Site-directed orthogonal biocombination modalities and their applications

Abstract

A manufacturing process for an immune ligand / payload complex is provided, and in particular, a novel enzyme site-specific complexing technique using a dual enzyme orthogonal catalyst, comprising the steps of: complexing a first payload with an immune ligand via the enzymatic catalyst of a first enzyme; and complexing a second payload with an immune ligand via the enzymatic catalyst of a second enzyme, wherein the first enzyme is different from the second enzyme.

Description

[Technical Field]

[0001] This disclosure relates to a manufacturing process for immune ligand / payload conjugates, and more particularly to a novel enzyme site-specific conjugation technique using a dual enzyme orthogonal catalyst, and to highly homogeneous and mutually orthogonal drug-loading antibody-drug conjugates, in particular dual-payload antibody-drug conjugates (dpADCs), prepared thereby. [Background technology]

[0002] Antibody-drug conjugates (ADCs) consist of three parts: an antibody, a linker, and a highly potent small-molecule drug. The small-molecule payload is linked to an antibody that specifically binds to an antigen overexpressed on the surface of tumor cells. Subsequently, the resulting ADC is internalized by the tumor cells and releases the cytotoxic payload into the tumor microenvironment (TME) or inside the tumor cells. The released payload then exerts an antitumor effect with minimal toxicity to normal tissue. Therefore, ADCs are also known as a class of targeted chemotherapy drugs with controllable side effects. As of 2023, there are 14 ADC drugs approved worldwide, and more than 100 are in clinical development, but most of these ADCs are based on a single type of payload. However, tumors are highly heterogeneous and prone to drug resistance. In conventional oncology chemotherapy, combinations of two or more chemotherapy agents are used to overcome drug resistance and improve antitumor efficacy (Non-Patent Literature 1). For example, the combination of docetaxel and gemcitabine has been used in clinical trials, and the synergistic effects of various mechanisms of action and non-overlapping tumor-killing effects have been validated by enhanced patient survival in the treatment of both drug-sensitive and drug-resistant tumor models (Non-Patent Literature 2). However, the combination of chemotherapeutic agents can also increase toxicity. To take this a step further, ADC-based combination therapies are gaining interest and are being investigated in preclinical models and clinical studies. Generally, the most attractive ADC partners are those that provide synergistic or additive effects on cancer cells or TMEs without any unacceptable toxicity, and current ADC partners such as chemotherapeutic agents, molecularly targeted agents and immunotherapeutic agents are precursors that begin to show increased antitumor efficacy with ADCs in a synergistic manner (Non-Patent Literature 3). Based on these existing research findings, the inventors hypothesize that a superior therapeutic solution for treating immunoresistant cancers with minimal off-target toxicity is a dpADC, which combines various MoA drugs in a single modality, allowing the two drugs to reach their targets simultaneously and exert their effects in a highly synergistic manner.

[0003] Currently, several types of conjugation techniques exist for constructing dual-payload antibody-drug conjugates (Non-Patent Literature 4): combinations of two chemical conjugation methods, combinations of chemical and enzymatic conjugation methods, and combinations of two enzymatic conjugation methods. The currently available methods for constructing dpADCs are mainly the first two methods, but dual enzyme-catalyzed orthogonal specific conjugation has rarely been reported.

[0004] Regarding the combination of two chemical complexing methods, for example, in 2016, Pfizer reported the first example of dual-payload complexing of a solid-phase immobilized antibody (by binding to protein A / L agarose beads) with a Fab fragment. Using a reduction-reoxidation strategy to cap recombinant non-natural cysteine ​​in the bead-bound Fab fragment, the capped cysteine ​​was then complexed with a first maleimide-functionalized linker-payload. After another washing step, the interchain disulfide bonds of Fab were then reduced with TCEP, and subsequently complexed with a second maleimide-based linker-payload to obtain a dual-payload antibody-fragment complex. They also demonstrated solid-phase dual-payload complexing based on enzymatic and chemical complexing relays. As a proof of concept, a bicyclo[6.1.0]nonine (BCN) group was introduced into an antibody by transglutaminase-mediated conjugation, followed by the introduction of a first linker-payload by a BCN-azidocrick reaction, and then the second linker-payload was linked via cystine-maleimide chemical conjugation. Both protocols require a multi-step (more than 6 steps) process, which can cause significant problems in CMC manufacturing. (Non-Patent Literature 5).

[0005] In 2017, Seagen reported a strategy for constructing dpADCs by stepwise building a trifunctional linker and chemical complexes. The researchers synthesized a trifunctional linker containing two orthogonally protected cysteine ​​groups and one maleimide connector. The maleimide functional group of the linker was coupled to the cysteine ​​sulfhydryl group of the antibody via a Michael addition reaction, and then the toxin was attached by two cycles of selective deprotection and Michael addition. A total of five chemical reactions were performed to obtain an MMAE / MMAF dual-payload ADC. This ADC showed stronger antigen-positive tumor cell killing activity than ADCs loaded with a single drug in cell activity studies, but did not show a synergistic effect in in vivo mouse activity studies, which may be due to the consistent mechanism of action of the two drugs. (Non-Patent Literature 6).

[0006] Regarding combinations of chemical and enzymatic site-specific complexing methods, for example, in 2019, Williams et al. reported successfully constructing a dual-payload antibody fluorescein conjugate with the potential to develop dpADCs based on a transpeptidase-catalyzed and perfluoroaryl nucleophilic substitution reaction strategy.

[0007] In 2022, Sutro also reported a site-directed dpADC complexing method in which a transglutaminase recognition motif (Qtag) having GLLQGA (SEQ ID NO: 48) at the C-terminus of an antibody light chain is designed, the Qtag-LC is then paired with a heavy chain containing the non-natural amino acid pAMF, the Qtag-LC is then targeted-coupled to a cytotoxic-PEG-primary amine compound via transglutaminase, and the pAMF group in the HC is coupled to the TLR agonist-PEG-DBCO by SPAAC to form a complex (Patent Document 1).

[0008] In summary, reported methods for constructing dpADCs are primarily based on either a bichemical complexing relay or an enzymatic and chemical complexing relay, both of which require a multi-step process where the CMC can be complex and time-consuming. Chemical complexing often results in heterogeneous DAR distribution, and furthermore, they are more prone to premature release in systemic circulation due to the unstable linker design derived from the intact cysteine-maleimide ring structure. All of these raise problems in CMC production and safety concerns in clinical applications. To address these issues, there is a great need for a more convenient, simple, and efficient complexing technique for the preparation of homogeneous cultured dpADCs. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] CN 114206392 A [Non-patent literature]

[0010] [Non-Patent Document 1] Jain S,Jain R,Das M,Agrawal AK,Thanki K,Kushwah V.Combinatorial bio-conjugation of gemcitabine and curcumin enables dual drug delivery with synergistic anticancer efficacy and reduced toxicity.RSC Adv 2014,4,29193. [Non-Patent Document 2] Hensley ML, Maki R, Venkatraman E, Geller G, Lovegren M, Aghajanian C, et al.Gemcitabine and docetaxel in patients with unresectable leiomyosarcoma: results of a phase II trial.J Clin Oncol2002,20,2824

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[0011] In a first embodiment, an enzymatic complexing process for preparing an immune ligand / payload complex, comprising the following steps: The steps include: conjugating the first payload to an immune ligand via enzymatic catalysis by the first enzyme; The process includes the step of conjugating a second payload to the immune ligand via enzymatic catalysis by a second enzyme, Here, the first enzyme is different from the second enzyme. Provide the process.

[0012] In some embodiments, the enzymatic catalysis step of the first enzyme and the enzymatic catalysis step of the second enzyme are performed simultaneously or sequentially. In one embodiment, the enzymatic complexation process includes simultaneously complexing a first payload and a second payload molecule with an immune ligand via the enzymatic catalysis of the first and second enzymes. In another embodiment, the enzymatic complexation process includes complexing the first payload with an immune ligand via the enzymatic catalysis of the first enzyme to obtain a first complex, and complexing the second payload with the first complex via the enzymatic catalysis of the second enzyme, thereby obtaining an immune ligand / payload complex.

[0013] In some embodiments, the first enzyme comprises a ligase and / or the second enzyme comprises an endoglucosidase.

[0014] In one embodiment, the endoglycosidase is an N-acetylglucosaminidase that covalently binds a donor containing an oxazoline oligosaccharide to an immune ligand containing a GlcNAc motif, and the donor containing the oxazoline oligosaccharide further contains a payload.

[0015] In another general embodiment, ligase fusion proteins and / or endoglycosidase fusion proteins are provided. In one embodiment, a ligase is covalently linked to a self-labeled protein tag to form a ligase fusion protein. In one embodiment, an endoglycosidase is covalently linked to a self-labeled protein tag to form an endoglycosidase fusion protein.

[0016] In a second embodiment, an immobilized enzyme is provided in which the first enzyme and the second enzyme are independently immobilized on a support.

[0017] In a third embodiment, an immune ligand / payload complex prepared by the process of the present disclosure is provided.

[0018] In a fourth embodiment, the use of an immune ligand / payload complex prepared by the process of the present disclosure is provided in the manufacture of a drug for treating proliferative disorders.

[0019] In a fifth embodiment, a method is provided for treating a proliferative disorder, comprising administering a therapeutically effective amount of an immune ligand / payload complex prepared in this disclosure to a subject in need thereof.

[0020] In a sixth embodiment, a dual-drug ADC is provided, comprising an antibody having a first linker payload conjugated to the C-terminus of the light chain and a second linker payload conjugated to the heavy chain Asn-297, wherein the payloads of the first and second linker payloads are different. [Brief explanation of the drawing]

[0021] [Figure 1] The SDS-PAGE electrophoresis maps of the purified endoglycosidase fusion protein (Halo-Endo S2-His) of this disclosure are shown. In the map, 1 indicates that the sample originated from cell saturation, 2 indicates that the sample originated from supernatant, 3 indicates that the sample originated from flow solution (the equilibrium buffer used was composed of 50 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 7.4), and 4 indicates that the sample originated from washing solution (the washing solution was composed of 50 mM Tris, 150 mM NaCl, 80 mM imidazole, pH 7.4). 5 indicates that the sample originates from the eluent (eluent composition: 50 mM Tris, 150 mM NaCl, 500 mM imidazole, p7.4), 6 indicates that the sample originates from the eluent (eluent composition: 50 mM Tris, 150 mM NaCl, 500 mM imidazole, pH7.4), and 7 indicates that the sample originates from magnetic beads. The molecular weight of the endoglycosidase fusion protein (Halo-Endo S2-His) is approximately 130 kDa. [Figure 2] The ring-opening molecular structure of the linker 2-DM1 intermediate is shown (n is an integer from 1 to 100, x is an -OH or -NH2 group, and A and B are isomers). [Figure 3] The SDS-PAGE electrophoresis maps of the supernatants of immobilized endoglycosidase fusion protein (Halo-Endo S2-His) at different incubation times are shown. [Figure 4] The results of the SDS-PAGE assay analysis of the ADC-1 antibody-drug conjugate are shown. [Figure 5] The results of the HIC-HPLC assay analysis of the ADC-1 antibody-drug conjugate are shown. [Figure 6] The results of the SEC-HPLC assay analysis of the ADC-1 antibody-drug conjugate are shown. [Figure 7] The results of the HIC-HPLC assay analysis of the ADC-2 antibody-drug conjugate are shown. [Figure 8] The results of SEC-HPLC assay analysis and antibody-drug conjugate analysis of ADC-2 are shown. [Figure 9]The results of SDS-PAGE assay analysis of dpADC-1 using the one-step and two-step methods are shown. [Figure 10] The results of the RP-HPLC assay analysis of the ADC drug dpADC-1 are shown. [Figure 11] The results of the SEC-HPLC assay analysis of the ADC drug dpADC-1 are shown. [Figure 12] The results of the analysis of the ADC drug dpADC-2 by RP-HPLC are shown. [Figure 13] The results of the analysis of the ADC drug dpADC-2 by SEC-HPLC are shown. [Figure 14] The results of inhibiting tumor cell proliferation by different drugs are shown. [Figure 15] This paper presents the results of the bystander killing effect of the ADC drug dpADC-1 against ErbB2 / Her2-negative tumor cells. [Figure 16] This paper presents the bystander killing effect of the ADC drug dpADC-2 against tumor cell proliferation with different ErbB2 / Her2 expression levels. [Figure 17] The results of the analysis of the ADC drug dpADC-3 by RP-HPLC are shown. [Figure 18] The results of the analysis of the ADC drug dpADC-3 by SEC-HPLC are shown. [Figure 19] This shows the effect of the ADC drug dpADC-3 on osimertinib-resistant NSCLC PDX model mice with moderate ErbB3 / Her3 expression levels in vivo. Figure 19A shows the inhibitory effect of dpADC-3 on osimertinib-resistant NSCLC PDX model mice with moderate ErbB3 / Her3 expression levels. Figure 19B shows the effect of dpADC-3 on body weight in osimertinib-resistant NSCLC PDX model mice with moderate ErbB3 / Her3 expression levels. [Figure 20]This shows the effect of the ADC drug dpADC-3 on osimertinib-sensitive NSCLC PC-9 model mice with low ErbB3 / Her3 expression levels in vivo. Figure 20A shows the inhibitory effect of dpADC-3 on osimertinib-sensitive NSCLC PC-9 model mice with low ErbB3 / Her3 expression levels. Figure 20B shows the effect of dpADC-3 on body weight in osimertinib-sensitive NSCLC PC-9 model mice with low ErbB3 / Her3 expression levels. [Modes for carrying out the invention]

[0022] general definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Furthermore, terms and experimental procedures relating to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are those commonly used in those fields. Where trade names are present herein, they mean the corresponding commodity or active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference. On the other hand, to aid in understanding this disclosure, definitions and explanations of relevant terms are provided below.

[0023] As used herein, the expressions “at least one” or “one or more” refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 or more, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more, etc. As used herein, “a” and “an” should be understood to mean “at least one” unless the opposite is explicitly stated.

[0024] Where a particular quantity, concentration, or other value or parameter is described in the form of a range, preferred range, preferred upper limit, or preferred lower limit, this should be understood as equivalent to specifically revealing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, whether or not such range is explicitly stated. Unless otherwise specified, the ranges of numbers listed herein are intended to include both ends of the range, as well as all integers and fractions (decimals) within that range. For example, the expression "i is an integer between 2 and 20" means that i is any integer between 2 and 20, for example, i may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions should also be understood in a similar manner.

[0025] When the terms "approximately" or "about" are used in relation to numerical variables such as concentration, isoelectric point (pI), pH, temperature, or a range, they generally mean that the value of the variable and all values ​​of the variable are within experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of a specified value, or within a wider range.

[0026] The terms "optional" or "optionally" mean that the events described below may occur, but do not necessarily occur, and this description includes cases in which the events or circumstances described above occur or do not occur.

[0027] The expression “comprising,” or similar expressions such as “including,” “containing,” and “having,” are non-exclusive and do not exclude additional elements, steps, or components not listed. The expression “consisting of,” excludes any elements, steps, or components not specified. The expression “essentially consisting of,” means that the scope is limited to the specified elements, steps, or components, plus any optional elements, steps, or components that do not substantially affect the essential and novel features of the claimed subject matter. The expression “comprising” should be understood to encompass the expressions “essentially consisting of” and “consisting of.”

[0028] As used herein, the definition of “biomolecule” includes proteins, nucleic acids, lipids, carbohydrates, small nucleotides, amino acids, and their derivatives.

[0029] As used herein, “nucleic acid” or “polynucleotide” refers to a polymer of at least two nucleotides or nucleotide derivatives linked together by a phosphodiester bond, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0030] As used herein, “vector” is a vehicle used to transfer an exogenous nucleic acid into a host cell, where the exogenous nucleic acid is amplified or expressed. As used herein, the definition of “vector” includes plasmids, linear plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. As used herein, a vector may be expressible and / or replicable in a host cell, meaning that the vector can express an RNA polynucleotide or polypeptide in a host cell and / or produce multiple copies of the vector. To be “expressible” or “replicable,” a vector may contain nucleic acid sequences or elements that are operably ligated to a promoter. As used herein, “operably ligated” with respect to nucleic acid sequences or elements means that these nucleic acid sequences are functionally related to one another. For example, a promoter may be operably ligated to a nucleic acid sequence encoding a polypeptide, thereby the promoter regulates or mediates the transcription of the nucleic acid. Those skilled in the art can select and use a vector appropriate for a particular purpose.

[0031] As used herein, “peptide,” “polypeptide,” or “protein” refers to two or more covalently linked amino acids. Unless otherwise specified, these terms are interchangeable.

[0032] As used herein, “sequence identity” has the meaning recognized in the art, and the percentage of sequence identity between two polypeptides can be calculated by aligning the two sequences using publicly available algorithms such as BLAST (Basic Local Alignment Search Tool) and FASTA (Fast Adaptive Shrinkage / Thresholding Algorithm) (see Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994, etc.). There are many methods for measuring the identity between two polypeptides, but the term "identity" is well known to those skilled in the art (Carrillo, H & Lipman, D 7 SIAM J Applied Math 48:1073 (1988)).

[0033] As used herein, the term “mutant” refers to a protein having one or more residue substitutions, deletions, or insertions compared to a reference protein. The reference protein may be a naturally occurring protein (i.e., a wild-type protein) that can be isolated from a natural source, or a recombinant protein. As used herein, the function or activity of a mutant such as a saltase A mutant or a Halo-tagged mutant is substantially the same as, comparable to, or higher than, the function or activity of the reference saltase A or Halo-tagged protein, respectively. As used herein, the function or activity of a mutant such as an Endo S2 mutant or a Halo-tagged mutant is substantially the same as, comparable to, or higher than, the function or activity of the reference Endo S2 or Halo-tagged protein, respectively.

[0034] As used herein, the term “endoglycosidase” refers to an enzyme having 1) activity that can be used to catalyze the hydrolysis of internal glycosidic bonds of oligosaccharides and polysaccharides, and to cleave polysaccharides from glycoproteins, and 2) activity that can transfer oligosaccharide portions to acceptors. In some embodiments, the “endoglycosidase” of this disclosure may be used to hydrolyze the β-(1→4) glycosidic bond between two N-acetylglucoses in the N-glycosylation site of the Fc region of an antibody, and may also be used to transfer an oxazoline oligosaccharide to a GlcNAc antibody acceptor. “Glycosidization activity” refers to the catalytic activation of sugars linked to various acceptor molecules such as proteins, nucleic acids, oligosaccharides, lipids, and small molecules. In this disclosure, a donor having an oxazoline oligosaccharide can be linked to the GlcNAc in the Fc region of an antibody.

[0035] As used herein, the term "Halo tag" refers to an alkyl halide (e.g., an alkyl halide moiety-(CH2)2- 30-X refers to a halogenated alkane dehalogenase or a variant thereof that removes a halogen from a drug containing X (wherein X is a halogen such as F, Cl, Br, I, and especially Cl or Br) and forms a covalent bond with the rest of the substrate. Variant haloalkane dehalogenases are described, for example, in WO2006 / 093529 and WO2008 / 054821, which are incorporated herein by reference. The mutant haloalkane dehalases that can be used in this disclosure include, but are not limited to, mutants of xanthobacter dehalases (e.g., xanthobacter autotropicus dehalase (DhIA)) or rhodococcus dehalases (e.g., rhodococcus halogens (DhaA)) that contain one or more substituents in the three catalytic residues, such as substituting His272 with Phe / Ala / Gly / Gln / Asn or Asp106 with Cys or other substitutes, as described in WO2008 / 054821. However, the mutant haloalkane dehalases may form covalent bonds with haloalkyl substances.

[0036] As used herein, the "His tag" refers to histidine, for example, His-His(His2), His-His-His(His3), His-His-His-His(His4) (SEQ ID NO: 49), His-His-His-His-His(His5) (SEQ ID NO: 50), His-His-His-His-His-His(His6) (SEQ ID NO: 51), His-His-His-His-His-His-His-His-His-His(His 10 This polypeptide is composed of (Sequence ID 52).

[0037] As used herein, the term “fusion protein” refers to a protein product obtained through DNA recombination techniques that is co-expressed by two or more genes. For example, co-expression of both genes can be achieved by deleting the stop codon of the gene encoding the first protein and then affixing the second protein gene to the stop codon. The endoglycosidase fusion protein may further contain one or more additional elements, such as additional peptides or tags, such as halogen tags and / or His tags. In some embodiments, the endoglycosidase fusion protein essentially retains the desired characteristics. The ligase fusion protein may further contain one or more additional elements, such as additional peptides or tags. In some embodiments, the ligase fusion protein essentially retains the desired characteristics.

[0038] In the specific context of this disclosure, the position of an amino acid in a protein is defined as follows: (i) starting from the N-terminus, and (ii) designating the first amino acid position at the N-terminus as 1. An amino acid (e.g., Ser) at a given position (e.g., site 34) can be represented as Ser34. An amino acid (e.g., His) at a given amino acid position (e.g., site 272) can be replaced by another amino acid (e.g., Phe), which can be represented as His272Phe. As used herein, “ligase” refers to an enzyme capable of catalyzing the covalent bonding of two or more molecules. A ligase can specifically catalyze the complexation between a first portion containing a recognition motif of a ligase donor substrate and a second portion containing a recognition motif of a ligase acceptor substrate to produce a target complex.

[0039] As used herein, the term “peptide transfer reaction” refers to a chemical reaction in which one or more amino acids (e.g., peptides) are transferred from one molecule to another. Transpeptidases are enzymes that can catalyze peptide transfer reactions between a donor substrate and an acceptor substrate. In a simplified peptide transfer reaction catalyzed by saltase, saltase first cleaves the ligase donor substrate recognition motif (also called a donor recognition motif, such as LPXTG (SEQ ID NO: 35) when SrtA is used), forming a substrate-enzyme intermediate via the formation of a thioester bond; then, the ligase acceptor substrate recognition motif (also called an acceptor recognition motif, such as GGG) nucleophilically attacks the thioester bond, releasing the enzyme and forming a new peptide bond between the two substrates. Peptide transfer reactions often result in the complexation of two parts to form a complex.

[0040] As used herein, the term “resin” refers to an organic polymer that has a softening or melting range after heating, tends to flow under external force during softening, and is solid, semi-solid, or liquid at room temperature. “Halogenated resin” refers to a novel compound formed by substituting at least one functional group in a resin with a halogenated group.

[0041] As used herein, the term “compounding” refers to a covalent bond between at least two parts (for example, at least two molecules or at least two ends of the same molecule).

[0042] As used herein, a “complex” can be prepared from at least two parts (e.g., at least two molecules or at least two ends / side chains of the same molecule) via covalent bonds.

[0043] As used herein, “biocomplex” refers to a complex in which at least one of the complexed parts is a biomolecule. Examples of biocomplexes include therapeutic molecules complexed with polymers, lipids, antibodies, peptides, aptamers, or small molecule ligands, such as siRNA complexes, peptide hormone complexes, peptide peptide complexes, peptide drug complexes, antibody drug complexes, and multispecific antibodies.

[0044] The term "target molecule" refers to a molecule that has affinity for a specific target (e.g., receptors, cell surface proteins, cytokines, etc.). Target molecules can deliver a payload to a specific site in vivo through targeted delivery. A target molecule can recognize one or more targets. A specific target site is defined by the target recognized by the target molecule. For example, a target molecule that targets receptors can deliver cytotoxins to a site containing numerous receptors. Examples of target molecules include, but are not limited to, antibodies, antibody fragments, proteins that bind to a given antigen, antibody mimetic compounds, scaffold proteins with affinity for a given target, and ligands.

[0045] As used herein, the term “antibody-drug conjugate (ADC)” refers to a conjugate comprising an antibody or antibody fragment covalently bound to a payload.

[0046] As used herein, the terms “activity,” “enzyme activity,” and “catalytic activity” of a ligase (e.g., saltase) are interchangeable and refer to the ability of the ligase to catalyze a complexation reaction. As used herein, the catalytic activity of saltase in a complexation reaction (e.g., a complexation reaction between an antibody and a payload) can be expressed as a complexation efficiency (complexation efficiency = (moles of complexed antibody : moles of total antibody) × 100%) or a DAR (drug-to-antibody ratio, the mean drug-to-antibody ratio of a given antibody-drug conjugate) distribution.

[0047] As used herein, the term “antibody (Ab)” refers to an immunoglobulin (Ig) molecule or a derivative thereof that specifically binds to an antigen via at least one antigen-binding site. A “conventional” or “full-length” antibody typically consists of four polypeptides: two heavy chains (HC) and two light chains (LC). As used herein, the definition of “antibody” encompasses conventional antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, diabodies, nanobodies (i.e., single-domain antibodies, VHH domains), and anti-idiotype antibodies. Any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2) or subclass (e.g., IgG2a and IgG2b), or any derivative thereof is also intended.

[0048] Those skilled in the art will understand that the heavy chain categories include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and that there are several subclasses within them (e.g., γ1-γ4). The properties of the chains determine the “type” of the antibody, which is IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized, and the functional specificity they provide is also known. All types of immunoglobulins are within the scope of this disclosure. In some embodiments, the immunoglobulin molecule is IgG. IgG typically contains two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of about 53,000-70,000. The four chains are linked by disulfide bonds in a “Y” configuration, with the light chains beginning with a “Y” opening and continuing to surround the heavy chains through a variable region.

[0049] The light chain can be divided into kappa (κ) or lambda (λ). Each heavy chain can be bound to either the κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or recombinant host cells, the light and heavy chains are covalently linked, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the branched end in a Y configuration to the C-terminus at the bottom of each chain. The variable region of the immunoglobulin κ light chain is Vκ, and the variable region of the immunoglobulin λ light chain is Vλ.

