Ligase fusion proteins and uses thereof

Abstract

The present invention relates to biotechnology. Specifically, the present invention provides a ligase fusion protein and an immobilized ligase comprising the same. The present invention also provides the use of the ligase fusion protein or the immobilized ligase in preparing a conjugate. The present invention further provides a method for preparing a conjugate using the ligase or the ligase unit.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the priority of International Patent Application No. PCT / CN2021 / 074082, filed on January 28, 2021, and all of its contents are incorporated herein by reference.

[0002] The present invention relates to the field of biotechnology, specifically to ligase fusion proteins and immobilized ligases containing the same. The present invention also provides uses in the preparation of conjugates of the above - mentioned ligase fusion proteins or immobilized ligases. The present invention further provides a method for preparing conjugates using ligases or ligase units.

Background Art

[0003] The demand for high - quality conjugates, especially for bioconjugates for purposes such as bioscience research, diagnosis, or treatment, is rapidly increasing. However, the current high - throughput production of bioconjugates is far from satisfactory. Part of the reason is that due to the complexity of biomolecules, it is difficult to meet the high - quality standards of bioconjugates.

[0004] Conventional conjugation methods are chemical. For example, in a typical production method of antibody-drug conjugates (ADCs), a drug is chemically conjugated to a lysine or cysteine ​​residue in an antibody via a linker. Before proceeding to the conjugation process, the antibody is prepared by upstream and downstream purification processes. After the conjugation step, another downstream purification process is required to remove aggregates, solvents, by-products and impurities in the ADC. Since there are multiple downstream steps in the method from antibody preparation to ADC production, the cost and time are significantly increased, and the yield is also reduced. In addition, from the viewpoint of safety, the conjugation reaction needs to be carried out in a chemical isolator, which makes it difficult to scale up the method. In short, the conventional method including multiple upstream and downstream purification steps is time-consuming, uneconomical, inflexible and lacks scalability.

[0005] Ligases, such as sortase enzymes, are intended to catalyze conjugation in a highly substrate-specific and efficient manner under mild conditions (e.g., WO2015 / 165413A1, WO2014 / 177042, and WO2014 / 140317), potentially saving time, reducing costs, and reducing waste. Although ligases offer many advantages, a number of challenges remain that limit their industrial application in conjugation reactions.

[0006] Problems such as poor operational stability and poor reusability of enzymes can be overcome by enzyme immobilization. Applications of conjugation include immobilized sortase A on cyanogen bromide-activated agarose gels (see, for example, Witte et al., Site-specific protein modification using immobilized sortase in batch and continuous-flow systems, Nat Protoc, (2015), 10(3):508-516) or His-immobilized on nickel-modified magnetic particles. 6Tagged sortase A (see, for example, Zhao et al., One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst, Sci Rep, (2017), 7:6561).

[0007] However, removing residual enzyme contaminants from upstream catalytic reactions remains a major concern for most enzyme-catalyzed conjugates, especially bioconjugates, because they can be difficult to remove (in the case of immobilized enzymes, free enzyme nonspecifically adsorbed to the support can still be shed).Therefore, there is a need for ligases that are cost-effective, stable, controllable, and easily removable from the conjugate product. Summary of the Invention

[0008] In one general aspect, the invention provides a ligase fusion protein comprising a ligase and a Halo tag.

[0009] In some embodiments, the ligase is a transpeptidase. In some embodiments, the ligase is a sortase. In some embodiments, the ligase is sortase A. In some preferred embodiments, sortase A comprises an amino acid sequence selected from SEQ ID NOs: 1-26, 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. In some other preferred embodiments, sortase A comprises SNAT, YNAT, WNDT, or VNNS amino acid substitutions at positions 34, 100, 105, and 136, preferably sortase A comprises 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.

[0010] In one embodiment, the Halo tag is a mutant haloalkane dehalogenase or variant thereof that removes a halogen from a haloalkyl substrate to form a covalent bond with the remaining alkyl group, hi some embodiments, the Halo tag comprises an amino acid sequence of SEQ 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.

[0011] In a preferred aspect, the present invention provides a ligase fusion protein having an altered isoelectric point (pI) compared to the ligase from which it is derived, wherein the ligase has a basic pI and the Halo tag has an acidic pI. In some embodiments, the ligase has an isoelectric point (pI) of about 7.5 to about 10.0, the Halo tag has an isoelectric point 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.

[0012] In another general aspect, the invention provides an immobilized ligase comprising a ligase fusion protein of the invention immobilized on a support.

