[0135] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 7. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 7, and optionally having an IL2 signal sequence and an MVRS amino acid sequence at its N-terminus and a 6-histidine tag at its C-terminus.

[0136] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 8. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 8, and optionally having an MVRS amino acid sequence at its N-terminus and a 6-histidine tag at its C-terminus.

[0137] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 9. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 9, and optionally having an IL2 signal sequence and an MVRS amino acid sequence at its N-terminus, two additional cysteines in the hinge region (at the position corresponding to the * position in SEQ ID NO: 9), and a 6-histidine tag at its C-terminus.

[0138] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 10. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 10, and optionally having an MVRS amino acid sequence at its N-terminus, a hinge region containing two additional cysteines (at positions corresponding to the * positions in SEQ ID NO: 10), and a 6-histidine tag at its C-terminus.

[0139] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 11. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 11, and optionally having a hinge region at its N-terminus containing an MVRS amino acid sequence and two additional cysteines (at positions corresponding to the * positions in SEQ ID NO: 11).

[0140] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 12. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 12, and optionally having an IL2 signal sequence at the N terminus, an asparagine-to-alanine substitution (at the position corresponding to the * position in SEQ ID NO: 12), and a 6-histidine tag at the C terminus.

[0141] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 13. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 13, and optionally comprises an asparagine (Asn) to alanine (Ala) substitution (at the position corresponding to the * position in SEQ ID NO: 13), and has a 6-histidine tag at the C-terminus.

[0142] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 14. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 14, and optionally having an IL2 signal sequence and an MVRS amino acid sequence at its N-terminus, including an asparagine to alanine substitution (at the position corresponding to the * position in SEQ ID NO: 14), and having a 6-histidine tag at its C-terminus.

[0143] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 15. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 15, and optionally having an MVRS amino acid sequence at its N-terminus, including an asparagine to alanine substitution (at the position corresponding to the * position in SEQ ID NO: 15), and having a 6-histidine tag at its C-terminus.

[0144] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 16. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 16, and optionally having a human serum albumin signal sequence and an N-terminal ISAMVRS amino acid sequence at its N-terminus.

[0145] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 17. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 17, and optionally having a human serum albumin signal sequence and an N-terminal ISAMVRS amino acid sequence at its N-terminus, and comprising a C-terminal G4S connector and a C-terminal c-Myc tag.

[0146] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 18. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 18, and optionally having an ISAMVRS amino acid sequence at its N-terminus, a G4S connector at its C-terminus, and a c-Myc tag at its C-terminus.

[0147] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 19. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 19, and optionally having a human serum albumin signaling sequence and an ISAMVRS amino acid sequence at the N-terminus, and including a lysine-to-serine substitution (corresponding to the * position in SEQ ID NO: 19) to prevent binding at this site.

[0148] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 20. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 20, and optionally having an ISAMVRS amino acid sequence at the N-terminus, and including a lysine-to-serine substitution (corresponding to the * position in SEQ ID NO: 20) to prevent binding at this site.

[0149] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 21. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 21, and optionally having a human serum albumin signaling sequence and an ISAMVRS amino acid sequence at the N-terminus, including a lysine-to-serine substitution (corresponding to the * position in SEQ ID NO: 21) to prevent binding at this site, and having a G4S connector and a c-Myc tag at the C-terminus.

[0150] In some embodiments, E comprises the amino acid sequence described in 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 with the amino acid sequence described in SEQ ID NO: 22, and optionally having an ISAMVRS amino acid sequence at its N-terminus, comprising a lysine-to-serine substitution (at the position corresponding to the * position in SEQ ID NO: 22) to prevent binding at this site, and having a G4S connector and a C-Myc tag at its C-terminus.

[0151] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 23. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 23, and optionally having a human serum albumin signaling sequence and an ISAMVRS amino acid sequence at the N-terminus, including an asparagine to alanine substitution (at the position corresponding to the * position in SEQ ID NO: 23), and having a G4S connector and a c-Myc tag at the C-terminus.

[0152] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 24. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 24, and optionally having an ISAMVRS amino acid sequence at the N-terminus, including an asparagine (Asn) to alanine (Ala) substitution (at the position corresponding to the * position in SEQ ID NO: 24), and having a G4S connector and a c-Myc tag at the C-terminus.

[0153] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 25. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 25, and optionally having a human serum albumin signaling sequence and an ISAMVRS amino acid sequence at the N-terminus, including H310A and H435A substitutions to prevent FcRn binding, and having a G4S connector and a c-Myc tag at the C-terminus.

[0154] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 26. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 26, and optionally having an ISAMVRS amino acid sequence at its N-terminus, including H310A and H435A substitutions to prevent FcRn binding, and having a G4S connector and a c-Myc tag at its C-terminus.

[0155] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 27. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 27, and optionally having a human serum albumin signaling sequence and an ISAMVRS amino acid sequence at its N-terminus, as well as a G4S connector and mutant (lysine to phenylalanine substitution, at the position corresponding to the bolded position in SEQ ID NO: 27), and a c-Myc tag at its C-terminus.

[0156] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 28. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 28, and optionally having an ISAMVRS amino acid sequence at the N-terminus, and further having a G4S connector and a variant (lysine to phenylalanine substitution at the position corresponding to the bolded position in SEQ ID NO: 28), and having a c-Myc tag at the C-terminus.

[0157] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 29. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 29, and optionally having a human serum albumin signaling sequence and an ISAMVRS amino acid sequence at the N-terminus, comprising an alanine (Ala) substitution of asparagine (Asn), having a G4S connector and a variant (a lysine to phenylalanine substitution at the position corresponding to the bolded position in SEQ ID NO: 29), and having a c-Myc tag at the C-terminus.

[0158] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 30. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 30, and optionally having an ISAMVRS amino acid sequence at the N-terminus, with asparagine (Asn) substituted for alanine (Ala) (at the position corresponding to the * position in SEQ ID NO: 30), a G4S connector and mutant (lysine to phenylalanine substitution, at the position corresponding to the bolded position in SEQ ID NO: 30), and a c-Myc tag at the C-terminus.

[0159] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 31. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 31, and optionally having a human serum albumin signaling sequence, allotype G1m(fa), and a G4S connector and mutant (lysine to phenylalanine substitution at the position corresponding to the bolded position in SEQ ID NO: 31) at the N-terminus, and a c-Myc tag at the C-terminus.

[0160] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 32. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 32, and optionally having a human serum albumin signaling sequence and allotype G1m(fa) at its N-terminus.

[0161] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 33. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 33, and optionally having an MVRS amino acid sequence at its N-terminus and containing a YTE triple mutation.

[0162] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 34. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 34, and optionally comprises an EPKSS amino acid sequence having a human serum albumin signal sequence at its N-terminus and being the hinge region of mature human Fc-IgG1, and further comprises a cysteine-to-serine substitution (corresponding to the # position in SEQ ID NO: 34) and allotype G1m(fa).

[0163] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 35. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 35, and optionally having a mouse IgG signal sequence at its N-terminus, with the hinge region EPKSSD amino acid sequence of mature human Fc-IgG removed, and having allotype G1m(fa).

[0164] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 36. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 36, and optionally having the EPKSDSD amino acid sequence of the hinge region of mature human Fc-IgG removed at its N-terminus, having allotype G1m(fa), and containing a YTE triple mutation.

[0165] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 37. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 37, and optionally having the EPKSDSD amino acid sequence of the hinge region of mature human Fc-IgG removed at the N-terminus, and possessing an LS double mutation and allotype G1m(fa).

[0166] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 38. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 38, and optionally having a human serum albumin signaling sequence at its N-terminus, containing a YTE triple mutation, having allotype G1m(fa), and having a G4S connector and a c-Myc tag at its C-terminus.

[0167] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 39. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 39, and optionally comprises an amino acid defined for the position corresponding to the position shown in SEQ ID NO: 39 at the position corresponding to the X position shown in SEQ ID NO: 39.

[0168] In some embodiments, E includes the amino acid sequence described in SEQ ID NO: 40. In some embodiments, E includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 40, and optionally includes an amino acid defined for the position corresponding to the position shown in SEQ ID NO: 40 at the position corresponding to the X position shown in SEQ ID NO: 40.

[0169] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 41. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 41, and optionally having a YTE triple mutation and an amino acid defined for the position corresponding to the position shown in SEQ ID NO: 41 at the position corresponding to the X position shown in SEQ ID NO: 41.

[0170] In some embodiments, E includes the amino acid sequence described in SEQ ID NO: 42. In some embodiments, E includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 42, and optionally having a YTE triple mutation and allotype G1m(fa).

[0171] In some embodiments, E includes the amino acid sequence described in SEQ ID NO: 43. In some embodiments, E includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 43, and optionally having a YTE triple mutation and allotype G1m(f).

[0172] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 44. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 44, and optionally having an LS double mutation and having an amino acid defined by SEQ ID NO: 44 at a position corresponding to the X position shown in SEQ ID NO: 44.

[0173] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 45. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 45, and optionally having an LS double mutation and allotype G1m(fa).

[0174] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 46. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 46, and optionally having an LS double mutation and allotype G1m(f).

[0175] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 47. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 47, and optionally having a mouse heavy chain IgG Vh signal sequence at its N-terminus, a cysteine-to-serine change (at the position corresponding to the # position in SEQ ID NO: 47), and an amino acid defined by SEQ ID NO: 47 at the position corresponding to the X position in SEQ ID NO: 47.

[0176] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 48. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 48, and optionally having a mouse heavy chain IgG Vh signal sequence at its N-terminus, a cysteine-to-serine substitution (at a position corresponding to the # position in SEQ ID NO: 48), and allotype G1m(fa).

[0177] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 49. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 49, and optionally having a mouse heavy chain IgG Vh signal sequence at its N-terminus, comprising a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 49), and comprising allotype G1m(f).

[0178] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 50. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 50, and optionally having a mouse heavy chain IgG Vh signal sequence at its N-terminus, comprising a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 50), having M428L and N434S mutations, and having allotype G1m(fa).

[0179] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 51. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 51, and optionally having a mouse heavy chain IgG Vh signal sequence at the N-terminus, a cysteine-to-serine substitution (at the # position corresponding to the # position in 51), M428L and N434S mutations, and being allotype G1m(f).

[0180] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 52. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 52, and optionally having a mouse heavy chain IgG Vh signal sequence at its N-terminus, a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 52), a YTE triple mutation, and allotype G1m(fa).

[0181] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 53. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 53, and optionally having a mouse heavy chain MIgGVh signal sequence at its N-terminus, a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 53), a YTE triple mutation, and allotype G1m(f).

[0182] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 54. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 54, and optionally having a mouse heavy chain IgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, having 428L and N434S mutations, having a G4S connector and a c-Myc tag at its C-terminus, and comprising allotype G1m(f).

[0183] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 55. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 55, and optionally having a mouse heavy chain MIgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, having M428L and N434S mutations, having a G4S connector and a c-Myc tag at its C-terminus, and comprising allotype G1m(fa).

[0184] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 56. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 56, and optionally having a mouse heavy chain MIgG Vh signaling sequence and an ISAMVRS amino acid sequence at its N-terminus, having a YTE triple mutation, having a c-Myc tag at its C-terminus, and comprising allotype G1m(f).

[0185] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 57. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 57, and optionally having a mouse heavy chain IgG Vh signal sequence and an ISAMVRS amino acid sequence at its N-terminus, having a YTE triple mutation, having a G4S connector and a c-Myc tag at its C-terminus, and comprising allotype G1m(fa).

[0186] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 58. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 58, and optionally having a mouse heavy chain IgG Vh signal sequence at its N-terminus, a cysteine-to-serine substitution (position #), a G4S connector and an IgA peptide tag at its C-terminus, and comprising allotype G1m(fa).

[0187] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 59. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 59, and optionally having a mouse heavy chain IgG Vh signal sequence at its N-terminus, a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 59), M428L and N434S mutations, a G4S connector and an IgA peptide tag at its C-terminus, and allotype G1m(fa).

[0188] In some embodiments, E includes the amino acid sequence described in SEQ ID NO: 60. In some embodiments, E includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 60, and optionally has an amino acid defined for the corresponding position by SEQ ID NO: 60 at a position corresponding to position Z or X shown in SEQ ID NO: 60.

[0189] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 61. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 61, and optionally having a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 61), and having an amino acid defined for the corresponding position by SEQ ID NO: 61 at the position corresponding to position X shown in SEQ ID NO: 61.

[0190] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 62. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 62, and optionally having a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 62), and having the amino acid defined by SEQ ID NO: 62 at the position corresponding to position X shown in SEQ ID NO: 62.

[0191] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 63. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 63, and optionally having a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 63), and having allotype G1m(f).

[0192] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 64. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 64, and optionally having a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 64), and having allotype G1m(fa).

[0193] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 65. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 65, and optionally having a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 65), M428L and N343S mutations, and allotype G1m(fa).

[0194] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 66. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 66, and optionally having a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 66), M428L and N343S mutations, and allotype G1m(f).

[0195] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 67. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 67, and optionally having a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 67) and a YTE triple mutation, and having allotype G1m(fa).

[0196] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 68. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 68, and optionally having a cysteine-to-serine substitution (at the position corresponding to the # position in SEQ ID NO: 68) and a YTE triple mutation, and having allotype G1m(f).

[0197] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 69. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 69, and optionally having a YTE triple mutation and allotype G1m(fa).

[0198] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 70. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 70, and optionally having a YTE triple mutation and allotype G1m(fa).

[0199] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 70. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 70, and optionally having the M428L and N343S mutations and allotype G1m(fa).

[0200] In some embodiments, E includes the amino acid sequence described in SEQ ID NO: 72. In some embodiments, E includes 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 described in SEQ ID NO: 72.

[0201] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 73. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 73, and optionally having the M428L and N343S mutations and allotype G1m(f).

[0202] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 74. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 74, and optionally having the GAALIE, M428L, N343S mutation and allotype G1m(fa).

[0203] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 75. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 75, and optionally having the GAALIE, M428L, N343S mutation and allotype G1m(f).

[0204] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 76. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 76, and optionally having a cysteine-to-serine substitution (#), GAALIE, M428L, or N343S mutation, and having allotype G1m(fa).

[0205] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 77. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 77, and optionally having a cysteine-to-serine substitution (#), GAALIE, M428L, or N343S mutation, and having allotype G1m(fa).

[0206] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 78. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 78, and optionally having a cysteine-to-serine substitution (#), a DHS triple mutation, and allotype G1m(fa).

[0207] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 79. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 79, and optionally having a cysteine-to-serine substitution (#), a DHS triple mutation, or allotype G1m(f).

[0208] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 80. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 80, and optionally having a DHS triple mutation and allotype G1m(fa).

[0209] In some embodiments, E comprises the amino acid sequence described in SEQ ID NO: 81. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 81, and optionally having a DHS triple mutation and allotype G1m(f).

[0210] In any embodiment described herein, E includes an Fc domain monomer containing a triple mutation corresponding to M252Y / S254T / T265E(YTE) (for example, having the sequence described in any of SEQ ID NOs: 1 to 81). Those skilled in the art will understand that "corresponding to" refers to a specific amino acid site at the corresponding position obtained after sequence homology alignment using sequence analysis software (including, but not limited to, DNA Star, Vector NTI, etc.). For example, any of SEQ ID NOs: 1 to 81 may contain a mutation containing YTE.

[0211] In any embodiment described herein, E comprises an Fc domain monomer (for example, having the sequence described in any of SEQ ID NOs: 1-81) and includes a double mutation corresponding to M428L / N434S(LS). Those skilled in the art will understand that "corresponding to" refers to a specific amino acid site at the corresponding position obtained after sequence homology alignment using sequence analysis software (including, but not limited to, DNA Star, Vector NTI, etc.). For example, any one of SEQ ID NOs: 1-81 may include a mutation containing LS.

[0212] In any embodiment described herein, if E comprises an Fc domain monomer (for example, having the sequence described in any of SEQ ID NOs: 1-81), it comprises a double mutation corresponding to N434H. Those skilled in the art will understand that "corresponding to" refers to a specific amino acid site at the corresponding position obtained after sequence homology alignment using sequence analysis software (including, but not limited to, DNA Star, Vector NTI, etc.). For example, any one of SEQ ID NOs: 1-81 may contain a mutation comprising N434H.

[0213] In any embodiment described herein, E comprises an Fc domain monomer (for example, having the sequence described in any of SEQ ID NOs: 1-81) and includes a double mutation corresponding to C220S. Those skilled in the art will understand that "corresponding to" refers to a specific amino acid site at the corresponding position obtained after sequence homology alignment using sequence analysis software (including, but not limited to, DNA Star, Vector NTI, etc.). For example, any of SEQ ID NOs: 1-81 may include a mutation containing C220S.

[0214] In any embodiment described herein, E comprises a fragment of an Fc domain monomer (e.g., a fragment of an Fc domain monomer from any sequence described in SEQ ID NOs: 1 to 81), and 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 It is a fragment of consecutive amino acids, consisting of 47, 48, 49, 50 or more, at least 50 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or more), or at least 75 (e.g., 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).

[0215] In one embodiment, the conjugate has the following structure:

[0216] [Table 8-1]

[0217] [Table 8-2]

[0218] Here, n is defined as described above.

[0219] In one aspect, the present invention provides linker-payload compounds, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled compounds thereof.

[0220] In some embodiments, the present invention provides compounds represented by the structural formulas I-5a or I-6a, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled compounds thereof.

[0221] [ka]

[0222] In the formula, each symbol (for example, L, R) 2a , R 3a , R 5a , R 6a , R 7a , R 8a , R 9a , R 10a , and w) are as defined herein.

[0223] [ka]

[0224] In the formula, each symbol (for example, L, z, q, R) 1’ , R 2’ , R 3’ , R 4’ and R 5’ ) is as defined herein.

[0225] In some embodiments, the present invention provides compounds having the structure of formula I-2, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled compounds thereof.

[0226] [ka]

[0227] Here, L 1 and D 1 This is defined by equation I-1 above.

[0228] L 3The following structures are selected:

[0229] [Table 9-1]

[0230] [Table 9-2]

[0231] [Table 9-3]

[0232] [Table 9-4]

[0233] Here, each variable such as Z, R, s, y, and t is the same as L mentioned above. 2 It is the same as defined in . In the structure of L3-1 to L3-14, L 1 The substructure is L 3 Not a component of (L 1 ) is L 1 It should be understood that this is used to indicate the end connected to something.

[0234] In some embodiments, compounds comprising the compound of formula I-2 preferably have the structures of formulas C-inter-1 to C-inter-12:

[0235] [Table 10-1]

[0236] [Table 10-2]

[0237] [Table 10-3]

[0238] In some embodiments,

[0239] [ka]

[0240] The carbon atoms to which the group is linked, or

[0241] [ka]

[0242] The carbon atom to which the corresponding group is linked has an R or S configuration, preferably an R configuration.

[0243] In some embodiments, in C-inter-1 to C-inter-6, the carbon atom to which the left-side NH2-CH2- is bonded is in an R or S configuration, preferably an R configuration.

[0244] In another embodiment, the present invention comprises, as described above, a conjugate of formula I-1, IA, or formula IB, a compound of formula I-2, I-5a, or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, and optionally 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), and optionally, pharmaceutically acceptable excipients.

[0245] In another embodiment, the present invention includes 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 compounds thereof, as described above, 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).

[0246] In another embodiment, the present invention provides a method for preventing or treating a patient infected with a virus or at risk of viral infection, the method comprising, for example, administering to the patient, for example, by injection, a conjugate of formula I-1, IA, or formula IB, a compound of formula I-2, I-5a, or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

[0247] To use 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 compounds thereof, in the manufacture of pharmaceuticals for the prevention or treatment of viral infections in patients infected with a virus or at risk of viral infection.