[0050] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used depending on the function. The variable regions of the light (VL) and heavy (VH) chains determine antigen recognition and specificity. The constant regions of the light and heavy chains give rise to important biological properties such as secretion, Fc receptor binding, and complement binding, although traditionally, the number of constant regions increases as the antibody moves further away from the antigen-binding site or amino terminus. The N-terminal portion is the variable region, and the C-terminal portion is the constant region, with the CH3 and CL domains containing the carboxyl terms of the heavy and light chains, respectively.

[0051] In naturally occurring antibodies, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous sequences of antigen-specific binding amino acids that form the antigen-binding domain, assuming the antibody adopts its three-dimensional structure in an aqueous environment. The remaining amino acids in the antigen-binding domain, known as the "framework" region, exhibit small intermolecular variability. The majority of the framework region is a β-folded structure, and the CDRs either form a cyclic structure to which they bind, or, in some cases, form part of the β-folded structure. Thus, the framework region orients the CDRs in the correct direction via interchain non-covalent interactions by forming a scaffold. An antigen-binding domain with CDRs in a specific position forms a surface complementary to the epitope on the antigen, which facilitates non-covalent binding of the antibody to that epitope. For a given heavy chain or light chain variable region, the amino acids containing the CDR and framework region can be identified by methods well known to those skilled in the art (Kabat, E. et al., USD Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0052] CDRs, as defined by Kabat and Chothia, may contain duplicates or subsets of amino acid residues when compared to one another. Nevertheless, CDRs that refer to an antibody or its variants in any sense are within the scope of this disclosure. The exact number of residues containing a particular CDR varies depending on the sequence and size of the CDR. Those skilled in the art can generally determine which particular residues a CDR contains based on the amino acid sequence of the antibody's variable region. A numbering system for variable region sequences applicable to any antibody has also been defined by Kabat et al. Those skilled in the art can apply the “Kabat numbering” system to any variable region sequence without relying on other experimental data besides the sequence itself. “Kabat numbering” refers to the numbering system proposed by Kabat et al, USDept. of Health and Human Services in “Sequence of Proteins of Immunological Interest” (1983). Antibodies may also use the EU numbering system.

[0053] The "light chain constant region" consists of a portion of the amino acid sequence derived from the antibody's light chain. Preferably, the light chain constant region (CL) includes at least one of a constant κ structure domain or a constant λ structure domain. A "light chain-heavy chain pair" is an aggregate of light and heavy chains that can form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0054] The "Fc region" is the tail region of an antibody that interacts with cell surface receptors and several proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA, and IgD antibody isoforms, the Fc region consists of two identical protein fragments derived from the second and third constant regions of the antibody's two heavy chains. In IgM and IgE antibody isoforms, the Fc region contains three heavy chain constant structure domains (CH2-4). The Fc region of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is necessary for Fc receptor-mediated activity, and different glycoforms have different effects on the pharmacological properties of therapeutic antibodies.

[0055] Antibodies can be prepared using conventional recombinant DNA technology. Cell lines for antibody production can be selected, constructed, and cultured using techniques well known to those skilled in the art. These techniques are described in various experimental manuals and major publications. In this regard, references to applicable techniques in this disclosure are listed below, including Current Protocols in Immunology, Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), edited by Coligan et al., and Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DLHacker, FMWurm, in Reference Module in Life Sciences, 2017. The entire content, including supplements, is incorporated herein by reference.

[0056] In some embodiments, the DNA encoding the antibody is synthesized by conventional methods according to the amino acid sequence of the antibody described herein, placed in an expression vector, and then transfected into a host cell, which is then cultured in culture medium to produce a monoclonal antibody. In some embodiments, the expression antibody vector comprises at least one promoter element, an antibody-coding sequence, a transcription termination signal, and a poly-A tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites on both sides of the insertion sequence to which RNA is spliced. Efficient transcription can be achieved by using pre and post-SV40 promoters, early promoters from retroviruses, e.g., RSV, HTLV1, HIVI long-term repeat sequences, and cytomegalovirus, as well as promoters from several other cells, e.g., the actin promoter. Suitable expression vectors include pIRES1neo, pRetro-Off, pRetro-On, PLXSN or Pncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS. Common mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells.

[0057] In some embodiments, the inserted gene fragment needs to contain a screening marker. Common screening markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, thaumatin resistance, and other screening genes to facilitate screening and isolation of successfully transfected cells. The constructed plasmid was transfected into host cells lacking the above genes, and the transfected cells were cultured in a selective medium to produce the desired protein.

[0058] As used herein, an "antibody fragment" of an antibody refers to any portion of an antibody that contains at least a part of the variable domain of the antibody (e.g., one or more CDRs), is an antigen-binding fragment that specifically binds to the same cognate antigen as the full-length antibody, or contains the heavy-chain constant region of the antibody and is an Fc fragment that binds to an Fc receptor on the cell surface, and contains fewer amino acid residues than the full-length antibody. Antibody fragments can be obtained by various methods such as chemical or enzymatic treatment, chemical synthesis, or recombinant DNA technology. Examples of antibody fragments include, but are not limited to, Fv (fragment variable region), scFv (single-chain Fv fragment), dsFv (disulfide-stabilized variable fragment), scdsFv (single-chain disulfide-stabilized variable fragment), diabody, Fd (fragment difficult), Fab (fragment antigen-binding), scFab (single-chain Fab), Fab’, F(ab’)2, Fc (fragment crystallizable region), and any derivatives thereof.

[0059] As used herein, the term "payload" refers to the functional part contained in a complex, for example, linked via a linker. Examples of payloads include small molecules (small molecule drugs such as inhibitors and toxins (e.g., cytotoxins)), radionuclides (e.g., 225 Ac, 211 At, 212 Bi, 213 Bi, 67 Ga, <A 123 I, 124 I, 125 I, 131 I, 111 In, 177 Lu, 191m Os, 195m Pt, 186 Re, 188 Re, 119 Sb, 153 Sm, 99m Tc, 227 Th, and 90Examples of payloads include, but are not limited to, Y), glycans, PEG moieties, nucleic acids and analogs (e.g., interfering RNA), tracer molecules (e.g., fluorophores and fluorescent molecules), polypeptides (e.g., protein tags, bioactive peptides, enzymes, antibodies and antibody fragments, and protein toxins), and peptide mimetic compounds. In use herein, payloads including linkers (such as linkers containing recognition motifs for ligase substrates) and the above-mentioned payloads are considered.

[0060] As used herein, the term “natural amino acids” refers to amino acids as building blocks of proteins, including the 20 most common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as the less common selenocysteine ​​and pyrrolicin.

[0061] As used herein, the term "non-natural amino acid" refers to an amino acid that is not a protein-forming amino acid. In particular, this term refers to an amino acid that is not a natural amino acid as defined above.

[0062] As used herein, the term "peptide mimetic" refers to a compound that mimics the structure and desired characteristics of a particular peptide.

[0063] As used herein, “receptor” refers to a structure inside or on the surface of a cell that binds to a specific substance and produces a specific effect in the cell. Receptors may include T cell receptors, B cell receptors, and receptors for signaling molecules, cell growth factors, and cytokines as described herein.

[0064] As used herein, the term “cell growth factor” refers to any substance that can stimulate cell growth, healing, proliferation, survival, and differentiation. Examples of growth factors include, but are not limited to, epidermal growth factor (EGF), fibroblast growth factor (FGF), transformation growth factor (TGF), platelet-derived growth factor (PDGF), teratoma-derived growth factor (TDGF), insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and erythropoietin (EPO).

[0065] As used herein, the term "cytokine" refers to any substance released by cells of the immune system that affects other cells. Examples of cytokines include chemokines, lymphokines, colony-stimulating factors (CSFs), monocyte chemotactic proteins (MCPs), angiogenic factors, interleukins, interferons, tumor necrosis factor (TNF), growth factors, and other secreted and cell surface molecules that signal other cells. Cytokines include, but are not limited to, INFα, INFβ, INFγ, IL-1, IL-2, IL-4, IL-6, IL-8 / CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-23, IP-10 / CXCL10, eotaxin / CCL11, MCP-1 / CCL2, MIP-1α / CCL4, RANTES / CCL5, TNFα, TNFβ, and growth factors.

[0066] Low molecular weight compounds refer to molecules whose size is comparable to that of organic molecules commonly used in pharmaceuticals. This term does not include biomacromolecules (e.g., proteins), but does include low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, and pentapeptides. Typically, the molecular weight of low molecular weight compounds can be, for example, about 100 to 2000 Da, 200 to 1000 Da, 200 to 900 Da, 200 to 800 Da, 200 to 700 Da, 200 to 600 Da, or 200 to 500 Da. As used herein, low molecular weight compounds may also be known as pharmaceuticals.

[0067] Cytotoxins are substances that inhibit or prevent cell expression activity, cell function, and / or cause cell destruction. In some cases, cytotoxins currently used in ADCs may be more toxic than commonly used chemotherapeutic agents. Examples of cytotoxins include, but are not limited to, drugs that target the microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and the regulation of apoptosis. Drugs that target the microtubule cytoskeleton may be, for example, microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of tubulin polymerization inhibitors include, but are not limited to, maytansinoids, auristatin, vinblastine, colchicine, and drastatin. DNA-targeted drugs may be, for example, drugs that directly disrupt DNA structure or topoisomerase inhibitors. Examples of drugs that directly disrupt DNA structure include, but are not limited to, DNA double-strand cleavers, DNA alkylating agents, and DNA intercalators. DNA double-strand cleavage agents can be engine antibiotics, including but not limited to dinemisin, esperamisin, neocarcinostatin, and unciaramycin. DNA alkylating agents can be, for example, DNA bisalkylating agents (i.e., DNA crosslinking agents) or DNA monoalkylating agents. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, dihydroindorobenzodiazepine (IGN) dimers, and duocalmycin-like compounds. Examples of topoisomerase inhibitors include, but are not limited to, camptothecin and anthracyclines. RNA-targeting agents can be, for example, agents that inhibit splicing, such as prazienolides.Drugs targeting kinesin-mediated protein transport may be mitotic kinesin inhibitors, including but not limited to kinesin spindle protein (KSP) inhibitors. Cytotoxins also include tyrosine kinase inhibitors (TKIs) that inhibit cell proliferation, including but not limited to lapatinib, neratinib, pyrorutinib, afatinib, gefitinib, erlotinib, and osimertinib, or their derivatives. In some preferred embodiments, the cytotoxin is osimertinib. In some preferred embodiments, the cytotoxin is a derivative of osimertinib, for example, having the following structure: [ka]

[0068] A spacer is a structure located between different structural modules and capable of spatially separating them. The definition of a spacer is not limited by whether or not it has a specific function or whether or not it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, where non-amino acid structures can be, but are not limited to, amino acid derivatives or analogs. A "spacer sequence" means an amino acid sequence that functions as a spacer, and examples include, but are not limited to, single amino acids such as Leu and Gin, sequences containing multiple amino acids, such as sequences containing two amino acids such as GA, such as GGGS (SEQ ID NO: 36) and GGGGSGGGGS (SEQ ID NO: 37). Other examples of spacers include, for example, self-destructing spacers such as PAB (p-aminobenzyl).

[0069] The term "alkyl" refers to a linear or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which are bonded to the rest of the molecule via single bonds. Alkyl groups can contain 1 to 20 carbon atoms, C1-C 20Alkyl groups refer to, for example, C1-C4 alkyl groups, C1-C3 alkyl groups, C1-C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, and C3-C6 alkyl groups. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers. A divalent radical refers to a group obtained from a corresponding monovalent radical by removing one hydrogen atom from a carbon atom having free valence electrons. A divalent radical has two bonding sites attached to the rest of the molecule. For example, "alkylene" or "alkylidene" refers to a saturated divalent hydrocarbon group that is either linear or branched. Examples of alkylene groups include methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), and pentylene (-C5H 10 -), Hexylene (-C6H 12 Examples include, but are not limited to, 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene, and ethylpropylene.

[0070] The term "cycloalkyl" refers to a cyclic saturated aliphatic group composed of carbon and hydrogen atoms, which are connected to the rest of the molecule by single bonds. Cycloalkyls have 3 to 10 carbon atoms, i.e., "C3-C 10 "Cycloalkyl" refers to divalent cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl. The term "cycloalkylene" refers to a divalent cycloalkyl group.

[0071] The term "heterocyclyl" refers to a cycloalkyl group in which one or more carbon atoms are substituted with heteroatoms selected from nitrogen, oxygen, and sulfur, such as aza, oxa or sulfur clopropyl, aza, oxa or sulfur clobutyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, or tetrahydrothiopyranyl. The term "heterocyclylene" refers to a divalent heterocyclyl group.

[0072] Where “substitution” is referred to herein, unless otherwise specified, the substituent in question is selected from alkyl, halogen, amino, monoalkylamino, dialkylamino, nitro, cyano, formyl, alkylcarbonyl, carboxyl, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, formylamino, alkylcarbonylamino, formyl(monoalkyl)amino, or alkylcarbonyl(monoalkyl)amino.

[0073] As used herein, when a group is combined with another group, the bond between these groups may be linear or branched, as long as a chemically stable structure is formed. The structure formed by such a combination can be bonded to other parts of the molecule via any suitable atom in the structure, preferably via a specified chemical bond. For example, C 1--4 When describing the combination of alkylene with one of the groups including -CH2-, -NH-, -(CO)-, -NH(CO)-, and -(CO)NH-, C 1--4 Alkylenes form linear connections with the above-mentioned groups, for example, C 1--4 Alkylene-CH2-,C 1--4 Alkylene-NH-,C 1--4 Alkylene-(CO)-, C 1--4 Alkylene-NH(CO)-, C 1--4 Alkylene-(CO)NH-,-CH2-C 1--4Alkylene, -NH-C 1--4 Alkylene, -(CO)-C 1--4 Alkylene, -NH(CO)-C 1--4 Alkylene, -(CO)NH-C 1--4 An alkylene can be formed. The resulting divalent structure can be further bonded to other parts of the molecule.

[0074] As used herein, the term "isoelectric point (pI)" refers to the pH (hydrogen potential) value of an aqueous solution of a molecule (e.g., a protein) at which the molecule has no net surface charge and is expressed in pH units. The pI of a protein can be experimentally measured using methods well known in the art, such as imaging capillary isoelectric focusing (iCIEF) and capillary isoelectric focusing (CIEF). Different biomolecules with different pIs (proteins, nucleic acids, polysaccharides, etc.) may have different charges at a given pH and can be separated by methods such as ion exchange chromatography or isoelectric focusing.

[0075] As used herein, a molecule having a "basic pI" means that the molecule's pI is greater than 7.0. As used herein, a molecule having an "acidic pI" means that the molecule's pI is less than 7.0.

[0076] As used herein, “protein tag” refers to a polypeptide that can be introduced into a molecule of interest to facilitate the detection, isolation, immobilization, or capture of that molecule, or to improve one or more properties of that molecule (e.g., expression level, solubility, and stability).

[0077] Ion exchange chromatography (IEX) separates biomolecules based on differences in their net surface charge and their affinity for ion exchangers (also called culture media, resins, or stationary phases). This is a commonly used technique for biomolecule purification. For example, in anion exchange chromatography, proteins with a pI lower than the buffer's pH have a negative net surface charge and bind to positively charged anion exchangers. However, other proteins with a pI higher than the buffer's pH have a positive net surface charge and do not bind to positively charged anion exchangers, thereby passing through the culture medium with the buffer.

[0078] As used herein, the term “support” refers to a reaction mixture in solid or semi-solid form, such as a surface, gel, polymer, matrix, particles, resin, beads, or membrane, which is a water-insoluble substance that can be isolated from the mixture.

[0079] The term "ultrafiltration" or "UF" refers to a membrane filtration technique that uses a semipermeable membrane with controlled pore size to concentrate or fractionate dissolved molecules. Molecules much larger than the pores are retained in the feed solution and are concentrated in direct proportion to the volume of liquid passing through the membrane. The pore size of ultrafiltration membranes is generally between 1 and 100 nm.

[0080] The term "diafiltration" or "DF" refers to the technique of using ultrafiltration membranes to completely remove, replace, or reduce the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules. This process selectively utilizes permeable (porous) membrane filters to separate components of solutions and suspensions based on their molecular size. Ultrafiltration and diafiltration can be used in combination and are referred to as UF / DF.

[0081] The following detailed description of the Disclosure is intended to illustrate non-limiting embodiments so that those skilled in the art can understand the technical proposals, principles and practical applications of the Disclosure, and those skilled in the art can modify and implement the Disclosure in various ways.

[0082] Dual enzyme-catalyzed site-specific complex formation process This disclosure relates to an enzymatic complexing process for preparing an immune ligand / payload complex, comprising the following steps: The steps include: conjugating the first payload to an immune ligand via enzymatic catalysis by the first enzyme; The process includes the step of conjugating a second payload to the immune ligand via enzymatic catalysis by a second enzyme, Here, the first enzyme is different from the second enzyme. Provide the process.

[0083] In some embodiments, the enzymatic catalysis step of the first enzyme and the enzymatic catalysis step of the second enzyme are performed simultaneously or sequentially. In one embodiment, the enzymatic complexation process includes simultaneously complexing a first payload and a second payload molecule with an immune ligand via the enzymatic catalysis of the first and second enzymes. In another embodiment, the enzymatic complexation process includes complexing the first payload with an immune ligand via the enzymatic catalysis of the first enzyme to obtain a first complex, and complexing the second payload with the first complex via the enzymatic catalysis of the second enzyme, thereby obtaining an immune ligand / payload complex.

[0084] In some embodiments, the first enzyme comprises a ligase and / or the second enzyme comprises an endoglucosidase. In some embodiments, the first enzyme comprises a ligase. In some embodiments, the second enzyme comprises an endoglucosidase. In some embodiments, the first enzyme comprises an endoglucosidase and / or the second enzyme comprises a ligase. In some embodiments, the first enzyme comprises an endoglucosidase. In some embodiments, the second enzyme comprises a ligase.

[0085] Ligauze The ligases of this disclosure may be ligases of any purpose. In particular, they can specifically catalyze the complexation between a first portion containing a recognition motif for a ligase donor substrate and a second portion containing a recognition motif for a ligase acceptor substrate to produce a target complex.

[0086] In some embodiments, the ligase is a transpeptidase or a variant thereof. The transpeptidase may be naturally occurring or may be recombinant. In some preferred embodiments, the ligase is a saltase such as saltase A (SrtA), saltase B (SrtB), saltase C (SrtC), saltase D (SrtD), saltase E (SrtE), or saltase F (SrtF), but is not limited to these. In this specification, "soltase" or "soltase enzyme" refers to an enzyme having sortase activity that catalyzes peptide transfer reactions, and includes, but is not limited to, class A, class B, class C, class D, class E, and class F sortases of the sortase enzyme superfamily (see, for example, Dramsi, et al., Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria, Research in Microbiology, (2005), 156:289-297; Bradshaw, et al., Molecular features of the sortase enzyme family, FEBS Journal, (2015), 282:2097-2114; Malik and Kim, A comprehensive in silico analysis of sortase superfamily, J Microbiol., (2019), 57(6):431-443; and EP3647419A1). Such enzymes may be named SrtA, SrtB, SrtC, SrtD, SrtE, or SrtF, but are not limited to these. Saltases may be naturally occurring or recombinant. Naturally occurring saltase enzymes may be found in any strain, species, or subspecies of various Gram-positive bacteria, such as Streptococcus (e.g., Streptococcus pneumoniae and Streptococcus pyogenes), Staphylococcus (e.g., Staphylococcus aureus and Staphylococcus aureus), Bacillus (e.g., Bacillus anthrax), and Listeria (e.g., Listeria monocytogenes).Engineered saltases, such as saltase mutants having one or more amino acid residue substitutions, deletions, or insertions, can be obtained from their natural counterparts by methods well known in the art, such as protein recombination and chemosynthesis. Other mutants of any wild-type saltase well known in the art (e.g., those having one or more active groups or labels) are also contemplated. The condition is that the mutant has the same or similar function as the wild-type saltase. Those skilled in the art can readily identify saltases based on their sequence and other characteristics and assign them to a particular class. However, the definition of saltase is not limited by any classification method or nomenclature.

[0087] In some specific embodiments, the ligase is saltase A (SrtA). SrtA is naturally occurring or can be recombinant. Examples of SrtA include those described in U.S. Patent No. 7,238,489 and Malik and Kim, 2019, derived from various strains, subspecies, or species of the genera Streptococcus (e.g., Streptococcus pneumoniae and Streptococcus pyogenes), Staphylococcus (e.g., Staphylococcus aureus and Staphylococcus aureus), Streptomyces (e.g., Streptomyces cericcolor), etc., as well as from the genera Bacillus (e.g., Bacillus anthrax), Lactobacillus (e.g., Lactobacillus plantarum), and Listeria (e.g., Listeria monocytogenes). Amino acid sequences of various SrtA can be found, for example, in U.S. Patent No. 7,238,489 or in public sequence databases (such as GenBank and Uniprot), the relevant information of which is incorporated herein by reference. Exemplary amino acid sequences of natural SrtA useful in this disclosure are Uniprot accession numbers: Q2FV99, A0A3S0JRJ4, A0A2T4Q430, A0A507SMZ3, A0A1F2JEX6, A0A364UNR7, A0A1J3ZU75, A0A0M2NSU2, A0A432A5V1, A0A1J4HB57, A0A4Q8MXV4, W1W5Z3, Recombined SrtA may also be, but not limited to, A0A2T4KDK7, A0A2K4DQX6, A0A2T4KHW3, A0A380FYB6, A0A2K4C0Y9, A0A4Q9WQB8, A0A121AFU6, A0A1Q8DH59, A0A5B2YTH7, A0A533IYI6, Q4L923, A0A1F1M8Z4, A0A2A1KC84, and A0A133Q671. Recombined SrtA have been reported in various publications, e.g., WO 2016 / 014501, the relevant content of which is incorporated herein by reference.For example, recombinant SrtA having one or more substitutions (such as Pro94Arg, Asp160Asn, Asp165Ala, Lys190Glu, Lys196Thr, Glu105Lys, and Glu108Gln) compared to Q2FV99, as described in WO 2016 / 014501, or cleaved SrtA having 59 amino acid deletions at the N-terminus compared to Q2FV99, may be considered. The amino acid sequences of SrtA mutants can have at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with any other amino acid sequences mentioned above. Also contemplated are any wild-type SrtA mutants known in the art (e.g., those having one or more active groups or labels). The condition is that the mutant has the same or similar function as wild-type SrtA.

[0088] In some embodiments, SrtA comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 (WT). In some other embodiments, SrtA comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26, and includes amino acid substitutions at positions 34, 100, 105, and 136. In some embodiments, the amino acid residues at positions 34, 100, 105, and 136 are substituted with Ser, Asn, Ala, and Thr (i.e., [Ser34][Asn100][Ala105][Thr136], SNAT (SEQ ID NO: 53)), Tyr, Asn, Ala, and Thr (i.e., [Tyr34][Asn100][Ala105][Thr136], YNAT (SEQ ID NO: 54)), Trp, Asn, Asp, and Thr (i.e., [Trp34][Asn100][Asp105][Thr136], WNDT (SEQ ID NO: 55)), or Val, Asn, Asn, and Ser (i.e., [Val34][Asn100][Asn105][Ser136], VNNS (SEQ ID NO: 56)). In certain embodiments, saltase A contains the amino acid sequence of SEQ ID NO: 27, which is the SNAT counterpart of SEQ ID NO: 1.

[0089] In some embodiments, Saltase A comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26.

[0090] In some embodiments, Saltase A comprises amino acids having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with amino acids selected from the group consisting of SEQ ID NOs: 1 to 26, and includes amino acid substitutions of SNAT, YNAT, WNDT, or VNNS at positions 34, 100, 105, and 136.

[0091] In another embodiment, SrtA is provided, comprising amino acids selected from the group consisting of SEQ ID NOs: 1 to 26, and having amino acid substitutions of SNAT, YNAT, WNDT, or VNNS at positions 34, 100, 105, and 136, or amino acids having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. In another specific embodiment, SrtA is provided, comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto.

[0092] In some embodiments, Saltase A comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 27, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto.

[0093] Ligase Fusion Protein In a general embodiment, a ligase fusion protein comprising a ligase and a self-labeled protein tag is provided. In some embodiments, the ligase fusion protein comprises a ligase and a Halo tag. In some embodiments, the ligase fusion protein comprises a ligase and one or more self-labeled protein tags. In some embodiments, the ligase fusion protein comprises a ligase, a Halo tag, and a His tag.

[0094] Halo Tag The Halo tag is a haloalkyl group (for example, the haloalkyl part -(CH2) 2-30 -X is a mutant haloalkane dehalogenase or a variant thereof that removes a halogen from a drug (wherein X is a halogen such as F, Cl, Br, I, or especially Cl or Br) and forms a covalent bond with the rest of the substrate. Mutant haloalkane dehalogenases are described, for example, in WO 2006 / 093529 and WO 2008 / 054821, the relevant contents of which are incorporated herein by reference. Mutant haloalkane dehalases useful to this disclosure include, but are not limited to, mutants of xanthobacter dehalases (e.g., xanthobacter autotropicus dehalase (DhIA)) or rhodococcus dehalases (e.g., rhodococcus rhodokuros dehalase (DhaA)) that include one or more substitutions in the three catalytic residues, such as substitution of His272 with Phe / Ala / Gly / Gln / Asn or substitution of Asp 106 with Cys, or other substitutions, as described in WO 2008 / 054821. The condition is that the mutant haloalkane dehalase can form a covalent bond with the haloalkyl substance.

[0095] In some preferred embodiments, the Halo tag includes the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Halo tag includes an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with SEQ ID NO: 28.

[0096] Embodiments further including additional elements and / or modifications Optionally, the ligase fusion protein may further contain one or more additional elements, such as additional polypeptides or labels. Preferably, the ligase fusion protein substantially retains the desired properties. Those skilled in the art can select appropriate elements based on the desired function or properties of the fusion protein. Methods for introducing these elements are well known in the art.