[0013] The present invention further provides the use of a ligase fusion protein or an immobilized ligase of the invention in the preparation of a conjugate.

[0014] In yet another general aspect, the invention provides a method of preparing a conjugate comprising a first moiety and a second moiety, the method comprising: (a) providing a system 1 including a first portion and providing a system 2 including a second portion; (b) contacting system 1 and system 2 of step (a) with a ligase unit to catalyze a conjugation reaction between the first moiety and the second moiety to obtain the conjugate; The ligase unit comprises a ligase, the first and second moieties each independently comprise a biomolecule, a protein, an antibody, an antibody fragment, a receptor, a signal transduction factor, a cell growth factor, a nucleic acid or a nucleic acid analog, a small molecule compound, a glycan, a PEG moiety, a radionuclide, a cytokine, an immunomodulator, a tracer molecule, a fluorophore, a fluorescent molecule, a peptide, a polypeptide, or a peptidomimetic; wherein one of the first and second portions further comprises a ligase donor substrate recognition motif, and the other of the first and second portions comprises a ligase acceptor substrate recognition motif.

[0015] In some embodiments, the ligase unit comprises a release ligase, preferably a transpeptidase, particularly preferably a sortase, more particularly preferably sortase A, and most preferably, the ligase unit comprises a ligase fusion protein of the invention. In some other embodiments, the ligase unit comprises a ligase immobilized to a support, preferably the ligase is covalently immobilized to a support, the ligase is preferably a transpeptidase, particularly preferably a sortase, more particularly preferably sortase A, and most preferably the ligase unit comprises an immobilized ligase of the invention.

[0016] In some embodiments, at least one of System 1 and System 2 in step (a) comprises one or more impurities. In other embodiments, at least one of System 1 and System 2 in step (a) is harvested clarified cell culture fluid (HCCF).

[0017] In some embodiments, the method further comprises: (1) prior to step (b), subjecting the system 1 of step (a) to one or more chromatographic steps to remove one or more impurities; and / or (2) prior to step (b), subjecting system 2 of step (a) to one or more chromatography steps to remove one or more impurities; and / or (3) reacting the conjugate obtained in step (b) with and performing one or more chromatography steps to remove one or more impurities.

[0018] The chromatography steps can be independently selected from the group consisting of affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, mixed mode chromatography, hydroxyapatite chromatography, and combinations thereof. Preferably, the chromatography steps are selected from affinity chromatography, ion exchange chromatography, and combinations thereof.

[0019] In some embodiments, at least one of the first and second portions comprises an antibody or an antibody fragment, and at least one of steps (1)-(3) comprises affinity chromatography, preferably, the antibody or antibody fragment comprises an Fc fragment, and the affinity chromatography is Protein A affinity chromatography. [Brief description of the drawings]