[0248] In some embodiments, the viral infection is caused by an influenza virus or a parainfluenza virus.

[0249] In some embodiments, the viral infection is caused by influenza virus A, B, or C, or a parainfluenza virus.

[0250] In some embodiments, patients who are infected with the virus or at risk of becoming infected with the virus may be immunocompromised.

[0251] In some embodiments, patients who are infected with the virus or at risk of becoming infected with the virus may be patients who are receiving or are scheduled to receive immunosuppressant therapy.

[0252] In some embodiments, patients who are infected with the virus or at risk of becoming infected with the virus may be patients diagnosed with an immunosuppressive disorder.

[0253] In some embodiments, patients diagnosed with an immunosuppressive disorder also have cancer or acquired immunodeficiency syndrome.

[0254] In some cases, patients diagnosed with an immunosuppressive cancer had leukemia, lymphoma, humoral immunodeficiency, T-cell deficiency, complement deficiency, or multiple myeloma.

[0255] In some embodiments, the patient is a patient who has received or is scheduled to receive a hematopoietic stem cell transplant.

[0256] In some embodiments, the patient is a patient who has received or is scheduled to receive an organ transplant.

[0257] In some embodiments, patients may be at risk of secondary infection.

[0258] A method for preventing the risk of secondary infection in a patient due to influenza virus infection, comprising, for example, administering to the patient, for example by injection, a conjugate of any of formulas I-1, IA, or IB, a compound of formula I-2, I-5a, or I-6a, or any of the pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled compounds described above.

[0259] In some embodiments, the secondary infection is a respiratory infection.

[0260] In some embodiments, secondary infection is associated with pneumonia.

[0261] In some embodiments, the secondary infection is a bacterial, viral, or fungal infection.

[0262] In some embodiments, the bacterial infection is caused by methicillin-resistant Staphylococcus aureus.

[0263] In some embodiments, the bacterial infection is an infection caused by Streptococcus pneumoniae.

[0264] In some embodiments, 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-labeled compound may be administered by intramuscular injection, intravenous injection, intradermal injection, intra-arterial injection, intraperitoneal injection, intrafocal injection, intracranial injection, intra-articular injection, intrapleural injection, intratracheal injection, intraprostatic injection, intranasal injection, intravitreous injection, intravaginal injection, intrarectal injection, local administration, intratumoral injection, intraperitoneal injection, subcutaneous injection, subconjunctival injection, intracapsular injection, mucosal injection, intrapericardial injection, intraumbilical injection, intraocular injection, oral administration, local inhalation, injection, or infusion.

[0265] In some embodiments, 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 compounds are administered in combination with additional therapeutic agents or used to prepare pharmaceuticals in combination with additional therapeutic agents.

[0266] In some embodiments, the additional therapeutic agent is an antiviral drug.

[0267] In some embodiments, the antiviral agent is baloxavir, pimozivir, oseltamivir, zanamivir, peramivir, laninamivir, amantadine, MEDI8852, or rimantadine.

[0268] In some embodiments, the additional medication used by the patient is an antiviral vaccine.

[0269] In some embodiments, the antiviral agent and the conjugate of formula I-1, IA, or formula IB, the compound of formula I-2, I-5a, or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound are administered sequentially to the patient, for example, by injection.

[0270] In some embodiments, an antiviral agent and a conjugate of a compound 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-labeled compound are administered simultaneously to the patient, for example, by injection.

[0271] Effects of the invention 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 compounds have the following advantages: It exhibits remarkable high antiviral activity, It shows high activity against drug-resistant virus strains; and / or It possesses excellent in vitro / in vivo pharmacokinetic properties and safety (e.g., long half-life and high systemic exposure), which allows for reduced administration frequency and improved patient compliance, indicating a promising outlook for clinical application.

[0272] Preparation of the Conjugate of the Present Invention In one embodiment, the present invention provides a method for preparing the conjugate described herein.

[0273] In some embodiments, the present invention provides a method for preparing the conjugate of formula IA described herein.

[0274] [ka]

[0275] In the formula, each symbol (for example, 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; The method includes the following steps: Step 1, (E) m Dissolve in a buffer solution. Here, E and m are as defined herein. Next, add a reducing agent (e.g., TCEP, DTT) to (E) m The disulfide bond in the compound is reduced to obtain a compound of formula II-1 containing a thiol residue. (HS) n -(E) m II-1 In the formula, E, m, and n are as defined herein;

[0276] Step 2: Add a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-5a to the compound of formula II-1.

[0277] [ka]

[0278] In the formula, each symbol (for example, L, R) 2a , R 3a , R 5a , R 6a , R 7a , R 8a , R 9a , R 10a , and w) are as defined by formula IA; The Michael addition reaction is carried out for 0.5 to 10 hours, preferably 0.5 to 2 hours, under conditions of pH 5.5 to 7.0 (e.g., 5.8 to 6.8, 6.0 to 6.6); then, 3 to 7 molar equivalents of the buffer solution of the compound of formula I-5a are added in several portions (e.g., 1 to 2 portions), and the reaction is continued for 0.5 to 10 hours (preferably 0.5 to 2 hours) to obtain the conjugate of formula I-3b.

[0279] [ka]

[0280] In the formula, each symbol (for example, 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.

[0281] Step 3: 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), then carry out the hydrolysis reaction to obtain the final conjugate of formula IA;

[0282] or, A method for preparing the conjugate of formula IB described herein is provided.

[0283] [ka]

[0284] In the formula, each symbol (for example, L, z, q, x, y, R) 1’ , R 2’ , R 3’ , R 4’ , R 5’ , R 8a , and E) are as defined herein;

[0285] The method includes the following steps: Step 1, (E) m Dissolve in a buffer solution. Here, E and m are as defined herein. Next, add a reducing agent (e.g., TCEP, DTT) m The disulfide bond in the compound is reduced to obtain a compound of formula II-1 containing a thiol residue. (HS) n -(E) m II-1 In the formula, E, m, and n are as defined herein.

[0286] Step 2: Add a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-6a to the compound of formula II-1.

[0287] [ka]

[0288] In the formula, each symbol (for example, L, z, q, R) 1’ , R 2’ , R 3’ , R 4’ , R 5’ , and R 8a ) is defined as in formula IB; The Michael addition reaction is carried out under pH 5.5–8 conditions (e.g., 5.8–7.6, 6.0–7.5) to obtain the conjugate of formula IB.

[0289] In some embodiments, the present invention provides two methods for preparing a conjugate of formula I-1, Method 1 includes the following steps:

[0290] [ka]

[0291] During the ceremony,

[0292] [ka]

[0293] L 3 Corresponds to, here (L 1 ) is L 2 is L 1 This indicates that it is connected to L 1 This is not part of the above structure; Here,

[0294] [ka]

[0295] L 2 Corresponds to; as mentioned above, the structure between the two dashes is L 2 This should be addressed; Here, n, m, E, D 1 , L 1 , L 2 , L 3 Each of these variables is as defined above. Preferably, L 3 is selected from equations L3-1 to L3-7, L 2 The value is selected from equations L2-1 to L2-7; Step 1, (E) m Dissolve in a buffer solution and add a reducing agent (TCEP, DTT, etc.) (E) m The disulfide bond in the compound is reduced to prepare a compound of formula II-1 containing a thiol residue; Step 2: Add a buffer solution containing 6 to 14 molar equivalents of the compound of formula I-2a to the compound of formula II-1. Carry out the Michael addition reaction for 0.5 to 10 hours, preferably 0.5 to 2 hours, under pH 5.5 to 7.0 (e.g., 5.8 to 6.8, 6.0 to 6.6); then add 3 to 7 molar equivalents of the buffer solution of the compound of formula I-2a in several portions (e.g., 1 to 2 times) and continue the reaction for 0.5 to 10 hours, preferably 0.5 to 2 hours, to obtain the conjugate of formula I-3; Step 3: Adjust the pH of the solution containing the conjugate of formula I-3 to 7-8 (e.g., 7.1-7.9, 7.3-7.7), then carry out the hydrolysis reaction to obtain the final conjugate of formula I-1a.

[0296] In some embodiments, the pH of the buffer solution is in the range of 5.0 to 7.5, for example, 5.5 to 7.0, 6.0 to 6.5, or 5.5 to 6.5.

[0297] In some embodiments, the buffer is selected from phosphate buffer, citrate buffer, acetate buffer, and histidine buffer.

[0298] In some embodiments, the reaction in the above step is carried out at 10-30°C, for example, 15-28°C, 20-25°C, for example, 22°C.

[0299] In some embodiments, a reducing agent and (E) m The molar ratios range from 30:1 to 1:1, for example, 25:1 to 2:1, 20:1 to 5:1, and 15:1 to 8:1, for example, 10:1.

[0300] In some embodiments, the compound of formula II-1 used in step 2 is present in the reaction solution obtained after the reaction in step 1 is complete.

[0301] In some embodiments, the solution containing the conjugate of formula I-3 in step 3 is a solution of the conjugate of formula I-3 in a buffer (e.g., the buffer described above).

[0302] In some embodiments, the solution containing the conjugate of formula I-3 in step 3 is the reaction solution obtained after the completion of the reaction in step 2.

[0303] The inventors unexpectedly discovered that by adding the raw material (compound of formula I-2a) in batches during step 2, the DAR value of the final product could be made 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 step 2 also contributed to improving the uniformity of the DAR value of the final product.

[0304] Method 2 includes the following steps:

[0305] [ka]

[0306] During the ceremony,

[0307] [ka]

[0308] L 3 In response to,

[0309] Here,

[0310] [ka]

[0311] L 2 In response to, Here, n, m, E, D 1 , L 1 , L 2 , and L 3 This is as defined above, and preferably L 3 is selected from equations L3-8 to L3-14, L 2 This is selected from equations L2-8 to L2-14; Here, in step 1, (E) m Dissolve in a buffer solution and add a reducing agent (TCEP, DTT, etc.) (E) m The disulfide bond in the compound is reduced to obtain a compound of formula II-1 containing a thiol residue; Step 2: 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 under pH 5.5 to 8 (e.g., 5.8 to 7.6, 6.0 to 7.5) to obtain the conjugate of formula I-1b.

[0312] In some embodiments, the buffer, reaction temperature, reducing agent and (E) mThe molar ratio is as defined in Method 1.

[0313] In some embodiments, the compound of formula II-1 used in step 2 is present in the reaction solution obtained after the reaction in step 1 is complete.

[0314] Pharmaceutical composition In some embodiments, the present invention provides pharmaceutical compositions comprising conjugates of formula I-1, IA, or IB, compounds of formula I-2, I-5a, or I-6a, or conjugates or compounds of their subformulas, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled compounds thereof. For simplicity, conjugates of formula I-1, IA, or IB, compounds of formula I-2, I-5a, or I-6a, or conjugates or compounds of their subformulas, or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled compounds thereof may simply be referred to as “compounds of the present invention.” In one embodiment, the composition further comprises pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition is a combination of a compound of the present invention with one or more other therapeutic agents.

[0315] As used herein, “pharmaceutically acceptable excipients” include any physiologically compatible solvent, dispersion medium, isotonic agent, absorption retarder, and the like.

[0316] For information on the use and application of pharmaceutically acceptable excipients, please also refer to "Handbook of Pharmaceutical Excipients" (8th edition, by RCRowe, PJSeskey, and SCOwen, Pharmaceutical Press, London and Chicago).

[0317] The pharmaceutical compositions of the present invention may exist in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, and include liquid solutions (e.g., injectable solutions and intravenous solutions), powders or suspensions, liposomal formulations, and suppositories. The preferred form depends on the intended method of administration and therapeutic use.

[0318] Pharmaceuticals containing the compound of the present invention can be prepared, preferably in the form of a lyophilized formulation or an aqueous solution, by mixing the compound of the present invention with one or more pharmaceutically acceptable excipients.

[0319] definition The following are definitions of terms related to the present invention. Furthermore, those skilled in the art can understand these terms in conjunction with the prior art. Terms that are not defined have the usual meanings understood by those skilled in the art related to the present invention.

[0320] In this specification, the term "inhibits neuraminidase activity" refers to IC50 of 1000 nM or less. 50 This refers to a value determined, for example, based on the neuraminidase inhibition test method described in the examples of this specification. In some examples, IC2 inhibits neuraminidase activity. 50 A value of 100 nM or less or 10 nM or less indicates that the compound inhibits neuraminidase activity.

[0321] In this specification, the term "inhibits viral replication" is defined as "EC 50 This refers to a value of 1000 nM or less. For example, it is determined by the experimental method for influenza virus-mediated cytopathic inhibitory activity in the examples of this specification. In some embodiments, the EC of viral replication inhibitory activity is used. 50 A value of 100 nM or less, or 10 nM or less, is used to indicate that the compound inhibits viral replication activity.

[0322] As used herein, the term “Fc domain monomer” refers to a polypeptide chain or a functional fragment thereof having the following characteristics (e.g., a fragment having the ability to dimerize with other Fc domain monomers and bind to an Fc receptor): 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 also comprises at least one hinge region or a portion of a hinge region. This Fc domain monomer can be any immunoglobulin antibody type, including IgG, IgE, IgM, IgA, and IgD. Furthermore, the Fc domain monomer can be any IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). For example, in natural antibodies, the immunoglobulin Fc domain consists of second and third constant regions (CH2 and CH3 domains) derived from two heavy chains of IgG, IgA, and IgD antibodies, or second, third, and fourth constant regions (CH2, CH3, and CH4 domains) derived from two heavy chains of IgM and IgE antibodies. Unless otherwise specified, the amino acid numbering of this IgG or Fc domain monomer follows the EC numbering system for antibodies (also known as Kabat EU or the EU numbering system, as described in Kabat et al., Sequences of Proteins of Immunological Interest, the 5th Edition, Public Health Services, National Institute of Health, Bethesda, MD, 1991). The Fc domain monomer can be derived from any species, including synthetic, human, mouse, rat, and llama. The Fc domain monomer may contain one or more non-natural amino acid sequences that function as site-specific binding sites for small molecule drugs. The Fc domain monomer contains one or more site-specifically designed, solvent-exposed cysteine ​​or lysine residues and can provide an additional binding site for small molecule drugs.

[0323] In this specification, the term "Fc domain" refers to a dimer formed by the interaction of two Fc domain monomers via a hinge region and / or CH2 and / or CH3 antibody constant regions. In some embodiments, one or more disulfide bonds are present between the monomers of the dimer. Fc domains can be derived from any species, including synthetic, human, mouse, rat, and llama. Fc domains may contain one or more non-natural amino acid sequences that function as site-specific binding sites for small molecules. Fc domains may contain one or more solvent-exposed cysteine ​​or lysine residues that are site-specifically designed to provide additional binding sites for small molecules.

[0324] In this specification, the term "covalent linkage" refers to a situation where two parts constituting a conjugate are linked to each other by a covalent bond formed between two atoms within the conjugate.

[0325] In this specification, “Fc-binding peptide” refers to a polypeptide having a continuous amino acid sequence of 5 to 50 amino acids (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 having affinity for and binding function to an Fc domain (e.g., any Fc domain as described herein). Fc-binding peptides may be derived from any species, including synthetic, human, mouse, rat, and llama. Fc-binding peptides may contain one or more non-natural amino acid sequences that function as site-specific binding sites for small molecular weight drugs. Fc-binding peptides may contain one or more solvent-exposed cysteine ​​or lysine residues that are site-specifically designed to provide additional binding sites for small molecular weight drugs.

[0326] In this specification, the term "solvent exposure" refers to an amino acid residue surrounded by solvent molecules that enclose a protein or polypeptide. Solvent-exposed amino acid molecules may be naturally occurring or artificially modified (including both natural and unnatural amino acids). In some embodiments, modification of this site does not affect the three-dimensional structure of the protein.

[0327] As used herein, the term "identity %" refers to the percentage of amino acid residues in a protein or polypeptide sequence that are identical to amino acid residues in a reference sequence after gaps have been introduced into the alignment sequence as necessary to achieve maximum identity % (i.e., gaps can be introduced into either or both of the candidate sequence and / or the reference sequence for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment for achieving identity percentage in sequence alignment can be achieved by various methods within the scope of the art of the art, including, but not limited to, publicly available computer software such as BLAST, ALIGN, and MegAlign (DNA Star).

[0328] As used herein, the terms “treatment” or “for treatment” refer to therapeutic measures taken by an individual after a viral infection. In some embodiments, therapeutic measures can slow the progression of a viral infection, alleviate the individual’s symptoms, and / or eliminate the viral infection.

[0329] The terms "effective dose" or "effective dosage" refer to the amount or dosage of the antibody, fragment, composition, or combination thereof of the present invention that, when administered once or multiple times to a patient in need of treatment or prevention, produces the desired effect.

[0330] "C1-C 20 The term "alkyl" can be used alone or in combination (e.g., C1-C 20Alkylaryls include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tertiary butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-2-butyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 2,3-dimethyl-2-butyl, 3-methyl-2-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., which contain 1 to 20 carbon atoms (e.g., 1 to 15, 1 to 10 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), especially 1 to 6 carbon atoms). Preferably, "C 1-20 "Alkyl" refers to one of the following: methyl, ethyl, isopropyl, or tert-butyl.

[0331] "C2-C 20 An "alkenyl" refers to a linear or branched alkyl group as defined above, but containing 1 to 10 double bonds (for example, 1, 2, 3, 4, or 5 double bonds), and does not include alkynyls. Representative alkenyls include vinyl, 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, and 2-methyl-2-pentenyl.

[0332] "C 3-20 The term "cycloalkyl" can be used alone or in combination (e.g., C3-C 20 This refers to saturated cycloalkylalkyl groups having 3 to 20 carbon atoms, for example, 3 to 15, 3 to 10 (for example, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), and especially 3 to 6 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. In particular, "C 3-7 "Cycloalkyl" refers to compounds such as cyclopropyl, cyclopentyl, and cyclohexyl.

[0333] "C3-C 20 A "cycloalkenyl" is a group containing 3 to 20 carbon atoms (e.g., 3 to 15 or 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), especially 3 to 6 carbon atoms. It contains 1 to 10 double bonds (e.g., 1, 2, 3, 4, or 5 double bonds), but does not contain an alkynyl group and is not an aromatic group. Representative cycloalkenyls include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl. In particular, "C 3-7 "Cycloalkenyl" refers to cyclopropenyl, cyclopentenyl, cyclohexenyl, etc.

[0334] "Halogen" refers to fluorine, chlorine, bromine, or iodine, either alone or in combination, and especially to fluorine, chlorine, or bromine.

[0335] "Haloalkyl" refers to a molecule in which one or more alkyl groups defined above are substituted with halogens (for example, 1, 2, 3, 4, or 5) either alone or in combination.

[0336] The term "amino," either alone or in combination, refers to a primary amino group (-NH2), a secondary amino group (-NH-), or a tertiary amino group.

[0337] The term "carbonyl," also known as "-C(=O)-," refers to a divalent group consisting of a carbon atom and an oxygen atom, where the carbon and oxygen atoms are linked by a double bond, and the carbon atom within the structure is further linked to the other two fragments via single bonds.