[0097] The additional polypeptide can be a protein tag having the desired properties. Examples of protein tags include, but are not limited to, reporter proteins, binding tags, and solubility-enhancing tags. Examples of reporter proteins include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein and its variants), AP (alkaline phosphatase), and HRP (horseradish peroxidase). Binding tags can effectively bind to their corresponding binding partners in a covalent or non-covalent manner. Examples of binding tags include polyhistidine tags (i.e., His tags, e.g., His6 or His8 tags), Fc tags (constant regions of immunoglobulin heavy chains (domains 3 and 4)), calmodulin tags, maltose-binding proteins (MBPs), glutathione-S-transferase (GST), S tags (interact with ribonuclease S-proteins), peptides that bind avidin / streptavidin / neutral avidin (e.g., SBP tags, Strep tags, and Strep tag II), Halo tags, SNAP tags, and CLIP tags (DNA repair protein O2). 6Examples include, but are not limited to, recombinant mutants of alkylguanine-DNA alkyltransferases and their mutants. When expressed as part of a recombinant protein, solubility-enhancing tags can typically enhance the expression level and solubility of the recombinant protein. Examples of solubility-enhancing tags include, but are not limited to, the GB1 tag (the B1 domain of Streptococcus protein G), the Z domain of Staphylococcus protein A, SUMO (small ubiquitin-related modifier), thioredoxin, GST, and MBP. The properties of a protein tag do not constitute any limitation to embodiments, and it is understood that a protein tag may have one or more properties, for example, a reporter protein or binding tag may also be a solubility-enhancing tag.

[0098] The additional polypeptide may also be a short peptide that can function as a linker, spacer, or enzymatically cleavable sequence (e.g., a TEV protease recognition motif or a thrombin recognition motif). In some embodiments, the linker peptide may be rigid or flexible (e.g., polyglycine stretch, (G4S) n The linker peptide (SEQ ID NO: 57), in which G is glycine, S is serine, and n is an integer from 1 to 6, preferably an integer from 2 to 5, can be inserted between the ligase and the Halo tag using methods well known in the art to ensure proper function of the fusion protein. In some embodiments, the linker peptide is (G4S)2. In some embodiments, the ligase fusion protein contains the amino acid sequence of SEQ ID NO: 29.

[0099] The ligase fusion protein may include one or more modifications, and the ligase, Halo tag, and additional polypeptide (where applicable) may be modified independently, for example, by substitution, deletion, addition, insertion of one or more amino acids, or introduction of a partial or active group at one or more suitable residues, insofar as the desired biological activity or function of the modified fusion protein is substantially similar to that of the corresponding fusion protein.

[0100] pI In some embodiments, the ligase has an isoelectric point (pI) of about 7.5 to about 10.0, the Halo tag has a pI of about 4.5 to about 5.0, and the pI of the ligase fusion protein is about 2.0 to about 4.5 pH units lower than the pI of the ligase. In these embodiments, one or more additional elements (e.g., additional polypeptides or labels as defined above, or combinations thereof) and / or modifications (e.g., amino acid substitutions, deletions, additions, insertions, or partial or active groups) are included, preferably, to achieve the desired pI difference between the ligase fusion protein and the ligase. In some embodiments, the additional polypeptide (if applicable) may have a specific pI that helps achieve the desired pI of the ligase fusion protein.

[0101] In some embodiments, the pI of the ligase fusion protein is about 4.5 to about 6.5, for example, about 4.5, 4.6, 4.7, 4.8, 4.9, 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, or 6.5. In some preferred embodiments, the pI of the ligase fusion protein is about 5.0 to about 6.0.

[0102] In some embodiments, the pI of the ligase is about 7.5 to about 8.5, for example, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pI of the ligase is about 8.6 to about 9.5, for example, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5. In some embodiments, the pI of the ligase is about 9.6 to about 10.0, for example, about 9.6, 9.7, 9.8, 9.9, or 10.0.

[0103] In some specific embodiments, the pI of the fusion protein is approximately 5.0 to 6.0, and the pI of the ligase is approximately 7.6 to 9.7.

[0104] In some embodiments, the ligase is a saltase. The saltase can be selected from SrtA, SrtB, SrtC, SrtD, SrtE, and SrtF.

[0105] In some preferred embodiments, SrtA comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 (WT). In some other embodiments, SrtA comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, and includes amino acid substitutions at positions 34, 100, 105, and 136. In some embodiments, the amino acid residues at positions 34, 100, 105, and 136 are substituted with Ser, Asn, Ala, and Thr (SNAT), Tyr, Asn, Ala, and Thr (YNAT), Trp, Asn, Asp, and Thr (WNDT), or Val, Asn, Asn, and Ser (VNNS), respectively. The pIs of these SrtA are listed in Table 1.

[0106] [Table 1]

[0107] In some embodiments, Saltase A comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

[0108] In some embodiments, Saltase A comprises amino acids having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with amino acids selected from the group consisting of SEQ ID NOs: 1 to 12, and includes amino acid substitutions of SNAT, YNAT, WNDT, or VNNS at positions 34, 100, 105, and 136.

[0109] In certain embodiments, saltase A contains the amino acid sequence of SEQ ID NO: 27. It is the SNAT counterpart of SEQ ID NO: 1 and has a pI of 8.508.

[0110] In some embodiments, a rigid or flexible linker peptide (e.g., polyglycine stretch, (G4S)) is used. n The linker peptide (wherein G is glycine, S is serine, and n is an integer from 1 to 6, preferably an integer from 2 to 5) can be inserted between the ligase and the Halo tag using methods well known in the art to ensure proper function of the fusion protein. In some embodiments, the linker peptide is (G4S) n In some embodiments, the ligase fusion protein contains the amino acid sequence of SEQ ID NO: 29.

[0111] Method for obtaining ligase fusion proteins The ligase, Halo tag, and additional polypeptides (if applicable) can be fused in any manner. In some embodiments, the ligase is located at the N-terminus of the Halo tag. In some embodiments, the Halo tag is located at the N-terminus of the ligase. In some embodiments, a rigid or flexible linker peptide (e.g., polyglycine stretch, (G4S)) may be used. n The linker peptide (wherein G is glycine, S is serine, and n is an integer from 1 to 6, preferably an integer from 2 to 5) can be inserted between the ligase and the Halo tag using methods well known in the art to ensure proper function of the fusion protein. In some embodiments, the linker peptide is (G4S) n In some embodiments, the ligase fusion protein contains the amino acid sequence of SEQ ID NO: 29.

[0112] Ligase fusion proteins can be obtained using various techniques well known in the art, such as recombinant DNA technology, chemical synthesis, expression from nucleic acids obtained by enzyme-catalyzed or chemical complexing methods. In some preferred embodiments, the ligase fusion protein is a recombinant protein encoded by a nucleic acid containing nucleic acid sequences encoding a ligase and a Halo tag. The recombinant protein can be expressed in a suitable host cell, such as mammalian cells, bacteria, yeast cells, or insect cells, preferably bacteria, such as Escherichia coli, and then purified.

[0113] Nucleic acids and vectors Also provided is a nucleic acid encoding a ligase fusion protein according to the Disclosure, comprising a first polynucleotide encoding the ligase according to the Disclosure and a second polynucleotide encoding a Halo tag, wherein the first and second polynucleotides are operably linked to a promoter. In some embodiments, the nucleic acid according to the Disclosure further comprises a third polynucleotide encoding an additional polypeptide operably linked to the ligase and the Halo tag. Examples of the third polypeptide are as described above.

[0114] In some embodiments, the first polynucleotide encodes saltase A, and the second polynucleotide encodes a Halo tag. In some embodiments, the first polynucleotide encodes saltase A having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26, and the second polynucleotide encodes a Halo tag having the amino acid sequence of SEQ ID NO: 28. In a particular embodiment, the first polynucleotide encodes SrtA having the amino acid sequence of SEQ ID NO: 27, and the second polynucleotide encodes a Halo tag having the amino acid sequence of SEQ ID NO: 28. In another particular embodiment, the nucleic acid encodes a ligase fusion protein having the amino acid sequence of SEQ ID NO: 29. It will be understood by those skilled in the art that one or more nucleotides in the nucleic acid can be optimized without departing from the spirit of this disclosure.

[0115] In some embodiments, the nucleic acids according to the Disclosure are prepared as recombinant nucleic acids that may further comprise one or more additional polynucleotides, such as polynucleotides encoding regulatory elements and protein tags. Such regulatory elements can modulate the expression of the fusion protein according to the Disclosure and include, but are not limited to, enhancers, insulators, and intrasequence ribosome entry sites (IRESs). Recombinant nucleic acids, including the nucleic acids according to the Disclosure, can be prepared using molecular cloning techniques well known in the Art, such as chemosynthesis, site-directed mutagenesis, and polymerase chain reaction (PCR) techniques (see Sambrook, J, E.F. Fritsch and T. Maniatis (1989), Molecular Cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0116] In some embodiments, the nucleic acids according to this disclosure are cloned into a vector, preferably an expression vector expressible in a host cell (e.g., bacterial, mammalian, yeast, or insect cell). Those skilled in the art can select an appropriate expression vector depending on the properties of the ligase fusion protein and the host cell used. In some embodiments, the vector is a bacterial expression vector expressible in bacteria such as Escherichia coli. In some embodiments, the expression vector may further contain one or more selectable marker genes, such as neomycin or puromycin resistance genes. After expression, the fusion protein may be purified using methods well known in the art, depending on the protein tag used. Depending on the type of host cell used and the purification strategy, those skilled in the art can select an appropriate expression vector, promoter, regulators, and protein tag.

[0117] Immobilized ligament In another general embodiment, an immobilized ligase is provided, comprising a ligase fusion protein according to the present disclosure immobilized on a support.

[0118] The support may be in solid or semi-solid form, made from any material. Non-limiting examples of supports include, but are not limited to, resins (e.g., agarose resin, silicone resin, polymethyl methacrylate resin, epoxy resin, or cellulose resin), gels (e.g., alginate hydrogel), beads / microspheres / particles (e.g., polystyrene beads, magnetic particles), plates, wells, tubes, films, membranes, matrices, and glass (e.g., glass slides).

[0119] In some preferred embodiments, the support is a resin. In some more preferred embodiments, the support is selected from the group consisting of agarose resin, silicone resin, polymethyl methacrylate resin, and cellulose resin. In certain embodiments, the support is a highly crosslinked agarose resin.

[0120] Methods for enzyme immobilization are well known in the art, including adsorption, covalent or non-covalent bonding, capture, encapsulation, and crosslinking. It is desirable that the maximum enzymatic activity of the ligase be preserved after immobilization, and that a minimum amount of free ligase be present in the complex product after the complex reaction. Preferably, the support is modified on its surface to include one or more functional groups so that the ligase fusion protein can be covalently immobilized on the support.

[0121] Preferably, the support contains one or more chemically active functional groups that can form covalent bonds with reactive groups (such as amines, thiols, and carboxylates) of the ligase fusion protein or reactive groups in the haloalkyl group, or contains one or more binding partners of the corresponding binding tag / affinity label contained in the ligase fusion protein. The correspondence between chemically active functional groups and reactive groups, or between binding tags / affinity labels and binding partners, is well known in the art. In some embodiments, the ligase is covalently immobilized on the support via a self-labeled protein tag, such as a SNAP tag, CLIP tag, His tag, Halo tag, or variants thereof. In one embodiment, the ligase is immobilized on the support by a covalent interaction between a haloalkyl linker and a Halo tag.

[0122] In some embodiments, the support comprises a chemically active functional group that can form a covalent bond with a reactive group on the ligase fusion protein (such as amines, thiols, and carboxylates) or with a reactive group in the haloalkyl group. In some specific embodiments, the support comprises a functional group selected from the group consisting of cyanates, isothiocyanates, isocyanates, carbodiimides, N-hydroxysuccinimide (NHS) esters, amines, carbonates, epoxides, maleimides, haloacetyls, aziridines, ethyl chloroformate, and aliphatic aldehydes.

[0123] In some embodiments, the support is an epoxy-activated resin, a CNBr (cyanide bromide)-activated resin, or an NHS-activated resin, preferably an epoxy-activated resin. In some specific embodiments, the support is an epoxy-activated agarose resin, preferably a highly crosslinked epoxy-activated agarose resin. In some preferred embodiments, the epoxy-activated resin is pre-treated to introduce an amino group before reacting with a haloalkyl substance. In some preferred embodiments, the pre-treatment of the epoxy-activated resin is carried out using ammonia. In some preferred embodiments, the pre-treatment of the epoxy-activated resin results in the introduction of an amino group onto the oxirane ring, and the ring-opening of the oxirane ring yields a hydroxyl group. Such a hydroxyl group is optionally terminal-capped in a subsequent step in the preparation of the support. In certain embodiments, the pre-treatment of the epoxy-activated resin results in the introduction of an amino group onto the oxirane ring, and the ring-opening of the oxirane ring yields a hydroxyl group, which is optionally esterified using an esterifying agent (e.g., an acetylating agent such as Ac2O) in a subsequent step in the preparation of the support. Such pre-treated epoxy-activated resins fall within the scope of “epoxy-activated resins” as defined above. In some preferred embodiments, the resin is an agarose resin (e.g., a highly crosslinked agarose resin) or a polymethyl methacrylate resin.

[0124] In some other embodiments, the support includes one or more binding partners of the corresponding binding tag / affinity label contained in the ligase fusion protein, such as additional tags or affinity labels. The correspondences between reactive groups or between binding tags / affinity labels and binding partners are well known in the art. Examples of binding tags / affinity labels and their corresponding binding partners include His tags and Ni 2+Examples include, but are not limited to, biotin / SPB tag / Strep tag / Strep tag II and streptavidin / avidin / neutral avidin, GST tag and glutathione, Fc tag and protein A, calmodulin tag and Ca, MBP and amylose, S tag and ribonuclease S-protein, SNAP tag and benzylguanine (BG) derivatives, and CLIP tag and benzylcytosine (BC) derivatives.

[0125] In some preferred embodiments, the support is functionalized to form a covalent interaction with the Halo tag by including a haloalkyl linker. A haloalkyl linker can be introduced into the support by covalently bonding one or more functional groups contained in the support to one or more reactive groups in the haloalkyl substance, and the support thus obtained is also known as a haloalkyl linker-modified support. A haloalkyl linker-modified support is within the scope of “support” as defined above. Examples of haloalkyl substances include, but are not limited to, those described in US20060024808A1 and W02006093529. Methods for preparing haloalkyl substances and such supports are described, for example, in U.S. Patents 7,429,472, 7,888,086 and 8,202,700 and Japanese Patent No. 4748685, the relevant contents of which are incorporated herein by reference.

[0126] The haloalkyl substance may include a haloalkyl moiety containing a primary or secondary halo group, preferably a primary halo group. The halo group of the haloalkyl moiety is selected from F, Cl, Br, and I, preferably from Cl and Br. In some embodiments, the haloalkyl substance has the structure of the following formula (I). (F1 a ―H1 b ) r ―Lh―(F2 b ―H2 a ) s (I) During the ceremony, F1 and F2 are moieties containing reactive groups that can independently form covalent bonds with chemically active functional groups contained in the support. H1 and H2 are independently Halo C 2-30 Selected from alkyl groups, Lh is either a chemical bond or C 3-200 It is an alkylene, where one or more (-CH2-) structures in the alkylene are optionally substituted with -O-, -NH-, -(CO)-, -NH(CO)-, and -(CO)NH-. Lh is -OC 1-10 Alkyl, -NH-C 1-10 Alkyl, -(CO)-C 1-10 Alkyl, -NH(CO)-C 1-10 Alkyl and -(CO)NH-C 1-10 Optionally substituted with one, two, or three substituents selected from alkyl groups, a is either 0 or 1, and b is either 0 or 1, provided that a and b are different. r is an integer between 1 and 100. s is an integer between 1 and 100.

[0127] In some embodiments, r is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, s is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the reactive group in F1 or F2 is selected from amino, amine, thiol groups, and active esters. In some embodiments, the active ester comprises one or more carboxylic acid groups (e.g., a suitable alcohol or phenolic carbonate monoester, such as an electron-deficient phenol like 4-nitrophenol, or, for example, an NHS ester or sulfo-NHS ester) or one or more sulfonic acid groups (e.g., a methanesulfonic acid active ester, for example, MsO-).

[0128] In a particular embodiment, F1 or F2 is [ka] That is the case.

[0129] In some embodiments, H1 and H2 are independently halo C 2-20 Alkyl, preferably halo C 2-10 Alkyl, particularly halo-C6 alkyl, is selected. In some specific embodiments, the alkyl in H1 or H2 is a linear alkyl. In certain embodiments, H1 or H2 is (CH2) 2-30 -X, preferably (CH2) 2-20 -X, more (CH2) 2-10 -X, in particular (CH2)6-X, where X is a halogen selected from F, Cl, Br and I.

[0130] In some preferred embodiments, the support is HaloLink TM It is made of resin (Promega).

[0131] In some more preferred embodiments, the support is -(CH2) 2-30 The resin may contain a haloalkyl linker having the structure -X, where X is a halogen selected from the group consisting of F, Cl, Br, and I. In certain embodiments, the support is a haloallyl linker-modified resin, preferably an agarose resin or a polymethyl methacrylate resin, more preferably a highly crosslinked agarose resin.

[0132] In some specific embodiments, a is 1, b is 0, r is 1, s is 1, and F1 is [ka] And Lh is, [ka] And H2 is (CH2) 2-20 -Cl, and the chloroalkyl group has the structure of formula (I-1). [ka] In the formula, u is an integer between 1 and 20, v is an integer between 0 and 20, and w is an integer between 1 and 19.

[0133] In a particular embodiment, u is 3, v is 2, w is 5, and the chloroalkyl group has the structure of the following formula (I-1-1). [ka]

[0134] In a particular embodiment, the support is a chloroalkyl linker-modified support having the structure of formula (II). [ka] In the formula, u is an integer between 1 and 20, v is an integer between 0 and 20, and w is an integer between 1 and 19. [ka] represents the support, which is a resin, beads, membrane, gel, matrix, film, plate, well, tube, glass slide, or surface, preferably a resin, more preferably an agarose resin, silicone resin, polymethyl methacrylate resin, or cellulose resin, most preferably a highly crosslinked agarose resin. For clarity, only one single chloroalkyl-linker moiety bound to the support is shown, but it should be noted that it is understood that there can be many such chloroalkyl-linker moieties bound to the support.

[0135] In one embodiment, the chloroalkyl linker-modified support represented by formula (II) is provided together with the chloroalkyl substance of formula (I-1), [ka] It is prepared using resins, beads, membranes, gels, matrices, films, plates, wells, tubes, glass slides, or surfaces indicated by [the specified symbol].

[0136] In certain embodiments, the chloroalkyl linker-modified support represented by formula (II) is prepared from a pre-treated epoxy-activated resin, which is prepared by introducing an amino group onto the oxirane ring of the epoxy-activated resin, the ring-opening of the oxirane ring during pre-treatment yields a hydroxyl group, which is optionally esterified with Ac2O in a subsequent step in the preparation of the support, and the support represented by formula (II) has the structure of formula (II-1). [ka] During the ceremony, substructure [ka] represents a pre-treated epoxy-activated resin, where, [ka] The portion represents an oxirane ring that reacts with an amino group to open its ring, giving an esterified hydroxyl group, and subsequently forming AcO-. [ka] The portion represents the other portion of the pre-treated epoxy-activated resin.

[0137] In some embodiments, the immobilized ligase has the following structure. Support ---- Linker ---- Halo tag ---- Ligauze During the ceremony, The support is a solid support selected from, for example, resins, beads, membranes, gels, matrices, films, plates, wells, tubes, glass slides, or surfaces, preferably resins, more preferably agarose resins, silicone resins, polymethyl methacrylate resins, or cellulose resins, most preferably highly crosslinked agarose resins.

[0138] The linker is a linker portion covalently bonded to the support, for example, comprising a chain of 10 to 60 carbon atoms, and optionally containing one or more ether, ester, carbamate, and / or amide bonds, for example, formula (II-1') or (II'). [ka] In the formula, u is an integer between 1 and 20, v is an integer between 0 and 20, and w is an integer between 1 and 19. The Halo tag is a Halo tag (haloalkane dehalogenase polypeptide) covalently bonded to the linker. Ligase is a ligauced polypeptide. Here, one or more "--linker--Halo tag--ligase" segments are bound to the same support.

[0139] In some embodiments, an immobilized ligase containing the linker moiety of formula (II-1') is obtained from the following reactions: 1) a reaction between one or more chloroalkyl materials and a support to form a chloroalkyl linker-modified support; and 2) a subsequent reaction between the chloroalkyl linker-modified support and a Halo tag (e.g., a Halo tag contained in a ligase fusion protein) to obtain an immobilized ligase.

[0140] Endoglycosidase The endoglycosidase of this disclosure may be an endoglycosidase for any purpose. In particular, the endoglycosidase is an N-acetylglucosaminidase that covalently binds a donor containing an oxazoline oligosaccharide to an immune ligand containing a GlcNAc motif, wherein the donor containing the oxazoline oligosaccharide further contains a payload.

[0141] In some embodiments, the oxazoline oligosaccharide is one or more selected from the group consisting of disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, hexsaccharide oxazoline, heptasaccharide oxazoline, octasaccharide oxazoline, nonusaccharide oxazoline, decasaccharide oxazoline, and elevensaccharide oxazoline.

[0142] In some embodiments, oxazoline oligosaccharides have the following structure.

[0143] First hexose group or its derivative - (Second hexose group or its derivative) f -β-D-glucopyranosyloxazoline, f is 0, 1, 2, 3, 4, 5 or 6, and β-D-glucopyranosyloxazoline is [ka] It has the structure of [the object].

[0144] In some embodiments, the first hexose group or derivative thereof in the oxazoline oligosaccharide is selected from glucosyl, mannosyl, galactosyl, or derivatives thereof, and / or the carbon at position 6 of the first hexose group is in the -C(O)- form.

[0145] In some embodiments, the second hexose group in the oxazoline oligosaccharide or its derivative is independently selected from glucosyl, mannosyl, galactosyl, or their derivatives in each presence.

[0146] In some embodiments, each monosaccharide portion of the oligosaccharide structure is linked by a β-(1→4) glycosidic bond.

[0147] In some embodiments, the first hexose group derivative and the second hexose group derivative in the oxazoline oligosaccharide are independently selected from derivatives in which the hydroxyl group of a uronic acid or monosaccharide is substituted with an acylamino group.

[0148] In some embodiments, oxazoline oligosaccharides have the following structure.

[0149] A first hexose group or its derivative -β-D-glucopyranosyloxazoline, where the first hexose group or its derivative is mannose or its derivative.

[0150] In some embodiments, oxazoline oligosaccharides have the following structure: A first hexose group or its derivative -β-D-glucopyranosyloxazoline, where the first hexose group or its derivative is galactose or its derivative.

[0151] In a preferred embodiment, the structure of the oxazoline oligosaccharide is as follows: [ka]

[0152] In some embodiments, the endoglycosidase is N-acetylglucosaminidase. In some embodiments, the N-acetylglucosaminidase is at least one selected from Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia myricola endoglycosidase-F3), Endo S2 (endoglycosidase S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo CC (endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC) or their variants. In some embodiments, the N-acetylglucosaminidase is at least one selected from Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2 or their variants. In some preferred embodiments, the N-acetylglucosaminidase is Endo S2 or its variant.

[0153] Endoglycosidase fusion protein In another general embodiment, an endoglycosidase fusion protein comprising an endoglycosidase and a Halo tag is provided. In some embodiments, the endoglycosidase fusion protein comprises covalently linked endoglycosidase and Halo. In some embodiments, the amino terminus of the endoglycosidase is covalently connected to the Halo tag. In some embodiments, one end of the endoglycosidase is covalently connected to the Halo tag and the other end is covalently connected to the His tag. In some embodiments, the amino terminus of the endoglycosidase is covalently connected to the Halo tag and the carboxyl terminus is covalently connected to the His tag. In some embodiments, the amino terminus of the endoglycosidase is covalently connected to the Halo tag and the carboxyl terminus is covalently connected to the His tag, and the endoglycosidase is Endo S2. In one embodiment, the endoglycosidase fusion protein comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 90% identity thereto, or an amino acid sequence having one or more conserved amino acid substitutions compared to SEQ ID NO: 30. In some embodiments, the endoglycosidase fusion protein contains or comprises the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the endoglycosidase fusion protein contains the amino acid sequence shown at positions 1 to 1150 of SEQ ID NO: 30, or has at least 90% identity with the amino acids shown at positions 1 to 1150 of SEQ ID NO: 30, or has one or more conserved amino acid substitutions with the amino acids shown at positions 1 to 1150 of SEQ ID NO: 30. In some embodiments, the endoglycosidase fusion protein contains or comprises the amino acid sequence shown at positions 1 to 1150 of SEQ ID NO: 30.

[0154] His tag In some embodiments, the His tag is a plurality of consecutive histidine residues. In some embodiments, the His tag is 3 histidine, 4 histidine, 5 histidine, 6 histidine, 7 histidine, 8 histidine, 9 histidine, or 10 amino acids. In one embodiment, the His tag is His6. In one embodiment, the His tag is His8. In one embodiment, the His tag is His 10 That is the case.

[0155] pI of endoglycosidase fusion protein In some embodiments, the pI of the endoglycosidase fusion protein is approximately 4–7. In some embodiments, the pI of the endoglycosidase fusion protein is approximately 4, 4.1, 4.3, 4.5, 4.7, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, or an interval between any two of these values ​​(including the extreme values).