[0020] [Figure 1] 1 shows sortase activity of an exemplary SrtA from Staphylococcus warneri (SEQ ID NO:3) and its SNAT mutants. [Diagram 2] The activities of purified (A) Halo-sortase, (B) His6-sortase and (C) GB1-sortase are shown. [Diagram 3] The enzyme loading of various chloro-resins is shown. [Figure 4]FIG. 1 shows the catalytic activity (expressed as DAR) of immobilized Halo-sortase prepared from various chloro-resins. [Diagram 5] 1 shows the solubility of ADC products catalyzed by GB1-sortase, His-sortase, or Halo-sortase at low temperature. [Figure 6] Chromatographic profiles of (A) ADC, (B) Halo-sortase, and (C) ADC+Halo-sortase in AEX using QSepharose FF media. [Figure 7] Chromatographic profiles of (A) ADC and (B) Halo-sortase during CEX using CaptoSImpAct media. [Figure 8] 1 shows the DAR composition of the conjugates contained in the crude conjugate mixture of Process 2 by HIC-HPLC analysis. [Figure 9] The amount of remaining impurities in the sample containing the target ADC after each chromatography step in process 2 is shown (in the figure, Protein A is the mAb eluate of Protein A affinity chromatography, AEX is the ADC flow-through of AEX, and CEX is the ADC eluate of CEX). [Figure 10] 1 shows the DAR composition of the conjugates contained in the crude conjugate mixture of Process 1 by HIC-HPLC analysis. [Figure 11] The amount of remaining impurities in the sample containing the target ADC after each chromatography step in process 1 is shown (in the figure, Protein A is the ADC eluate of Protein A affinity chromatography, AEX is the ADC flow-through of AEX, and CEX is the ADC eluate of CEX). [Figure 12]The amount of remaining impurities in the sample containing the target ADC after each chromatography step in process 3 is shown (in the figure, 1st Protein A is the mAb eluate from Protein A affinity chromatography, 2nd Protein A is the ADC eluate from Protein A affinity chromatography, AEX is the ADC flow-through from AEX, and CEX is the ADC eluate from CEX). [Figure 13] The amount of residual Halo-sortase in samples containing target ADC is shown (in the figure, Conjugation is the crude conjugate mixture collected from the flow-through of the Halo-sortase column, Protein A is the ADC eluate of Protein A affinity chromatography, AEX is the ADC flow-through of AEX, and CEX is the ADC eluate of CEX). [Figure 14] Optimized chromatographic profiles for linker-toxin (linker-payload intermediate) removal are shown: (A) with Biomax Protein A media, (B) with GE Protein A media, and (C) with GE CEX media. [Figure 15] FIG. 1 shows a step flow chart of ADC preparation using conventional methods (conventional ADC process) and the methods of the present invention (ADC process 1, ADC process 2, ADC process 3, and ADC process 4). In the figure, Protein A is Protein A chromatography, Low pH is low pH treatment, UF / DF is ultrafiltration / diafiltration, AEX is anion exchange chromatography, CEX is cation exchange chromatography, HIC is hydrophobic interaction chromatography, and MabDS is downstream process of monoclonal antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] General definition Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition, the terms and experimental methods related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology and immunology are terms and common methods widely used in the art. When a trade name is mentioned in this specification, it is intended to refer to the corresponding product or its active ingredient. All patents, published patent applications and publications cited in this specification are incorporated by reference in their entirety into this application. In addition, definitions and explanations of related terms are provided below to better understand the present invention.

[0022] As used herein, the phrase "at least one" or "one or more" means 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, unless expressly stated to the contrary, "one" and "one" should be understood as "at least one."

[0023] It should be understood that the indication of a particular amount, concentration, or other value or parameter as a range, a preferred range, or a preferred upper value or a preferred lower value is equivalent to specifically disclosing all ranges combining any upper or preferred value with any lower or preferred value, whether or not expressly stated. Unless otherwise stated, the ranges recited herein are intended to include the end points of the range and all integers and fractions (decimals) within the range. For example, the expression "i is an integer from 2 to 20" should be understood to mean that i is any integer from 2 to 20, for example, i can 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 be understood in the same manner.

[0024] The terms "about" and "approximately," when used in conjunction with a numerical variable, such as concentration, isoelectric point (pI), pH, temperature, or a specified range, generally mean that the value of the variable, and all values ​​of the variable, are within experimental error (e.g., within a 95% confidence interval of the mean) or within ±10% of the specified value, or within a broader range.

[0025] The terms "optional" or "optionally" mean that the event described thereafter may, but does not necessarily, occur, and are meant to include cases where the event or circumstance occurs or does not occur.

[0026] The expression "comprises" or similar expressions such as "comprising," "containing," and "having" are open ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of" excludes any element, step, or ingredient not expressly recited. The expression "consisting essentially of" means limiting the scope to the specified elements, steps, or ingredients, and to any optionally present elements, steps, or ingredients that do not materially affect the key and novel characteristics of the claimed subject matter. The expression "comprising" should be understood to encompass the expressions "consisting essentially of" and "consisting of."

[0027] As used herein, the definition of "biomolecule" includes proteins, nucleic acids, lipids, carbohydrates, small nucleotides, amino acids and their derivatives.

[0028] As used herein, "nucleic acid" or "polynucleotide" refers to a polymer of at least two nucleotides or nucleotide derivatives linked by phosphodiester bonds, and includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0029] As used herein, a "vector" is a vehicle for introducing an exogenous nucleic acid into a host cell, where the exogenous nucleic acid is amplified or expressed in the host cell. As used herein, the definition of "vector" encompasses plasmids (e.g., linearized plasmids), viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like. As used herein, a vector capable of expression and / or replication in a host cell means that the vector is capable of expressing an RNA polynucleotide or polypeptide in the host cell and / or capable of generating multiple copies of the vector. For "expressible" or "replicable", a vector may include a nucleic acid sequence or element operably linked to a promoter. As used herein, "operably linked" with respect to a nucleic acid sequence or element means that these nucleic acid sequences are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid sequence encoding a polypeptide to regulate or mediate transcription of the nucleic acid. Those skilled in the art can select and use an appropriate vector depending on a particular purpose.