[0338] The term "heterocycloalkyl," also known as "heterocyclo group," refers to a saturated or partially unsaturated (containing one or two double bonds) non-aromatic cyclo group consisting of carbon and heteroatoms such as nitrogen, oxygen, and sulfur. This cyclo group may be monocyclic or bicyclic. In the present invention, the heterocycloalkyl can be a 3-20 membered ring heterocycloalkyl, for example, a 3-15 membered ring, a 3-10 membered ring, a 3-7 membered ring, a 3-6 membered ring, a 5-7 membered ring, or a 4-6 membered ring heterocycloalkyl. The number of carbon atoms in the heterocycloalkyl ranges from 2 to 16, for example, from 2 to 11, and the number of heteroatoms is one or more, preferably 1, 2, 3, or 4. The nitrogen, carbon, or sulfur atoms in the heterocycloalkyl may optionally be oxidized. The hydrogen atoms in the "heterocyclic alkyl" may be independently and optionally substituted with one or more substituents described in the present invention. The "heterocyclic alkyl" may be bonded to the parent molecule via any ring atom within the ring. The terms "3-6 membered heterocycloalkyl" and "3-7 membered heterocycloalkyl" refer to saturated or partially unsaturated monocyclic or polycyclic heterocycloalkyls containing 3-6 and 3-7 membered ring members (selected from carbon and heteroatoms or heteroatomic groups), respectively, where the heteroatoms or heteroatomic groups are N, O, S(O) m (where m is any integer from 0 to 2) are selected. For example, aziridinyl, azetidinyl, oxetanyl, tetrahydropyrrolyl, oxopyrrolidinyl, tetrahydrofuranil, tetrahydrothienyl, piperidinyl, morpholinyl, piperazinyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxothiomorpholinyl, etc.

[0339] "Aryl" refers to a stable 6- to 15-membered, for example, 6- to 10-membered monocyclic or bicyclic aromatic carboncyclic hydrocarbon group, including phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindenyl, and biphenyl. The hydrogen atoms in the "aryl" may be independently and optionally substituted with one or more substituents described in the present invention.

[0340] A "heteroaryl" or "heterocyclic aryl" refers to an aromatic ring group formed by substituting a carbon in the ring with at least one heteroatom selected from sulfur, oxygen, or nitrogen. This aromatic ring group may be a 5-15 membered ring, such as a 5-7 membered ring, a 5-6 membered monocyclic ring, or a 7-12 membered bicyclic ring. This includes, but is not limited to, 5, 6, 7, 8, 9, or 12-membered heteroaryl rings. In the present invention, the number of heteroatoms in the heteroaryl is preferably 1, 2, 3, or 4. Typical heteroaryl groups can be selected from thienyl, pyridyl, pyrimidinyl, pyrazinyl, pyridadinyl, pyridine-2(1H)-one group, pyridine-4(1H)-one group, pyrrolyl, pyrazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, imidazolyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, benzothienyl, indolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, quinolinelyl, isoquinolinelyl, quinazolinyl, etc. The hydrogen atoms in the "heteroaryl" may be independently and arbitrarily substituted with one or more substituents described in the present invention.

[0341] "C 6-15 The term "aryl" refers to an aryl molecule containing 6-15 carbon atoms, where "aryl" is defined above.

[0342] A "5-15 membered heteroaryl ring" refers to an aromatic heterocyclic group containing 5 to 15 ring atoms, where "heteroaryl" is defined as described above.

[0343] "Cyano" refers to the -CN group.

[0344] "Carboxyl" refers to the -COOH group.

[0345] "Hydroxyl" refers to the -OH group.

[0346] The term "substituted" means having 1 to 25, 1 to 20, 1 to 10, or 1 to 5 substituents, including, for example, having 1, 2, 3, 4, or 5 substituents. Substituents include alkyl, cycloalkyl, alkenyl, alkynyl, aryl, alkaryl, acyl, heteroaryl, heteroalkyl, heterocycloalkyl, heteroalkenyl, heteroalkynyl, heteroalkaryl, halogen, oxo, cyano, nitro, amino, alkylamino, hydroxyl, alkoxy, alkanoyl, carbonyl, carbamoyl, guanidino, ureido, amidino, and combinations of the above groups or substructures. The substituents are F, Cl, methyl, phenyl, benzyl, OR, NR2, SR, SOR, SO2R, OCOR, NRCOR, NRCONR2, NRCOOR, OCONR2, RCO, COOR, alkyl-OOCR, SO3R, CONR2, SO2NR2, NRSO2NR2, CN, CF3, OCF3, SiR3, and NO2, where each R is independently selected from H, alkyl, alkenyl, aryl, heteroalkyl, heteroalkenyl, or heteroaryl. Furthermore, any two substituents on the same or adjacent atoms may be bonded to form an optionally substituted aromatic or non-aromatic, saturated or unsaturated ring containing 3 to 8 atoms. Alternatively, any two substituents on the same atom may be bonded to form an optionally substituted aromatic or non-aromatic, saturated or unsaturated ring containing 3 to 8 atoms.

[0347] The phrase "depending on the circumstances" indicates that a subsequent event or situation may occur / exist, or may not occur / exist. For example, "depending on the circumstances" indicates that the relevant element may be substituted or not substituted.

[0348] The terms "comprising" or "including" also encompass situations that are composed of, or essentially constitute, the features or elements related to them.

[0349] The term "stereoisomer" encompasses all forms of isomerism, including enantiomers, diastereomers, and geometric isomers (cis / trans isomers). Therefore, individual stereochemical isomers of the compounds designed in this invention, or mixtures of their enantiomers, diastereomers, geometric isomers (or cis / trans isomers), are all included within the scope of this invention.

[0350] The term "pharmaceutically acceptable salt" refers to the compounds of the present invention in pharmaceutically useful salt forms, including acid addition salts and base addition salts. Pharmaceutically acceptable, non-toxic acid addition salts in the present invention include, but are 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, and malic acid. Pharmaceutically acceptable, non-toxic base addition salts refer to, but are not limited to, salts formed between the compounds of the present invention and an organic or inorganic base, including alkali metal salts such as lithium salts, sodium salts, or potassium salts; alkaline earth metal salts such as calcium salts or magnesium salts; and N formed by reaction with ammonium salts or N-containing organic bases. + (C 1-6 This includes organic base salts such as alkyl(4) salts.

[0351] A "solvate" refers to a complex formed by one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide.

[0352] "Hydrate" refers to a complex formed by water and the compound of the present invention.

[0353] A "prodrug" refers to a chemical derivative of the compound of the present invention that is converted into a compound represented by general formula I-1 or I-2 through a chemical reaction in a living organism.

[0354] An "isotope-labeled compound" is an isotope-labeled compound obtained by substituting one or more hydrogen atoms, such as 1, 2, 3, 4, or 5 hydrogen atoms, with deuterium in general formulas I-1 and I-2, and one or more carbon atoms (e.g., 1 to 3) of carbon-14 atoms. 14 This refers to isotope-labeled compounds obtained by substitution with C).

[0355] "Combination drug formulation" refers to a non-fixed combination formulation or a fixed combination formulation, and includes, but is not limited to, drug kits and pharmaceutical compositions. "Non-fixed combination" means that the active ingredients (for example, (i) the compounds of the present invention (including 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 compounds), and (ii) other therapeutic agents) are administered to the patient simultaneously or sequentially at the same or different time intervals, as separate units, without any specific time limit, thereby providing the patient with two or more active ingredients at a level that is prophylactically or therapeutically effective. In some embodiments, the compounds of the present invention and other therapeutic agents used in the combination drug formulation are not administered in doses exceeding those that would be used when they are used individually. "Fixed combination formulation" refers to the simultaneous administration of two or more active ingredients to the patient as a single dosage form. The dosages and / or time intervals of two or more active ingredients are preferably selected so that the combined use of each ingredient produces a greater effect in treating the disease or condition than any single ingredient alone. Each ingredient may be in the same or different formulation form.

[0356] The terms "drug:antibody ratio" or "DAR" refer to the small molecule drug portion (D) bound to the E portion (antibody, Fc region, or albumin, etc.) as described herein. 1 and D 2DAR refers to the ratio of the D portion to the E portion (e.g., antibody, Fc domain, or albumin). In some embodiments described herein, DAR is represented as n:m in formula I-1 and is in the range of 1 to 20, preferably in the range of 2 to 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10. DAR is also determined by detection method (e.g., MS) to the small molecule drug portion (D) bound to the E portion (e.g., antibody, Fc domain, or albumin) as described herein. 1 and D 2 ) can also be calculated as the average DAR of the molecular population in the product, as the overall ratio to the E portion. This DAR is referred to as the average DAR in this text. In some embodiments, the average DAR value of the conjugates of the present invention is between 0.5 and 10.0, for example, 1.0 to 8.0, specifically 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, The range is defined as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, and the range with any two of these values ​​as endpoints.

[0357] In context, when variables n and y are integers within a specific range, or are selected from that range, it means that all integers within that range are considered in this invention and are treated as if they were individually listed herein. For example, when n is described as an integer from 1 to 8, it is equivalent to describing n as any integer of 1, 2, 3, 4, 5, 6, 7, or 8.

[0358] Sequence IDs 1-81 and their descriptions Sequence ID 1: Mouse Fc-IgG2a with IL2 signal sequence (bold) at its N-terminus (bold)

[0359] [ka]

[0360] Sequence ID 2: Mature mouse Fc-IgG2a

[0361] [ka]

[0362] Sequence ID 3: Human Fc-IgG1 having an N-terminal (bold) IL2 signal sequence and an N-terminal MVRS amino acid sequence (underlined)

[0363] [ka]

[0364] Sequence ID 4: Mature human Fc-IgG1 with MVRS amino acid sequence (underlined) at the N-terminus

[0365] [ka]

[0366] Sequence ID 5: Mouse Fc-IgG2a with an IL2 signal sequence (bold) attached to the N-terminus and a 6-histidine tag (italic) attached to the C-terminus.

[0367] [ka]

[0368] Sequence ID 6: Mature mouse Fc-IgG2a with a C-terminal 6-histidine tag (italicized)

[0369] [ka]

[0370] Sequence ID 7: Human Fc-IgG1 with an IL2 signal sequence (bold) and MVRS amino acid sequence (underlined) attached to the N-terminus and a 6-histidine tag (italicized) attached to the C-terminus.

[0371] [ka]

[0372] Sequence ID 8: Mature human Fc-IgG1 with an MVRS amino acid sequence (underlined) added to the N-terminus and a 6-histidine tag (italicized) added to the C-terminus.

[0373] [ka]

[0374] Sequence ID 9: Human Fc-IgG1 having an IL2 signal sequence (bold) and MVRS amino acid sequence (underlined) attached to the N-terminus, two additional cysteine ​​(*) in the hinge region, and a 6-histidine tag (italicized) attached to the C-terminus.

[0375] [ka]

[0376] Sequence ID 10: Mature human Fc-IgG1 with an MVRS amino acid sequence (underlined) added to the N-terminus, two additional cysteine ​​molecules (*) in the hinge region, and a 6-histidine tag (italicized) added to the C-terminus.

[0377] [ka]

[0378] Sequence ID 11: Mature human Fc-IgG1 with an MVRS amino acid sequence (underlined) added to the N-terminus and two additional cysteine ​​molecules (*) in the hinge region.

[0379] [ka]

[0380] Sequence ID 12: Mouse Fc-IgG2a with an N-terminal IL2 signal sequence (bold), an Asn→Ala substitution (*), and a C-terminal 6-histidine tag (italic).

[0381] [ka]

[0382] Sequence ID 13: Mouse Fc-IgG2a with Asn→Ala substitution (*) and a 6-histidine tag (italicized) added to the C-terminus.

[0383] [ka]

[0384] Sequence ID 14: Human Fc-IgG1 with an IL2 signal sequence (bold) and MVRS amino acid sequence (underlined) added to the N-terminus, an Asn→Ala substitution (*), and a 6-histidine tag (italic) added to the C-terminus.

[0385] [ka]

[0386] Sequence ID 15: Human Fc-IgG1 with an MVRS amino acid sequence (underlined) with an Asn→Ala substitution (*) at the N-terminus and a 6-histidine tag (italicized) at the C-terminus.

[0387] [ka]

[0388] Sequence ID 16: Human Fc-IgG1 with an N-terminally added human serum albumin signaling sequence (bold) and an N-terminal ISAMVRS amino acid sequence (underlined).

[0389] [ka]

[0390] Sequence ID No. 17: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) appended to the N-terminus, an N-terminal ISAMVRS amino acid sequence (underlined), a C-terminal G4S connector (italicized), and a C-terminal c-Myc tag (underlined and italicized).

[0391] [ka]

[0392] Sequence ID 18: Mature human Fc-IgG1 having an ISAMVRS amino acid sequence (underlined) at the N-terminus, a G4S connector (italicized) at the C-terminus, and a c-Myc tag (underlined and italicized) at the C-terminus.

[0393] [ka]

[0394] Sequence ID 19: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) and an ISAMVRS amino acid sequence (underlined) added to the N-terminus, and containing a lysine → serine substitution (*) to prevent binding at this site.

[0395] [ka]

[0396] Sequence ID 20: Mature human Fc-IgG1 having the N-terminal ISAMVRS amino acid sequence (underlined) and a lysine → serine substitution (*) to prevent binding at this site.

[0397] [ka]

[0398] Sequence ID No. 21: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) and an ISAMVRS amino acid sequence (underlined) added to the N-terminus, a lysine-to-serine substitution (*) to prevent binding at this site, and a G4S connector (italic) and a c-Myc tag (underlined and italicized) added to the C-terminus.

[0399] [ka]

[0400] Sequence ID 22: Mature human Fc-IgG1 having the ISAMVRS amino acid sequence (underlined) added to the N-terminus, including a lysine to serine substitution (*) to prevent binding at this site, and a G4S connector (italicized) and c-Myc tag (underlined and italicized) added to the C-terminus.

[0401] [ka]

[0402] Sequence ID 23: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, including an Asn→Ala substitution (*), and a G4S connector (italic) and c-Myc tag (underlined and italicized) added to the C-terminus.

[0403] [ka]

[0404] Sequence ID No. 24: Mature human Fc-IgG1 having the ISAMVRS amino acid sequence (underlined) added to the N-terminus, including an Asn→Ala substitution (*), and having a G4S connector (italicized) and a c-Myc tag (underlined and italicized) added to the C-terminus.

[0405] [ka]

[0406] Sequence ID 25: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, H310A(*) and H435A(*) modifications to prevent FcRn binding, and a G4S connector (italic) and c-Myc tag (underlined and italicized) added to the C-terminus.

[0407] [ka]

[0408] Sequence ID No. 26: Human Fc-IgG1 having the ISAMVRS amino acid sequence (underlined) added to the N-terminus, containing H310A(*) and H435A(*) variants to inhibit FcRn binding, and containing a G4S connector (italic) and c-Myc tag (underlined, italic) added to the C-terminus.

[0409] [ka]

[0410] Sequence ID No. 27: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, a G4S connector (italic) added to the C-terminus, and a mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italic).

[0411] [ka]

[0412] Sequence ID 28: Mature human Fc-IgG1 having an ISAMVRS amino acid sequence (underlined) added to the N-terminus, a G4S connector (italicized) added to the C-terminus, and a mutation (lysine to phenylalanine, bold) c-Myc tag (underlined, italicized).

[0413] [ka]

[0414] Sequence ID No. 29: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) and ISAMVRS amino acid sequence (underlined) added to the N-terminus, including an Asn→Ala substitution (*), a G4S connector (italic) added to the C-terminus, and a mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italic).

[0415] [ka]

[0416] Sequence ID 30: Mature human Fc-IgG1 having the ISAMVRS amino acid sequence (underlined) added to the N-terminus, including an Asn→Ala substitution (*), a G4S connector (italicized) added to the C-terminus, and a mutant (lysine to phenylalanine, bold) c-Myc tag (underlined, italicized).

[0417] [ka]

[0418] Sequence ID 31: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) added to the N-terminus, allotype G1m(fa) (bold and italic), a G4S connector (italic) added to the C-terminus, and a mutation (lysine to phenylalanine, bold) c-Myc tag (underlined and italic).

[0419] [ka]

[0420] Sequence ID 32: Human Fc-IgG1 containing a human serum albumin signaling sequence (bold) added to the N-terminus and allotype G1m(fa) (bold and italicized).

[0421] [ka]

[0422] Sequence ID 33: Mature human Fc-IgG1 with an MVRS amino acid sequence added to the N-terminus (underlined) and containing a YTE triple mutation (bold, underlined).

[0423] [ka]

[0424] Sequence ID 34: Human Fc-IgG1 containing a human serum albumin signaling sequence (bold) at its N-terminus, the EPKSS amino acid sequence (underlined) of the hinge region of mature human Fc-IgG1, and the cysteine-to-serine conversion (#) and allotype G1m(fa) (bold and italicized).

[0425] [ka]

[0426] Sequence ID 35: Human Fc-IgG1 having a mouse IgG signal sequence (bold) added to the N-terminus, with the amino acid sequence of the hinge region EPKSSD of mature human Fc-IgG removed, and allotype G1m(fa) (bold and italicized).

[0427] [ka]

[0428] Sequence ID 36: Mature human Fc-IgG1 in which the EPKSSD amino acid sequence of the hinge region is removed at the N-terminus, possessing allotype G1m(fa) (bold and italicized), and containing a YTE triple mutation (bold and underlined).

[0429] [ka]

[0430] Sequence ID 37: Mature human Fc-IgG1 in which the EPKSSD amino acid sequence of the hinge region of mature human Fc-IgG1 is removed at the N-terminus, has an LS double mutation (bold, underlined), and retains allotype G1m(fa) (italic).

[0431] [ka]

[0432] Sequence ID 38: Human Fc-IgG1 having a human serum albumin signaling sequence (bold) appended to the N-terminus, containing a YTE triple mutation (bold and underlined), allotype G1m(fa) (bold and italicized), and a G4S connector (italicized) and c-Myc tag (underlined) appended to the C-terminus.

[0433] [ka]

[0434] Sequence ID 39: Mature human Fc-IgG1 where X1 is Met or Trp, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser.

[0435] [ka]

[0436] Sequence ID 40: Mature human Fc-IgG1 where X4 is Asp or Glu and X5 is Leu or Met

[0437] [ka]

[0438] Sequence ID No. 41: Mature human Fc-IgG1 with YTE triple mutation (bold, underlined), where X4 is Asp or Glu and X5 is Leu or Met.

[0439] [ka]

[0440] Sequence ID No. 42: Mature human Fc-IgG1 with YTE triple mutation (bold, underlined) and allotype G1m(fa) (bold and italicized)

[0441] [ka]

[0442] Sequence ID No. 43: Mature human Fc-IgG1 with YTE triple mutation (bold, underlined) and allotype G1m(f) (bold and italicized)

[0443] [ka]

[0444] Sequence ID No. 44: Mature human Fc-IgG1 with LS double mutation (bold, underlined), where X4 is Asp or Glu and X5 is Leu or Met.

[0445] [ka]

[0446] Sequence ID No. 45: Mature human Fc-IgG1 with LS double mutation (bold, underlined) and allotype G1m(fa) (bold and italicized)

[0447] [ka]

[0448] Sequence ID 46: Mature human Fc-IgG1 with LS double mutation (bold, underlined) and allotype G1m(f) (bold and italicized).

[0449] [ka]

[0450] Sequence ID 47: Mature human Fc-IgG1 having a mouse heavy chain IgG Vh signal sequence (bold) attached to the N-terminus, a cysteine-to-serine conversion (#), where X1 is Met or Trp, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser.

[0451] [ka]

[0452] Sequence ID 48: Mature human Fc-IgG1 having a mouse heavy chain MIgG Vh signaling sequence (bold) attached to the N-terminus, cysteine-to-serine conversion (#), and allotype G1m(fa) (bold and italicized).

[0453] [ka]

[0454] Sequence ID 49: Mature human Fc-IgG1 having a mouse heavy chain MIgG Vh signaling sequence (bold) attached to the N-terminus, cysteine-to-serine conversion (#), and allotype G1m(f) (bold and italicized).