[0156] Method for obtaining endoglycosidase fusion proteins In one embodiment, a process for preparing an endoglycosidase fusion protein of the present disclosure is provided, comprising: (a) providing an endoglycosidase nucleic acid sequence; (b) ligating one end of the endoglycosidase nucleic acid sequence with a Halo tag nucleic acid sequence; cloning the resulting nucleic acid sequence into a suitable vector; then transforming the vector into a suitable host cell to express the endoglycosidase fusion protein of the present disclosure in the host cell. In some embodiments, a method for preparing an endoglycosidase fusion protein of the present disclosure comprises: (a) providing an endoglycosidase nucleic acid sequence; (b) ligating one end of the endoglycosidase nucleic acid sequence with a Halo tag nucleic acid sequence; and (c) ligating the other end of the endoglycosidase nucleic acid sequence with a His tag nucleic acid sequence.

[0157] Immobilized Endoglycosidase In one aspect, the present disclosure provides an immobilized endoglycosidase fusion protein comprising the endoglycosidase fusion protein of the present disclosure immobilized on a support.

[0158] In some embodiments, the endoglycosidase is covalently immobilized on the support via a self-labeling protein tag, such as a SNAP tag, a CLIP tag, a His tag, a Halo tag, or variants thereof.

[0159] In some embodiments, the support comprises a haloalkyl linker, whereby the endoglycosidase fusion protein is immobilized on the support by a covalent interaction between the haloalkyl linker and the Halo tag.

[0160] In some embodiments, the support comprises a chloroalkyl linker, and the endoglycosidase fusion protein is immobilized on the support by a covalent interaction between the chloroalkyl linker and the Halo tag. In some embodiments, the support on which the endoglycosidase fusion protein is immobilized is the same as those described in the above portion of "Immobilized Ligase".

[0161] In some embodiments, the immobilized endoglycosidase fusion protein has the following structure. Support----Linker---Halo tag----EndoS / EndoS2----His tag, or Support----Linker----Halo tag----EndoS / EndoS2 wherein The support is a solid support selected from, for example, resins, beads, membranes, gels, matrices, films, plates, wells, tubes, glass slides, or surfaces. In one embodiment, the support is a resin. In one embodiment, the support is an agarose resin, a silicone resin, a polymethyl methacrylate resin, or a cellulose resin. In a particular embodiment, the support is a highly crosslinked agarose resin. In some embodiments, the support is selected from the group consisting of NHS activated resins, CNBr activated resins, and epoxy activated resins. The linker is a linker portion covalently bonded to the support, for example, comprising a chain of 10 to 60 carbon atoms, and optionally containing one or more ether, ester, carbamate, and / or amide bonds, for example, formula (II-1') or (II'). [ka] In the formula, u is an integer between 1 and 20, v is an integer between 0 and 20, and w is an integer between 1 and 19. The Halo tag is a Halo tag (haloalkane dehalogenase polypeptide) covalently bonded to the linker. EndoS / EndoS2 is an endoglycosidase, Here, one or more "----linker----Halo tag----EndoS----His tag" segments are linked to the same support.

[0162] In some embodiments, the immobilized endoglycosidase fusion protein has the following structure. Supporter----Linker----Halo Tag----EndoS / EndoS2 Here, the support, linker, Halo tag, and EndoS / EndoS2 are as defined above. Here, one or more "----linker----Halo tag----EndoS" segments are linked to the same support.

[0163] Ligauze-mediated compounding Composite formation using ligase site-specific catalysts In some embodiments, the complex has the structure of formula (III), the first part comprising T, and the second part comprising a linker-payload intermediate of formula (IV), [ka] T comprises an immune ligand that is optionally modified to have one of the following: a recognition motif for a ligase donor substrate and a recognition motif for a ligase acceptor substrate. L includes a linker containing the other of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. P includes the payload, z is an integer between 1 and 20. t1 is an integer between 1 and 20.

[0164] t1 represents the number of payloads that combine with a single linker to form the linker-payload intermediate of formula (IV). z represents the number of compounds of formula (IV) that combine with a single T to form the compound of formula (III).

[0165] In one embodiment, z is selected from integers 1 to 10, 1 to 8, 1 to 6, or 1 to 4. In another embodiment, z is 1 or 2. In a very special embodiment, z is 2.

[0166] In some embodiments, the immune ligand is selected from an antibody, a modified antibody format, an antibody derivative or fragment, and / or a mimetic antibody, and preferably the immune ligand contains an Fc terminus that is optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. In another embodiment, T is a receptor, antibody, or antibody fragment that is optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. In a preferred embodiment, T is a molecule comprising an Fc fragment and an antigen-binding fragment of an antibody, optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. In another embodiment, T is a soluble receptor that is optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif.

[0167] In some embodiments, T is a target molecule that is optionally modified to have one of the recognition motifs of a ligase donor substrate and a ligase acceptor substrate. Targets recognized by the target molecule (e.g., an antibody or its antigen-binding fragment) include CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD Examples include, but are not limited to, 174, CD227 / MUC1, CD352, CLDN1&2, DLL3, ErbB2 / HER2, ErbB3 / HER3, GPNMB, ENPP3, Nectin-4, EGFRvTTT, SLC44A4 / AGS-5, Mesoserine, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin-4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin-6, FGFR2, FGFR3, CA6, CanAg, Integnn ccCV, TDGF1, Ephnn A4, Trop2, PTK7, NOTCH3, C44A, and FLT3.

[0168] In one embodiment, the target molecule is an anti-human HER2 antibody or its antigen-binding fragment, which is optionally modified to have one of the following: a recognition motif for a ligase donor substrate and a recognition motif for a ligase acceptor substrate. Examples of anti-human HER2 antibodies include, but are not limited to, pertuzumab and trastuzumab.

[0169] In a preferred embodiment, the anti-human HER2 antibody is a recombinant antibody selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetic compounds. In one embodiment, the antibody mimetic compound is selected from the group consisting of scFv, minibodies, diabodies, and nanobodies.

[0170] In one embodiment, the target molecule is an anti-human HER3 antibody or its antigen-binding fragment, which is optionally modified to have one of the following: a recognition motif for a ligase donor substrate and a recognition motif for a ligase acceptor substrate. Examples of anti-human HER3 antibodies include, but are not limited to, patritumab.

[0171] In a preferred embodiment, the anti-human HER3 antibody is a recombinant antibody selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetic compounds. In one embodiment, the antibody mimetic compound is selected from the group consisting of scFv, minibodies, diabodies, and nanobodies.

[0172] For complexation with the compound of formula (IV), the target molecule of this disclosure may include a modified portion for linking to D1 or D2 in the compound of formula (V), i.e., a portion in the linker containing a ligase acceptor or donor substrate recognition motif (see below). The location of such a modified portion is not limited; for example, if the target molecule is an antibody, its location is not limited, but it may be located at the C-terminus or N-terminus of the heavy or light chain of the antibody.

[0173] In an alternative embodiment, the modification moiety for complexation with D1 or D2 in the compound of formula (V) can be introduced, for example, to the non-terminal position of the heavy or light chain of the antibody using a chemical modification method.

[0174] In one embodiment, the target molecule of the present disclosure is an antibody or an antigen-binding fragment thereof that may include a terminal modification. The terminal modification refers to a modification at the C-terminus or N-terminus of the heavy chain or light chain of the antibody, which includes, for example, a ligase recognition motif. In another embodiment, the terminal modification can further include a spacer Sp2 containing 2 to 100 amino acids, where the antibody, Sp2, and the ligase recognition motif are sequentially linked. In a preferred embodiment, Sp2 is a spacer sequence containing 2 to 20 amino acids. In a specific embodiment, Sp2 is a spacer sequence selected from the group consisting of GA, GGGS, and GGGGSGGGGS, particularly GA.

[0175] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment has three types: wild type (LC), C-terminal modified light chain (LCCT) modified by direct introduction of the ligase recognition motif LPXTG, and a short peptide spacer, in addition, C-terminal modified light chain (LCCT L ) modified by introduction of the ligase donor substrate recognition motif LPXTG. The heavy chain of the antibody or its antigen-binding fragment has three types: wild type (HC), C-terminal modified heavy chain (HCCT) modified by direct introduction of the ligase recognition motif LPXTG, and a short peptide spacer, in addition, C-terminal modified heavy chain (HCCT L ) modified by introduction of the ligase donor substrate recognition motif LPXTG. X can be any natural or unnatural single amino acid. When z in the compound of formula (IV) is 1 or 2, eight preferred antibody molecules can be formed by the combination of the above heavy and light chains. For these, refer to the amino acid sequence listing.

[0176] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment has three types: wild type (LC), N-terminal modified light chain (LCNT) modified by direct introduction of the ligase recognition motif GGG, and a short peptide spacer, in addition, N-terminal modified light chain (LCNT LThe heavy chain of the antibody or its antigen-binding fragment is of three types: wild type (HC), N-terminal modified heavy chain (HCNT) modified by direct introduction of the ligase recognition motif GGG, and N-terminal modified heavy chain (HCNT) modified by the introduction of a short peptide spacer and the ligase acceptor substrate recognition motif GGG. L ) includes.

[0177] The composite of this disclosure may further include a payload, as described in this disclosure.

[0178] Ligauze Linker In one embodiment, the linker, i.e., L in formula (III), is the compound of formula (V). (A1 p ―D1 q ―Y) t2 ―Lk―(W―A2 q ―D2 p ) t2 (V) During the ceremony, D1 and D2 independently comprise regions containing recognition motifs for ligase acceptors or donor substrates. A1 and A2 independently represent a portion containing a reactive group that can be bonded to or compounded with the payload. Lk is a chemical bond, L1-L2-L3 or L1-L2-L3-L4 or L4-L1-L2-L3 or L4. L1 and L3 are each independently selected from the following groups: -CH2-, -NH-, -(CO)-, -NH(CO)-, -(CO)NH-; and C 1-4 A combination of alkylene and one of the following: -CH2-, -NH-, -(CO)-, -NH(CO)-, -(CO)NH- L2 does not exist, or C 7-34 It is an alkylene, and one or more (-CH2-) structures in the alkylene are optionally substituted by -O-. L1, L2, and L3 are each independently and optionally substituted with one, two, or three substituents selected from -OR1 and -NR1R2. R1 and R2 are independently hydrogen and -C. 1-6 Alkyl, -(CO)-C 1-6 Alkyl and -S(=O)2-C 1-6 Selected from the group consisting of alkyl groups, L4 is a peptide sequence containing either optionally derivatized Lysine (numbers 1-100) or optionally derivatized Cysine (numbers 1-100) (the amide bond is formed by a condensation reaction between an α-amino group and a carboxyl group). Y and W are each independently selected from the group consisting of non-existent or cleavable sequences, spacers Sp1, and combinations thereof. The above cleavable sequence includes an amino acid sequence that can be cleaved by an enzyme, and the above cleavable sequence includes 1 to 10 amino acids. Sp1 is selected from the group consisting of spacer sequences containing 1 to 20 amino acids, PABs, and combinations thereof. p is either 0 or 1, and q is either 0 or 1, provided that p and q are different. t2 is defined as shown in equation (III).

[0179] In one embodiment, the linker of formula (V) is connected to the payload via A1 or A2, and is connected to the immune ligand T via the complexation of D1 or D2 to a ligase acceptor substrate recognition motif contained in the immune ligand T, and optionally, the ligase recognition motif in the immune ligand T exists in the form of a modified moiety introduced into the immune ligand, for example, by recombinant or chemical modification.

[0180] In one embodiment, L1, L2, and L3 are each independently substituted with one, two, or three substituents selected from -OR1 and -NR1R2. The substitutions are, for example, (-CH3), (-CH2-), or [ka] This occurs particularly with (-CH2-).

[0181] In one embodiment, L2 is C 7-34 an alkylene, where the alkylene is a straight-chain or branched-chain alkylene group, and optionally, one or more of the (-CH2-) structures in the alkylene can be replaced by -O-, and the alkylene is optionally substituted with 1, 2, or 3 substituents selected from -OR1 and -NR1R2. In yet another embodiment, L2 is selected from groups optionally substituted with 1, 2, or 3 substituents selected from -OR1 and -NR1R2, where the above groups are methylene, ethylene, propylene, butylene, pentylene, hexylene, 1-methylethylene, 2-methylethylene, 2-methylpropylene, and 2-ethylpropylene. In another embodiment, L2 is -(C2H4-O) i -C 1-4 an alkylene, and i is an integer from 2 to 10. “-(C2H4-O) i -” represents a structure formed by the polymerization of PEG units, where i represents the number of PEG units. In another embodiment, L2 is -(C2H4-O) i -C 1-2 an alkylene. In a specific embodiment, L2 is -(C2H4-O) i -C2H4-. In another embodiment, L2 is -C 1-4 an alkylene-(O-C2H4) i -. In another embodiment, L2 is -C 1-2 an alkylene-(O-C2H4) i -. In a specific embodiment, L2 is -C2H4-(O-C2H4) i -. In one embodiment, i is selected from the values of 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 4 to 6. In a specific embodiment, i is 4.

[0182] In another embodiment of L4 based on the desired degree of conjugation, the ε-amino of lysine can be used to introduce a maleimide functional group into the Al or A2 moiety with a suitable bifunctional crosslinker, or the ε-amino of lysine can be used to form an amide bond with the α-carboxyl group of another lysine to form a branched chain, and then the α- and ε-amino of lysine in the branched chain can be used to introduce a maleimide group with a suitable bifunctional crosslinker. Also, by increasing the number of lysines in the main chain and / or the branched side chains, the number of A1 or A2 moieties introduced by such a partial L4 can reach from 1 to 1000.

[0183] In another embodiment of L4, based on the desired degree of conjugation, the mercapto group of each cysteine can be used to react with the maleimide functional group in A1 or A2. Thus, A1 or A2 can be conjugated to Lk. Al and A2 each further contain a reactive group that can conjugate with the payload. In L4, for example, by increasing the number of cysteines in the main chain and / or the branched side chains of L4, the number of A1 or A2 moieties introduced by such a partial L4 can reach from 1 to 1000.

[0184] In one embodiment, L4 is optionally derivatized lysine.

[0185] In a preferred embodiment, the lysine derivatization is selected from the group consisting of: 1) amidation of the carboxyl group where the resulting amide NH2 is optionally substituted with an alkyl group; 2) attachment of the carboxyl group and / or the amino group to an amino acid fragment containing 1 to 10 amino acids or a nucleotide fragment containing 1 to 10 nucleotides (where the amino acid fragment is preferably Gly). 1-6

[0186] ​In one embodiment, Y and W are each independently selected from the group consisting of absent or cleavable sequences, spacer Sp1, and combinations thereof. In a particular embodiment, Y is absent. In another particular embodiment, W is absent. In yet another particular embodiment, both Y and W are absent. In one embodiment, the cleavable sequence includes an amino acid sequence that can be recognized as an enzyme substrate and can be cleaved by an enzyme. In a particular embodiment, the cleavable sequence can be enzymatically cleaved in the lysosome of a cell. In another particular embodiment, the cleavable sequence can be cleaved by a protease, particularly by a cathepsin. In yet another particular embodiment, the cleavable sequence can be cleaved by a glutaminase. In one embodiment, the cleavable sequence is selected from the group consisting of a cathepsin restriction site, a glutaminase restriction site, and combinations thereof. In one embodiment, the cleavable sequences are selected from Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly (SEQ ID NO: 58), Ala-Leu-Ala-Leu (SEQ ID NO: 59), Val-Ala-PABC, and combinations thereof.

[0187] In one embodiment, Y and W are each independently absent or selected from the spacer Sp1. In another embodiment, Sp1 is a spacer sequence containing 1 to 10, preferably 1 to 6, more preferably 1 to 4 amino acids. In a particular embodiment, Sp1 is Leu. In another particular embodiment, Sp1 is Gln. In one embodiment, Sp1 is PAB. In one embodiment, Sp is PABC. In yet another embodiment, Y and W are each independently selected from Phe-Lys-PAB, Val-Cit-PAB, Val-Ala-PABC, and Val-Lys-PAB.

[0188] In one embodiment, the amino acids contained in Y and / or W may be natural or unnatural. In a particular embodiment, Y is absent or amino acid fragment 1. Amino acid fragment 1 comprises 1 to 30 natural or unnatural amino acids, each independently identical or distinct. Amino acid fragment 1 is selected from the group consisting of cleavable sequences containing 1 to 10 amino acids, spacer sequences containing 1 to 20 amino acids, and combinations thereof. In another particular embodiment, W is absent or amino acid fragment 2. Amino acid fragment 2 comprises 1 to 30 natural or unnatural amino acids, each independently identical or distinct. Amino acid fragment 2 is selected from the group consisting of cleavable sequences containing 1 to 10 amino acids, spacer sequences containing 1 to 20 amino acids, and combinations thereof.

[0189] In one embodiment, p=0 and q=1, and the structure of the compound of formula (V) is as shown in formula (V-1) below. D1―Y―Lk―(W―A2) t2 (V-1), In the formula, A2, D1, Y, Lk, and W are defined as shown in formula (V), respectively.

[0190] In another embodiment, p=1 and q=0, and the structure of the compound of formula (III) is as shown in formula (V-2) below. (A1-Y) ―t2 Lk-W-D2 (V-2), In the formula, A1, D2, Y, Lk, and W are defined as shown in formula (V), respectively.

[0191] In one embodiment, a suitable linker can be selected from any one of Figures 13-16 of WO 2014177042 A. In yet another embodiment, a suitable linker can be selected from any one of Figures 7-10 of WO2015165413A.

[0192] In one embodiment, a suitable linker can be selected from any one of Figures 1 to 12 of WO 2014177042 A. In yet another embodiment, a suitable linker can be selected from any one of Figures 3 to 6 of WO2015165413A.

[0193] In one embodiment, a suitable linker can be selected from any one of the figures on pages 16 to 29 of WO2022218331A1.

[0194] In one embodiment, a suitable linker has the structure of formula (V'). [ka]

[0195] A portion containing a recognition motif of a ligase acceptor or donor substrate. In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of natural transpeptidases, non-natural transpeptidases, their variants, and combinations thereof. The non-natural transpeptidase enzyme may, but is not limited to, be obtained by recombinant natural transpeptidase.

[0196] In a preferred embodiment, the ligase is selected from the group consisting of natural saltases, non-natural saltases, and combinations thereof. Examples of natural saltases include SrtA, SrtB, SrtC, SrtD, SrtE, and SrtF (see, for example, US20110321183A1 and EP3647419A1). The type of ligase corresponds to a ligase recognition motif, which is then used to achieve specific coupling between different molecules or structural fragments.

[0197] In one embodiment, the recognition motif of the ligase acceptor substrate is selected from the group consisting of oligomeric glycine, oligomeric alanine, and oligomeric glycine / alanine mixtures with a degree of polymerization of 3 to 10. In a particular embodiment, the recognition motif of the ligase acceptor substrate is G n Here, G is glycine (Gly) and n is an integer between 3 and 10.

[0198] In some embodiments, the ligase is SrtA and the donor recognition motif may be LPXTG, where X is any natural or non-natural amino acid. In some embodiments, the ligase is SrtB and the donor recognition motif may be NPXTG (SEQ ID NO: 38), where X is any natural or non-natural amino acid. In some embodiments, the ligase is SrtC and the donor recognition motif may be LPXTG, where X is any natural or non-natural amino acid. In some other embodiments, the ligase is SrtD and the donor recognition motif may be LPXTA (SEQ ID NO: 39), where X is any natural or non-natural amino acid. In yet another embodiment, the ligase is SrtE and the donor recognition motif may be LAXTG (SEQ ID NO: 40), where X is any natural or non-natural amino acid. In some other embodiments, the ligase is SrtF and the donor recognition motif may be LPXTG, where X is selected from A, R, N, D, Q, I, L, and K.

[0199] In another specific embodiment, the ligase is SrtA derived from Staphylococcus aureus. Thus, the ligase recognition motif can be LPXTG, a typical recognition motif of the enzyme. In yet another specific embodiment, the recognition motif of the ligase donor substrate is LPXTGJ (SEQ ID NO: 41), and the recognition motif of the ligase acceptor substrate is G nHere, X may be any single amino acid, natural or non-natural, and J is either absent or optionally labeled, an amino acid fragment containing 1 to 10 amino acids. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment containing 1 to 10 amino acids, where each amino acid is independently any natural or non-natural amino acid. In yet another embodiment, J is G m Here, m is an integer from 1 to 10. In yet another specific embodiment, the ligase donor substrate recognition motif is LPETG (SEQ ID NO: 42). In yet another specific embodiment, the ligase donor substrate recognition motif is LPETGG (SEQ ID NO: 43). In one embodiment, the ligase may be SrtB derived from Staphylococcus aureus, and the corresponding donor substrate recognition motif may be NPQTN (SEQ ID NO: 44). In another embodiment, the ligase may be SrtB derived from Bacillus anthrax, and the corresponding donor substrate recognition motif may be NPKTG (SEQ ID NO: 45). In yet another embodiment, the ligase may be saltase A derived from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence may be LPXTGJ, where J is as defined above. In yet another embodiment, the ligase may be SrtE derived from Streptomyces coericolor, and the corresponding donor substrate recognition motif may be LAXTG. In yet another embodiment, the ligase may be SrtA derived from Lactobacillus plantarum, and the corresponding donor substrate recognition motif may be LPQTSEQ (SEQ ID NO: 46). The ligase recognition motif may also be another entirely novel recognition sequence for transpeptidases, optimized by manual screening.

[0200] LPXTGJ to G n When coupled with, the upstream peptide bond of glycine in the LPXTGJ sequence is cleaved by saltase A, and the resulting intermediate is G n It ligates to the free N-terminus, generating a new peptide bond. The resulting amino acid sequence is LPXTG n (Sequence ID 47). Sequence G nAnd LPXTGJ is as defined above.

[0201] In some specific embodiments, the ligase is SrtA derived from Staphylococcus aureus, the donor recognition motif is LPETGG, and the acceptor recognition motif is GGG.

[0202] part containing a reactive group In one embodiment, A1 and A2 in formula (V) are each independently selected from the group consisting of amino compounds, maleimides and their derivatives, thiol compounds, pyridyldithiol compounds, haloacetic acids (haloacetyl acids), and isocyanates. In another embodiment, the reactive groups in A1 and A2 are each independently selected from the group consisting of amino groups, maleimide groups, thiol groups, pyridyldithio groups, haloacetyl groups, and isocyanate groups.

[0203] In one embodiment, depending on the structure of the reactive group therein, A1 and A2 can each be independently covalently bonded to a Michael acceptor (a Michael addition acceptor molecule) via a disulfide bond, a thioether bond, a thioester bond, or a urethane bond. In a particular embodiment, A1 and A2 are each independently and optionally selected from derivatized cysteine.

[0204] In another specific embodiment, A1 and A2 are each independently and optionally selected from derivatized cysteines. In a preferred embodiment, the derivatization of cysteine ​​is performed such that 1) the resulting amide NH2 is C 1-6 The derivatization of cysteine ​​is selected from the group consisting of: 1) amidation of the carboxyl group, which is optionally substituted with an alkyl group; 2) acylation of the amino group; and 3) bonding of the carboxyl group and / or amino group to an amino acid fragment containing 1 to 10 amino acids or a nucleotide fragment containing 1 to 10 nucleotides (wherein the amino acid fragment is preferably glycine). In certain embodiments, derivatization of cysteine ​​refers to the amidation or bonding of the carboxyl group of cysteine ​​to glycine.

[0205] In one embodiment, A2 is [ka] In the formula, x is selected from the group consisting of hydrogen, OH, NH2, an amino acid fragment containing 1 to 10 amino acids, and a nucleotide fragment containing 1 to 10 nucleotides. In one embodiment, A1 is [ka] In the formula, x is selected from the group consisting of hydrogen, an amino acid fragment containing 1 to 10 amino acids, and a nucleotide fragment containing 1 to 10 nucleotides. In one embodiment, the acylation of the amino group is performed by converting the amino group of cysteine ​​to C 1-6 This refers to substitution with an alkylcarbonyl group.

[0206] In some embodiments of the linked unit of formula (V-1), t2 is 1 and D1 is G n G is glycine, and A2 is [ka] The structure of the compound of formula (V-1) is as shown in formula (V-1-1) below. [ka] In the formula, n is an integer between 3 and 10. x is selected from the group consisting of hydrogen, OH, NH2, amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides. Lk is L1-L2-L3, L1, L2, L3, t, Y, and W are defined as shown in equation (V), respectively.

[0207] In a preferred embodiment, in formula (V-1-1), X is selected from OH, NH2, and Gly.

[0208] In a particular embodiment, in formula (V-1-1), neither Y nor W is present, Lk is L1-L2-L3, L1 is -NH-, L3 is -(CO)-, and L2 is -(C2H4-O) i The compound is -C2H4-, i=4, and the structure of the compound of formula (V-1-1) is as shown in the following formula (V-1-1-1). [ka]

[0209] In a particular embodiment, in formula (V-1-1), W is absent, Y is L, L is leucine (Leu), Lk is L1-L2-L3, L1 is -NH-, L3 is -(CO)-, and L2 is -(C2H4-O). i The compound is -C2H4-, i=4, and the structure of the linked unit is as follows (V-1-1-2). [ka]

[0210] In yet another specific embodiment, in formula (V-1-1), W is absent, Y is Q, Q is glutamine (Gln), Lk is L1-L2-L3, L1 is -NH-, L3 is -(CO)-, and L2 is -(C2H4-O) i The compound is -C2H4-, i=4, and the structure of the linked unit is as follows (V-1-1-3). [ka]

[0211] In a particular embodiment, in formula (V-1-1), neither Y nor W is present, Lk is L1-L2-L3, L1 is -NH-, L3 is -(CO)-, and L2 is -C5H 10 -The structure of the connecting unit is as follows (V-1-1-4). [ka]

[0212] In a further specific embodiment, in formula (V-1-1), neither Y nor W is present, Lk is L1-L2-L3, L1 is -NH-, L3 is -(CO)-, and L2 is -C5H substituted with one -NR1R2 group. 10 -It is a group, R1 is hydrogen, R2 is -(CO)CH3, and the structure of the linking unit is as follows (V-1-1-5). [ka]

[0213] In some embodiments of the linked unit of formula (V-2), t2 is 1, D2 is LPXTG, and A1 is [ka] In that case, the structure of the compound of formula (V-2) is as shown in formula (V-2-1) below. [ka] In the formula, x is selected from hydrogen, an amino acid fragment containing 1 to 10 amino acids, and a nucleotide fragment containing 1 to 10 nucleotides. Lk is L1-L2-L3, L1, L2, L3, Y, and W are defined as shown in equation (V), respectively.