[0455] [ka]

[0456] Sequence ID 50: Mature human Fc-IgG1 with a mouse heavy chain MIgG Vh signaling sequence (bold) added to the N-terminus, cysteine-to-serine conversion (#), M428L and N434S mutations (bold, underlined), and allotype G1m(fa) (bold and italicized).

[0457] [ka]

[0458] Sequence ID 51: Mature human Fc-IgG1 having a mouse heavy chain MIgG Vh signaling sequence (bold) added to the N-terminus, cysteine-to-serine conversion (#), M428L and N434S mutations (bold, underlined), and allotype G1m(f) (bold and italicized).

[0459] [ka]

[0460] Sequence ID 52: Mature human Fc-IgG1 with a mouse heavy chain MIgG Vh signaling sequence (bold) added to the N-terminus, cysteine-to-serine conversion (#), YTE triple mutation (bold, underlined), and allotype G1m(fa) (bold and italicized).

[0461] [ka]

[0462] Sequence ID 53: Mature human Fc-IgG1 with a mouse heavy chain MIgG Vh signaling sequence (bold) added to the N-terminus, cysteine-to-serine conversion (#), YTE triple mutation (bold, underlined), and allotype G1m(f) (bold and italicized).

[0463] [ka]

[0464] Sequence ID 54: Mature human Fc-IgG1 having a mouse heavy chain MIgG Vh signaling sequence (bold) and ISAMVRS amino acid sequence (italic) attached to the N-terminus, M428L and N434S mutations (bold, underlined), a G4S connector (italic) and c-Myc tag (underlined) attached to the C-terminus, and containing allotype G1m(f) (bold and italic).

[0465] [ka]

[0466] Sequence ID 55: Mature human Fc-IgG1 having a mouse heavy chain MIgG Vh signaling sequence (bold) and ISAMVRS amino acid sequence (italic) attached to the N-terminus, M428L and N434S mutations (bold, underlined), a G4S connector (italic) and c-Myc tag (underlined) attached to the C-terminus, and containing allotype G1m(fa) (bold and italic).

[0467] [ka]

[0468] Sequence ID 56: Mature human Fc-IgG1 having a mouse heavy chain MIgG Vh signaling sequence (bold) and ISAMVRS amino acid sequence (italic) attached to the N-terminus, a YTE triple mutation (bold, underlined), a G4S connector (italic) and c-Myc tag (underlined) attached to the C-terminus, and containing allotype G1m(f) (bold and italic).

[0469] [ka]

[0470] Sequence ID 57: Mature human Fc-IgG1 having a mouse heavy chain MIgG Vh signaling sequence (bold) and ISAMVRS amino acid sequence (italic) attached to the N-terminus, a YTE triple mutation (bold, underlined), a G4S connector (italic) and c-Myc tag (underlined) attached to the C-terminus, and containing allotype G1m(fa) (bold and italic).

[0471] [ka]

[0472] Sequence ID 58: Mature human Fc-IgG1 containing a mouse heavy chain MIgG Vh signal sequence (bold) attached to the N-terminus, a cysteine-to-serine conversion (#), a G4S connector (italic) and an IgA peptide tag (underlined) attached to the C-terminus, and allotype G1m(fa) (bold and italic).

[0473] [ka]

[0474] Sequence ID 59: Mature human Fc-IgG1 containing a mouse heavy chain MIgG Vh signaling sequence (bold) attached to the N-terminus, cysteine-to-serine conversion (#), M428L and N434S mutations (bold, underlined), a G4S connector (italic) and IgA peptide tag (underlined) attached to the C-terminus, and allotype G1m(fa) (bold and italic).

[0475] [ka]

[0476] Sequence ID 60: Mature human Fc-IgG1, where Z1 is Cys or Ser, X1 is Met or Trp, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, and X7 is Asn or Ser.

[0477] [ka]

[0478] Sequence ID 61: Mature human Fc-IgG1 having a cysteine-to-serine conversion (#), where X1 is Met or Trp, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, X6 is Met or Leu, and X7 is Thr.

[0479] [ka]

[0480] Sequence ID 62: Mature human Fc-IgG1 having a cysteine-to-serine conversion (#), where X4 is Asp or Glu and X5 is Leu or Met.

[0481] [ka]

[0482] Sequence ID 63: Mature human Fc-IgG1 with cysteine-to-serine conversion (#) and allotype G1m(f) (bold and italicized).

[0483] [ka]

[0484] Sequence ID 64: Mature human Fc-IgG1 with cysteine-to-serine conversion (#) and allotype G1m(fa) (bold and italicized).

[0485] [ka]

[0486] Sequence ID 65: Mature human Fc-IgG1 with cysteine-to-serine mutation (#), M428L and N434S mutations (bold, underlined), and allotype G1m(fa) (bold and italicized).

[0487] [ka]

[0488] Sequence ID 66: Mature human Fc-IgG1 with cysteine-to-serine mutation (#), M428L and N434S mutations (bold, underlined), and allotype G1m(f) (bold and italicized).

[0489] [ka]

[0490] Sequence ID 67: Mature human Fc-IgG1 with cysteine-to-serine mutation (#), YTE triple mutation (bold, underlined), and allotype G1m(fa) (bold and italicized).

[0491] [ka]

[0492] Sequence ID 68: Mature human Fc-IgG1 with cysteine-to-serine mutation (#), YTE triple mutation (bold, underlined), and allotype G1m(f) (bold and italicized).

[0493] [ka]

[0494] Sequence ID No. 69: Mature human Fc-IgG1 with YTE triple mutation (bold, underlined) and allotype G1m(fa) (bold and italicized)

[0495] [ka]

[0496] Sequence ID 70: Mature human Fc-IgG1 with glycine-to-alanine mutation (#), alanine-to-leucine mutation ($), isoleucine-to-glutamic acid mutation (*), YTE triple mutation (bold, underlined), and allotype G1m(fa) (bold and italicized).

[0497] [ka]

[0498] Sequence ID No. 71: Mature human Fc-IgG1 with M428L and N434S mutations (bold, underlined) and allotype G1m(fa) (bold and italicized).

[0499] [ka]

[0500] Sequence ID 72: (Wild type)

[0501] [ka]

[0502] Sequence ID No. 73: Mature human Fc-IgG1 with M428L and N434S mutations (bold, underlined) and allotype G1m(f) (bold and italicized).

[0503] [ka]

[0504] Sequence ID 74: Mature human Fc-IgG1 with GAALIE, M428L and N434S mutations (bold, underlined), and allotype G1m(fa) (bold and italicized).

[0505] [ka]

[0506] Sequence ID 75: Mature human Fc-IgG1 with GAALIE, M428L and N434S mutations (bold, underlined), and allotype G1m(f) (bold and italicized).

[0507] [ka]

[0508] Sequence ID 76: Mature human Fc-IgG1 with cysteine-to-serine mutation (#), GAALIE, M428L and N434S mutations (bold, underlined), and allotype G1m(fa) (bold and italicized).

[0509] [ka]

[0510] Sequence ID 77: Mature human Fc-IgG1 with cysteine-to-serine mutation (#), GAALIE, M428L and N434S mutations (bold, underlined), and allotype G1m(f) (bold and italicized).

[0511] [ka]

[0512] Sequence ID 78: Mature human Fc-IgG1 with cysteine-to-serine mutation (#), DHS triple mutation (bold, underlined), and allotype G1m(fa) (bold and italicized).

[0513] [ka]

[0514] Sequence ID 79: Mature human Fc-IgG1 with cysteine-to-serine mutation (#), DHS triple mutation (bold, underlined), and allotype G1m(f) (bold and italicized).

[0515] [ka]

[0516] Sequence ID No. 80: Mature human Fc-IgG1 with DHS triple mutation (bold, underlined) and allotype G1m(fa) (bold and italicized)

[0517] [ka]

[0518] Sequence ID No. 81: Mature human Fc-IgG1 with DHS triple mutation (bold, underlined) and allotype G1m(f) (bold and italicized)

[0519] [ka]

[0520] In the sequences listed in sequence numbers 1-81, C(!) is L 2 It is a cysteine ​​that can be linked to. [Examples]

[0521] The above and other aspects and embodiments of the present invention are illustrated by the following examples. All or part of the features described throughout this specification can be combined in various embodiments of the present invention. The following examples further illustrate the present invention. However, it should be understood that these examples are illustrative and not limiting, and those skilled in the art can make various modifications.

[0522] Example 1: Preparation of the Fc portion The protein amino acid sequences (SEQ ID NOs: 1-68) were back-translated to synthesize the corresponding nucleotide sequences. Appropriate restriction enzyme cleavage sites (XbaI+SalI) were added to both ends of the nucleotide sequences, and the data was cloned into a pWX4.1 expression vector (WuXi Biologics, Shanghai). Each constructed vector incorporated either a signal peptide sequence of human interleukin-2 or a signal peptide sequence of human serum albumin. The pWX4.1 plasmid was transfected into E. coli Top 10 strains (Life Technologies) to amplify the DNA, which was then purified using the PURELINK® HiPURE Plasmid Filter Maxiprep Kit (Life Technologies). Next, the plasmid was transfected into CHO cells (ATCC) using the EXPIFECTAMINE® 293 transfection kit (Life Technologies). After culturing the transfected cells for 7 days, centrifugation and filtration were performed, and the supernatant was collected. Subsequently, the sample was purified using MabSelect Sure Resin (GE Healthcare, Chicago, IL, USA) to obtain purified h-IgG Fc. The purified sample was subjected to 4-12% BisTris SDS-PAGE electrophoresis, with 1-2 μg of sample loaded and stained with Coomassie Brilliant Blue. Each sample was subjected to both reduction (R) and non-reduction (NR) treatments.

[0523] Example 2: Preparation of the Fc portion The amino acid sequences of the proteins (SEQ ID NOs. 69-81) were back-translated to synthesize the corresponding nucleotide sequences. Next, these sequences were recombined into homologous pCDNA3.4 vectors, and the signal peptide sequence of human interleukin-10 was introduced into each constructed vector. The constructed plasmids were electroporated into CHO cells, and the transfected cells were cultured for 7 days. After centrifugation, the precipitate was collected, and the supernatant was collected by filtration. Purification was performed using a protein A affinity chromatography column to obtain purified h-IgG Fc. The purified samples were subjected to Bistrice SDS-PAGE electrophoresis and then stained with coumarcine blue. Each sample was subjected to reduction (R) and non-reduction (NR) treatments.

[0524] Example 3: General synthesis method for h-IgG1 Fc-SH

[0525] [ka]

[0526] In a centrifuge tube, combine 50 mM PB, pH 6.5 buffer, and antibody ((E) m ) solution (30 mg), and 10 mM reducing agent (TCEP) solution (reducing agent / (E) m The molar equivalent ratios were sequentially added (E). m The reaction concentration was controlled to 5 mg / mL, and the reaction was carried out in a metal water bath at a controlled temperature of 22°C for 2 hours. (HS) has partially open disulfide bonds. n -(E) m The following was obtained, where E, m, and n are as defined herein.

[0527] Example 4: General method I for preparing conjugates

[0528] [ka]

[0529] Here, each variable is defined as described herein.

[0530] To the reaction solution containing II-1, 50 mM PB buffer (pH 6.5) and a solution of the compound of formula I-2a (I-2 / II-1 = 7 to 12) were added. The mixture was placed in a metal bath, the reaction temperature was controlled to 22°C, and the mixture was reacted for 1 hour. After the reaction time, the compound of formula I-2a was added to the reaction solution (I-2 / II-1 = 3.5 to 6). The mixture was placed in a metal bath, the reaction temperature was controlled to 22°C, and the mixture was reacted for a further 0.5 hours. After the reaction time, the compound of formula I-2a was added to the reaction solution (I-2 / II-1 = 3.5 to 6). The mixture was placed in a metal bath, the reaction temperature was controlled to 22°C, and the mixture was reacted for a further 1.0 hour to obtain I-3.

[0531] The reaction solution was adjusted to pH=7.5 with 0.5M NaOH, and the reaction temperature was controlled to 22°C in a metal bath. The reaction was carried out for 2 hours until all of I-3 was hydrolyzed to I-1a. After ultrafiltration and centrifugal concentration of the reaction solution, ultrafiltration was performed for solution exchange to prepare the purified target-binding compound. The mean DAR (drug / antibody ratio) of the conjugate was determined by MS analysis.

[0532] Example 5: Synthesis of a drug intermediate (INT-DRUG)

[0533] [ka]

[0534] [ka]

[0535] INT-DRUG-1 (3.0 g, 6.57 mmol) was added to a reaction flask, followed by the addition of tetrahydrofuran (30 mL) and triphenylphosphine (1.9 g, 7.23 mmol). The mixture was then reacted at 30°C for 2 hours with stirring. After the reaction was complete, lithium hydroxide monohydrate (28 mg) and water (1.5 mL) were added, and the resulting solution was stirred at 30°C for a further 16 hours. After confirming the completion of the reaction by TLC, N,N'-di-Boc-1H-1-guanidinylpyrazole (2.1 g, 6.91 mmol) was added, and the mixture was stirred at 30°C for 48 hours. After the reaction was complete, the reaction solution was concentrated and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:1). After purification, INT-DRUG-2 (2.2 g) was obtained as a colorless oily substance. MS:[M+1] + Detected value: 673.40.

[0536] [ka]

[0537] INT-DRUG-2 (2.19 g, 3.25 mmol) was placed in a reaction flask, protected with nitrogen, and then a methanol (8 mL) solution of dry sodium methanol (0.65 mmol) was added. After stirring at room temperature for 10 minutes, the reaction was stopped with hydrochloric acid (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%~100%) to obtain INT-DRUG-3 (1.15 g) as a white solid. MS:[M+1] + Detected value: 547.33.

[0538] [ka]

[0539] 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 mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated, the residue was separated, and the residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, 0%~45%) to obtain INT-DRUG-4 (242.8 mg) as a white solid. MS:[M+1] + Detected value: 573.35.

[0540] [ka]

[0541] 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 the reaction flask. After purging with nitrogen, the reaction was stirred at 40°C for 4 hours. After more than 90% conversion was confirmed by LC-MS detection, water (20 mL) was added to stop the reaction (it could not be left for a long time). The reaction products were separated by reverse-phase column chromatography (acetonitrile / water 0%-90%) and freeze-dried to obtain a white solid INT-DRUG (173 mg). MS:[M+1] + 738.33 was detected.

[0542] Example 6: Synthesis of Intermediate A (inter-A)

[0543] [ka]

[0544] [ka]

[0545] Compound 1 (150 g, 1.43 mol) and triethylamine (289 g, 2.85 mol) were added to the reaction flask, followed by the addition of tetrahydrofuran (1 L). After purging with nitrogen, the reaction was stirred 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 overnight at room temperature. After the reaction was complete, water (3 L) was added to stop the reaction, and the mixture was extracted with ethyl acetate (300 mL x 2). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to recover the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain compound 2 (255 g) as a yellow oil.

[0546] [ka]

[0547] Compound 2 (245 g, 1.02 mol) and carbon tetrabromide (509 g, 1.54 mol) were added to a reaction flask, and methane dichloride (2 L) was added. After purging with nitrogen, 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 hours. After the reaction was complete, the reaction solution was concentrated to recover the 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 oily substance.

[0548] [ka]

[0549] Compound 3 (200 g, crude product) was added to the reaction flask, and then N,N-dimethylformamide (2 L) was added. Benzylamine (28.3 g, 264 mmol) and sodium carbonate (73.0 g, 528 mmol) were added to the reaction solution. The reaction solution was stirred overnight at 60°C. After the reaction was complete, it was cooled to room temperature, water (3 L) was added, and the resulting solution was extracted with ethyl acetate (300 mL x 2). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to recover the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain compound 4 (45 g) as a yellow oily substance.

[0550] [ka]

[0551] Compound 4 (43.0 g, 78.2 mmol) and tetrahydrofuran (450 ml) were added to the reaction flask, and after purging with nitrogen, the mixture was stirred at 0°C. LiAlH (23.7 g, 62.6 mmol) was added to the reaction solution. The reaction solution was stirred at room temperature for 1.5 hours, then heated to reflux and stirred for a further 6 hours. After the reaction was complete, the solution was cooled to 0°C, and sodium decahydrate sulfate (90 g) was added to stop the reaction. The reaction solution was stirred at room temperature for 1 hour. Triethylamine (31.7 g, 31.3 mmol) and Boc anhydrous (68.3 g, 31.3 mmol) were added to the reaction solution, and the resulting solution was stirred at room temperature overnight. After the reaction was complete, water (2 L) was added, followed by extraction with ethyl acetate (300 mL x 2). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the 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] + 510.2.

[0552] [ka]

[0553] Under a nitrogen atmosphere, compound 6 (19 g, 3.73 mmol), Pd / C (2 g), and methanol (400 mL) were added to the reaction flask. The mixture was purged with hydrogen gas several times. The reaction solution was stirred overnight at room temperature. After the reaction was complete, the reaction solution was filtered, and the filter cake was washed with methanol (50 mL). The filtrate was concentrated to recover the 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 oily substance. MS:[M+1] + Detection: 420.2.

[0554] Example 7: Synthesis of intermediate B

[0555] [ka]

[0556] At 0°C, compound 8 (8.4 g, 54 mmol) was gradually added to 110 ml of saturated sodium bicarbonate aqueous solution of compound 7 (5.5 g, 27 mmol). The mixture was stirred at room temperature for 18 hours. After the reaction was complete, the pH of the reaction solution was adjusted to weak acidity using saturated potassium bisulfate aqueous solution. After acid adjustment, the resulting solution was extracted by DCM, and the extract was concentrated by rotation and dried. Purification was performed using a reversed-phase column (water / acetonitrile), and after lyophilization, product 9 (470 mg) was obtained. MS[M+1] + :285.1

[0557] [ka]

[0558] At 0°C, EDCI (201 mg, 1.05 mmol) was added to a solution of 9 (150 mg, 0.53 mmol) and 10 (149 mg, 0.79 mmol) in DCM (20 ml). The reaction solution was stirred at room temperature for 4 hours. After the reaction was complete, the solvent was removed using a rotary vacuum evaporator, and the mixture was purified by silica gel column chromatography (PE:EA = 3:1) to obtain intermediate B (110 mg) as a white solid. MS[M+1] + :451.32

[0559] Example 8: Synthesis of inter C

[0560] [ka]

[0561] 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 was complete, the reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. This was purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain intermediate C (1.34 g) as a colorless oily substance.

[0562] Example 9: Synthesis of Linker 1

[0563] [ka]

[0564] [ka]

[0565] Chlorosulfonyl isocyanate (400 mg, 2.83 mmol) was dissolved in dichloromethane (10 mL), protected with nitrogen, and stirred at 0°C. Bromoethanol (353 mg, 2.83 mmol) dissolved in dichloromethane (5 mL) was added dropwise, and the resulting solution was stirred at 0°C for 1 hour. Then, L1-1 (2.83 mmol) dissolved in dichloromethane (5 mL) was added dropwise. The reaction was stirred at 0°C for 1 hour. After the reaction was complete, dichloromethane (10 mL) was added, and the reaction solution was washed with dilute hydrochloric acid (1 M, 20 mL). The organic phase was dried over anhydrous sodium sulfate. The solvent was removed using a rotary evaporator, and the residue was purified by silica gel column chromatography (PE:EA = 1:1) to obtain crude L1-2 (800 mg) as a yellow oily substance.

[0566] [ka]

[0567] Crude 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 the reaction was complete, dichloromethane (10 mL) was added, and the resulting mixture was washed with dilute hydrochloric acid (1 M, 20 mL). The organic phase was dried over anhydrous sodium sulfate. The solvent was removed using a rotary evaporator, and the residue was purified by silica gel column chromatography (PE:EA = 1:3) to obtain yellow, oily L1-3 (670 mg).

[0568] [ka]

[0569] L1-3 (300 mg, 0.79 mmol), intermediate 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 using a rotary vacuum evaporator, and the residue was purified by reverse-phase column chromatography (acetonitrile / water) to obtain yellow oily L1-4 (190 mg). MS[M+1] + :713.43

[0570] [ka]

[0571] L1-4 (107 mg, 0.15 mmol) was placed in a 5 ml solution of 1,4-dioxane in hydrochloric acid and stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated to obtain crude linker 1. This was used directly in the next step. MS[M+1] + :513.51.

[0572] Example 10: Synthesis of Linker 2

[0573] [ka]

[0574] [ka]

[0575] L2-1 (464 mg, 1.57 mmol), intermediate A (600 mg, 4.06 mmol), potassium carbonate (395 mg, 2.86 mmol), and acetonitrile (10 mL) were added to the reaction flask and stirred at 85°C for 18 hours. After the reaction was complete, the reaction solution was concentrated, and the crude product was purified by reverse-phase column chromatography to obtain colorless oily L2-2 (320 mg). MS[M+1] + :634.31

[0576] [ka]

[0577] L2-2 (320 mg, 0.51 mmol) was dissolved in dichloromethane (10 mL), and hydrochloric acid (4 mL, 4 M in 1,4-dioxane) was added. The reaction was stirred at room temperature for 4 hours, and after the reaction was complete, the mixture was concentrated to obtain Linker 2 (219 mg) as a colorless liquid. MS[M+1] + :434.53

[0578] Example 11: Synthesis of Linker 3

[0579] [ka]

[0580] [ka]

[0581] L3-1 (1 g, 2.7 mmol) and NaH (216 mg, 5.4 mmol) were added to the reaction flask, followed by the addition of tetrahydrofuran (20 mL). After purging with nitrogen, the reaction was stirred at 25°C for 3 hours. After the reaction was complete, water (5 mL) was added to stop the reaction, and the resulting mixture was concentrated to recover the crude product. This was purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain L3-2 (400 mg) as a yellow oily substance.

[0582] [ka]

[0583] L3-2 (1.7 g, 4.16 mmol) and DMAP (1.01 g, 8.32 mmol) were placed in a reaction flask, and dichloromethane (20 mL) was added. After purging with nitrogen, 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 system. The reaction solution was stirred at room temperature for 15 hours. After the reaction was complete, the crude product was recovered by concentration and purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1) to obtain yellow, oily L3-3 (1.1, crude product).

[0584] [ka]

[0585] L3-3 (440 mg, 0.78 mmol) was dissolved in acetonitrile (10 mL) and stirred. Intermediate A (326 mg, 0.78 mmol) and DIPEA (201 mg, 1.56 mmol) were added to the reaction solution and stirred at 80°C for 10 hours. After the reaction was complete, the solvent was removed using a rotary vacuum evaporator. The solution was purified by reverse-phase column chromatography (acetonitrile / water) to obtain L3-4 (310 mg) as a yellow oily substance.

[0586] [ka]

[0587] Compound 4 (247 mg, 0.3 mmol) was dissolved in dichloromethane (10 mL), and a 1,4-dioxane solution in hydrochloric acid (3 mL) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was concentrated to obtain Linker 3 (182 mg) as a colorless oil. MS[M+1] + :610.52

[0588] Example 12: Synthesis of C-Inter1

[0589] [ka]

[0590] [ka]

[0591] 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), added dropwise to the mixture, and stirred at room temperature for 16 hours. The mixture was concentrated under reduced pressure to recover the crude product, 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

[0592] [ka]

[0593] C-Inter-1-1 (240 mg, 0.146 mmol) was dissolved in methanol (5 mL), and an aqueous lithium hydroxide solution (0.55 mmol, 1 mL) was added. After stirring at room temperature for 1 hour, acetic acid (0.3 mL) was added. After low-temperature concentration, the solution was purified by reverse-phase column chromatography (acetonitrile / water) to obtain C-Inter-1-2 (213 mg, 0.131 mmol) as a white solid. MS[M / 2+1] + :815.4

[0594] [ka]

[0595] At 0°C, a solution of C-Inter-1-2 (213 mg, 0.131 mmol) and azido-tetraethylene glycolamine (137 mg, 0.523 mmol) dissolved in MeCN (2 ml) was added to a solution of THPTA (45.5 mg, 0.105 mmol), sodium ascorbate (51.8 mg, 0.262 mmol), and CuSO4 (51.8 mg, 0.262 mmol) dissolved in purified water (2 ml). The reaction solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. After the reaction was complete, C-Inter-1-3 (187 mg) was purified by preparative high-pressure liquid chromatography. MS[M / 2+1] + :946.4

[0596] [ka]

[0597] At 0°C, Inter B (77.3 mg, 0.171 mmol) and DIEA (57.56 mg, 0.445 mmol) were added to a solution of C-Inter-1-3 (270 mg, 0.143 mmol) dissolved in DMF (3 ml), and the reaction solution was stirred at room temperature for 1 hour. After the reaction was complete, 1.5 ml of trifluoroacetic acid was added to the reaction solution, and the mixture was freeze-dried to obtain crude C-Inter-1-4. MS[M / 2+1] + :1079.5

[0598] [ka]

[0599] The crude product of C-Inter-1-4 was dissolved in 30 ml of 10% aqueous trifluoroacetic acid solution, stirred at room temperature for 15 minutes, and then dried using a rotary evaporator. Purification by preparative high-pressure liquid chromatography yielded C-Inter-1 (85 mg). MS[M / 2+1] + :829.4

[0600] Example 13: Synthesis of C-Inter-3

[0601] [ka]

[0602] [ka]

[0603] Linker 2 (219 mg, 0.5 mmol) and DIEA (396 mg, 3 mmol) were dissolved in dichloromethane (10 mL). Next, INT-DRUG (885 mg, 1.2 mmol) was dissolved in dichloromethane (5 mL) and added dropwise to the reaction solution. The reaction solution was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated and purified by reverse-phase column chromatography to obtain C-Inter-3-1 (730 mg) as a white solid. MS[M / 2+1] + :815.8

[0604] [ka]

[0605] C-Inter-3-1 (730 mg, 0.45 mmol) was dissolved in methanol (10 mL). Next, lithium hydroxide monohydrate (112.7 mg, 2.69 mmol) was dissolved in water (3 mL) and added dropwise to the reaction solution. The reaction solution was stirred at room temperature for 4 hours. After the reaction was complete, acetic acid (1 mL) was added. The reaction solution was concentrated and purified by reverse-phase column chromatography to obtain C-Inter-3-2 (290 mg) as a white solid. MS[M / 2+1] + :775.8

[0606] [ka]

[0607] C-Inter-3-2 (290 mg) and azido-tetraethylene glycolamine (196 mg, 0.75 mmol) were dissolved in acetonitrile. Subsequently, 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 solution. After purging with nitrogen, the mixture was stirred at room temperature for 2 hours. The reaction mixture was purified using a reversed-phase column to obtain C-Inter-3-3 (123 mg) as a white solid. MS[M / 2+1] + :907.0

[0608] [ka]

[0609] C-Inter-3-3 (123 mg) and intermediate B (36.6 mg, 0.081 mmol) were dissolved in DMF (7 mL), and DIEA (27.36 mg) was added. The reaction was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was freeze-dried to obtain the crude product of C-Inter-3-4. MS[M+1] + :1040.0

[0610] [ka]

[0611] C-Inter-3-4 (crude product), trifluoroacetic acid (10 mL), and water (0.5 mL) were added to a reaction flask and stirred at room temperature for 30 minutes. The reaction solution was concentrated at low temperature and the solvent was removed. The crude product was purified by preparative high-pressure liquid chromatography to obtain C-Inter-3 (65 mg). MS[M / 2+1] + :789.8

[0612] Example 14: Synthesis of C-Inter-6

[0613] [ka]

[0614] [ka]

[0615] 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 resulting solution was stirred at room temperature for 16 hours, then concentrated under reduced pressure to recover the crude product. This was purified by reverse-phase column chromatography (acetonitrile / water) and lyophilized to obtain a white solid C-Inter-6-1 (397 mg). MS[M / 2+1] + :903.9

[0616] [ka]

[0617] C-Inter-6-1 (440 mg, 0.24 mmol) was dissolved in methanol (6 mL), and an aqueous lithium hydroxide solution (0.48 mmol, 1 mL) was added. After stirring the reaction mixture at room temperature for 1 hour, acetic acid (0.2 mL) was added. After low-temperature concentration, the reaction mixture was purified by reverse-phase column chromatography (acetonitrile / water) to obtain C-Inter-6-2 (310 mg) as a white solid. MS[M / 2+1] + :863.9

[0618] [ka]

[0619] At 0°C, a solution of C-Inter-6-2 (310 mg, 0.18 mmol) and azido-octaethylene glycolamine (188 mg, 0.72 mmol) dissolved in MeCN (2 ml) was added to a solution of THPTA (62.6 mg, 0.14 mmol), sodium ascorbate (71.28 mg, 0.36 mmol), and CuSO4 (57.2 mg, 0.36 mmol) dissolved in purified water (2 ml). The reaction solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. After the reaction was complete, C-Inter-6-3 (176 mg) was purified by preparative high-pressure liquid chromatography. MS[M / 2+1] + :1083.0

[0620] [ka]

[0621] At 0°C, C-Inter-6-3 (176 mg, 0.08 mmol) dissolved in DMF (3 ml) was mixed with Inter B (43.04 mg, 0.09 mmol) and DIEA (30.96 mg, 0.24 mmol), and the reaction solution was stirred at room temperature for 1 hour. After the reaction was complete, 1.5 ml of trifluoroacetic acid was added to the reaction solution, and the mixture was freeze-dried to obtain crude C-Inter-6-4. MS[M / 2+1] + :1216.1

[0622] [ka]

[0623] C-Inter-6-4 was dissolved in 10 ml of 5% aqueous trifluoroacetic acid solution, stirred at room temperature for 15 minutes, and then dried using a rotary vacuum evaporator. Purification by preparative high-pressure liquid chromatography yielded C-Inter-6 (25 mg). MS[M / 2+1] + :967.4

[0624] Example 15: Synthesis of C-Inter-9

[0625] [ka]

[0626] [ka]

[0627] C-Inter-3-3 (155 mg), Inter-C (40.7 mg), and DIEA (34.89 mg) were dissolved in DMF and stirred at room temperature for 2 hours. After the reaction was complete, the solution was freeze-dried to obtain crude C-Inter-9-1. MS[M / 2+1] + :1013.5

[0628] [ka]

[0629] Crude product C-Inter-9-1, trifluoroacetic acid (10 mL), and water (0.5 mL) were added to a reaction flask and stirred at room temperature for 30 minutes. After the reaction was complete, the reaction solution was concentrated at low temperature to remove the solvent, and purified by preparative high-pressure liquid chromatography to obtain C-Inter-9 (26 mg). MS[M / 2+1]+:813.5

[0630] Example 16: Expression and purification of hIgG1-Fc 1. Experimental materials

[0631] [Table 11]

[0632] 2. Expression of hIgG1-Fc 1) The day before transfection (-1 day), cells are measured to the final concentration of 3-4 × 10⁶ 6 The solution was diluted to a viable cell / mL concentration and cultured overnight. 2) Cell density on the day of transfection is 5-6 × 10 6 The cell density should be expressed as cells / mL. The cell viability must be 95% or higher. 3) Following the protocol, 0.8 μg of the pcDNA3.1 plasmid expressing hIgG1-Fc was transfected per 1 mL of cells. 0.8 μg of plasmid was diluted in 40 μL of OptiPRO® SFM medium (4°C) and gently mixed. 4) 3.2 μL of ExpiFectamine® CHO was diluted with 37 μL of OptiPRO® SFM medium (4°C) and gently mixed. 5) Diluted ExpiFectamine® CHO reagent was added to the diluted DNA, gently mixed, and incubated at room temperature for 1-5 minutes. 6) Slowly add the above complex to each shaking flask. 7) The cells were placed in a shaker under conditions of 37°C and 8% CO2, and the rotation speed was set to 125 rpm. 8) After incubating the cells for 20 hours, 10 μL of ExpiCHO transfection enhancer and 400 μL of ExpiCHO feed were added to each 1 ml of cells, homogeneously mixed, and then added to each culture flask. 9) Cell supernatant was collected after 8 days of culture.

[0633] 3. Purification of hIgG1-Fc 1) Collection of the supernatant The transfected Fc protein fermentation broth, having reached harvest conditions, was transferred to a 50 ml centrifuge tube labeled with the order number and name, and centrifuged at 10,000 rpm for 5 minutes. After centrifugation, the supernatant was poured into a new 50 ml centrifuge tube labeled with the name, and this process was repeated until all supernatant from the same Fc fermentation broth had been centrifuged and collected. 2) Pretreatment The gravity column was immersed in a 0.5 M NaOH solution. Pro A packing solution was added to the empty gravity column. Once the solution in the gravity column had finished dripping, a 0.1 M NaOH solution was added to the column. Once the solution in the gravity column had finished dripping, pure water was added to the column. 3) Equilibriumization and sample injection Equilibration was performed by adding 5-10 column volumes of Pro A equilibration buffer to a Pro A column. The supernatant (to be purified) after centrifugation was loaded onto the Pro A column. The supernatant that passed through the column was collected in a 250 ml clean shake flask. After the entire supernatant had passed through the column, 5-10 column volumes of Pro A equilibration buffer were added and held until all the buffer flowed out from the bottom of the column. 4) Protein elution The bottom of the washed column was sealed with a clean stopper. Two to three times the column volume of Pro-A / G elution buffer was added to the washed, bottom-sealed column. Using a pipette, the packing material in the column filled with elution buffer was completely suspended and allowed to stand for 5 minutes. As preparation for elution, a new recovery tube was taken out, and a certain amount of neutralizing Tris solution was added to prevent protein denaturation. The plug was removed, and the liquid was collected in the recovery tube. After measuring the protein concentration, the liquid was stored in a refrigerator at -80°C.

[0634] 4. Characterization of hIgG1 Fc Based on the Fc sequence information described herein, the target Fc was obtained through the expression and purification steps provided in this example.

[0635] [Table 12]

[0636] Example 17: Expression and purification of hIgG1-Fc hIgG1-Fc (SEQ ID NO: 69) was prepared and expressed by the method described in Example 16. 2. Purification of hIgG1-Fc Experimental method: Purification of protein A using affinity chromatography column 1) Equilibration of the chromatography column: 1×PBS, flow rate 1 ml / min, 20 ml 2) Sample injection: Flow rate 1 ml / min 3) Column washing: 1×PBS, flow rate 1 ml / min, 20 ml 4) Elution: Sodium acetate buffer (pH 3.2) was eluted at a rate of 1 ml / min and collected in separate tubes (approximately 500 μl per tube). A total of 10 tubes were collected, and the absorbance values ​​at 280 nm were measured using a NanoDrop device. 5) Dialysis: The high-concentration protein was transferred to a dialysis bag and immersed in a beaker filled with 1x PBS for dialysis.

[0637] 3. Characterization of hIgG1 Fc Based on the Fc sequence information described herein, the target Fc was obtained through the expression and purification steps mentioned in this example.

[0638] [Table 13]

[0639] Example 18: Synthesis and Characterization of Conjugate 1 1. Experimental materials and methods 1.1 Equipment / Equipment

[0640] [Table 14]

[0641] 1.2 Reagents / Solutions

[0642] [Table 15]

[0643] 1.3 Experimental Method

[0644] [ka]

[0645] 1.3.1 Ultrafiltration-1 1) Pretreatment of centrifuge tubes for ultrafiltration Two 15 mL 10 kDa ultrafiltration centrifuge tubes were filled with 0.1 M NaOH solution, and the tubes were centrifuged at 4000 G for 10 minutes. This process was repeated three times. Ultrapure water was added to an ultrafiltration centrifuge tube and centrifuged twice at 4000G for 10 minutes each time. A 50 mM PB, pH 6.5 buffer solution was added to an ultrafiltration centrifuge tube and centrifuged at 4000 G for 10 minutes. This process was repeated until the pH of the permeate reached approximately 6.5. 2) Antibody ultrafiltration and liquid exchange Fc(SEQ ID NO: 67) antibody was added to a processed ultrafiltration centrifuge tube. The solution volume in the ultrafiltration tube was replenished to 12 mL with 50 mM PB, pH 6.5 buffer, and the tube was centrifuged at 4000 G for 10 minutes. This procedure was repeated four times. After the solution change, the sample was collected and the antibody concentration was detected. 3) Antibody concentration detection Detection was performed using the Protein A280 method in NanoDrop One, with the sample category "Other Proteins E 1%" selected. The extinction coefficient E 1% L / gm-cm was set to 13.5, and the baseline correction wavelength was 340 nm.

[0646] A 50 mM PB, pH 6.5 buffer solution was used as a blank. 2.5 μL of the sample was collected and tested, and the average of the three detected results was taken as the sample concentration.

[0647] 1.3.2 Reduction Reaction 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 sequentially added to a centrifuge tube to control the antibody reaction concentration to 5 mg / mL. The reaction was then placed in a metal water bath, the reaction temperature was controlled to 22°C, and the reaction was allowed to proceed for 2 hours.

[0648] 1.3.3 Coupling Reactions After allowing the reduction reaction to proceed for 2 hours, 50 mM PBS, pH 6.5 buffer, and 10 mg / mL C-Inter-1 solution (C-Inter / Fc molar equivalent ratio 7) were added to the reaction solution. The final concentration of the reaction was adjusted to 2 mg / mL, and the reaction was carried out in a metal water bath for 1 hour while controlling the reaction temperature at 22°C.

[0649] After the reaction time had elapsed, 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. The reaction was then carried out in a metal bath, with the reaction temperature controlled at 22°C for a further 0.5 hours.

[0650] After the reaction time had elapsed, a 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, and the reaction was carried out in a metal bath for a further 1.0 hour while controlling the reaction temperature at 22°C.

[0651] 1.3.4 Hydrolysis reaction A 0.5 M NaOH solution was added to the reaction solution to adjust the pH to approximately 7.5. The reaction was then placed in a metal bath, and the reaction was allowed to proceed for another 2 hours while controlling the reaction temperature at 22°C to obtain conjugate 1.

[0652] 1.3.5 Ultrafiltration-2 1) Pretreatment of centrifuge tubes for ultrafiltration Two 15 mL 10 kDa ultrafiltration centrifuge tubes were filled with 0.1 M NaOH solution, and the mixture was centrifuged at 4000 G for 10 minutes three times. Ultrapure water was added to an ultrafiltration centrifuge tube and centrifuged at 4000G for 10 minutes, and this was repeated twice. 10 mM His-HCl, pH 5.5 buffer was added to the ultrafiltration centrifuge tube and centrifuged at 4000G for 10 minutes. This was repeated until the pH of the permeate was approximately 5.6. 2) Ultrafiltration and exchange of antibodies Conjugate 1 was added to a pre-treated ultrafiltration centrifuge tube. The solution volume in the ultrafiltration tube was replenished to 12 mL with 10 mM His-HCl, pH 5.5 buffer, and the tube was centrifuged at 4000 G for 10 minutes. This procedure was repeated six times. The solution after ultrafiltration was filtered through a 0.22 μm PES filter membrane. After the solution was changed, the antibody concentration was detected. 3) Antibody concentration detection Measurements were performed using the Protein A280 method with the sample category set to "Other Proteins E 1%" on NanoDrop One. The extinction coefficient E 1% L / gm-cm was set to 13.5, and the baseline correction wavelength was set to 340 nm.