[0214] In one embodiment, X is hydrogen.

[0215] In one embodiment, A1 and A2 are each independently maleimide functional groups. The maleimide functional groups are introduced into the molecule of formula (V) by a suitable bifunctional crosslinking agent.

[0216] In preferred embodiments, the bifunctional crosslinking agents for introducing maleimide functional groups include N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), the "long-chain" SMCC analog N-[alpha-maleimidoacetoxy]succinimidate (AMAS), N-gamma-maleimidobutyryl-oxysuccinimidate (GMBS), 3-maleimidobenzoate N-hydroxysuccinimidate (MBS), and 6-maleimidohexanoate N-hydroxysuccinimidate. Cysuccinimide ester (EMCS), N-succinimidyl 4-(4-maleimidophenyl)butyrate (SMPB), succinimidyl 6-[(β-maleimidopropionamide)hexanoate (SMPH), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidecaproate) (LC-SMCC), N-succinimidyl 11-(maleimide)undecanoate (KMUS), and N-hydroxysuccinimide-(polyethylene glycol alcohol) n (SM(PEG) n Examples of bifunctional crosslinking agents include, but are not limited to, those containing ), where n is 2, 4, 6, 8, 12, or 24 polyethylene glycol (PEG) units. Exemplary maleimide functional groups introduced into A1 or A2 immediately after reaction with the bifunctional crosslinking agent may be those listed in the table below.

[0217] [Table 2]

[0218] In one embodiment, A1 and A2 are each independently selected from mc and mcc.

[0219] In some embodiments of the linker of formula (V-1), t2 is 1 and D1 is G n Here, G is glycine, A2 is mcc, W is absent, Lk is L4, and L4 is optionally derivatized lysine, and the structure of the compound of formula (V-1) is as shown in formula (V-1-2) below. [ka] In the formula, n is an integer between 3 and 10. x is selected from the group consisting of hydrogen, OH, NH2, amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides. Y is defined as shown in equation (V).

[0220] In one embodiment, Y does not exist in the above formula (V-1-2), and the linker structure is as follows (linker 2). [ka]

[0221] In a particular embodiment, in equation (V-1-2), Y is absent, n=3, x is OH, and the linker structure is as follows (linker LU104). [ka]

[0222] Endoglycosidase linker-payload compounds Linker-payload compounds having formula (VI) are also provided. [ka] During the ceremony, P' is the payload, DC(O)-L'- is a linker, DC(O)- represents a disaccharide structure. [ka] And, L' is a connecting portion. For example, it can be cleaved from P' chemically (e.g., by hydrolysis) or biologically (e.g., by enzyme catalysis) to release P'. L' is directly connected to the carbonyl group in DC(O)- via -NH- in it, where if L' is a linear connecting portion, it is connected to one P' and t3 is 1; if L' is a branched connecting portion, each branch can be connected to one P' and t3 is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0223] payload In this disclosure, the payload may be selected from small molecule compounds (e.g., small molecules with various mechanisms of action, e.g., various conventional small molecule drugs, photoacoustic therapy drugs, photothermal therapy drugs, etc., e.g., chemotherapeutic drugs, small molecule targeted drugs, immune agonists, etc., e.g., conventional cytotoxic drugs such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine; small molecule targeted drugs such as imatinib mesylate, gefitinib and analotinib; immune agonists such as STING agonists and TLR agonists), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radiolabels, etc.), short-chain peptides, polypeptides, peptide mimes and proteins. In one embodiment, the payload is selected from the group consisting of small molecule compounds and nucleic acid molecules. In a preferred embodiment, the payload is selected from small molecule compounds. In a more preferred embodiment, the payload is selected from the group consisting of cytotoxins and fragments thereof.

[0224] In one embodiment, the payload is a cytotoxin or a fragment thereof, subject to optional derivatization for connection to a linker.

[0225] In one embodiment, the cytotoxin is taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole sulfonamides, vinblastines, for example, vinblastine, vincristine, vindesine, vinorelbine, vinflunin, vinglycinate, anhydrovinblastine, drastatin 10 and its analogues, halichondrin B, eribulin, indole-3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2' -Benzoxazolyl)-coumarin (DBC), discodermorid, laurimalide, camptothecin and its derivatives, mitoxantrone, mitoganidine hydrazone, nitrogen mustard, nitrosourea, aziridines, benzodopa, carbocon, metsuredepa, uredepa, arasinomycin, actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, carminomycin, sarcomycin, actinomycin D, daunorubicin, detrubicin, adriamycin, epirubicin, esorubicin Idarubicin, maceromycin, mitomycins, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, ferricadriamycin, rhodorubicin, rufochromycin, streptozocin, dinostatin, zolubicin, trichothecene, T-2 toxin, beraclin A, basilosporine A, anguidin, ubenimex, azacerin, 6-diazo-5-oxo-L-norleucine, dimethylfolic acid, methotrexate, pteropterin, trimethrexate, edatrexate, fludarabine, The following are selected from the group consisting of 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine, carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, protein kinase inhibitors, and proteasome inhibitors.In one embodiment, the cytotoxin is selected from vinblastines, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxorubicin, duocalmycin, SN-38, cryptophycin analogs, deruxtecan, duocalmazine, centanamycin, drastancine, pyrrolobenzodiazepines, and exatecan and their derivatives. In one embodiment, the cytotoxin is selected from auristatins, particularly MMAE, MMAF, or MMAD. In another embodiment, the cytotoxin is selected from exatecan and its derivatives, for example, DX8951f. In one embodiment, the cytotoxin is selected from DXd-(1) and DXd-(2), preferably DXd-(1).

[0226] In one embodiment, the cytotoxin is a maytansinoid such as DM1. It should be noted that when using a cytotoxin containing a sulfhydryl moiety, the sulfhydryl moiety can react with the maleimide moiety to form thiosuccinimide (e.g., a maytansinoid such as DM1), and the cytotoxin can be directly linked via thiosuccinimide. In this case, in some embodiments, the payload and the sulfhydryl moiety together constitute the cytotoxin, and therefore, in this case, the payload may represent the remainder of the cytotoxin molecule excluding the sulfhydryl moiety.

[0227] In one embodiment, the cytotoxin is a compound of the following formula (i). [ka] In the formula, g is an integer between 1 and 6.

[0228] In one embodiment, g is an integer between 1 and 3, preferably 1.

[0229] In one embodiment, the cytotoxin is selected from the following compounds 1 to 15, where the wavy bond represents the linkage site to the linker. [ka]

[0230] In some embodiments, the payload is selected from DX8951f, DXd-(1), and DXd-(2), and compound 15, preferably DX8951f or DXd-(1), more preferably DXd-(1).

[0231] In some embodiments, the cytotoxin includes those that inhibit cell proliferation. In some embodiments, the cytotoxin is a tyrosine kinase inhibitor (TKI). In some embodiments, the cytotoxin is selected from lapatinib, neratinib, pirotinib, afatinib, gefitinib, erlotinib, and osimertinib, or derivatives thereof. In one embodiment, the cytotoxin is osimertinib. In a preferred embodiment, the cytotoxin is a derivative of osimertinib. In a preferred embodiment, the cytotoxin has the structure of formula (A). [ka]

[0232] Immune ligands The immune ligand is selected from antibodies, modified antibody formats, antibody derivatives or fragments, and / or mimic antibodies. In some embodiments, the immune ligand contains an Fc terminus. In some embodiments, the immune ligand contains a GlcNAc motif. In one embodiment, the immune ligand is an anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30 / TNFRSF8 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD44v6 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD117 / KITk antibody, and anti-CD123 antibody, anti-CD138 antibody, anti-CD142 antibody, anti-CD174 antibody, anti-CD227 / MU C1 antibody, anti-CD352 antibody, anti-CLDN18.2 antibody, anti-DLL3 antibody, anti-ErbB2 / HER2 antibody, anti-GPNMB antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRvIII antibody, anti-SLC44A4 / AGS-5 antibody, anti-CEACAM5 antibody, anti-PSMA antibody, anti-TIM1 antibody, anti-LY6E antibody, anti-LIV1 antibody, anti-SLITRK6 antibody, anti-HGFR / cMet antibody, anti-SLAMF7 / CS1 antibody, anti-EGFR antibody , anti-BCMA antibody, anti-AXL antibody, anti-NaPi2B antibody, anti-GCC antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-mesothelin antibody, anti-ETBR antibody, anti-EphA2 antibody, anti-5T4 antibody, anti-FOLR1 antibody, anti-L AMP1 antibody, anti-cadherin 6 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-CA6 antibody, anti-CanAg antibody, anti-integrin αV antibody, anti-TDGF1 antibody, anti-ephrinA4 antibody, anti-TROP2 antibody, anti-PTK7 antibody The antibody is an antibody selected from anti-NOTCH3 antibody, anti-C4.4A antibody, anti-FLT3 antibody, anti-B7H3 / 4 antibody, anti-TF (tissue factor) antibody, anti-ROR1 / 2 antibody, preferably anti-CD19 antibody, anti-ErbB2 / HER2 antibody, anti-ErbB3 / HER3 antibody, anti-CLDN18.2 antibody, anti-nectin-4 antibody, anti-FGFR3 antibody, and anti-Trop2 antibody, more preferably an antibody selected from anti-ErbB2 / HER2 antibody and anti-ErbB3 / HER3 antibody.

[0233] In one embodiment, the anti-ErbB2 / HER2 antibody comprises a light chain having the amino acid sequence shown in SEQ ID NO: 31 and / or a heavy chain having the amino acid sequence shown in SEQ ID NO: 32.

[0234] In one embodiment, the anti-ErbB3 / HER3 antibody comprises a light chain having the amino acid sequence shown in SEQ ID NO: 33 and / or a heavy chain having the amino acid sequence shown in SEQ ID NO: 34.

[0235] Immune ligand / payload complex In further embodiments, immune ligand / payload complexes prepared by the process of the first embodiment are provided. In one embodiment, the immune ligand is an antibody. In one embodiment, the payload is a drug. In one embodiment, the immune ligand / payload complex is an antibody-drug conjugate. In one embodiment, the immune ligand / payload complex is a bidrug-antibody-drug conjugate (dpADC). In one embodiment, the drug-antibody ratio (DAR) of the immune ligand / payload is 2 to 16, preferably 4 to 12, more preferably 4 to 8, and most preferably 6 to 8. In some embodiments, the first drug-antibody ratio of the immune ligand / payload is 2 to 4. In some embodiments, the second drug-antibody ratio of the immune ligand / payload is 2 to 4.

[0236] In some embodiments, the immune ligand / payload comprises an antibody having a first linker payload conjugated to the C-terminus of the light chain and a second linker payload conjugated to Asn-297 of the heavy chain, wherein the payloads of the first and second linker payloads are different.

[0237] In some embodiments, the linker payload for forming an immune ligand / payload complex is conjugated to an antibody catalyzed by a transpeptidase or its variants, such as saltase A, saltase B, saltase C, saltase D, saltase E, or saltase F and their variants, preferably saltase, more preferably saltase A and its variants. In some embodiments, the linker payload for forming an immune ligand / payload complex is conjugated to an antibody catalyzed by an N-acetylglucosaminidase, such as Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2, and their variants.

[0238] In some embodiments, the antibody in the immune ligand / payload complex is IgG, where the antibody binds to ErbB2 / Her2. In some embodiments, the antibody in the immune ligand / payload complex is modified with GALPETGG (SEQ ID NO: 60) to conjugate to the linker payload. In some embodiments, the antibody in the immune ligand / payload complex contains the amino acid sequence of SEQ ID NO: 31.

[0239] In some embodiments, the antibody in the immune ligand / payload complex is IgG, where the antibody binds to ErbB3 / Her3. In some embodiments, the antibody in the immune ligand / payload complex is modified with GALPETGG to conjugate to the linker payload. In some embodiments, the antibody in the immune ligand / payload complex contains the amino acid sequence of SEQ ID NO: 33.

[0240] In some embodiments, the linker payload used to form the immune ligand / payload complex has the following formula: [ka]

[0241] In some embodiments, the linker payload used to form the immune ligand / payload complex has the following formula: [ka]

[0242] In some embodiments, the immune ligand / payload complex comprises an antibody having a linker payload conjugated to the C-terminus of its light chain, wherein the linker payload is conjugated to an antibody catalyzed by a transpeptidase or its variants, such as saltase A, saltase B, saltase C, saltase D, saltase E, or saltase F and their variants, preferably saltase, more preferably saltase A and its variants, and the linker payload has the following formula: [ka]

[0243] In some embodiments, the immune ligand / payload complex comprises an antibody having a linker payload conjugated to a heavy chain Asn-297, wherein the linker payload is conjugated to an antibody catalyzed by N-acetylglucosaminidases, such as Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2, and their variants, and the linker payload has the following formula: [ka]

[0244] In some embodiments, the immune ligand / payload is a first linker payload conjugated to the C-terminus of the light chain, conjugated to an antibody catalyzed by a transpeptidase or its variants, such as saltase A, saltase B, saltase C, saltase D, saltase E, or saltase F and their variants, preferably saltase, more preferably saltase A and its variants, and having the following formula: [ka] A first linker payload and a second linker payload conjugated to the heavy chain Asn-297, which is conjugated to antibodies catalyzed by N-acetylglucosaminidases, such as Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2, and their variants, and has the following formula: [ka] It includes a second linker payload and an antibody having a second linker payload.

[0245] In some embodiments, the immune ligand / payload is a first linker payload conjugated to a heavy chain Asn-297, conjugated to an antibody catalyzed by N-acetylglucosaminidases, such as Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2, and their variants, and having the following formula: [ka] A first linker payload and a second linker payload conjugated to the C-terminus of the light chain, conjugated to an antibody catalyzed by a transpeptidase or its variants, such as saltase A, saltase B, saltase C, saltase D, saltase E, saltase F and their variants, preferably saltase, more preferably saltase A and its variants, and having the following formula: [ka] It includes a second linker payload and an antibody having a second linker payload.

[0246] Use of immune ligand / payload complexes In yet another embodiment, the use of an immune ligand / payload complex by a process of the first embodiment is provided in the manufacture of a drug for treating a proliferative disorder. In one embodiment, the proliferative disorder is selected from bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck neoplasms, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, thyroid cancer, skin cancer, leukemia, multiple myeloma, chronic lymphocytic lymphoma, adult T-cell leukemia, B-cell lymphoma, acute myeloid leukemia, Hodgkin lymphoma or non-Hodgkin lymphoma, Waldenström macroglobulinemia, hairy cell lymphoma, cellular lymphoma, Burkitt lymphoma, glioblastoma, melanoma, or rhabdomyosarcoma.

[0247] Beneficial effects Site-directed orthogonal biocomplexing modalities have solved the following technical problems: Methods reported for the construction of dpADCs are primarily based on either a bi-chemical complexing relay or an enzymatic and chemical complexing relay, both of which require a multi-step process where the CMC can be complex and time-consuming; chemical complexing often results in uncertainty of the complexing site and heterogeneous DAR distribution; and furthermore, complexes based on conventional cysteine-maleimide chemical reactions are also more prone to premature release in systemic circulation due to unstable linker design derived from the intact cysteine-maleimide ring structure. All of these raise problems in CMC production and safety concerns in clinical applications. To address this, we hereby report a highly site-directed orthogonal enzymatic complexing method for providing homogeneous bi-payload antibody complexes in a simple and efficient single-step manner.

[0248] Compared to combinations of two ADC drugs, dual-payload ADCs can significantly reduce antigen epitope occupation and competition, thus increasing potential efficacy and maximum dose. Dual-payload ADCs can also be conveniently administered regardless of differences in endocytosis efficiency, dose ratio, and the intervals between the two drugs reaching the target. Since multiple drug loadings require a single antibody modality, the cost in antibody administration can be significantly reduced. As a result, this dpADC platform and design can significantly improve the efficiency of drug candidates through the manipulation of drug synergies, increase the cost-effectiveness of dpADC development, and, more importantly, increase the success rate of drug development.

[0249] The dual enzyme orthogonal complexing technology platform is suitable for rapidly, efficiently, and high-throughput preparation of candidate ADC molecules targeting different targets, and for rapidly screening ADC drugs with novel structures and mechanisms of action (MOAs) that exhibit synergistic effects between toxins by coupling various toxins with different linkers.

[0250] Examples Embodiments of this disclosure are described in detail below. The embodiments described below are illustrative and should not be understood as limiting, but rather as serving to interpret this disclosure. Unless otherwise indicated, proportions, percentages, etc., referred to herein are calculated by weight.

[0251] Example 1. Preparation of fusion protein enzyme 1.1 Preparation of Endoglycosidase Fusion Proteins 1.1.1 Amino acid sequence of Halo-Endo S2-His [ka] Here, the Halo tag is indicated by a single underscore, and the His tag is indicated in italics.

[0252] 1.1.2 Cloning of Halo-Endo S2-His The nucleic acid sequence encoding Halo-Endo S2-His was synthesized using a standard gene synthesis method and inserted into a pET expression vector. The Halo-EndoS2-His expression plasmid was used to transform E. coli BL21(DE3). The corresponding antibiotic was added to LB (Luria-Bertani) medium, and the cells were cultured at 37°C until the OD600 reached 0.5-1.0, after which the bacteria were preserved.

[0253] 1.1.3 Purification of Halo-Endo S2-His tags (bioreactors) Glycerol bacteria were harvested and inoculated into LB liquid medium containing the corresponding antibiotic, and cultured at 100-300 rpm and 37°C for 2-8 hours. When the OD600 reached 0.5-1.0, it was inoculated into a 10 L bioreactor and cultured for 5-10 hours. Expression was induced overnight by adding 0.2 mM IPTG at 16°C. The bacterial precipitate was collected by centrifugation at 3000-5000 rpm for 10-30 minutes at 2-8°C, the bacteria were resuspended in equilibrium buffer, disrupted under pressure, and the supernatant and precipitate were separated by centrifugation at 5000-10,000 rpm for 10-60 minutes at 2-8°C.

[0254] Halo-Endo S2-His protein was purified by Ni affinity chromatography. The Ni affinity column was connected to a protein purification system, and pure water and equilibrium buffer were used for complete flushing. The supernatant sample was loaded at low flow rate. Equilibrium buffer and 80 mM imidazole buffer were used for washing, and the Halo-Endo S2-His protein was eluted with 500 mM imidazole buffer. SDS-PAGE was used for analysis.

[0255] As shown in Figure 1, Halo-Endo S2-His bound to a Ni affinity column, no target protein was present in the flow-through solution, and Halo-Endo S2-His was eluted under 500 mM imidazole conditions. The results indicate that Halo-Endo S2-His can be expressed by E. coli, cultured in a bioreactor, and purified by a Ni affinity column. Both upstream and downstream processes are easily scaled up.

[0256] 1.2 Preparation of ligase fusion proteins 1.2.1 Cloning and Purification of SrtA Nucleic acids encoding SrtA, containing amino acids selected from SEQ ID NOs: 1-26, and their variants ([Ser34][Asn100][Ala105][Thr136], SNAT; [Tyr34][Asn100][Ala105][Thr136], YNAT; [Trp34][Asn100][Asp105][Thr136], WNDT; [Val34][Asn100][Asn105][Ser136], VNNS) were synthesized using standard gene synthesis methods and subcloned into expression vector pET-21a(+) using NdeI and EcoRI via Gibson Assembly. A HiS6 tag was then inserted into the N-terminus of the SrtA open reading frame.

[0257] SrtA expression plasmids were used to transform E. coli BL21(DE3). After culturing in LB containing 50 μg / mL ampicillin at 37°C, IPTG was added to a final concentration of 0.2 mM until the OD600 reached 0.5-0.8, and saltase expression was induced at 25°C for 12 hours. Target cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl), then lysed by sonication, and the supernatant was purified on Ni-NTA agarose according to the manufacturer's instructions. The purity of saltase, as determined by SDS-PAGE, was >90%. The concentration of SrtA was calculated from A280, measured using the extinction coefficient method.

[0258] 1.2.2 Evaluation of Saltase Activity The recombinant SrtA prepared in Example 1.2.1 was subjected to an assay, and their saltase activity was measured. The reaction in a 96-well plate (total volume 100 μL, 0.085 mM Abz-LPETGK-Dnp and 18 mM triglycine in buffer A (buffer A: 5 mM CaCl2, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5)) was started at 37°C by adding 0.625 μM purified SrtA or the mutant. Abz-LPETGK-Dnp was an internal quenching peptide having 2-aminobenzimidazole (Abz) as a fluorophor and 2,4-dinitrophenyl (Dnp) as a quencher. During the cleavage of peptide LPETGK by saltase, Dnp and Abz were separated, and saltase activity could be indicated by detecting the fluorescence signal from Abz. The increase in fluorescence signal was collected continuously after 1 hour (λ exc / λ em (320nm / 420nm, Gain=85, Biotek Cytation3 plate reader).

[0259] 1.2.3 Cloning of ligase fusion proteins (Halo saltase) The nucleic acids encoding the ligase fusion proteins according to this disclosure were obtained by cloning SrtA and its variants ([Ser34][Asn100][Ala105][Thr136], SNAT;[Tyr34][Asnl00][Ala105][Thr136], YNAT;[Trp34][Asnl00][Aspl05][Thr136], WNDT;[Val34][Asnl00][Asn105][Serl36], VNNS) having amino acid sequences selected from SEQ ID NOs. 26, and Halo tags (Halo saltase) having the amino acid sequence of SEQ ID NO. 28, into bacterial expression vectors pET21a or pET24d. In the following examples, Halo saltase having the amino acid sequence of SEQ ID NO. 29, including a SrtA variant (SEQ ID NO. 27) and a Halo tag (SEQ ID NO. 28) derived from Staphylococcus aureus, was used.

[0260] 1.2.4 Purification of Halo Saltase Halo saltase was expressed in E. coli BL21(DE3), purified, and stored in 5%-10% glycerol at -80°C. For comparison, His saltase with a His6 tag and GBl saltase with a GB1 tag were prepared in the same manner.

[0261] 1.2.5 Halo-Saltase Activity procedure: (1) Purified ErbB2 / HER2 antibodies T-LCCTL-HC (ErbB2 / HER2 SEQ ID NO: 31 and HER2 SEQ ID NO: 32) were mixed in a complexing buffer with a linker-payload intermediate having the structure shown in (A) or (B) in Figure 2 at an optimal molar ratio (Ab:linker-payload intermediate = 1:1 to 1:100). (2) The Halo saltase, His saltase, or GBl saltase prepared as in Example 1.2.4 was incubated with the mixture from step (1) at 4-40°C for 0.5-20 hours. (3) The product from step (2) was stored at 4°C or -80°C. (4) The products were subjected to 12% SDS-PAGE electrophoresis to determine the compounding efficiency.

[0262] Example 2. Preparation of immobilized enzymes 2.1 Preparation of chloroalkyl-linker modified resins (chlorinated resins) Methods for preparing chlorinated resins are described, for example, in U.S. Patents 7,429,472, 7,888,086, and 8,202,700, which are incorporated herein by reference in their entirety. The resins used in the preparation of the chlorinated resins are shown in Table 2.

[0263] [Table 3]

[0264] procedure: (1) Pretreatment The resin was filtered with isopropyl alcohol, the filter cake was washed once with DMF, and then vacuum-dried. The filter cake was transferred to a flask using DMF and stirred. Ethylenediamine was then added to the mixture and stirred for 10-15 hours. The filter cake was filtered, washed with DMF, and the liquid was drained. About epoxy-activated resins: The resin was filtered with isopropyl alcohol, the filter cake was washed once with H2O, and then vacuum-dried. The filter cake was transferred to a flask and stirred. Next, 25%–28% aqueous ammonia was added to the mixture, and the system was slowly heated to 40–50°C, reacting at 40–50°C with stirring. The temperature of the system was lowered to 20–30°C, and the mixture was filtered. The filter cake was washed with H2O until the pH of the filtrate reached approximately 7–8. Next, the filter cake was washed with DMF, and the liquid was drained.

[0265] (2) The filtration cake was transferred from step (1) to a flask and stirred. Then, DMF containing a chloroalkyl substance having the structure of formula (I-1-1) and triethylamine was added to the system. The reaction was carried out with stirring. The system was then filtered, the filtration cake was washed with DMF, and finally the liquid was drained. [ka]

[0266] (3) The filtered cake was transferred from step (2) to a flask. Stirring was started. Ac2O and triethylamine were added sequentially to the mixture. The mixture was reacted while stirring. The mixture was then filtered and the filtered cake was washed with DMF. Next, the cake was washed with H2O and the liquid was drained. Finally, the mixture was transferred to a container with 20% ethanol and stored. As a result, a chlorinated resin having the structure of formula (II-1) was obtained: [ka] During the ceremony, [ka] This is a highly crosslinked agarose resin or polymethyl methacrylate resin.

[0267] 2.2 Preparation of immobilized Halo-Endo S2-His 2.2.1 Immobilization of Halo-Endo S2-His onto chlorinated resin Halo-Endo S2-His was incubated with a chlorinated resin at room temperature for 10 min to 24 hours, and washed three times with 20 mM Tris-HCl, 150 mM NaCl, and a buffer solution with a pH of 6.0 to 10.0. The activity of the immobilized endoglycosidase fusion protein resin (Halo-Endo S2-His covalently bound to a chlorinated resin ligand) was tested. After passing the test, the resin was washed with 20 mM Tris-HCl, 150 mM NaCl, and a buffer solution with a pH of 6.0 to 10.0, and finally stored at 2 to 8°C.