[0653] A 10 mM His-HCl, pH 5.5 buffer solution was used as a blank, and a 2.5 μL sample was taken and detected. The average of three detection results was used as the sample concentration.

[0654] Based on the detected sample concentration and volume, the sample was diluted to a concentration of approximately 10 mg / mL using 10 mM His-HCl, pH 5.5 buffer, and the diluted sample concentration was re-detected.

[0655] 2. Experimental Results and Analysis 2.1 Detection of DAR values ​​using LC-MS The DAR value of the sample was detected by LC-MS. The relevant method conditions are shown in the table below.

[0656] [Table 16]

[0657] [Table 17]

[0658] The MS-DAR values ​​of the samples are shown in the table below. According to the MS detection results, the MS-DAR value of conjugate sample 1 was 4.0. After sugar cleavage and reduction treatment, the sample peak appeared as a single-chain peak. C0, C1, C2, and C3 represent the molecular weights of the conjugates with 0, 1, 2, and 3 low-molecular-weight molecules attached, respectively.

[0659] [Table 18]

[0660] The MS detection spectrum of the sample is shown in Figure 1.

[0661] 2.2 Purity testing by SEC-HPLC The purity of the sample was tested by SEC-HPLC, and the relevant method conditions are shown in the table below.

[0662] [Table 19]

[0663] The results of detecting low molecular weight residues in the sample are shown in Figure 2. Based on the SEC detection results, the purity of conjugate 1 was 98.21%.

[0664] 2.3 Detection of Residual Low Molecules The residual free drug levels (mol / mol, free drug / bound drug) in the samples were measured by RP-HPLC. First, two standards (2% and 5%) containing free drug and Fc were prepared. The proportion of free drug was determined by comparing the EIC signal peak area with the peak area of ​​residual free drug in the conjugated sample. The relevant method conditions are outlined in the table below.

[0665] [Table 20]

[0666] Preparation of standard solutions: C Fc(配列番号67) = 1 mg / mL, C Fc(配列番号69) =1 mg / mL; the DAR values ​​of the samples were 4.0 and 6.0, respectively. MW Fc(配列番号67) = 55414 g / mol, MW Fc(配列番号69) = 56000 g / mol, MW C-Inter-1 = 1656 g / mol; M mAb = 1 / MW mAb M 総薬物 =DAR / MW mAb ; Molar concentration of a 2% standard solution = DAR × M mAb ×2%; Molar concentration of a 5% standard solution = DAR × M mAb ×5%; The weight of the low molecular weight compound to be added to 100 μL of 2% standard solution (2% m C-Inter-1 ) = number of moles of 2% standard solution × 100 μL × MW C-Inter-1 ; The weight of the low molecular weight compound added to 100 μL of 5% standard solution (5% m C-Inter-1 ) = number of moles of 5% standard solution × 100 μL × MW C-Inter-1

[0667] The calculated weight of the low molecular weight was added to 100 μL of 1 mg / mL antibody solution to obtain a standard solution.

[0668] Figure 3 shows the results of detecting small molecule residues in the samples. Here, the AKSZ-2627-0.01 mg / mL sample is the AKSZ2627 small molecule sample, AKSZ-2627-2% and AKSZ-2627-5% are samples obtained by adding 2% and 5% mol / mol of the low molecule compound to the corresponding Fc, respectively, and C060-7607B-230424 is the conjugate 1 sample. From the RP detection results, no peaks of the low molecule compound were detected by RP-HPLC in either sample. Compared with antibody samples to which 2 mol% and 5 mol% of the low molecule compound were added, the residue in both samples was less than 2 mol%.

[0669] 2.4 Detection of endotoxin residues The amount of endotoxin residue in the sample was detected using the following procedure: 1) Add 1 vial of endotoxin work standard solution to 1 mL of BET water, vortex stir for 15 minutes, and then prepare for future use by 4λ(E 4λ ) and 2λ(E 2λ It was diluted in stages. 2) Preparation of test solution: Dilute the sample solution stepwise with Tris buffer in a 1:80 ratio (S) 80 ) and 1:160(S 160 It was diluted to the ratio shown and used. 3) Solution preparation for each group Test Group A:S 160 solution; Test sample positive control group B:S 80 E in 1 volume of solution 4λ Add 1 volume of solution and mix well. 160 E 2λ Prepared; Positive control group C:E 2λ solution; Negative control group D: BET water. 4) Reconstitution of Limulus reagent: Each vial of Limulus reagent was prepared by adding 0.1 mL of BET water. 5) After reconstituting the Limulus reagent, 0.1 mL of each of the four group solutions prepared in step 3) was added to the Limulus reagent. Each group received two Limulus reagents in parallel. 6) Culturing: The mixed ampoule was placed in a test tube incubator preheated to 37°C and incubated for 60 minutes.

[0670] Test results: The concentration of conjugate 1 was 10.13 mg / mL, and the amount of endotoxin residue in the sample was less than 1 EU / mg.

[0671] Example 19: Synthesis and Characterization of Conjugate 2 1. Experimental materials and methods 1.1 Equipment / Equipment This is the same as in Example 18. 1.2 Reagents / Test Solutions This is the same as in Example 18. 1.3 Experimental Method

[0672] [ka]

[0673] 1.3.1 Ultrafiltration-1 1) Pretreatment of centrifuge tubes for ultrafiltration Two 15 mL 10 kDa ultrafiltration centrifuge tubes were filled with 0.1 M NaOH solution, and the tubes were centrifuged at 4000 G for 10 minutes. This was repeated three times. Ultrapure water was added to the ultrafiltration centrifuge tubes, and the tubes were centrifuged at 4000 G for 10 minutes twice. A 50 mM PB, pH 6.5 buffer solution was added to the ultrafiltration centrifuge tubes, and the tubes were centrifuged at 4000 G for 10 minutes. This was repeated until the pH of the permeate was approximately 6.5. 2) Antibody ultrafiltration and liquid exchange Fc(SEQ ID NO: 67) antibody was added to a processed ultrafiltration centrifuge tube. The solution volume in the ultrafiltration tube was replenished to 12 mL with 50 mM PB, pH 6.5 buffer, and the tube was centrifuged at 4000 G for 10 minutes. This procedure was repeated four times. After the solution change, the sample was collected and the antibody concentration was detected. 3) Antibody concentration detection Detection was performed using the Protein A280 method with NanoDrop One, and the sample category "Other Proteins E 1%" was selected. The extinction coefficient E 1% L / gm-cm was set to 13.44, and the baseline correction wavelength was set to 340 nm. A 50 mM PB, pH 6.5 buffer solution was used as a blank, and a 2.5 μL sample was taken and detected. The average of three detection results was used as the sample concentration.

[0674] 1.3.2 Reduction Reaction 50 mM PBS, pH 6.5 buffer, Fc (SEQ ID NO: 67) antibody solution (30 mg), and 10 mM TCEP solution (TCEP / Fc molar equivalent ratio of 10) were sequentially added to a centrifuge tube to control the antibody reaction concentration to 5 mg / mL. The reaction was then placed in a metal water bath, the reaction temperature was controlled to 22°C, and the reaction was allowed to proceed for 2 hours.

[0675] 1.3.3 Coupling Reactions After allowing the reduction reaction to proceed for 2 hours, 50 mM PBS, a pH 6.5 buffer, and a 10 mg / mL C-Inter-1 solution (with a molar equivalent ratio of C-Inter-1 / Fc of 12) were added to the reaction solution. The final concentration of the reaction was adjusted to 2 mg / mL, and the reaction was placed in a metal bath to control the reaction temperature to 22°C and allowed to proceed for 1 hour.

[0676] After the reaction time had elapsed, a 10 mg / mL C-Inter-1 solution with a C-Inter-1 / Fc molar equivalent ratio of 6.0 was further added to the reaction solution. The reaction was carried out in a metal bath, the reaction temperature was controlled to 22°C, and the reaction was allowed to proceed for a further 0.5 hours.

[0677] After the reaction time had elapsed, 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 was carried out in a metal bath, with the reaction temperature controlled at 22°C, for a further 1.0 hour.

[0678] 1.3.4 Hydrolysis reaction A 0.5 M NaOH solution was added to the reaction solution to adjust the pH to approximately 7.5. The reaction was then placed in a metal bath, the reaction temperature was controlled to 22°C, and the reaction was allowed to continue for another 2 hours to obtain conjugate 2.

[0679] 1.3.5 Ultrafiltration-2 1) Pretreatment of centrifuge tubes for ultrafiltration Two 15 mL 10 kDa ultrafiltration centrifuge tubes were filled with 0.1 M NaOH solution, and the tubes were centrifuged at 4000 G for 10 minutes. This was repeated three times. Ultrapure water was added to the ultrafiltration centrifuge tubes, and the tubes were centrifuged at 4000 G for 10 minutes. This was repeated twice. A 10 mM His-HCl, pH 5.5 buffer solution was added to the ultrafiltration centrifuge tubes, and the tubes were centrifuged at 4000 G for 10 minutes. This was repeated until the pH of the permeate was approximately 5.6. 2) Ultrafiltration and exchange of antibodies Conjugate 2 was added to a processed ultrafiltration centrifuge tube. The solution volume in the ultrafiltration tube was replenished to 12 mL with 10 mM His-HCl buffer (pH 5.5), and the tube was centrifuged at 4000 G for 10 minutes. This procedure was repeated six times. The ultrafiltrate was filtered through a 0.22 μm PES filter membrane. After the solution was changed, the antibody concentration was detected. 3) Antibody concentration detection Detection was performed using the NanoDrop One Protein A280 method, and the sample category "Other Proteins E 1%" was selected. The extinction coefficient E 1% L / gm-cm was set to 13.44, and the baseline correction wavelength was set to 340 nm.

[0680] A 10 mM His-HCl, pH 5.5 buffer solution was used as a blank, and a 2.5 μL sample was taken and detected. The average of three detection results was used as the sample concentration.

[0681] Based on the detected sample concentration and volume, the sample was diluted with 10 mM His-HCl, pH 5.5 buffer to a concentration of approximately 10 mg / mL, and the diluted sample concentration was re-detected.

[0682] 2. Experimental Results and Analysis 2.1 Detection of LC-MS DAR values The method was the same as in Example 18, and the MS-DAR value of the two conjugate samples was 6.0.

[0683] The MS detection spectrum of the sample is shown in Figure 4. After sugar removal and reduction treatment, the sample peak appeared as a single-chain peak. C0, C1, C2, and C3 represent the molecular weight of the sample with 0, 1, 2, and 3 low-molecular-weight molecules attached, respectively.

[0684] [Table 21]

[0685] 2.2 Purity detection by SEC-HPLC The method was the same as in Example 18. The SEC detection results are shown in Figure 5. The purity of Conjugate 2 was 94.47%.

[0686] 2.3 Detection of low molecular weight residues by RP-HPLC The method was the same as in Example 18, and as shown in Figure 6, the low molecular weight residue was less than 2%.

[0687] 2.4 Detection of endotoxin residues The method was the same as in Example 18. The concentration of Conjugate 2 was 10.26 mg / mL, and the endotoxin residue in the sample was less than 1 EU / mg.

[0688] Example 20: Preparation of Conjugate 3 1. Experimental materials and methods 1) Experimental apparatus

[0689] [Table 22]

[0690] 2) Reagents / Solutions

[0691] [Table 23]

[0692] 2. Experimental Method

[0693] [ka]

[0694] 1) Ultrafiltration-1 (1) Antibody ultrafiltration and solution exchange Fc (SEQ ID NO: 71) was added to an ultrafiltration centrifuge tube. The solution volume in the ultrafiltration tube was replenished to 12 mL with 50 mM PB buffer (pH 6.5), and centrifugation was repeated four times at 4500 G for 10 minutes each. The sample was collected, and the antibody concentration was detected. (2) Detection of antibody concentration Detection was performed using the Protein A280 method with NanoDrop One, selecting the sample category "Other Proteins E 1%". The extinction coefficient E 1% L / gm-cm was set to 13.44 (7607H), and the baseline correction wavelength was set to 340 nm. A 2.5 μL sample was taken from a 50 mM PB, pH 6.5 buffer as a blank, and detection was performed. The average of three detection results was taken as the sample concentration.

[0695] 2) Reduction reaction 50 mM PB, pH 6.5 buffer, the corresponding antibody solution (28 mg), and 10 mM TCEP solution (TCEP / mAb molar equivalent ratio of 20) were sequentially added to a centrifuge tube to control the antibody reaction concentration to 5 mg / mL. The tube was placed in a metal water bath, the reaction temperature was controlled to 25°C, and the reaction was allowed to proceed for 2 hours. In this specification, the term "equivalent" refers to molar equivalent unless otherwise specified or unless it is inconsistent with the context.

[0696] 3) Coupling reaction 1. After allowing the reduction reaction to proceed for 2 hours, a 10 mg / mL C-Inter-3 solution (drug / mAb equivalent ratio 10) was added to the reaction solution. The reaction was placed in a metal water bath and allowed to proceed for 1 hour while controlling the reaction temperature at 25°C. A 10 mg / mL C-Inter-3 aqueous solution (AKSZ2665 aqueous solution) was repeatedly added at 1-hour intervals (drug / mAb equivalent ratio 10). Addition was continued until the total reaction equivalent reached 50 (0.5 M NaOH was added to adjust the pH to approximately 6.5). After the reaction was complete, the pH of the reaction solution was adjusted to 7.0-7.5 with 0.5 M NaOH, and the reaction was placed in a metal bath and allowed to proceed for a further 2 hours at 25°C.

[0697] 4) Ultrafiltration (1) Antibody ultrafiltration and liquid exchange 3. The conjugate sample was added to the processed ultrafiltration centrifuge tube. The solution volume in the ultrafiltration tube was replenished to 12 mL with 10 mM His-HCl buffer (pH 5.5), and the tube was centrifuged at 4000 G for 10 minutes. This procedure was repeated 12 times. The ultrafiltrate was filtered through a 0.22 μm PES filter membrane. The antibody concentration was then detected after the solution change. (2) Detection of antibody concentration Detection was performed using the NanoDrop One Protein A280 method, and the sample category "Other Proteins E 1%" was selected. The extinction coefficient E 1% L / gm-cm was set to 13.44, and the baseline correction wavelength was set to 340 nm.

[0698] A 10 mM His-HCl, pH 5.5 buffer solution was used as a blank, and a 2.5 μL sample was taken and detected. The average of three detection results was used as the sample concentration.

[0699] 5) Experimental results and analysis (1) Detection of DAR values ​​by LC-MS The DAR values ​​of the samples were detected by LC-MS, and the relevant method conditions are shown in the table below.

[0700] [Table 24]

[0701] [Table 25]

[0702] The MS-DAR value detection results for the samples are shown in the table (figure) below. From the MS detection results, the MS-DAR value of sample C060-7607H-2665-240125 (conjugate 3) was 5.9.

[0703] [Table 26]

[0704] Note: Formula for calculating drug load distribution: Drug load distribution (%) = (Single-chain peak area with drug load n / Sum of two single-chain peak areas) × 100 (%) Here, n represents the drug loading capacity, and a single chain consists of heavy chain constant domain 2, heavy chain constant domain 3, and a 1 / 2 hinge region (CH2+CH3+1 / 2Hi); DAR value calculation formula: Single-chain weighted peak area = (drug loading n × drug load distribution rate %) Drug-to-antibody ratio = 2 × Σ (weighted peak area of ​​single chain)

[0705] The MS detection spectrum of the sample is shown in Figure 8:

[0706] (2) Purity test by SEC-HPLC The purity of the sample was tested by SEC-HPLC, and the relevant method conditions are shown in the table below.

[0707] [Table 27]

[0708] The purity test results of the sample are shown in Figure 9. From the SEC test results, it was confirmed that the purity of C060-7607H-2665-240125 (Conjugate 3) is 97.50%.

[0709] (3) Detection of low molecular weight residues by RP-HPLC The residual free drug levels (mol / mol, free drug / bound drug) in the samples were measured by RP-HPLC. First, two standards (2% and 5%) containing free drug and Fc were prepared. The proportion of free drug was determined by comparing the EIC signal peak area with the peak area of ​​residual free drug in the conjugated sample. The relevant method conditions are outlined in the table below.

[0710] [Table 28]

[0711] Preparation of standard solutions: C 7607H = 1 mg / mL; the DAR value of the sample is 5.9; MW 7607H = 56000 g / mol, MW 薬物 = 1578.63 g / mol; M mAb = 1 / MW mAb M 総薬物 =DAR / MW mAb ; Number of moles in a 2% standard solution = DAR × MmAb × 2%; Number of moles in a 5% standard solution = DAR × MmAb × 5%; The weight of low molecular weight (2% m) added to 100 μL of 2% standard solution 薬物 ) = number of moles of 2% standard solution × 100 μL × molecular weight 薬物 ; The weight of low molecular weight (5% m) added to 100 μL of 5% standard solution 薬物 ) = number of moles of 5% standard solution × 100 μL × molecular weight 薬物

[0712] The calculated weight of the low molecular weight compound was added to 100 μL of 1 mg / mL antibody solution to obtain a standard solution.

[0713] Figure 10 shows the detection results for low molecular weight residues in the sample. The RP detection results showed that the residue in this sample was less than 2% mol / mol, compared to antibody samples to which 2% mol / mol and 5% mol / mol of low molecular weights were added by RP-HPLC.

[0714] Example 21: Preparation of Conjugate 4 1. Experiment Overview In this experiment, disulfide bonds between antibody chains were cleaved using the reducing agent TCEP. Subsequently, a Michael addition reaction was performed between -SH and maleimide on the linker-payload to obtain the desired coupling product DAR6. The DAR value of the coupling product was detected by LC-MS, the purity of the coupling product was tested by SEC-HPLC, and low molecular weight residues in the coupling product were detected by RP-HPLC.

[0715] [ka]

[0716] 2. Summary of experimental results

[0717] [Table 29]

[0718] 3. Experimental materials and methods 3.1 Equipment / Equipment

[0719] [Table 30]

[0720] 3.2 Reagents / Solutions

[0721] [Table 31]

[0722] 3.3 Experimental Method 3.3.1 Ultrafiltration-1 1) Pretreatment of centrifuge tubes for ultrafiltration A 0.1 M NaOH solution was added to a 15 mL 10 kDa ultrafiltration centrifuge tube, and the mixture was centrifuged at 4500 G for 10 minutes three times. Ultrapure water was added to the ultrafiltration centrifuge tube, and the mixture was centrifuged at 4500 G for 10 minutes, repeating this process twice. 50 mM phosphate buffer (PB) with a pH of 6.5 was added to the ultrafiltration centrifuge tube, and the mixture was centrifuged at 4500 G for 10 minutes. This process was repeated until the pH of the permeate reached approximately 6.5. 2) Ultrafiltration and exchange of antibodies 7607H antibody (SEQ ID NO: 71) was added to a pre-treated ultrafiltration centrifuge tube. The solution volume in the ultrafiltration tube was replenished to 12 mL with 50 mM PB, pH 6.5 buffer, and the tube was centrifuged at 4500 G for 10 minutes. This procedure was repeated four times, and the samples were filtered through a 0.22 μm filter membrane. The samples were collected, and the antibody concentration was detected. 3) Antibody concentration detection Detection was performed using the NanoDrop One Protein A280 method, with the sample category "Other Protein E 1%" selected. The extinction coefficient E 1% L / gm-cm was set to 13.44 (7607H), and the baseline correction wavelength was set to 340 nm. A 2.5 μL sample was taken from a 50 mM PB, pH 6.5 buffer as a blank, and detection was performed. The average of three detection results was taken as the sample concentration.

[0723] 3.3.2 Reduction reaction 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 sequentially added to a centrifuge tube to achieve an antibody reaction concentration of 5 mg / mL. The reaction was placed in a metal water bath and the reaction temperature was controlled at 22°C for 2 hours.

[0724] 3.3.3 Coupling Reactions and Hydrolysis Reactions After the reduction reaction proceeded for 2 hours, 50 mM PB, pH 6.5 buffer, and a 10 mg / mL aqueous solution of C-Inter-1 (AKSZ2627 aqueous solution) with a low molecular weight / mAb equivalent ratio of 10 (drug / mAb equivalent ratio of 10) were added to the reaction solution. The final concentration of the reaction was controlled to 2 mg / mL, and the reaction was placed in a metal bath, with the reaction temperature controlled to 22°C, and allowed to react for 1 hour. After the reaction time, a 10 mg / mL aqueous solution of C-Inter-1 (AKSZ2627 solution) with a drug / mAb equivalent ratio of 5 was further added to the 7607H antibody reaction solution. The reaction was placed in a metal bath, with the reaction temperature controlled to 22°C, and allowed to react for a further 2 hours.

[0725] 3.3.4 Ultrafiltration-2 1) Pretreatment of centrifuge tubes for ultrafiltration 0.1 M NaOH solution was added to a 15 mL 10 kDa ultrafiltration centrifuge tube, and the mixture was centrifuged at 4500 G for 10 minutes three times. Ultrapure water was added to the ultrafiltration centrifuge tube, and the mixture was centrifuged at 4500 G for 10 minutes, repeating this process twice. 10 mM His-HCl buffer with a p of 5.5 was added to the ultrafiltration centrifuge tube, and the mixture was centrifuged at 4500 G for 10 minutes. This process was repeated until the pH of the permeate reached approximately 5.5. 2) Ultrafiltration and exchange of antibodies The conjugated sample was added to a pre-treated ultrafiltration centrifuge tube. The solution volume in the ultrafiltration tube was replenished to 12 mL with 10 mM His-HCl, pH 5.5 buffer, and the tube was centrifuged at 4500 G for 10 minutes. This procedure was repeated eight times. The ultrafiltrate was filtered through a 0.22 μm PES filter. The antibody concentration was then detected after the solution exchange. 3) Antibody concentration detection Detection was performed using the Protein A280 method of NanoDrop One, and the sample category "Other Protein E 1%" was selected. The extinction coefficient E 1% L / gm-cm was set to 13.44, and the baseline correction wavelength was set to 340 nm. A 2.5 μL sample was taken using 10 mM His-HCl, pH 5.5 buffer as a blank, and detection was performed. The average of the three detection results was taken as the sample concentration. Based on the detected sample concentration and sample volume, the sample was diluted to a concentration of approximately 10 mg / mL using 10 mM His-HCl, pH 5.5 buffer, and the diluted sample concentration was re-detected.

[0726] 4. Experimental Results and Analysis 4.1 Detection of DAR values ​​using LC-MS The DAR value of the sample was detected by LC-MS, and the relevant method conditions are shown in the table below.

[0727] [Table 32]

[0728] [Table 33]

[0729] The detection results of the MS-DAR values ​​of the samples are shown in the table below. According to the MS detection results, the MS-DAR value of conjugate sample 4 (internal number C060-7607H-231008) was 5.9.

[0730] [Table 34]

[0731] Note: Formula for calculating drug load distribution: Drug load distribution (%) = (Single-chain peak area with drug load n / Sum of two single-chain peak areas) × 100 (%) Here, n represents the drug loading capacity, and a single chain consists of heavy chain constant domain 2, heavy chain constant domain 3, and a 1 / 2 hinge region (CH2+CH3+1 / 2Hi). DAR value calculation formula: Single-chain weighted peak area = (drug loading n × drug load distribution rate %) Antibody-to-drug ratio = 2 × Σ (single-chain weighted peak area)

[0732] The MS detection spectrum of the sample is shown in Figure 11.

[0733] 4.2 Purity testing by SEC-HPLC The purity of the sample was tested by SEC-HPLC, and the relevant method conditions are shown in the table below.

[0734] [Table 35]

[0735] The purity test results of the sample are shown in Figure 12. The SEC test results showed that the purity of Conjugate 4 (C060-7607H-231008) was 98.57%.

[0736] 4.3 Detection of low molecular weight residues by RP-HPLC The residual free drug levels (mol / mol, free drug / bound drug) in the samples were measured by RP-HPLC. First, two standards (2% and 5%) containing free drug and Fc were prepared. The proportion of free drug was determined by comparing the EIC signal peak area with the peak area of ​​residual free drug in the conjugated sample. The relevant method conditions are outlined in the table below.

[0737] [Table 36]

[0738] Preparation of standard solutions: C 7607H = 1 mg / mL; the DAR value of the sample is 5.9; MW 7607H = 56000 g / mol, MW 薬物 = 1590.79 g / mol; M mAb = 1 / MW mAb M 総薬物 =DAR / MW mAb ; Number of moles in a 2% standard solution = DAR × M mAb ×2%; Number of moles in a 5% standard solution = DAR × M mAb ×5%; The weight of the low molecular weight compound added to 100 μL of 2% standard solution (2% m 薬物 ) = number of moles of 2% standard solution × 100 μL × MW 薬物 ; The weight of the low molecular weight compound added to 100 μL of 5% standard solution (5% m 薬物 ) = number of moles of 5% standard solution × 100 μL × MW 薬物

[0739] The calculated weight of the low molecular weight compound was added to 100 μL of 1 mg / mL antibody solution to obtain a standard solution.

[0740] Figure 13 shows the detection results for low molecular weight residues in the sample. No low molecular weight peaks were detected from the RP detection results, and it was confirmed that the sample residue was less than 2% mol / mol compared to antibody samples to which 2% mol / mol and 5% mol / mol of low molecular weights were added by RP-HPLC.

[0741] 4.4 Detection of endotoxin residues Test results: Based on comparison with a 1 EU / mg positive control, the endotoxin residue in the sample was less than 1 EU / mg.

[0742] Example 22: In vitro anti-influenza virus activity of the conjugate (inhibition test of cell damage caused by influenza virus H1N1) 1. Experimental Method An in vitro anti-IAV / IBV activity assay based on cytopathic effects quantitatively detected the activity of compounds against IAV / IBV-induced cytopathic effects using MDCK cell lines. 2. Experimental materials

[0743] [Table 37]

[0744] 3. Experimental Procedure 1) Digest MDCK cells and prepare 2 × 10⁶ cells in DMEM medium containing 2% FBS and 1% P / S. 5 The solution was diluted to a concentration of / mL. Then, cells were seeded at 50 μL / well into a 96-well plate and cultured overnight in an incubator. 2) First, the compound was diluted to 2 μM, and then repeatedly diluted threefold from 2 μM, for a total of eight dilutions. 5 μL of the diluted compound was added to each well and incubated in an incubator for 1 hour. 3) Influenza virus was diluted to 2222 pfu / mL in DMEM medium containing 2% FBS, 1% P / S, and 4 μg / mL TPCK-treated trypsin, and 45 μL / well was added to each well. Simultaneously, a control group containing the virus but no compound and a cell control group containing neither the compound nor the virus were established. The plates were placed in an incubator and cultured for 4 days. 4) 50 μL of Cell-titer Glo was added to each well, and chemiluminescence was detected using a microplate reader.

[0745] 4. Data analysis: The viral inhibition rate of the compounds was calculated. Inhibition rate = (Measured value of sample - Mean value of virus control group) / (Mean value of cell control group - Mean value of virus control group) × 100%

[0746] Using the calculated inhibition rate, the inhibition curve was fitted using the log(inhibitor) vs. response-variable gradient (4 parameters) function of the GraphPad Prism 8 software's nonlinear regression (curve fitting) feature, and the EC (Effective Computation) was calculated. 50 The value was calculated.

[0747] Reference molecules A and B were prepared according to the specific procedures described in Example 156 (conjugate 45b) and Example 188 (conjugate 46) of Patent WO2021046549A1.

[0748] The purity of reference molecule A was 99.2% (SEC-HPLC), and its DAR value was 4.26.

[0749] The purity of reference molecule B was 98.8% (SEC-HPLC), and its DAR value was 5.87.

[0750] Summary of results: Summary of Results

[0751] [Table 38]

[0752] The data revealed that the tested conjugate molecules exhibited superior in vitro anti-H1N1 virus activity. Compared to reference molecules A and B, complex 1 showed an 8.8-fold and 5.9-fold increase in activity, respectively. Complex 2 showed a 15.8-fold and 10.5-fold increase in activity, respectively. Complexes 3 and 4 showed a 10.0-fold and 6.7-fold increase in activity, respectively.

[0753] This high in vitro antiviral activity suggests that the conjugate molecule of the present invention exhibits excellent antiviral activity in animal and clinical human studies.

[0754] Example 23: Screening test of coupling conditions for conjugate 1 Aside from the different coupling conditions, the experimental materials, experimental methods, and detection methods were all the same as in Example 18.

[0755] Coupling reaction with batch addition of C-Inter-1: After the reduction reaction reached 2 hours, 50 mM PBS, pH 6.5 buffer, and 10 mg / mL C-Inter-1 solution (C-Inter-1 / Fc molar equivalent ratio 7) were added to the reaction solution in one go. The final concentration of the reaction was controlled to 1 mg / mL. The reaction was placed in a metal bath and allowed to proceed for 2 hours at a controlled reaction temperature of 22°C.

[0756] The DAR values ​​detected by LC-MS were as follows:

[0757] [Table 39]

[0758] The MS detection results are shown in Figure 7.

[0759] Example 24: Pharmacokinetic study of conjugate 1 in mice 1. Experiment Information Pharmacokinetic studies were conducted using male CD-1(ICR) mice. Blood samples were analyzed by ELISA, and specific experimental information and design are detailed in the table below.

[0760] [Table 40]

[0761] 2. Main experimental materials

[0762] [Table 41]

[0763] 3. Detection Procedure Method Introduction: Conjugate concentrations in CD-1 mouse plasma were measured by 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 procedure was as follows: 1) Blood samples were collected at predetermined time points, and corresponding test samples were prepared using EDTA-K2 as an anticoagulant. 2) 100 μL of coating solution was added to a 96-well microplate, the plate was sealed, and incubated overnight at 2-8°C before use. 3) The plate was washed five times with washing buffer (300 μL / well). 4) Add 300 μL of blocking buffer to each well and block the plate. Seal the plate and incubate at room temperature for 2 hours without shaking. 5) Repeat step 2. 6) 100 μL of standard solution and test sample were added to each well. The plate was sealed and incubated at room temperature with shaking at 450 RPM for 2 hours ± 10 minutes. 7) Repeat step 2. 8) 100 μL of HRP solution was added to each well. The plate was sealed and incubated at room temperature with shaking at 450 RPM for 1 hour ± 10 minutes. 9) Repeat step 2. 10) Add 100 μL of TMB to each well. Incubate the plate at room temperature for 5–20 minutes. 11) 100 μL of stop solution was added to each well. 12) Within 30 minutes, the plate was read at wavelengths of 450 nm and 630 nm using a SpectraMax M5e / M5 / Plus 384 microplate reader. 13) The measured values ​​of the standard solution were converted to the corresponding conjugate concentrations. 14) Data processing was performed using Watson LIMS V7.6 or SoftMax Pro GxP 7.0.3, and Microsoft Excel 2007 or later.

[0764] 4. Summary of Results

[0765] [Table 42]

[0766] Pharmacokinetic data from mice revealed that conjugate 1 has a relatively long half-life (165 hours). Simultaneously, conjugate 1 showed good in vivo exposure. These data suggest that conjugate 1, with its relatively long half-life and high exposure potential, may exhibit extremely excellent antiviral activity in clinical settings.

[0767] Example 25: Pharmacokinetic study of conjugate 2 in mice The pharmacokinetic study of Conjugate 2 was conducted according to the method described in Example 22, and the results are summarized below:

[0768] [Table 43]

[0769] Pharmacokinetic data from mice revealed that conjugate 2 exhibited a relatively long half-life, reaching 140 hours. Conjugate 1, on the other hand, showed favorable in vivo exposure. These data suggest that conjugate 1 may exhibit excellent antiviral activity in a clinical setting, possessing a relatively long half-life and high exposure.

[0770] Example 26: Pharmacokinetic study of conjugate 4 in mice 1. Experiment Information Pharmacokinetic studies were conducted using male CD-1(ICR) mice. Blood samples were analyzed by ELISA, and specific experimental information and design are detailed in the table below.

[0771] [Table 44]

[0772] 2. Testing of conjugate drug (ADC) concentrations in animal plasma using the ELISA method. 1) Main reagents

[0773] [Table 45]

[0774] 2) Key experimental parameters

[0775] [Table 46]

[0776] 3. Experimental Procedure (1) 100 μL of coating solution (Solarbio) was added to a 96-well microplate, the plate was sealed, and incubated overnight at 2-8°C before use. (2) The plate was washed five times with washing buffer (300 μL / well). (3) Add 300 μL of blocking solution to each well and seal the plate. The plate was sealed and incubated at room temperature for 2 hours without shaking. (4) Repeat step (2). (5) 100 μL each of the standard / quality control sample and the sample was added to each well. The plate was sealed and incubated at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes. (6) Repeat step 5.2. (7) 100 μL of HRP solution was added to each well. The plate was sealed and incubated at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes. (8) Repeat step 5.2. (9) 100 μL of TMB was added to each well. The plate was incubated at room temperature for 5–20 minutes. (10) 100 μL of 2N H2SO4 was added to each well. (11) The absorbance at 450 nm was measured using a plate reader within 30 minutes.

[0777] 3. Measurement of conjugated drugs (total drug concentration) in animal plasma using the ELISA method. 1) Main reagents

[0778] [Table 47]

[0779] 2) Key experimental parameters

[0780] [Table 48]

[0781] 3. Experimental Procedure (1) 100 μL of coating solution was added to a 96-well microplate, the plate was sealed, and incubated overnight at 2-8°C before use. (2) The plate was washed five times with washing buffer (300 μL / well). (3) Add 300 μL of blocking solution to each well and seal the plate. The plate was sealed and incubated at room temperature for 2 hours without shaking. (4) Repeat step (2). (5) 100 μL each of the standard / quality control sample and the sample was added to each well. The plate was sealed and incubated at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes. (6) Repeat step 5.2. (7) 100 μL of HRP solution was added to each well. The plate was sealed and incubated at room temperature with shaking (450 rpm) for 1 hour ± 10 minutes. (8) Repeat step 5.2. (9) 100 μL of TMB was added to each well. The plate was incubated at room temperature for 5–20 minutes. (10) 100 μL of 2N H2SO4 was added to each well. (11) Absorbance at 450 nm and 630 nm was measured within 30 minutes using a plate reader.

[0782] 4. Experimental Results

[0783] [Table 49]

[0784] [Table 50]

[0785] Pharmacokinetic data from mice revealed that conjugate 4 has a relatively long half-life, exceeding 190 hours across all three administration routes (intravenous injection (IV), subcutaneous injection (SC), and intraperitoneal injection (IM)). Conjugate 4 also showed high exposure levels in vivo. These data suggest that conjugate 4 has a relatively long half-life and high exposure levels in a clinical setting, and may exhibit extremely excellent antiviral activity.

[0786] Example 27: Pharmacokinetic study of conjugate 4 in rats 1. Experiment Information Pharmacokinetic studies were conducted using male SD rats. Blood samples were analyzed by ELISA, and specific experimental information and design are detailed in the table below.

[0787] [Table 51]

[0788] 2. Measurement of conjugated drug concentrations in animal plasma using ELISA method Referring to the method for testing conjugates in animal plasma in Example 26, the ADC drug concentration and total drug concentration in rat plasma were measured.

[0789] 3. Experimental Results

[0790] [Table 52]

[0791] [Table 53]

[0792] Pharmacokinetic data from rats revealed that conjugate 4 exhibited a relatively long half-life, reaching over 190 hours. Furthermore, conjugate 4 showed good in vivo exposure. These data suggest that conjugate 4 may exhibit excellent antiviral activity in a clinical setting, possessing a relatively long half-life and high exposure.

[0793] Example 28: Evaluation of the in vivo antiviral effect of the test substance (conjugate 4) using an influenza virus mouse infection model. 1. Experimental materials and methods 1) Animals: Female BALB / c mice (6-7 weeks old, 18-20g weight) free of specific pathogens were purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd. They arrived at the animal laboratory of the Department of Biology, Nantong Wuxi Aptech Pharmaceutical Technology Co., Ltd. on November 3, 2023, and were housed in individual ventilated cages. The quality certificate number for the laboratory animals is 20220009014323. The mice were housed and used in accordance with the Wuxi Aptech IACUC-QD approved experimental protocol (animal use protocol number: ID01-QD031-2022v1.0). After providing an environmental adaptation period of at least 3 days, qualified individuals were used in the experiment.

[0794] 2) Solvents and test substances 10 mM histidine buffer (pH=5.5). As an example of preparing a 1 L solution, 0.372 g of histidine and 1.593 g of histidine hydrochloride were added to 1 L of ddH2O, vortex-mixed until homogeneous, and then used later.

[0795] [Table 54]

[0796] 3) Virus Influenza A virus, A / California / 07 / 2009 (H1N1). Original strain purchased from ATCC (Catalog No.: VR1843). Working strain amplified by WuXi AppTec Biologics (Lot No.: 20201228YNR). Titer: 7.0E+06 PFU / mL.

[0797] 4) Reagents and equipment

[0798] [Table 55]

[0799] [Table 56]

[0800] 2. Experimental Methods and Procedures 1) The day of the first drug administration is defined as day 0 of the experiment, the day before is defined as day -1, and the day after is defined as day 1. The same applies hereafter. 2) Viral inoculation: On day 28, mice were deeply anesthetized by intraperitoneal injection of Zoletil 50 (30 mg / kg) and xylazine hydrochloride (6 mg / kg) (injection volume 6 mL / kg). Subsequently, the virus was inoculated by nasal drip method, with an inoculation volume of 500 PFU and 50 μL per mouse. 3) Grouping: Based on the experimental design, the animals were randomly divided into 11 groups, with 10 animals in each group. Adjustments were made based on animal weight to ensure that there were no statistically significant differences in average body weight between groups. 4) Drug administration: Mice in each group, excluding the healthy control group (Group 1), were administered the vehicle or test substance by subcutaneous injection. The administration was performed as a single dose 28 days before viral inoculation.

[0801] [Table 57]

[0802] 5) Sample Collection: For mice in groups 4-9, blood samples were collected from 3 mice in each group at 48, 336, 672, 885, or 1008 hours after administration, and serum samples were collected. The collected whole blood samples were incubated at 4°C for 8 hours, and then centrifuged at 4°C at 3000g for 10 minutes to separate the serum. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected from groups 4 and 7 at 1008 hours after administration. All samples were stored in a refrigerator at -80°C. 6) Health monitoring: During the experiment, mice underwent weight measurements three times a week from day 0 to day 27, and once a day from day 28 to day 42. Their condition was observed and their weight was recorded. 7) Median survival time: The survival time corresponding to a 50% cumulative survival rate. 8) Animal welfare endpoint: Based on the IACUC protocol, mice that lost more than 35% (including 35%) of their initial body weight during the experiment, or / or showed symptoms of imminent death, were euthanized and recorded as deceased animals in the results. The above criteria were also followed in the actual experimental procedures.