[0268] 2.2.2 Immobilized enzyme loading 1) 250 μL of chlorinated resin was taken for testing, excess glycosidase (Halo-Endo S2-His) was added, and the mixture was placed in a rotary mixer and fixed for 2 hours. 2) During the immobilization of Halo-Endo S2-His, the supernatant of the immobilized enzyme was collected at 15 min, 30 min, 1 h, and 2 h. The resin solution was centrifuged at 3000 g for 3 min each time at room temperature, the supernatant was aspirated, and the enzyme concentration in the supernatant was measured using a Nanodrop UV spectrophotometer. 3) The endoglycosidase concentration at each time point was calculated, and the amount of enzyme immobilized at each time point was calculated by subtracting the enzyme concentration at each time point from the starting concentration. The load change curve of the culture medium immobilized Halo-Endo S2-His process was then plotted.

[0269] SDS-PAGE detected changes in the protein profile in the supernatant, as shown in Figure 3. The amount of Halo-Endo S2-His in the supernatant decreased over time, indicating that it was specifically immobilized on the chlorinated resin.

[0270] 2.3 Preparation of immobilized Halo-soltase 2.3.1 Immobilization of Halo-soltase onto chlorinated resin procedure: (1) The purified Halo saltase prepared in Example 1.2.4 and the chlorinated resin prepared in Example 2.1 were incubated at room temperature for 10 min to 24 hours; (2) The resin was washed three times with 20 mM Tris-HCl and 150 mM NaCl (pH 6.0-10.0); (3) The enzymatic activity of immobilized Halo-soltase was determined; (4) Optionally, a column was packed with immobilized Halo saltase to obtain a Halo saltase column; (5) The immobilized Halo-soltase from step (3) or the Halo-soltase column from step (4) was washed with 20 mM Tns-HCl, 1-3 M NaCl (pH 6.0-10.0), and 0.1-1.0 M NaOH, and stored at 4°C.

[0271] 2.3.2 Characterization of Chlorinated Resins procedure: (1) 250 μL of each chlorine resin was taken and tested, an excess amount of Halo-Saltase was added, the tubes were placed on a rotor and incubated at room temperature for 2 hours; (2) At different time points in the immobilization reaction (15 min, 30 min, 1 h, and 2 h, respectively), the supernatant of each chlorinated resin was collected at room temperature for 3 minutes using a 3000 g centrifuge tube, and the Halo-soltase concentration in the supernatant was measured using a Nanodrop spectrophotometer. (3) The amount of Halo-soltase at each time point was calculated, and then subtracted from the initial concentration of Halo-soltase to obtain the amount of immobilized Halo-soltase at each time point. A curve showing the amount of immobilized Halo-soltase on the chlorinated resin was then plotted as a function of compounding time.

[0272] 2.3.3 Characterization of Immobilized Halo Saltase procedure: (1) A fixed amount of Halo-soltase was immobilized on the chlorinated resin in the same manner as in Example 2.3.1. (2) The immobilized enzyme was washed three times with 5-10 volumes of resin in 1× storage buffer, and each time it was centrifuged at 3000g for 3 minutes at room temperature to pelletize the immobilized enzyme resin, and the immobilized enzyme was resuspended in the compounding buffer. (3) 25 μL of each immobilized enzyme resin was collected, and 200 μL of GFP protein containing the donor recognition motif LPETGG and a low molecular weight reaction buffer containing a linker-payload intermediate containing the acceptor recognition motif GGG complexed with a low molecular weight compound were added to initiate the complexing reaction. (4) Supernatant samples were collected for HIC-HPLC analysis at different time points in the compounding reaction (15 min, 30 min, 1 h, and 2 h, respectively). (5) The compounding efficiency was determined by HIC-HPLC.

[0273] Example 3. Linker-payload synthesis 3.1 Synthesis of Disaccharide Compound 1 3.1.1 Synthesis of Compound 1c [ka] A newly activated 4 Å MS was added to an oven-dried two-necked flask (100 mL) equipped with a Teflon-coated magnetic stirring rod, capped with a sleeve stopper, then the system was heated under vacuum with a heat gun, cooled to ambient temperature, and then refilled with nitrogen. Compound 1a (4.41 g, 7.96 mmol) and anhydrous dichloromethane (30 mL) were added and stirred at room temperature (rt) for 0.5 hours. Compound 1b (1.89 g, 3.98 mmol) was stirred according to the same protocol and pre-dried in 20 mL of anhydrous dichloromethane in the presence of an activated molecular sieve. To the resulting reaction mixture of compound 1a, 1-(phenylsulfinyl)piperidine (BSP, 1.37 g, 6.56 mmol) and 2,4,6-tri-tert-butylpyrimidine (TTBP, 2.94 g, 11.94 mmol) were added at rt and stirred at the same temperature for 20 minutes. Next, the reaction flask was cooled to -65°C, and then trifluoromethanesulfonic anhydride (1.2 mL, 7.16 mmol) was added dropwise. After 2 minutes, a dichloromethane solution of compound 1b, which had been dried beforehand, was added to the system, and the resulting reaction mixture was stirred at -65°C until complete, while being monitored by TLC (eluent: toluene / PE = 1 / 8). The reaction mixture was then quenched with saturated sodium bicarbonate solution (20 mL), extracted with dichloromethane (150 mL x 3), and the combined organic layers were sequentially washed with water and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (eluent: toluene / PE = 1 / 12 to 1 / 10) to obtain compound 1c as a colorless viscous oily liquid (2.72 g, yield 75.4%). 11H NMR (400 MHz, chloroform-d) δ 7.55 (dd, J = 7.6, 2.1 Hz, 2H), 7.51 - 7.28 (m, 28H), 5.60 (s, 1H), 5.14 (d, J = 10.4 Hz, 1H), 5.00 (d, J = 12.0 Hz, 1H), 4.94 (d, J = 11.8 Hz, 1H), 4.90 - 4.80 (m, 2H), 4.76 (d, J = 12.0 Hz, 1H), 4.74 - 4.63 (m, 3H), 4.57 (s, 1H), 4.48 (d, J = 12.1 Hz, 1H), 4.37 (d, J = 8.1 Hz, 1H), 4.22 - 4.10 (m, 2H), 4.05 (t, J = 9.3 Hz, 1H), 3.80 (d, J = 3.1 Hz, 1H), 3.72 (dd, J = 11.2, 2.2 Hz, 1H), 3.67 - 3.52 (m, 3H), 3.49 (dd, J = 9.8, 3.1 Hz, 1H), 3.41 (t, J = 9.3 Hz, 1H), 3.35 (dt, J = 9.8, 2.9 Hz, 1H), 3.16 (td, J = 9.7, 4.8 Hz, 1H). C 54 H 56 N3O 10 + Calculated value of MS (ESI) for [M + H] + : 906.4. Observed value: 906.7.

[0274] 3.1.2 Synthesis of Compound 1d

Chemical formula

[0275] 3.1.3 Synthesis of Compound 1e [ka] To a solution of compound 1d (168 mg, 0.168 mmol) in methanol / tetrahydrofuran (1:1, 10 mL), p-toluenesulfonic acid monohydrate (35 mg, 0.168 mmol) was added, and the resulting mixture was stirred overnight under rt. After the reaction was complete, the mixture was quenched with saturated sodium bicarbonate solution (20 mL), extracted with dichloromethane (20 mL x 3), and the combined organic layer was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (eluent: MeOH / DCM = 1 / 20) to obtain compound 1e as a white solid (136 mg, yield 89%). 1 H NMR(400 MHz,chloroform-d)δ7.43-7.24(m,29H),5.76(d,J=7.9 Hz,1H),5.01(d,J=6.9 Hz,1H),4.94(dd,J=11.8,2.3 Hz,2H),4.85(d,J=11.7 Hz,1H),4.77(d,J=11.8 Hz,1H),4.70-4.59(m,3H),4.58-4.47(m,3H),4.36(d,J=11.7 Hz,1H),4.20 (t,J=7.9 Hz,1H),3.94(t,J=7.5 Hz,1H),3.91-3.80(m,3H),3.80-3.71(m,3H),3.63-3.50(m,2H),3.22-3.14(m,2H),1.78(s,3H). C 49 H 56 NO 11 + The calculated MS(ESI) value for [M+H]+ is 834.4. The measured value is 834.5.

[0276] 3.1.4 Synthesis of compound 1f [ka] Compound 1e (130 mg, 0.156 mmol), dichloromethane, tert-butanol, water, iodobenzene diacetate (0.5-10 equivalents), and 2,2,6,6-tetramethyl-1-piperidine oxide (0.01-1 equivalent) were added to a round-bottom flask (100 mL), and the resulting reaction mixture was stirred overnight with rt. After the reaction was complete, the reaction mixture was extracted with dichloromethane, the combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain compound 1f as a white solid (100 mg, yield 75.1%). 1 H NMR(400 MHz,chloroform-d)δ7.42-7.31(m,18H),7.30-7.27(m,2H),7.25-7.20(m,3H),7.15(dd,J=6.7,2.9 Hz,2H),6.06(d,J=8.3 Hz,1H),4.96(d,J=6.6 Hz,1H),4.93(d,J=11.9 Hz,1H),4.86(d,J=11.1 Hz,1H),4.81(d,J=12.1 Hz,1H),4.73(d,J=11.1 Hz,1H),4.71-4.65(m,3H),4.65-4.57(m,3H),4.49(s,1H),4.46(d,J=12.0 Hz,1H),4.23(t,J=7.1 Hz,1H),4.15(t,J=9.5 Hz,1H),3.89(t,J=6.5 Hz,1H),3.86-3.65(m,6H),3.54(d,J=9.7 Hz,1H),3.30(dd,J=9.3,2.8 Hz,1H),1.65(s,3H). C 49 H 52 NO 12 - Calculated MS(ESI) value for [MH] - :846.3. Measured value: 846.3.

[0277] 3.1.5 Synthesis of Compound 1 [ka] To a solution of compound 1f (160 mg, 0.189 mmol) in tetrahydrofuran / methanol (1:1, 10 mL), palladium carbon (10 wt.%) was added, and H2 was blown into the solution for 5 minutes. The mixture was then stirred under a hydrogen atmosphere. After the reaction was complete, the reaction mixture was filtered through Celite, washed with methanol, and the filtrate was concentrated under reduced pressure and dried under high vacuum to obtain compound 1 as a white solid (75 mg, 100% yield). 14 H 22 NO 12 - Calculated MS(ESI) value for [MH] - :396.1. Measured value: 396.1.

[0278] 3.2 Synthesis of Compound 2 [ka]

[0279] Step A: Synthesis of Compound 2b To a stirred solution of compound 2a (1 equivalent) in DMF (5 v / v), DIEA (3 equivalents) was added dropwise at 0°C under a nitrogen atmosphere, and the resulting mixture was stirred at 0°C for 10 minutes. Then, benzyl bromide (1.3 equivalents) was added dropwise, and the reaction mixture was allowed to rise naturally to room temperature and stirred for a further 16 hours. Next, the reaction mixture was slowly poured into ice water and extracted with MTBE (4 times). The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by silica gel flash column chromatography (eluent:PE / EA=6:1) to obtain compound 2b as a pale yellow oily substance (quantitative yield).

[0280] Step B: Synthesis of compound 2d To a solution of compound 2b (2.0 equivalents) and compound 2c (1 equivalent) in THF (10 v / v), TsOH (0.1 equivalent) was added, and the resulting mixture was stirred at rt for 4 hours. The reaction solution was then slowly poured into ice water and extracted three times with EA. The combined organic layer was sequentially washed with saturated sodium bicarbonate aqueous solution, water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography (eluent:PE / EA=1:1) to obtain compound 2d as a white solid (yield 40%).

[0281] Step C: Synthesis of Compound 2e To a stirred solution of compound 2d in DMAc (10 v / v), DBU (0.5 equivalents) was added dropwise at 15°C, and the reaction mixture was stirred at the same temperature for 1.5 hours. After the reaction was complete, the reaction mixture was cooled to 0°C, and the crude product, compound 2e, was used directly in the next step without purification.

[0282] Step D: Synthesis of 2g of compound PPTS (0.5 equivalents), EDCI (1 equivalent), HOBT (1 equivalent), and compound 2f (0.85 equivalents) were added to the reaction mixture in the final step at 0°C, and the resulting mixture was stirred at 0°C for 3-4 hours. After the reaction was complete, the reaction mixture was treated with ice water and extracted with 2-methyltetrahydrofuran (3 times). The combined organic layers were sequentially washed with 0.5 M hydrochloric acid, saturated NaHCO3 aqueous solution, water, and brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash chromatography (eluent: DCM / MeOH) to obtain 2 g of the compound as a white solid (yield 78%).

[0283] Step E: Synthesis of compound 2h To a stirred solution of 2 g of compound in DMAc (10 v / v), 0.5 equivalents of DBU were added dropwise at 15°C, and the reaction mixture was stirred at the same temperature for 1.5 hours. After the reaction was complete, the reaction mixture was cooled to 0°C, and 2 h of the crude product compound was used directly in the next step without purification.

[0284] Step F: Synthesis of compound 2j PPTS (0.5 equivalents), EDCI (1 equivalent), HOBT (1 equivalent), and compound 2i (0.85 equivalents) were added to the reaction mixture in the final step at 0°C, and the resulting mixture was stirred at 0°C for 3-4 hours. After the reaction was complete, the reaction mixture was treated with ice water and extracted with 2-methyltetrahydrofuran (3 times). The combined organic layers were sequentially washed with 0.5 M hydrochloric acid, saturated NaHCO3 aqueous solution, water, and brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash chromatography (eluent: DCM / MeOH) to obtain compound 2j as a white solid (yield 50%).

[0285] Step G: Synthesis of Compound 2 To a stirred solution of compound 2j in DCM (15v / v), DBU (0.5 equivalents) was added dropwise under a nitrogen atmosphere at 20°C, and the reaction mixture was stirred at the same temperature for 5 hours. After the reaction was complete, the reaction solution was diluted with DCM and subjected to silica gel column chromatography (eluent: DCM / MeOH) to obtain compound 2 as a white solid (yield 82%).

[0286] 3.3 Synthesis of Compound 3 [ka]

[0287] Step 3.3.1 Synthesis of NH2-Asp(OtBu)-Rink amide resin 400 g of Rink amide resin was weighed and completely swollen with 2400 mL of DCM. 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin was blue.

[0288] 88.87 g of Fmoc-Asp(OtBu)-OH and 29.19 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80 mL of DIC solution. After standing in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to a reaction kettle containing the resin, and the reaction was stirred at room temperature under a nitrogen atmosphere for 2-5 hours, followed by filtration. The resin was sequentially washed with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0289] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin turned blue.

[0290] Step 3.3.2 Synthesis of NH2-PEG4-Asp(OtBu)-Rink amide resin 131.64 g of Fmoc-PEG4-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After standing in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to a reaction kettle containing the resin, and the reaction was stirred at room temperature under a nitrogen atmosphere for 2-4 hours, followed by filtration. The resin was sequentially washed with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0291] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin turned blue.

[0292] Step 3.3.3 Synthesis of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 222.18 g of Fmoc-Asp(OtBu)-OH and 72.96 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80 mL of DIC solution. After standing in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to a reaction kettle containing the resin, and the reaction was stirred at room temperature under a nitrogen atmosphere for 2-4 hours, followed by filtration. The resin was sequentially washed with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0293] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin turned blue.

[0294] Step 3.3.4 Synthesis of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 191.75 g of Dde-Lys(Fmoc)-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After standing in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to a reaction kettle containing the resin, and the reaction was stirred at room temperature under a nitrogen atmosphere for 2-4 hours, followed by filtration. The resin was sequentially washed with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0295] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin turned blue.

[0296] Step 3.3.5 Synthesis of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 170.84 g of m-PEG12-CH2CH2COOH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After standing in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to a reaction kettle containing the resin, and the reaction was stirred at room temperature under a nitrogen atmosphere for 2-4 hours, followed by filtration. The resin was sequentially washed with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0297] Step 3.3.6 NH 2- Synthesis of Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 2400 mL of de-Dde reagent was added, and the reaction was stirred in nitrogen at room temperature for 0.5 hours, followed by filtration. This procedure was repeated three times, after which the resin was sequentially washed with DMF and DCM, and subsequently showed a blue color in the ninhydrin test.

[0298] Step 3.3.7 Synthesis of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 111.08 g of Fmoc-Gly-Gly-Gly-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After standing in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to a reaction kettle containing the resin, and the reaction was stirred at room temperature under a nitrogen atmosphere for 2-4 hours, followed by filtration. The resin was sequentially washed with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow. The resin peptide was washed three times with anhydrous ethanol, filtered, and allowed to cleave.

[0299] Step 3.3.8 Synthesis of Compound 3 10,000 mL of cutting reagent (TFA:TIS:H2O = 95:2.5:2.5) was added to a 10 L reactor and cooled to -10 ± 2°C. The dried and weighed resin was added. The reaction mixture was warmed to room temperature and stirred under nitrogen for 2-3 hours. The resin was then filtered and washed once with 100 mL of TFA. The filtrate and washing solution were combined.

[0300] 40 L of pre-cooled (below -10°C) cold ether was added to the product solution. The mixture was stirred for 10 minutes, and then the precipitate was centrifuged. After centrifugation, the supernatant was discarded, the precipitate was collected and washed with cold ether, and the precipitate was centrifuged again (each time the centrifugation speed was 3600 rpm, the centrifugation time was 5 minutes, and the temperature of the centrifugation cavity was -5°C).

[0301] The precipitate was purified by preparative HPLC and freeze-dried to obtain compound 3.

[0302] 3.4 Synthesis of Compound 4 [ka]

[0303] Step A: Synthesis of Compound 4a To a stirred solution of compound 3 (400 mg, 0.245 mmol) and compound 2 (377 mg, 0.539 mmol) in DMF (6 mL), DIPEA (159 mg, 1.23 mmol) was added, followed by HATU (233 mg, 0.613 mmol). The resulting reaction mixture was stirred at room temperature for 2 hours. After monitoring the completion of the reaction by HPLC, the reaction mixture was purified by preparative HPLC to obtain compound 4a as a white solid (380 mg, yield 52%). 142 H 207 O 49 N 21 Calculated MS(ESI) for [(M+3H) / 3] + :997.8. Measured value: 875.9 (fragment mass)

[0304] Step B: Synthesis of intermediate compound 4b [ka]

[0305] Compound 4a (380 mg, 0.245 mmol) was stirred in water (80 mL), to which palladium carbon hydroxide (5 wt.% packed, 38 mg) was added. The air in the system was replaced three times with hydrogen gas, and the resulting reaction mixture was stirred at room temperature. After completion, the reaction solution was filtered and purified by preparative HPLC to obtain compound 4b as a white solid (270 mg, yield 76%). 128 H 195 O 49 N 21 Calculated MS(ESI) for [(M+3H) / 3] + :937.8. Measured value: 875.9 (fragment mass)

[0306] Step C: Synthesis of intermediate compound 4d [ka]

[0307] To a solution of compound 4b (270 mg, 0.096 mmol) and compound 4c (120 mg, 0.211 mmol) in DMF (5 mL), DIPEA (62 mg, 0.48 mmol) and HATU (92 mg, 0.24 mmol) were added, and the resulting reaction solution was stirred at rt. After monitoring the completion of the reaction by HPLC, the reaction mixture was directly purified by preparative HPLC to obtain compound 4d as a pale yellow solid (235 mg, yield 66%). 174 H 229 O 55 Cl2F2N 27 Calculated MS(ESI) for [(M+3H) / 3] + : 1229.2. Measured value: 1229.3.

[0308] The synthesis of compound 4c is shown below: [ka]

[0309] Step 4c-A: N-(2-bromo-5-fluorophenyl)acetamide: To a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL), concentrated H2SO4 (3 mL) was added, followed by the gradual addition of 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) at room temperature. The mixture was stirred for 3 hours and then poured into 2000 mL of ice water. A precipitate formed, which was collected by filtration and vacuum-dried at room temperature to obtain N-(2-bromo-5-fluorophenyl)acetamide (105 g) as a yellow solid. 1 H NMR(400 MHz,DMSO-d6) δ 7.68(dd,J=8.9,6.0 Hz,1H),7.61(ddd,J=10.7,5.3,3.1 Hz,1H),7.02(ddd,J=8.9,8.0,3.1 Hz,1H),2.11(s,3H). MS m / z 232.0(M+H).

[0310] Step 4c-B: N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide: To a stirred solution of N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) in THF (1000 mL), n-BuLi (594 mL, 1.6 M in N-hexane, 950.22 mmol) was added dropwise over 1 hour at -78°C. After completion, the mixture was stirred under N2 for 0.5 hours. Next, a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise over 0.5 hours at -78°C, and the mixture was stirred at -78°C to room temperature for 6 hours. The mixture was poured into 500 mL of saturated NH4Cl aqueous solution at 0°C. Extraction was performed with ethyl acetate (500 mL x 3), washed with saline solution (250 mL x 2), dried over Na2SO4, and concentrated. The mixture was ground in (PE / EA = 1:1, 100 mL) for 10 minutes, filtered, and the filtered cake was collected and dried under vacuum to obtain N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g) as a yellow solid. MS m / z 206.1 (M-18+H), 246.1 (M+Na).

[0311] Step 4c-C:N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide:N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g, 107.50 mmol) was stirred in CH2Cl2 (170 mL) and water (170 mL). Silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol) were added, and the mixture was stirred at 30°C for 6 hours. The mixture was filtered through Celite, washed with CH2Cl2 (100 mL), the filtrate was concentrated, and purified by FCC (EA / PE = 0-40%) to obtain N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g) as a pale yellow solid. MS m / z 222.1 (M+H).

[0312] Step 4c-D: N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g, 63.28 mmol) was stirred in THF (500 mL) and butyl nitrite (8.48 g, 63.28 mmol) was added at 0°C, followed by t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0°C for 2 hours. After completion, the mixture was acidified with HCl (2N) to adjust the pH to 3. The mixture was extracted with ethyl acetate (200 mL x 3), washed with saline solution (100 mL x 2), dried over Na2SO4, and concentrated under reduced pressure. The crude mixture was ground with tert-butyl methyl ether (200 mL) for 10 min, filtered, the cake was recovered, and vacuum-dried to obtain N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12 g) as a yellow solid. MS m / z 251.1 (M+H).

[0313] Step 4c-E:N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide:N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (12 g, 47.96 mmol) was dissolved in acetic anhydride (90 mL) and THF (90 mL). 10% Pd / C (1 g) was added to the solution, and the mixture was stirred at 25°C under an H2 atmosphere for 16 hours. After cooling to 0°C, Et3N (20 mL) was added dropwise, and the mixture was stirred at 0°C for 1 hour. The solution was filtered through Celite, and the filtrate was poured into ice water (500 mL). Extraction was performed with ethyl acetate (500 mL x 3), washed with saline solution (250 mL x 2), dried over Na2SO4, and concentrated. The residue was ground with tert-butyl methyl ether (120 mL) for 10 min, filtered, and the cake was recovered. Vacuum drying was performed to obtain N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (7.9 g) as a yellow solid. MS m / z 279.1 (M+H).

[0314] Step 4c-F: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (7.9 g, 28.39 mmol) in MeOH (150 mL), aqueous HCl (2N, 150 mL) was added, and the mixture was stirred at 50°C for 7 hours. After cooling to 0°C, saturated aqueous NaHCO3 was added dropwise to adjust the pH to 8. The solution was extracted with ethyl acetate (200 mL x 3), washed with saline solution (200 mL x 2), dried over Na2SO4, and concentrated under reduced pressure to obtain N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (6.0 g) as a yellow solid. 1 ¹H NMR (400 MHz, chloroform-d): δ 6.57 (s, 3H), 6.18 (td, J=11.1, 2.4 Hz, 2H), 4.52 (dt, J=13.3, 5.0 Hz, 1H), 3.13 (ddd, J=17.5, 13.0, 4.6 Hz, 1H), 3.00-2.81 (m, 1H), 2.69 (dtd, J=9.4, 4.6, 2.5 Hz, 1H), 2.09 (s, 3H), 1.79 (qd, J=13.0, 4.3 Hz, 1H). MS m / z 237.1 (M+H).

[0315] Step 4c-G: N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1,7-diyl)diacetamide (4.0 g, 16.93 mmol) in DMF (80 mL), NCS (2.26 g, 16.93 mmol) was gradually added at 0°C and the mixture was stirred at room temperature for 16 hours. The mixture was poured into 200 mL of ice water. A precipitate formed, which was collected by filtration and vacuum-dried at room temperature to obtain N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g) as a yellow solid. 1H NMR(400 MHz,DMSO-d6) δ 8.11(d,J=8.0 Hz,1H),7.71(s,2H),6.62(d,J=11.9 Hz,1H),4.53(ddd,J=13.0,8.0,4.7 Hz,1H),3.18-3.04(m,1H),2.91(ddd,J=17.5,12.4,4.8 Hz,1H),2.21-2.08(m,1H),1.99-1.83(m,4H). MS m / z 271.0(M+H).

[0316] Step 4c-H:N-(9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)acetamide:N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acet To a mixture of amide (4.0 g, 14.78 mmol) in toluene (400 mL), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolidine-3,6,10(4H)-trione (4.28 g, 16.25 mmol), pyridinium p-toluenesulfonate (1.11 g, 4.43 mmol), and o-cresol (10 mL) were added, and the mixture was heated under N2 reflux for 24 hours. The solvent was removed under reduced pressure, and the mixture was purified by FCC (THF / CH2Cl2 = 0-60%) to obtain N-(9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)acetamide (4.1 g) as a brown solid. MS m / z 498.1 (M+H).

[0317] Step 4c-I: The mixture of (9S)1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione:N-(9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)acetamide (2.0 g, 4.02 mmol) in 20 mL of concentrated HCl aqueous solution was stirred at 70°C under N2 for 36 hours. The mixture was concentrated under reduced pressure to obtain crude (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione hydrochloride (2g) as a brown solid. MS (ESI) m / z 456.1(M+H).

[0318] Step 4c-J: Preparation of the intermediate salt form of compound 4c Compounds 4c and 4c' were prepared by preparative HPLC from (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione hydrochloride as a TFA salt.

[0319] [Table 4]

[0320] The above HPLC conditions: Apparatus: Agilent 1200; Chromatography column: Waters Xbridge C18 4.6×50mm, 3.5μm; Flow rate: 2.0 mL / min; Gradient elution: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00 min-1.50 min-2.50 min-2.52 min-3.00 min; Temperature: 40℃; Phase: A: Acetonitrile, B: H2O (0.05% TFA); Wavelength: 214 nm / 254 nm.

[0321] Step D: Synthesis of Compound 4 (H0058) [ka]

[0322] To a solution of compound 4d (210 mg, 0.057 mmol) in DMF (5 mL), diethylamine (0.5 mL) was added, and the resulting mixture was stirred at rt for 15 min. The system was neutralized at 0°C with 10% TFA aqueous solution, and the reaction mixture was then purified by preparative HPLC and freeze-dried to obtain compound 4 as a pale yellow solid (145 mg, yield 73%). 159 H 219 O 53 Cl2F2N 27 Calculated MS(ESI) for [(M+3H) / 3] + : 1155.2. Measured value: 1155.3.

[0323] 3.5 Synthesis of Compound 5 [ka]

[0324] Step A: DIEA and HATU were added to the stirred solution of compound 1 and compound 4, and the resulting mixture was stirred at a specific temperature (e.g., -20°C to 40°C). After the reaction was complete, the reactants were directly purified by preparative HPLC to obtain compound 5a as a pale yellow solid (337 mg, yield 63%). 173 H 243 Cl2F2N 28 O 64MS(ESI) calculation value for [(M+3H) / 3] + : 1281.5. Measured value: 1281.7.

[0325] Step B: Et3N and 2-chloro-1,3-dimethylimidazolidinium chloride (DMC, CAS: 37091-73-9) were added to a stirred solution of compound 5a in water. The resulting mixture was stirred at a specific temperature (e.g., -20°C to 40°C), and the completion of the reaction was monitored by HPLC. The reaction mixture was then directly purified by preparative HPLC to obtain compound 5 as a pale yellow solid (130 mg, yield 74%). 173 H 243 Cl2F2N 28 O 64 Calculated MS(ESI) value for [(M+H2O+3H) / 3] + : 1281.5. Measured value: 1281.7.

[0326] 3.6 Synthesis of Compound 7 [ka]

[0327] Step A: Synthesis of compound 7a DIEA and HATU were added to the stirred solution of compound 3 and compound 6 (PEG2-GGFG-OBn) in DMF, and the resulting reaction mixture was stirred with rt. After completion, the reaction mixture was used directly in the next step without purification.

[0328] Step B: Synthesis of compound 7b Et2NH was added to the reaction mixture in the final step, the resulting mixture was stirred at rt for 15 minutes, then neutralized with 30% HOAc, and directly purified by preparative HPLC to obtain compound 7b as a white solid (560 mg, 2-step yield 62%). 117 H 183 N 19 O 43 Calculated MS(ESI) for [(M+2H) / 2] + : 1272.1. Measured value: 1272.2.

[0329] Step C: Synthesis of Compound 7c DIEA and HATU were added to a stirred solution of compound 7b and compound 1 in DMF. The resulting reaction mixture was stirred with rt, and after completion, the reaction mixture was purified by preparative HPLC to obtain compound 7c as a white solid (400 mg, yield 87%). 131 H 204 N 20 O 54 MS calculation value for [(M+3H+H2O) / 3] + : 980.8. Measured value: 981.0.

[0330] Step D: Synthesis of Compound 7 To a stirred solution of compound 7c (400 mg) in water, palladium-carbon hydroxide (5 wt.%) was added. The air in the system was replaced three times with hydrogen gas, and the resulting reaction mixture was stirred at room temperature. After the reaction was complete, the reaction solution was filtered and purified by preparative HPLC to obtain compound 7 as a white solid (240 mg, yield 64%). 117 H 192 N 20 O 54 Calculated MS(ESI) for [(M+2H) / 2] + : 1372.1. Measured value: 1372.29.

[0331] 3.7 Synthesis of Compound 8 [ka]

[0332] Step A: Synthesis of compound 8a DIEA and HATU were added to a stirred solution of compound 7 and eribulin in DMF, and the resulting reaction mixture was stirred with rt. After the reaction was complete, the reaction mixture was purified by preparative HPLC to obtain compound 8a as a white solid (245 mg, yield 67%). 197 H 306 N 22 O 74 MS(ESI) calculation value for [(M+3H) / 3] + : 1389.0. Measured value: 1389.1.

[0333] Step B: Synthesis of Compound 8 Et3N and 2-chloro-1,3-dimethylimidazolidinium chloride (DMC, CAS: 37091-73-9) were added to a stirred solution of compound 8a in water. The resulting mixture was stirred at a specific temperature (e.g., -20°C to 40°C), and the completion of the reaction was monitored by HPLC. The reaction mixture was then directly purified by preparative HPLC to obtain compound 8 as a white solid (139 mg, yield 63%). 197 H 304 N 22 O 73 Calculated MS(ESI) for [(M+3H+H2O) / 3] + : 1389.0. Measured value: 1389.1.

[0334] 3.8 Synthesis of the linker-payload of compound 9 and ligase The preparation of compound 9 (linker 2-DM1 intermediate (n=3, ring-opened)) is described in reference to Example 2 of US 20170112944 A1, the entire example of which is incorporated herein. The structure of the linker 2-DM1 intermediate is shown below: [ka]

[0335] 3.9 Synthesis of the linker-payload of compound 10 and ligase Compound 10(C 94 H 148 N 16 O 29 Calculated MS(ESI) for [(M+2H) / 2] + The measured value (983.53) was synthesized using the intermediates of formulas (V') and (A) by a general method.

[0336] Example 4: Antibody preparation Anti-human ErbB2 / Her2 antibody T-LCCT L- For the production, purification, and characterization of HC, refer to Example 1 of US 20170112944 A1. For the production and purification of anti-human ErbB3 / Her3 antibody, refer to anti-human ErbB2 / Her2 antibody T-LCCT. L Refer to the method of Example 1 in US 20170112944 A1 by using anti-human ErbB3 / Her3 antibodies (SEQ ID NOs. 33 and 34) to replace -HC (SEQ ID NOs. 31 and 32).

[0337] Example 5 Preparation of antibody-drug conjugates having bienzyme-catalyzed site-specific complexization This disclosure describes how an ADC drug was formed by the specific recognition and cleavage of the Fc glycan located at Asn-297 of the antibody, followed by site-specific conjugation of a linker-payload to the Asn-297 glycan residue mediated by an immobilized endoglycosidase.

[0338] 5.1 Preparation and Analysis of ADC-1 5.1.1 Solid-phase preparation of ADC drug ADC-1 The preparation of the ADC drug ADC-1 was based on glycan remodeling complexation of antibody and linker-payload mediated by immobilized endoglycosidase catalyst. The antibody was thoroughly mixed with the linker-payload (compound 5) in an appropriate molar ratio (1:1 to 1:100) in 1× endoglycosidase buffer, and then immobilized endoglycosidase medium was added and mixed. The complexation reaction in the mixed suspension was carried out at a specific temperature (4 to 40°C) for a specific time (0.5 to 20 hours). After completion, the solid-phase complexed mixture was centrifuged, purified, ultrafiltered, or dialyzed to remove unreacted drug intermediates. The purified ADC drug ADC-1 was stored in 1× PBS pH 7.4 at 2 to 8°C or -80°C.

[0339] 5.1.2 Analysis of ADC-1 by SDS-PAGE After conjugation, the purity and conjugation efficiency of ADC-1 were detected by SDS-PAGE. The SDS-PAGE results for ADC-1 are shown in Figure 4. Conjugation occurred on the antibody heavy chain, and the ADC-1 heavy chain conjugated with the linker-payload showed a significant increase in molecular weight compared to the heavy chain of the monoclonal antibody from which the sugar chains had been removed, demonstrating that the linker-payload was successfully and site-specifically conjugated to the mAb heavy chain. In essence, the unconjugated linker-payload antibody was not observed in the conjugated product, the conjugation efficiency exceeded 95%, and the purity of the conjugated product was consistent with expectations.

[0340] 5.1.3 Analysis of ADC-1 by HIC-HPLC Protomix HIC Butyl-NP5 4.6×100mm 5μm Non-Porous column (Manufacturer: Sefen, PN: 431NP5-4610); Mobile phase A: 1.5M ammonium sulfate + 20mM phosphate buffer (pH 7.0); Mobile phase B: 20mM phosphate buffer (pH 7.0):isopropanol = 7:3 (v / v); Flow rate: 0.8 mL / min; Gradient method: Phase B was increased from 10% to 100% over 8 mins; DAR distribution of the ADC drug ADC-1 was detected using a detection wavelength of 280 nm.

[0341] The assay results are shown in Figure 5, where the non-complexed antibody was <5%, the DAR of the complexed product was mainly 4, and the overall DAR value for the ADC drug was approximately 3.9.

[0342] 5.1.4 Analysis of ADC-1 by SEC-HPLC TSKgel G3000SW XL The degree of high molecular weight aggregation of ADC drugs was analyzed and detected using a 7.8 mm ID × 30 cm, 5 μm column (manufacturer: DongCao, PN: 0008541); mobile phase: 2 × PBS: methanol = 9:1 (v / v); room temperature; isocratic; flow rate: 1.0 mL / min; run time: 15 min; detection wavelength: 280 nm.

[0343] The assay results are shown in Figure 6. High molecular weight aggregation in the ADC drug was less than 5%, and the ADC sample at 8.1 min was mainly in monomeric form.

[0344] 5.2 Preparation and Analysis of ADC-2 5.2.1 Preparation of ADC-2 Refer to the general preparation protocol (i.e., ADC-1) described in Example 5.1 of this disclosure for supplying ADC-2, except that compound 8 is used as the linker-payload.

[0345] 5.2.2 Analysis of ADC-2 by HIC-HPLC Protomix HIC Butyl-NP5 4.6×100 mm 5 μm Non-Porous column (Manufacturer: Sefen, PN: 431NP5-4610); Mobile phase A: 1.5 M ammonium sulfate + 20 mM phosphate buffer (pH 7.0), pH 7.0; Mobile phase B: 20 ​​mM phosphate buffer (pH 7.0): isopropanol = 7:3 (v / v); Flow rate: 0.8 mL / min; Gradient method: Phase B was increased from 10% to 85% over 8 mins; DAR distribution of the ADC drug ADC-2 was detected using a detection wavelength of 280 nm.

[0346] The assay results are shown in Figure 7, where the non-compounded cytotoxic antibody was <5%, the DAR of the compounded product was mainly 4, and the overall DAR value for the ADC drug was approximately 3.52.

[0347] 5.2.3 Analysis of ADC-2 by SEC-HPLC The degree of high molecular weight aggregation of ADC drugs was analyzed and detected using a TSKgel G3000SWXL 7.8mm ID×30cm, 5μm column (manufacturer: DongCao, PN:0008541); mobile phase: 2×PBS:methanol=9:1(v / v); room temperature; isocratic; flow rate: 1.0 mL / min; run time: 15 min; detection wavelength: 280 nm.

[0348] The assay results are shown in Figure 8. High molecular weight aggregation in the ADC drug was less than 6%, and the ADC sample at 7.87 min was mainly in monomeric form.

[0349] 5.3 Preparation and Analysis of Ligase-Catalyzed Antibody-Drug Conjugates (ADC-3) For the preparation and analysis of ligase-catalyzed antibody-drug conjugates, refer to Examples 3-7 of US 20170112944 A1.

[0350] This disclosure describes the linker 2-DM1 intermediate (n=3, ring-opened) and the ErbB2 / Her2 antibody ErbB2 / Her2 T-LCCT. L -HC was site-specifically conjugated with transpeptidase to form the following ADC drug: [ka] Here, n is 3, d is 2, and X in the ligase recognition sequence LPXT is glutamate (E).

[0351] 5.4 Preparation and Analysis of Antibody-Drug Conjugate dpADC-1 Having Bi-Enzyme Catalytic Site-Specific Complexation This disclosure describes a drug catalyzed by two enzymes that act independently to specifically complex at two different sites to form a unique dual-payload ADC drug (DAR2+DAR4).

[0352] The immobilized transpeptidase specifically recognizes and cleaves the LPXTGG enzyme recognition peptide sequence at the C-terminal end of the antibody's light chain (LC), and then site-specifically links the modified peptide sequence on the mAb to the linker-payload compound 9 to form an ADC intermediate.

[0353] The immobilized endoglycosidase specifically recognizes and cleaves the Fc glycan located at position Asn-297 of the antibody (heavy chain), and then connects the glycan remodeling mAb to the linker-payload (compound 5) to form an ADC intermediate.

[0354] Changes to the sample solution: ErbB2 / Her2 antibodies were treated using ultrafiltration, dialysis, or desalting columns, and their storage buffers were replaced with 50 mM Tris-HCl (pH 5-8) and 150 mM NaCl.

[0355] 5.4.1 One-Step Method The ADC drug dpADC-1 was prepared based on immobilized transpeptidase saltase and endoglycosidase, which catalyzed the conjugation reaction of the antibody with two linker-payloads (compound 9 and compound 5). In 1× buffer, the antibody was thoroughly mixed with each of the two linker-payloads (compound 9 and compound 5) in an appropriate molar ratio (1:1 to 1:100), and the immobilized transpeptidase medium and endoglycosidase medium were added and mixed. The two solid-phase enzyme media, each containing immobilized transpeptidase and endoglycosidase on the substrate, catalyzed the conjugation of the ErbB2 / Her2 antibody with the two linker-payloads (compound 9 and compound 5). The conjugation reaction in the mixed state was carried out at 4 to 40°C for 0.5 to 20 hours. After the reaction was complete, the solid-phase reaction mixture was centrifuged, purified, ultrafiltered, or dialyzed to remove unreacted drug intermediates. The purified ADC drug dpADC-1 was stored in 1×PBS pH 7.4 at 2-8°C or -80°C.

[0356] 5.4.2 Two-Step Method In the first step, the conjugated drug ADC-3 was initially prepared using transpeptidase to catalyze the conjugation reaction of the ErbB2 / Her2 antibody with the linker payload (compound 9). The antibody was thoroughly mixed with the linker payload (compound 9) in an appropriate molar ratio (1:1 to 1:100) in 1× transpeptidase buffer, and immobilized transpeptidase medium was added and mixed. The solid-phase transpeptidase medium, which had the transpeptidase immobilized on the substrate, catalyzed the conjugation reaction of the ErbB2 / Her2 antibody with the linker payload (compound 9). The conjugation reaction in the mixed state was carried out at 4 to 40°C for 0.5 to 20 hours. After the reaction was complete, the solid-phase conjugated reaction mixture was removed by centrifugation to obtain ADC-3.

[0357] In the second step, the complexation reaction between ADC-3 (DAR2) and the linker-payload (compound 5) was catalyzed using endoglycosidase to prepare the complex drug dpADC-1. ADC-3 was thoroughly mixed with the linker-payload (compound 5) in an appropriate molar ratio (1:1 to 1:100) in 1× endoglycosidase buffer, and immobilized endoglycosidase medium was added and mixed. The endoglycosidase-immobilized solid-phase endoglycosidase medium catalyzed the complexation reaction of ADC-3 with the linker-payload (compound 5). The complexation reaction in the mixed state was carried out at 4 to 40°C for 0.5 to 20 hours. After the reaction was complete, the solid-phase complexed reaction mixture was removed by centrifugation to obtain the ADC drug dpADC-1.

[0358] There is no difference in the order of the first and second steps. The second step can be completed before proceeding to the first step.

[0359] dpADC-1 was purified, ultrafiltered, or dialyzed to remove unreacted drug intermediates. The purified ADC drug dpADC-1 was stored in 1×PBS pH 7.4 at 2-8°C or -80°C.

[0360] 5.4.3 Analysis of dpADC-1 by SDS-PAGE (1-step and 2-step methods) After the complexation reaction, the purity and complexation efficiency of dpADC-1 were detected by SDS-PAGE. The results of SDS-PAGE (1-step and 2-step methods) of dpADC-1 are shown in Figure 9. Site-specific complexation occurred on the antibody's light chain (transpeptidase complexation) and heavy chain (endoglycosidase complexation). The dpADC-1 light chain complexed on the linker-payload showed a significant molecular weight transfer compared to the monoclonal antibody light chain, demonstrating the success of targeted complexation of the linker-payload to the monoclonal antibody light chain molecule. The dpADC-1 heavy chain complexed on the linker-payload also showed a significant molecular weight transfer compared to the monoclonal antibody heavy chain from which the sugar chains had been removed, demonstrating the success of targeted complexation of the linker-payload to the monoclonal antibody heavy chain molecule. In general, the uncomplexed linker-payload antibody was not observed in the complexed product, the complexation efficiency exceeded 95%, and the purity of the complexed product was not in line with expectations. Furthermore, there was no difference in complexation efficiency between the one-step and two-step methods, demonstrating excellent bioorthogonality. This allows for a significant reduction in the number of operating steps, substantial cost savings, and the realization of "one-step" two-enzyme orthogonal complexation that achieves homogeneous and controllable quality at CMC.

[0361] 5.4.4 Analysis of the ADC drug dpADC-1 by RP-HPLC An XBridge® Protein BEH C4 column, 300A, 3.5 μm, 4.6 mm × 150 mm column (manufacturer: Waters, PN: 186004504) was used; mobile phase A consisted of 0.1% HCOOH and 0.025% TFA in Waters; mobile phase B consisted of 0.1% HCOOH and 0.025% TFA in ACN; flow rate 0.4 mL / min; column temperature 80°C; gradient method: phase B was increased from 25% to 35% over 38 mins; the DAR distribution of the ADC drug dpADC-1 was analyzed using a detection wavelength of 280 nm.

[0362] The detection results are shown in Figure 10. The sum of the relative peak areas of the heavy and light chains of the non-complexed cytotoxic antibody was <7%, and the majority of the complexed products were products with a DAR of 2+4. The overall DAR value of the ADC drug was approximately 5.62 (LC+HC=1.89+3.73).

[0363] 5.4.5 Analysis of the ADC drug dpADC-1 by SEC-HPLC TSKgel G3000SW XL The degree of high molecular weight aggregation of ADC drugs was analyzed and detected using a 7.8 mm ID × 30 cm, 5 μm column (manufacturer: DongCao, PN: 0008541); mobile phase: 2 × PBS: methanol = 9:1 (v / v); room temperature; isocratic; flow rate: 1.0 mL / min; run time: 15 min; detection wavelength: 280 nm.

[0364] The assay results are shown in Figure 11. High molecular weight polymers in the ADC drug were <5%, and the ADC sample at 8.1 min was mainly in monomeric form.

[0365] 5.5 Preparation and Analysis of Antibody-Drug Conjugate dpADC-2 Having Bi-Enzyme Catalytic Site-Specific Complexation 5.5.1 Preparation of antibody-drug conjugate dpADC-2 For the preparation of dpADC-2, please refer to the general preparation protocol described in Example 5.4. Compounds 8 and 9 are used as linker-payloads for the complexation of dpADC-2.

[0366] 5.5.2 Analysis of the ADC drug dpADC-2 by RP-HPLC The test method is the same as the general preparation protocol described in Example 5.4.

[0367] The results are shown in Figure 12. The sum of the relative peak areas of the heavy and light chains of the non-complexed cytotoxic antibody was <7%, and the complexed product was mainly a product with a DAR of 2+4. The overall DAR value of the ADC drug was approximately 5.6 (LC+HC=1.87+3.73).

[0368] 5.5.3 Analysis of the ADC drug dpADC-2 by SEC-HPLC The test method is the same as the general preparation protocol described in Example 5.4 of this disclosure.

[0369] The detection results are shown in Figure 13, where high molecular weight polymers in the ADC drug were <5%, and at 8.02 min, the ADC sample was mainly in monomeric form.

[0370] 5.6 Preparation and Analysis of Antibody-Drug Conjugate dpADC-3 Having Bi-Enzyme Catalytic Site-Specific Complexation 5.6.1 Preparation of antibody-drug conjugate dpADC-3 For the preparation of dpADC-3, please refer to the general preparation protocol described in Example 5.4. Compounds 5 and 10 were used as linker-payloads for the complexation of dpADC-3, and in the preparation, the ErbB2 / Her2 antibody was substituted with the ErbB3 / Her3 antibody.

[0371] 5.6.2 Analysis of the ADC drug dpADC-3 by RP-HPLC The detection results are shown in the table and Figure 17 below. The sum of the relative peak areas of the heavy and light chains of the non-complexed cytotoxic antibodies was <8%, and the majority of the non-complexed products were products with a DAR of 2+4. The overall DAR value of the dpADC drug was approximately 5.76 (LC+HC=1.93+3.83).

[0372] [Table 5]

[0373] 5.6.3 Analysis of the ADC drug dpADC-3 by SEC-HPLC The assay results are shown in Figure 18. High molecular weight polymers in the ADC drug were <1%, and the dADC sample at 7.81 min was mainly in monomeric form.

[0374] Example 6: Effect of the ADC drug dpADC-1 on tumor cell proliferation with different ErbB2 / Her2 expression levels 1) The activity of dpADC-1 was tested using ErbB2 / Her2-positive human breast cancer cells SK-BR-3, HCC1954, BT-474, and ErbB2 / Her2-negative human breast cancer cells MDA-MB-468. 2) The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Tables 3 and 4 and Figures 14A-D. dpADC-1, ADC-3, and ADC-1 all exhibit clear inhibitory effects on the proliferation of ERB2 / HER2-positive cells. The inhibitory effect of dpADC-1 on the proliferation of ErbB2 / Her2-positive cells is similar to or slightly weaker than ADC-3, but better than ADC-1. dpADC-1 shows a slight inhibitory effect on ErbB2 / Her2-negative cells only at ≥200 nM, demonstrating its good stability, which is consistent with ADC-3, and there is no decrease in stability caused by further compounding with compound 5.

[0375] [Table 6]

[0376] [Table 7]

[0377] Example 7: Bystander killing effect of the ADC drug dpADC-1 against ErbB2 / Her2-negative tumor cells 1) The bystander-killing effect of dpADC-1 was tested using ErbB2 / Her2-positive human breast cancer cells SK-BR-3 and ErbB2 / Her2-negative human breast cancer cells MDA-MB-468. 2) The proportion of ErbB2 / Her2-positive and ErbB2-negative cell populations relative to the total cell population was detected by Beckman flow cytometry. The number of viable cells in each well was calculated for both ErbB2 / Her2-positive and ErbB2-negative cell populations and plotted using GraphPad software. The results are shown in Figures 15A-C.

[0378] As shown in Figure 15A, the overall cancer cell killing efficacy of dpADC-1 is significantly stronger than that of ADC-1 and ADC-3, demonstrating a synergistic effect of the two payloads. As shown in Figure 15B, in antigen-negative cancer cells (e.g., ErbB2 / Her2-negative human breast cancer cells MDA-MB-468), the killing effect of dpADC-1 is similar to that of ADC-1, and both are significantly stronger than that of ADC-3. As shown in Figure 15C, in antigen-positive cancer cells (e.g., ErbB2 / Her2-positive human breast cancer cells SK-BR-3), the killing effect of dpADC-1 is slightly lower than that of ADC-3, but significantly stronger than that of ADC-1. In general, the combined drug dpADC-1, which contains two drugs, has the strongest killing effect against cancer cells, and this is stronger than that of a single drug.

[0379] Example 8: Effect of the ADC drug dpADC-2 on tumor cell proliferation with different ErbB2 / Her2 expression levels 1) Human breast cancer cells SK-BR-3 and BT-474, which express ErbB2 / Her2, human breast cancer cells Jimt-1, which express it moderately, human non-small cell lung cancer cells NCI-H2110, which express it low, and human hepatocellular carcinoma cells HepG2, which are negative for ErbB2 / Her2, were used in the activity test. 2) The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 5. dpADC-2, ADC-2, and ADC-3 all showed significant inhibitory effects on the proliferation of ErbB2 / HER2-positive cells. dpADC-2 showed a similar or slightly stronger inhibitory effect on the proliferation of ErbB2 / HER2-positive cells as ADC-1, which was significantly better than ADC-3. dpADC-2 showed no inhibitory effect on ErbB2 / HER2-negative cells and, like ADC-3, had good stability without any decrease in stability due to additional compounding with compound 8.

[0380] [Table 8]

[0381] Example 9: Bystander killing effect of the ADC drug dpADC-2 against ErbB2 / Her2-negative tumor cells 1) The bystander-killing effect of dpADC-2 was tested using ErbB2 / HER2-positive human breast cancer cells SK-BR-3 and ErbB2 / HER2-negative human breast cancer cells MDA-MB-468 in a ratio of SK-BR-3:MDA-MB-468 = 4:1 / well. 2) The proportion of ErbB2 / HER2-positive and ErbB2-negative cell populations relative to the total cell population was detected by Beckman flow cytometry. The number of viable cells in each well was calculated for both ErbB2 / HER2-positive and ErbB2-negative cell populations and plotted using GraphPad software.

[0382] The results of the bystander killing effects of different drugs against tumor cells are shown in Figure 16. Both dpADC-2 and ADC-2 showed significant bystander killing effects against ErbB2 / HER2-negative cells. dpADC-2 showed slightly better bystander killing effects than ADC-2 and significantly better than ADC-3 against ErbB2 / HER2-negative cells, indicating a certain synergistic effect.