[0803] 3. Sample Analysis ELISA experiment to detect the concentration of test substance in a sample: 1) Dilute ADC capture antibody with coating solution in a ratio of 1:400 and coat a 96-well plate with 100 μL / well at 4°C overnight; 2) Discard the liquid from the microplate and wash three times with PBST (300 μL / well); 3) Coat each well with PBST containing 5% BSA. 4) Add 200 μL and block at room temperature for 2 hours; 5) Discard the liquid in the microplate and wash 3 times with PBST (300 μL / well); 6) Add 100 μL of the test sample to each well and incubate at room temperature for 2 hours; 7) Discard the liquid in the microplate and wash 3 times with PBST (300 μL / well); 8) Add 100 μL of HRP (see Example 24) (1:20000 dilution) to each well and incubate at room temperature for 40 minutes; 9) Discard the liquid in the microplate and wash 3 times with PBST (300 μL / well); 10) Add 100 μL of TMB chromogenic substrate to each well and incubate in the dark at room temperature for 5-10 minutes. Once the solution has clearly turned blue, add 100 μL of stop solution to each well to stop the color development; 11) Measure the absorbance at a dual wavelength of 450 / 620 nm using a microplate reader and fit a standard curve using the 4-parameter method. The content of the test substance in the sample was measured using the method described above.

[0804] 4. Data Analysis The data were statistically analyzed using Prism, and the survival status of the mice was analyzed using the Log-rank (Mantel-Cox) test.

[0805] 5.Results The in vivo effects of the test compound in an influenza A virus-infected mouse model were evaluated based on changes in mouse body weight and survival rate. The pharmacokinetic properties of the test compound were analyzed by measuring its concentration in serum, lung tissue, and surface fluid of lung epithelial cells. The tolerance of the test compound in mice was evaluated by monitoring the animals' body weight.

[0806] 1) Weight-protective effect of test substance in model mice Healthy control group: During the experiment, the weight of the healthy control group remained stable, and no significant decrease was observed. Vehicle group: Weight began to decrease significantly from day 31 and continued to decrease until death or euthanasia, with an average maximum weight loss of 37.77% (euthanasia performed). hIgG1-Fc(LS)(2.5mg / kg) group: Body weight began to decrease significantly from day 31 of the experiment, with an average maximum decrease of 36.08%. Body weight of surviving mice began to recover from day 39. Conjugate 4 (2.5 mg / kg) group: Body weight began to decrease significantly from day 31 of the experiment, with an average maximum decrease of 16.83%. The body weight of surviving mice began to recover from day 36 and reached normal levels. Conjugate 4 (0.5 mg / kg) group: Body weight began to decrease significantly from day 31 of the experiment, with an average maximum decrease of 34.58%. Body weight of surviving mice began to recover from day 38. Conjugate 4 (0.1 mg / kg) group: Mouse body weight began to decrease significantly from day 31 and continued to decrease until death or euthanasia, with an average maximum decrease of 33.00%. Reference conjugate (0.5 mg / kg) group: Mouse body weight began to decrease significantly from day 31 and continued to decrease until death or euthanasia, with a mean maximum decrease of 37.03% (euthanasia performed).

[0807] 2) Protective effect of the test substance on the survival rate of model mice The survival status of mice in each group after virus inoculation is summarized below. Healthy group: No deaths, 100% final survival rate, median survival time > 42 days. Control group: Death began on day 35, and all animals died by day 37. Final survival rate was 0%, and the median survival time was 36 days. hIgG-Fc(LS)(2.5mg / kg) group: Death was confirmed from day 36, with a final survival rate of 10% and a median survival time of 36.5 days. Conjugate 4 (2.5 mg / kg) group: Death was confirmed from day 38, final survival rate 90%, median survival time >42 days. Conjugate 4 (0.5 mg / kg) group: Mice began dying from day 36, with a final survival rate of 50% and a median survival time of 39.5 days. Conjugate 4 (0.1 mg / kg) group: Mice began dying from day 34, and all died by day 37. Final survival rate was 0%, median survival time was 36 days. Reference conjugate (0.5 mg / kg) group: Mice began dying from day 36, and all died by day 38. Final survival rate was 0%, median survival time was 37 days.

[0808] [Table 58]

[0809] Analysis of the results shows that Conjugate 4, obtained by the thiol site-specific binding technology described in this patent, exhibited excellent antiviral activity in a mouse model. At high doses (2.5 mg / kg), the survival rate of mice reached 90%. Compared to the control conjugate, at a dose of 0.5 mg / kg, the survival rate of mice administered with Conjugate 4 reached 50%, while the survival rate of mice administered with the control conjugate molecule was 0%. This confirms that Conjugate 4 exhibits superior antiviral activity and shows better clinical efficacy in the prevention and / or treatment of influenza in clinical settings.

[0810] The present invention has been illustrated by various examples. However, those skilled in the art will understand that the present invention is not limited to these examples. Those skilled in the art can make various modifications or variations within the scope of the present invention, and the various technical features described herein can be combined with one another without departing from the spirit and scope of the invention. All such modifications and variations are within the scope of the present invention.

Claims

1. Structural formula I-A or structural formula I-B: 【Chemistry 1】 【Chemistry 2】 [During the ceremony, m is either 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 the linker; E is selected from an antibody or antibody fragment, an Fc domain monomer, an Fc domain, an Fc-binding peptide, albumin, or an albumin-binding peptide; R １ａ 、R ２ａ 、R ３ａ 、R ５ａ 、R ６ａ 、R ７ａ 、R ９ａ and R １０ａ each independently is selected from hydrogen, deuterium, hydroxyl, -O-C １－６ alkyl, hydroxyl C １－６ alkyl-, C １－６ alkyl-, C ３－８ cycloalkyl-, C ３－８ heterocycloalkyl-, C ６ -C １５ aryl, 5- to 15-membered heteroaryl, halogen, cyano, and amino; R １ａ and R ２ａ together can form C ３－８ cycloalkyl or C ３－８ heterocycloalkyl? or R ５ａ and R ６ａ They are together C ３－８ Cycloalkyl or C ３－８ Can it form heterocycloalkyl groups? or R ９ａ and R １０ａ They are together C ３－８ Cycloalkyl or C ３－８ A heterocycloalkyl can be formed, where the C ３－８ Cycloalkyl or C ３－８ Heterocycloalkyl groups may be optionally substituted with substituents selected from halogens and hydroxyls; R ４ａ and R ８ａ Each of these is independently H, C １－６ Alkyl, C ３－８ Cycloalkyl or C ３－８ Selected from heterocycloalkyl groups; x is either 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’ Each of these is independently hydrogen, deuterium, hydroxyl, -O-C １－６ Alkyl, Hydroxyl C １－６ Alkyl, C １－６ Alkyl, C ３－８ Cycloalkyl, C ３－８ Heterocycloalkyl, C ６ -C １５ Selected from aryl, 5-15 member heteroaryl, halogen, cyano, and amino; or R １’ and R ２’ They are together C ３－８ Cycloalkyl or C ３－８ Can form a heterocycloalkyl group; or two adjacent R groups ３’ is C ３－８ Cycloalkyl, C ３－８ Heterocycloalkyl, C ６ -C １５ [May form an aryl or a 5-15 membered heteroaryl ring] A conjugate represented by , or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

2. L is the structure of formula I-3a: 【Transformation 3】 This is a linker represented by L １ and L ２ａ Includes L １ The left and right ends are connected to the drug portion, L ２ａ The upper end is -NR ８ａ Connected to, During the ceremony, L １ The structure is as follows: 【Chemistry 4】 During the ceremony, W stands for O, S, NR ｂ ,CH ２ - or selected from lack; R ａ and R ｂ Each of these is independently replaced by H and C of any choice. １ -C ２０ Alkyl and optionally substituted C ２ -C ２０ Selected from alkenyls, preferably selected from H and methyl, more preferably methyl; y １ and y ２ Each of them is independently 0, 1, 2, 3, 4, 5, or 6; Preferably L １ The structure is as follows: Table 1-1 Table 1-2 A conjugate according to claim 1, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, selected from the above.

3. L ２ａ The structure of equations L2-1a to L2-7a is: Table 2-1 Table 2-2 It has, During the ceremony, Z is selected from NR, S, and O; Each R group can be independently substituted with hydrogen, deuterium, or optionally substituted with C １ -C ２０ Alkyl, optionally substituted C ２ -C ２０ Alkenyl, C substituted by choice ３ -C ２０ Cycloalkyl groups, optionally substituted 3- to 20-membered heterocycloalkyl groups, optionally substituted C groups ６ -C １５ Selected from aryls and optionally substituted 5- to 15-membered heteroaryls; s and t are integers from 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; The conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, wherein y is 0 or 1.

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

5. The sulfur atom on E originates from a cysteine ​​(cys) residue or disulfide bond in 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-binding peptide, for example, an Fc domain monomer containing or comprising any of the amino acid sequences shown in SEQ ID NOs: 1 to 81, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto; Preferably, a conjugate according to any one of claims 1 to 4, comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 9, 10, 11, 33, 36, 37, 38, 41, 42, 43, 44, 45, 46, 51, 52, 53, 54, 55, 56, 57, 59, 65, 66, 67, 68, 69, 70, 71, and 72, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

6. A conjugate according to any one of claims 1 to 5, wherein the conjugate has the structure of formula I-A, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

7. The conjugate according to any one of claims 1 to 5, wherein the conjugate has the structure of formula I-B, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

8. The structure between L and E is as follows: 【Transformation 5】 Selected from, wherein, R １ａ , R ２ａ , R ３ａ , R ５ａ , R ６ａ , R ７ａ , R ９ａ and R １０ａ each independently is selected from hydrogen, deuterium, -O-C １－６ alkyl, hydroxyl C １－６ alkyl-, C １－６ alkyl-, and C ３－８ cycloalkyl-; or R １ａ and R ２ａ together may form C ３－８ cycloalkyl; or R ５ａ and R ６ａ They are together C ３－８ Can it form a cycloalkyl group? or R ９ａ and R １０ａ They are together C ３－８ A cycloalkyl group may be formed, where the C ３－８ The cycloalkyl group is optionally substituted with substituents selected from halogens and hydroxyls; R ４ａ and R ８ａ Each of these is independently H, C １－６ Alkyl and C ３－８ Selected from cycloalkyl; and w is 0, 1, or 2; Preferably, R １ａ , R ３ａ , R ４ａ , R ５ａ , R ７ａ , R ９ａ , and R １０ａ Each of them is independently a hydrogen; R ２ａ , R ６ａ and R ８ａ Each of them independently contains hydrogen and C １－６ Alkyl and C ３－８ Selected from cycloalkyl; w is 0; More preferably, the structure of formula I-4a is as follows: 【Transformation 6】 A conjugate according to claim 6, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, selected from the above.

9. The conjugate has structural formulas C-1a to C-27a: Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 A compound is selected from those represented by the following: In the formula, m, n, x, y, and E are the conjugate, pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound described in any one of claims 1 to 5.

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

11. A conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, having an average DAR value of 0.5 to 10.0, for example, 2.0 to 4.5 or 4.8 to 8.

5.

12. A conjugate according to any one of claims 1 to 11, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, wherein E comprises an Fc domain monomer or an Fc domain containing the Fc domain monomer, and the Fc domain monomer comprises or consists of one of the amino acid sequences shown in SEQ ID NOs: 1 to 81, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity therewith.

13. Is E capable of recognizing viral surface antigens such as CR6261, CR8020, MEDI8897, palivizumab, and SD38? Fc domain monomers can be used with any antibody subtype of immunoglobulin (e.g., IGHG1*01 (e.g., G1m(za)), IGHG1*07 (e.g., G1m(zax)), IGHG1*04 (e.g., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (e.g., G1m(fa)), IGHG2*01, IGHG2*02, IGHG2*06, IGHG3*01, IGHG3*04, IGHG3*05, IGHG3*09, IGHG3*10, IGHG3*11, IGH A conjugate according to any one of claims 1 to 12, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, which may be G3*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).

14. The conjugate has the following structure: Table 4 It has, In the formula, E is defined in any one of claims 1 to 13; Preferably, E is sequence number 67, sequence number 69, or sequence number 71; A conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, wherein m and n are defined in claim 1, and m is preferably 1. 【Request Item 15】 【Chemistry 7】 The carbon atoms to which the group is linked, or 【Transformation 8】 A conjugate according to any one of claims 1 to 14, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, wherein the carbon to which the corresponding group is linked is in an R or S configuration, preferably in an R configuration.

16. Formula I-5a: 【Chemistry 9】 (Each symbol in the formula is defined in any one of claims 1 to 15.) Or I-6a: 【Chemistry 10】 (Each symbol in the formula is defined in any one of claims 1 to 15.) A compound represented by the structure of, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

17. The compound has structures from formula C-Inter-1 to formula C-Inter-12: Table 5-1 Table 5-2 Table 5-3 A conjugate according to claim 16, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

18. Formula I-A according to any one of claims 1 to 15: 【Chemistry 11】 (Each symbol in the formula is defined in any one of claims 1 to 15.) A method for preparing a conjugate, The method involves the following steps: Step 1, in a buffer solution (E) ｍ Dissolve E and m as defined in any one of claims 1 to 15; then add a reducing agent (such as TCEP, DTT, etc.) ｍ The disulfide bond inside is reduced, resulting in formula II-1 having a thiol residue: (HS)) ｎ -(E) ｍ II-1 (wherein E, m, and n are defined in any one of claims 1 to 15) Steps to obtain the compound; Step 2, Formula I-5a: 【Chemistry 12】 (Each symbol in the formula is defined in formula I-A.) The step involves adding a buffer solution containing 6 to 14 molar equivalents of the compound to the compound of formula II-1, The Michael addition reaction is carried out for 0.5 to 10 hours, preferably 0.5 to 2 hours, under conditions of pH 5.5 to 7.0 (e.g., 5.8 to 6.8, 6.0 to 6.6); then, 3 to 7 molar equivalents of the buffer solution of the compound of formula I-5a are added in several portions (e.g., 1 to 2 portions), and the reaction is continued for 0.5 to 10 hours (preferably 0.5 to 2 hours) to obtain the compound of formula I-3b: 【Chemistry 13】 (Each symbol in the formula is defined in formula I-A.) Steps to obtain the conjugate; Step 3: 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 then carry out the hydrolysis reaction to obtain the final conjugate of formula I-A. A method including; or, Formula I-B according to any one of claims 1 to 15: 【Chemistry 14】 (Each symbol in the formula is defined in any one of claims 1 to 15.) A method for preparing a conjugate, The method involves the following steps: Step 1, in a buffer solution (E) ｍ Dissolve E and m as defined in any one of claims 1 to 15, and add a reducing agent (such as TCEP, DTT, etc.) to (E) ｍ The disulfide bond inside is reduced, resulting in formula II-1 having a thiol residue: (HS)) ｎ -(E) ｍ II-1 (wherein E, m, and n are defined in any one of claims 1 to 15) Steps to obtain the compound; Step 2, Formula I-6a: 【Chemistry 15】 (Each symbol in the formula is defined in formula I-B.) In the step of adding a buffer solution containing 6 to 14 molar equivalents of the compound to the compound of formula II-1, The Michael addition reaction is carried out under conditions of pH 5.5–8 (e.g., 5.8–7.6, 6.0–7.5) to obtain the conjugate of formula I–B. A method that includes this.

19. A pharmaceutical composition comprising a conjugate of formula I-A or formula I-B 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 claim 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, one or more other therapeutic agents, optionally such as a chemotherapeutic agent, angiogenesis inhibitor, cytokine, cytotoxic agent, other antibody, small molecule drug, or immunomodulator (e.g., immune checkpoint inhibitor or agonist), and optionally a pharmaceutically acceptable excipient.

20. A method for preventing or treating a patient infected with a virus or at risk of viral infection, comprising the step of administering to the patient, for example by injection, an effective amount of a conjugate of formula I-A or formula I-B as described in any one of claims 1 to 15, or a compound of formula I-5a or formula I-6a as described in claim 16 or claim 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof.

21. The use of a conjugate of formula I-A or formula I-B as described in any one of claims 1 to 15, or a compound of formula I-5a or formula I-6a as described in claim 16 or claim 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotopically labeled compound thereof, in the manufacture of a pharmaceutical product for the prevention or treatment of viral infection in a patient infected with a virus or a patient at risk of viral infection, Preferably, the viral infection is caused by an influenza virus or a parainfluenza virus; Preferably, the viral infection is caused by influenza virus A, B, or C, or parainfluenza virus; Preferably, patients who are infected with the virus or at risk of becoming infected with the virus may be immunocompromised patients; Preferably, a patient who is infected with the virus or at risk of becoming infected with the virus may be a patient who is receiving or scheduled to receive immunosuppressant therapy; Preferably, a patient who is infected with a virus or at risk of becoming infected with a virus may be a patient diagnosed with an immunosuppressive disorder; Preferably, patients diagnosed with an immunosuppressive disorder also have cancer or acquired immunodeficiency syndrome; Preferably, the cancer of a patient diagnosed with an immunosuppressive disorder is selected from leukemia, lymphoma, humoral immunodeficiency, T-cell deficiency, complement deficiency, or multiple myeloma; Preferably, the patient is a patient who has received or is scheduled to receive a hematopoietic stem cell transplant; Preferably, the patient is a patient who has received or is scheduled to receive an organ transplant; and / or Preferably, use if the patient may be at risk of secondary infection.

22. A method for preventing the risk of secondary infection due to influenza virus infection in a patient, comprising, for example, administering to the patient an effective amount of any of the following by injection: a conjugate of formula I-A or formula I-B described in any one of claims 1 to 15, a compound of formula I-5a or I-6a described in claim 16 or claim 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof; Preferably, the secondary infection is a respiratory 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 caused by methicillin-resistant Staphylococcus aureus (MRSA); preferably, the bacterial infection is caused by Streptococcus pneumoniae; Preferably, the conjugate of formula I-A or formula I-B, the compound of formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound may be administered by intramuscular injection, intravenous injection, intradermal injection, intra-arterial injection, intraperitoneal injection, intrafocal injection, intracranial injection, intra-articular injection, intrapleural injection, intratracheal injection, intraprostatic injection, intranasal injection, intravitreal injection, intravaginal injection, intrarectal injection, local administration, intratumoral injection, intraperitoneal injection, subcutaneous injection, subconjunctival injection, intracapsular injection, mucosal injection, intrapericardial injection, intraumbilical injection, intraocular injection, oral administration, local inhalation, injection, or infusion; Preferably, the conjugate of formula I-A or formula I-B, the compound of formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound is administered in combination with an additional therapeutic agent or used to prepare a pharmaceutical product in combination with an additional therapeutic agent; Preferably, the additional therapeutic agent is an antiviral drug; Preferably, the antiviral agent is baloxavir, pimozivir, oseltamivir, zanamivir, peramivir, laninamivir, amantadine, MEDI8852, or rimantadine; Preferably, the additional treatment agent used by the patient is an antiviral vaccine; Preferably, an antiviral agent and a conjugate of a compound of formula I-A or formula I-B, a compound of formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound are administered sequentially to the patient, for example by injection; and / or Preferably, a method of simultaneously administering an antiviral agent and a conjugate of formula I-A or formula I-B, a compound of formula I-5a or I-6a, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound to a patient, for example by injection.

23. A drug combination agent comprising a conjugate of formula I-A or formula I-B as described in any one of claims 1 to 15, or a compound of formula I-5a or formula I-6a as described in claim 16 or claim 17, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled compound thereof, and optionally one or more other therapeutic agents such as a chemotherapeutic agent, angiogenesis inhibitor, cytokine, cytotoxic agent, other antibody, small molecule drug, or immunomodulator (e.g., immune checkpoint inhibitor or agonist).

Images