[0383] Example 10: Efficacy of the ADC drug dpADC-3 in osimertinib-resistant NSCLC PDX model mice with moderate ErbB3 / Her3 expression levels in vivo. Human lung cancer tumors transplanted in vivo were divided into tumor tissue pieces approximately 3mm x 3mm x 3mm (approximately 45-60mg) in size and subcutaneously inoculated into NU / NU mice. The average tumor volume was 163mm². 3 In this case, mice were administered the vehicle, ADC-4, dpADC-3, and osimertinib, respectively. ADC-4 is an endoglycosidase-catalyzed antibody-drug conjugate obtained by coupling the ErbB3 / Her3 antibody with compound 5. The results are shown in Figures 19A-B.

[0384] As shown in Figure 19A, the inhibitory effect of dpADC-3 on tumors in mice was better than that of ADC-4 and osimertinib alone. This indicates that dpADC-3 has a significant advantage over single-loading ADCs in the treatment of osimertinib-resistant NSCLC. As shown in Figure 19B, there was no significant difference in body weight change in mice in all drug treatment groups compared to the control group, indicating that dpADC-3 has good safety.

[0385] Example 11: Effect of the ADC drug dpADC-3 on osimertinib-sensitive NSCLC PC-9 model mice with low ErbB3 / Her3 expression levels in vivo. PC-9 tumor cells (10 × 10) in 0.2 mL of PBS (1:1) containing Matrigel 6 A BALB / c (mouse) was subcutaneously inoculated into the right side of each female BALB / c nude mouse to induce tumor development. The average tumor volume was 172 mm². 3 In this case, mice were administered the vehicle, ADC-4, dpADC-3, and osimertinib, respectively. The results are shown in Figures 20A-B.

[0386] As shown in Figure 20A, the inhibitory effect of dpADC-3 on tumors in mice was better than that of ADC-4 and osimertinib alone. This indicates that dpADC-3 has a significant advantage over single-loaded ADCs in the treatment of osimertinib-sensitive NSCLC. As shown in Figure 20B, there was no significant difference in body weight change in mice in any of the drug treatment groups compared to the control group, indicating that dpADC-3 has good safety.

[0387] The embodiments described above are intended solely to illustrate the technical ideas and features of the Disclosure and to enable those skilled in the art to understand and implement the Disclosure, and are not intended to limit the scope of protection of the Disclosure. All equivalent variations or modifications made in accordance with the spirit and substance of the Disclosure shall be included within the scope of protection of the Disclosure.

[0388] Sequence List Sequence ID 1 (Saltase A) AKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK Sequence ID 2 (Saltase A) KTPEIPKDKSKMAGYIKVPDAEIEEPVYPGPATPEQLNRGVSFAEGNESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVDPSDVKVLDEHKGEKNQLTLITCDNYNKETGVWEKRKIFVAKEIK Sequence ID 3 (Saltase A) KAPAIPKDKSKMAGYIKVPDAEIEEPVYPGPATPEQLNRGVSFAEGNESLTDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRNVDPSDVKVLDEHKGEKNQLTLITCDNYNKNTGVWEKRKIFVAKQIN Sequence ID 4 (Saltase A) KTPTIPKDKSKMAGYIEVPDAEIKEPVYPGPATLEQLNRGVSFAEGDESLDQQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNQTRKYKMTKIHDVNPSDEVLDEQKGKKNQLTLITCDDYNEKTGVWEKRKIFIATQVN Sequence ID 5 (Saltase A) KAVEIPKDKSKMAGYIKIPDAEIEEPVYPGPATPEQLNRGVSFAEGNESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVDPSDVKVLDEHKGEKNQLTLITCDNYNKETGVWEKRKIFVAKEIK Sequence ID 6 (Saltase A) KAPEIPKDKSKMAGYIKVPDAEIEEPVYPGPATPEQLNRGVSFAEGNESLTDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVDPSDVKVLDEHKGEKNQLTLITCDNYNKNTGVWEKRKIFVAKQIN Sequence ID 7 (Saltase A) KKPTIPKDKSKMAGYIEVPDAEIKEPVYPGPATPEQLNRGVSFAEGDESLDQQNIAIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNEVRKYKMTKIHDVDPTEVKVLDEHKGKKNQLTLITCDDYNEQTGVWEKRKIFVATQVN Sequence ID 8 (Saltase A) KAVEIPKDKSKMVGYIKVPDAEIEEPVYPGPATPEQLNRGVSFAEGNESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVDPSDVKVLDEHKGEKNQLTLITCDNYNKETGVWEKRKIFVAKEIK Sequence ID 9 (Saltase A) ESPQIPKDKAKMAGYIEIPDAQIKEPVYPGPATPQQLNRGVSFAEGDESLNQQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGNQTRKYKITKIHDVKPTEVKVLDEHPSKKNQLTLITCDDYNEQTGVWETRKIFVATQMN Sequence ID 10 (Saltase A) STPKIPSDKSKMAGYIEVPDAQIKEPVYPGPATPEQLNRGVSFAEGDESLNQQNISIAGHTFTDRPHYQFTNLKSAKIGSKVYFKTGNQTRKYKITKIRDVKPTEVKVLDEHPNKKNQLTLITCDDYNEETGVWETRKIFIATQIN Sequence ID 11 (Saltase A) ERPQIPKDKAKMAGYIEIPDAQIKEPVYPGPATPQQLNRGVSFAEGDESLYQQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVRNQTRKYKITKIHDVKPTEVKVLDEHPSKKNQLTLITCDDYNEQTGVWETRKIFVATQMN Sequence ID 12 (Saltase A) STPKIPSDKSKMAGYIEVPDAQIKEPVYPGPATPEQLNRGVSFAEGDESLNQQNISIAGHTFTDRSHYQFTNLKSAKIGSKVYFKTGNQTRKYKITKIRDVKPTEVKVLDEHPNKKNQLTLITCDDYNEETGVWETRKIFIATQIN Sequence ID 13 (Saltase A) EKPTISKDKSKMTGYISIPDADIKEPVYPGPATPEQLNRGVSFAEEDESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSKVTFKIGNETRKYKMTSIRDVNPEDVEVLDEHKGKKNQLTLITCDDYNENTGVWEKRKIFVAEEVK Sequence ID 14 (Saltase A) EKPTISKDKSKMTGYISIPDADIKEPVYPGPATPEQLNRGVSFAEEDESLDDQNISIAGHTFTDRPNYQFTNLKAAKKGSKVTFKIGNETRKYKMTSIRDVDPDAVEVLDENKGKKNQLTLITCDDYNENTGVWEKRKIFVAEQIK Sequence ID 15 (Saltase A) EKPTISKDKSKMTGYISIPDADIKEPVYPGPATPEQLNRGVSFAEEDESLDDQNISIAGHTFTDRPNYQFTNLKAAKKGSKVTFKTGNETRKYKMTSIRDVDPDAVEVLDENKGKKNQLTLITCDDYNENTGVWEKRKIFVAEQIK Sequence ID 16 (Saltase A) ETPTIPKDKSKMAGYISIPDAEIKEPVYPGPATPEQLNRGVSFAEEDEKLDDQNISIAGHTFIDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVNPDDVKVLDEHKGETNQLTLITCDNYNEQTGIWEKRKIFVAKQIN Sequence ID 17 (Saltase A) ETPTIPKDKSKMAGYISIPDAEIKEPVYPGPATPEQLDRGVSFAEEDEKLDDQNISIAGHTFIDRPHYQFTNLKAAKKGSKVYFKVGNETRKYKMTSIRDVNPDDVKVLDEHKGETNQLTLITCDNYNEQTGIWEKRKIFIAKQIN Sequence ID 18 (Saltase A) EKPTIPKDKSKMAGYISVPDAEIKEPVYPGPATPEQLNRGVSFAEGDESLDDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGDETREYKMTSIRDVDPEDVQVLDEHKGETNQLTLITCDNYNQQTGVWEKRKIFVAKQIK Sequence ID 19 (Saltase A) ERPTIPKNKSEMAGYISIPDAEIKEPVYPGPATLEQLNRGVSFAEGDESLDDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGDETREYKMTSIRDVDPEDVQVLDEHKGETNQLTLITCDNYNQQTGVWEKRKIFVAKQIK Sequence ID 20 (Saltase A) QTPTIPKDKSKMAGYISVPDAEIKEPVYPGPATPEQLNRGVSFAEEDESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKIGNETREYKMTSIRDVNPDQVEVLNEHKGEKNQLTLITCDDYNEQTGVWEKRKIFVAKQVK Sequence ID 21 (Saltase A) QTPTIPKDKTKMAGYISVPDAEIKEPVYPGPATPEQLNRGVSFAEKDESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKIGNETREYKMTSIRDVNPDEVEVLDEHKGEKNQLTLITCDDYNEQTGVWEKRKIFVAKQVK Sequence ID 22 (Saltase A) ERPTIPKDKSKMAGYISVPDAEIKEPVYPGPATLEQLNRGVSFAEGDESLDDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKVGDETREYKMTSIRDVNPEDVQVLDEHEGETNQLTLITCDNYNQQTGVWEKRKIFVAKQIK Sequence ID 23 (Saltase A) QTPTIPKDKTKMAGYISVPDAEIKEPVYPGPATPEQLNRGVSFAEKDESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKIGNETREYKMTSIRDVNPDEVEVLDEHKGEKNQLTLITCDDYNEQTGVWEKRKIFVAKQVN Sequence ID 24 (Saltase A) DKPTIPKDKAEMAGYLRIPDADINEPVYPGPATPEQLNRGVSFAEEQESLDDQNIAIAGHTYIGRPHYQFTNLKAAKKGSKVYFKVGNETREYKMTTIRDVNPDEIDVLDEHRGDKNRLTLITCDDYNEKTGVWEKRKIFIAEQIK Sequence ID 25 (Saltase A) QTPTIPKDKTKMAGYISVPDAEIKEPVYPGPATPEQLNRGVSFTEKDESLSDQNISIAGHTFTDRPHYQFTNLKAAKKGSKVYFKIGNETREYKMTSIRDVNPDEVEVLDEHKGEKNQLTLITCDDYNEQTGVWEKRKIFVAKQVN Sequence ID 26 (Saltase A) DKPTIPKDKAEMAGYLRIPDADINEPVYPGPATPEQLNRGVSFAEEQESLDDQNIAIAGHTYIGRPHYQFTNLKAAKKGSKVYFKVGNETREYKMTTIRDVDPDEIDVLDEHRGDKNRLTLITCDDYNEKTGVWEKRKIFIAEQIK Sequence ID 27 (Saltase A, SNAT counterpart to Sequence ID 1) AKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVGVLDEQKGKDKQLTLITCDDYNEKTGVWETRKIFVATEVK Sequence ID 28 (Halo tag) MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARET FQAFRTTDVGRKLIIDQNVFIEGTLMGVVRPLTEEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG Sequence ID 29 (Halo Saltase) MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSYVWRNIIPHVAPTHRCIAPDLIGMKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLRFPNELPIAGEPANIVALVEYM DWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGGGSGGGGSAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKGCSMVYFKVGNETRKYKMTSIRNVKPTAVGVLDEQKGKDKQLTLITCDDYNEKTGVWETRKIVATEVK sequence number 30 (Halo-Endo S2-His) Sequence ID 31 (ErbB2 / Her2 T-LCCT) L -HC) Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETG Sequence ID 32 (ErbB2 / Her2 T-LCCT) L -HC) Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 33 (ErbB3 / Her3 T-LCCT) L -HC) Light chain: DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETG Sequence ID 34 (ErbB3 / Her3 T-LCCT) L -HC) Heavy chain: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 35: LPXTG Sequence ID 36: GGGS Sequence ID 37: GGGGSGGGGS Sequence ID 38: NPXTG Sequence ID 39: LPXTA Sequence ID 40: LAXTG Sequence ID 41: LPXTGJ Sequence ID 42: LPETG Sequence ID 43: LPETGG Sequence ID 44: NPQTN Sequence ID 45: NPKTG Sequence ID 46: LPQTSeq Sequence ID 47: LPXTG n Sequence ID 48: GLLQGA Sequence ID 49: HHHH Sequence ID 50: HHHHH Sequence ID 51: HHHHHH Sequence ID 52: HHHHHHHHHH Sequence ID 53: SNAT Sequence ID 54: YNAT Sequence ID 55: WNDT Sequence ID 56: VNNS Sequence ID 57: (GGGGS) n Sequence ID 58: GFLG Sequence ID 59: ALAL Sequence ID 60: GALPETGG

Claims

1. An enzymatic complexing process for preparing an immune ligand / payload complex, comprising the following steps: The steps include: conjugating the first payload to an immune ligand via enzymatic catalysis by the first enzyme; The process includes the step of conjugating a second payload to the immune ligand via an enzymatic catalyst of a second enzyme, Here, the first enzyme is different from the second enzyme. process.

2. The first enzyme catalysis step and the second enzyme catalysis step are performed simultaneously or sequentially. The process according to claim 1.

3. The method includes simultaneously conjugating the first payload and the second payload to the immune ligand via the enzymatic catalysts of the first and second enzymes, The process according to claim 1 or 2.

4. The steps include: obtaining a first complex by conjugating the first payload with the immune ligand via the enzyme catalyst of the first enzyme; The method includes conjugating the second payload to the first complex via the enzyme catalyst of the second enzyme, thereby obtaining the immune ligand / payload complex. The process according to claim 1 or 2.

5. The first enzyme comprises a ligase, and / or the second enzyme comprises an endoglycosidase. The process according to any one of claims 1 to 4.

6. The first enzyme comprises an endoglycosidase, and / or the second enzyme comprises a ligase. The process according to any one of claims 1 to 4.

7. The ligase is a transpeptidase or a variant thereof, such as saltase A, saltase B, saltase C, saltase D, saltase E, or saltase F and their variants, preferably saltase A. and / or The recognition motif of the ligated donor substrate is LPXTGJ, NPXTG, LPXTA, or LAXTG, preferably LPXTG or LPETGG. and / or The recognition motif of the ligase acceptor substrate is G ｎ Here, G is glycine and n is an integer between 3 and 10. X is any natural or non-natural amino acid, J is either absent or an amino acid fragment containing 1 to 10 amino acids, where each amino acid is independently any natural or non-natural amino acid, preferably J is absent or G ｍ Here, m is an integer from 1 to 10. The process according to claim 5 or 6.

8. The aforementioned saltase A comprises an amino acid sequence selected from any one of sequence numbers 1 to 27, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with them. Preferably, the saltase A contains the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. The process according to claim 7.

9. The endoglycosidase is an N-acetylglucosaminidase that covalently binds a donor containing an oxazoline oligosaccharide to an immune ligand containing a GlcNAc motif, and the donor containing the oxazoline oligosaccharide further contains a payload. The process according to claim 5 or 6.

10. The N-acetamide glucosidase is at least one selected from Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia myricola endoglycosidase-F3), Endo S2 (endoglycosidase S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo CC (endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC) or their variants. The N-acetylglucosaminidase is at least one selected from the group consisting of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2 or their variants. Preferably, the N-acetylglucosaminidase is Endo S2 or a variant thereof. The process according to claim 9.

11. The oxazoline oligosaccharide is one or more selected from the group consisting of disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, hexsaccharide oxazoline, heptasaccharide oxazoline, octsaccharide oxazoline, nonusaccharide oxazoline, decasaccharide oxazoline, and elevensaccharide oxazoline. The process according to claim 9.

12. The oxazoline oligosaccharide has the following structure: The first hexose group or its derivative - (the second hexose group or its derivative) ｆ -β-D-glucopyranosyloxazoline, f is 0, 1, 2, 3, 4, 5, or 6, and β-D-glucopyranosyloxazoline has the following structure: 【Chemistry 1】 The process according to claim 9.

13. The first hexose group or its derivative is selected from glucosyl, mannosyl, galactosyl or its derivatives, and / or The carbon at position 6 of the first hexose group is in the -C(O)- form, and / or The second hexose group or its derivative is independently selected from glucosyl, mannosyl, galactosyl or its derivatives in each presence, and / or Each monosaccharide portion of the oligosaccharide structure is linked by a β-(1→4) glycosidic bond, and / or The first hexose group derivative and the second hexose group derivative are independently selected from derivatives in which the hydroxyl group of a uronic acid or monosaccharide is substituted with an acylamide group. The process method according to claim 12.

14. The oxazoline oligosaccharide has the following structure: The first hexose group or its derivative -β-D-glucopyranosyloxazoline, where the first hexose group or its derivative is mannose or its derivative, or The first hexose group or its derivative -β-D-glucopyranosyloxazoline, where the first hexose group or its derivative is galactose or its derivative, Preferably, the structure of the oxazoline oligosaccharide is as follows: 【Chemistry 2】 The process according to claim 12 or 13.

15. The ligase and / or endoglycosidase covalently ligates to a self-labeled protein tag to form a ligase fusion protein and / or endoglycosidase fusion protein. The process according to claim 5 or 6.

16. The self-labeled protein tag includes a SNAP tag, CLIP tag, His tag, Halo tag, or variants thereof. The process according to claim 15.

17. In the ligase fusion protein, the ligase has an isoelectric point (pI) of approximately 7.5 to approximately 10.0, the self-labeled protein tag is a Halo tag having an isoelectric point of approximately 4.5 to approximately 5.0, and the pI of the ligase fusion protein is approximately 2.0 to approximately 4.5 pH units lower than the pI of the ligase. The process according to claim 15.

18. The Halo tag includes the amino acid sequence of Sequence ID No. 28, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. The process according to claim 17.

19. The ligase of the ligase fusion protein is a saltase A comprising an amino acid sequence selected from any one of Sequence IDs 1 to 27, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. Preferably, the saltase A contains the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. The process according to claim 18.

20. The ligase fusion protein includes the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto. The process according to claim 19.

21. In the endoglycosidase fusion protein, the self-labeled protein tag is one or more selected from the His tag and the Halo tag. The process according to claim 15.

22. The amino terminus of the endoglycosidase is shared with the Halo tag, or One end of the endoglycosidase is shared with the Halo tag, and the other end is shared with the His tag, or The amino terminus of the endoglycosidase is covalently connected to the Halo tag, and the carboxyl terminus is covalently connected to the His tag, or The amino terminus of the endoglycosidase is covalently connected to the Halo tag, and the carboxyl terminus is covalently connected to the His tag, and the endoglycosidase is Endo S2. The process according to claim 21.

23. The endoglycosidase fusion protein has an isoelectric point (pI) of about 4 to about 7. The process according to claim 21.

24. The endoglycosidase fusion protein comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 90% identity thereto, or an amino acid sequence having one or more conserved amino acid substitutions compared to SEQ ID NO:

30. The process according to claim 21.

25. The first enzyme and the second enzyme are immobilized independently on a support. The process according to any one of claims 1 to 24.

26. The first enzyme and the second enzyme are covalently immobilized on the support via self-labeled protein tags, such as SNAP tags, CLIP tags, His tags, Halo tags, or variants thereof. Preferably, the support comprises a haloalkyl linker, preferably a chloroalkyl linker, and as a result, the first enzyme and the second enzyme are immobilized on the support by a covalent interaction between the haloalkyl linker and the Halo tag. The process according to claim 25.

27. The support has the structure of formula (II), 【Transformation 3】 In the formula, u is an integer between 1 and 20, v is an integer between 0 and 20, and w is an integer between 1 and 19. 【Chemistry 4】 This refers to a resin, beads, membrane, gel, matrix, film, plate, well, tube, slide or surface, preferably a resin, more preferably an agarose resin, silicone resin, polymethyl methacrylate resin or cellulose resin, most preferably a highly crosslinked agarose resin or polymethyl methacrylate resin. The process according to claim 25 or 26.

28. The immune ligand is selected from antibodies, modified antibody formats, antibody derivatives or fragments and / or mimic antibodies, and preferably the immune ligand contains an Fc terminus. The process according to any one of claims 1 to 27.

29. The antibodies are anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30 / TNFRSF8 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD44v6 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD117 / KITk antibody and anti-CD123 antibody, anti-CD138 antibody, anti-CD142 antibody, anti-CD174 antibody, anti-CD227 / MUC1 antibody, anti-CD352 antibody, anti-CLDN18.2 antibody, anti-DLL3 antibody, anti-ErbB2 / HER2 antibody, anti-GPNMB antibody, anti-ENPP3 antibody, anti-nectin-4 antibody, anti-EGFRvIII antibody, anti-SLC44A4 / AGS-5 antibody, anti-CEACAM5 antibody, anti-PSMA antibody, anti-TIM1 antibody, anti-LY6E antibody, anti-LIV1 antibody, anti-SLITRK6 antibody, anti-HGFR / cMet antibody, anti-SLAMF7 / CS1 antibody, anti-EGFR antibody, anti-BCMA antibody, anti-AXL antibody, anti-NaPi2B antibody, anti-GCC antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-mesothelin antibody, anti-ETBR antibody, anti-EphA2 antibody, anti-5T4 antibody, anti-FOLR1 antibody, anti-LAMP1 antibody, anti-cadherin 6 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-CA6 antibody, anti-CanAg antibody, anti-integrin αV antibody, anti-TDGF1 antibody, anti-ephrin A4 antibody, anti-TROP2 antibody, anti-PTK7 antibody, anti-NOTCH3 antibody, anti-C4.4A antibody, anti-FLT3 antibody, anti-B7H3 / 4 antibody, anti-TF (tissue factor) antibody, anti-ROR1 / 2 antibody, preferably, anti-CD19 antibody, anti-ErbB2 / HER2 antibody, anti-CLDN18.2 antibody, anti-nectin-4 antibody, anti-FGFR3 antibody and anti-Trop2 antibody, more preferably, selected from anti-ErbB2 / HER2 antibody, The process according to claim 28.

30. The first payload and the second payload are independently selected from small molecule compounds (e.g., small molecules with various mechanisms of action, e.g., various conventional small molecule drugs, photoacoustic therapy drugs, photothermal therapy drugs, etc., e.g., chemotherapeutic drugs, small molecule targeted drugs, immune agonists, etc., e.g., conventional cytotoxic drugs such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine; small molecule targeted drugs such as imatinib mesylate, gefitinib and anilotinib; immune agonists such as STING agonists and TLR agonists), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radiolabels, etc.), short-chain peptides, polypeptides, peptide mimes and proteins. The process according to any one of claims 1 to 29.

31. The first and second payloads are each independently a cytotoxin or a fragment thereof, preferably the cytotoxin is a taxane, maytansinoid, auristatin, epothilone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole sulfonamides, vinblastines, for example, vinblastine, vincristine, vindesine, vinorelbine, vinflunin, vinglycinate, anhydrovinblastine, drastatin 10 and its analogues, halichondrin B, eribulin, indole -3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermorid, laurimalide, camptothecin and its derivatives, mitoxantrone, mitoganidine hydrazone, nitrogen mustard, nitrosourea, aziridines, benzodopa, carbocon, metsuredepa, uredepa, arasinomycin, actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, carminomycin, sarcomycin, actinomycin D Daunorubicin, Detrubicin, Adriamycin, Epirubicin, Esolubicin, Idarubicin, Maceromycin, Mitomycins, Nogaramycin, Olibomycin, Peplomycin, Porphyromycin, Puromycin, Ferric Adriamycin, Rhodolubicin, Rufochromycin, Streptozocin, Dinostatin, Zorubicin, Trichothecene, T-2 Toxin, Beraclin A, Basilosporin A, Anguidine, Ubenimex, Azaserin, 6-Diazo-5-Oxo-L-Norleucine, Dimethylfolate, Methotrexate Pteropterin, Trimethrexate, Edatrexate, Fludarabine, 6-Mercaptopurine, Thiamiprine, Thioguanine, Ancitabine, Gemcitabine, Enocitabine, Azacitidine, 6-Azauridine, Carmofur, Cytarabine, Dideoxyuridine, Doxyfluridine, Phloxuridine, Carsterone, Dromostanolone propionate, Epithiostanol, Mepithiostan, Testolactone, Aminoglutethimide, Mitotane, Trilostane, Flutamide, Niltamide, Bicalutamide, Leuprorelin acetate,Selected from the group consisting of protein kinase inhibitors and proteasome inhibitors, and / or The cytotoxin is selected from and / or from vinblastines, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxorubicin, duocalmycin, SN-38, cryptophycin analogs, deruxtecan, duocalmazine, centanamycin, drastacin, pyrrolobenzodiazepines and exatecan and their derivatives. The cytotoxin is selected from auristatin, particularly MMAE, MMAF, or MMAD, and / or The cytotoxin is selected from exatecan and its derivatives, for example, DX8951f, and / or The cytotoxin is selected from DXd-(1) and DXd-(2), preferably DXd-(1). The process according to claim 30.

32. The immune ligand is an antibody, the payload is a drug, the drug-antibody ratio (DAR) is 2 to 16, preferably 4 to 12, more preferably 4 to 8, most preferably 6 to 8, preferably the first drug-antibody ratio is 2 to 4, and the second drug-antibody ratio is 2 to 4. The process according to any one of claims 1 to 31.

33. An immune ligand / payload complex prepared by the process described in claims 1 to 32, preferably an antibody-drug conjugate (ADC), more preferably a bidrug antibody-drug conjugate (dpADC), Immune ligand / payload complex.

34. Use of the immune ligand / payload complex according to claim 33 in the manufacture of a drug for treating a proliferative disorder (e.g., cancer).

35. A method for treating a proliferative disorder (e.g., cancer), comprising administering a therapeutically effective amount of the immune ligand / payload complex described in claim 33 to a subject in need thereof. method.

36. The aforementioned proliferative disorders are bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck neoplasms, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, thyroid cancer, skin cancer, leukemia, multiple myeloma, chronic lymphocytic lymphoma, adult T-cell leukemia, B-cell lymphoma, acute myeloid leukemia, Hodgkin lymphoma or non-Hodgkin lymphoma, Waldenström macroglobulinemia, hairy cell lymphoma, cellular lymphoma, Burkitt lymphoma, glioblastoma, melanoma, or rhabdomyosarcoma. The use described in claim 34 or the method described in claim 35.

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