[0030] As used herein, "peptide," "polypeptide," or "protein" means two or more amino acids covalently linked together. Unless otherwise specified, these terms can be used interchangeably.

[0031] As used herein, "sequence identity" has its meaning generally recognized in the art, and the percentage of sequence identity between two polypeptides can be calculated by comparing two sequences using publicly available algorithms, such as the basic local alignment search tool (BLAST), and the fast adaptive shrinkage / thresholding algorithm (FASTA) (see, e.g., 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). Although there are various methods for measuring identity between two polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0032] As used herein, the term "variant" refers to a protein that has one or more residue substitutions, deletions, or insertions compared to a reference protein. A reference protein may be a naturally occurring protein isolatable from a natural source (i.e., a wild-type protein), or an engineered protein. As used herein, the function or activity of a variant, such as a sortase A variant or a Halo tag variant, is substantially similar or equal to or greater than the function or activity of the reference sortase A or Halo tag, respectively.

[0033] In the specific context of this specification, amino acid positions in a protein are defined as follows: (i) starting from the N-terminus, and (ii) the first amino acid position at the N-terminus is designated as 1. An amino acid (e.g., Ser) at a given position (e.g., position 34) may be represented as Ser34. An amino acid (e.g., His) at a given amino acid position (e.g., position 272) substituted with another amino acid (e.g., Phe) may be represented as His272Phe.

[0034] As used herein, a "ligase" refers to an enzyme capable of catalyzing the covalent joining of two or more molecules. A ligase can specifically catalyze the conjugation between a first moiety that contains a ligase donor substrate recognition motif and a second moiety that contains a ligase acceptor substrate recognition motif to generate a targeted conjugate.

[0035] As used herein, the term "transpeptidation reaction" refers to a chemical reaction in which one or more amino acids (e.g., a peptide) are transferred from one molecule to another. A transpeptidase is an enzyme that can catalyze a transpeptidation reaction between a donor substrate and an acceptor substrate. In a simplified transpeptidation reaction catalyzed by a resident sortase, the sortase first cleaves the recognition motif of the ligase donor substrate (also called the donor recognition motif, e.g., LPXTG when SrtA is used) and generates a substrate-enzyme intermediate by forming a thioester bond. Then, the acceptor substrate recognition motif of the ligase (also called the acceptor recognition motif, e.g., GGG) nucleophilically attacks the thioester bond to release the enzyme and form a new peptide bond between the two substrates. In a transpeptidation reaction, both usually conjugate to form a conjugate.

[0036] As used herein, the term "conjugation" means the covalent joining of at least two moieties (eg, at least two molecules or at least two termini of the same molecule).

[0037] As used herein, a "conjugate" can be prepared by the covalent attachment of at least two moieties (eg, at least two molecules or at least two termini / side chains of the same molecule).

[0038] As used herein, "bioconjugate" refers to a conjugate in which at least one of the attached moieties is a biological molecule. Examples of bioconjugates include therapeutic molecules attached to polymers, lipids, antibodies, peptides, aptamers, or small molecule ligands, such as siRNA conjugates, peptide hormone conjugates, peptide-peptide conjugates, peptide-drug conjugates, antibody-drug conjugates, and multispecific antibodies.

[0039] The term "targeting molecule" refers to a molecule that has affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, etc.). A targeting molecule can deliver a payload to a specific site in the body by targeted delivery. A targeting molecule can recognize one or more targets. A specific target site is defined by the target recognized by the targeting molecule. For example, a targeting molecule that targets a receptor can deliver a cytotoxin to a site that contains a large amount of said receptor. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins of a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc.

[0040] As used herein, the term "antibody-drug conjugate (ADC)" means a conjugate comprising an antibody or antibody fragment covalently attached to a payload. As used herein, the terms "activity," "enzymatic activity," and "catalytic activity" of a ligase (e.g., a sortase) refer to the ability of the ligase to catalyze a conjugation reaction, and can be used interchangeably. As used herein, the catalytic activity of a sortase in a conjugation reaction, e.g., a conjugation reaction between an antibody and a payload, can be expressed as a conjugation efficiency (conjugation efficiency = (moles of conjugated antibody: moles of total antibody) x 100%) or a DAR (drug-antibody ratio, i.e., the average drug-antibody ratio of a drug conjugate formulation of a given antibody) distribution.

[0041] As used herein, the term "antibody (Ab)" refers to an immunoglobulin (Ig) molecule or derivative thereof that specifically binds to an antigen through at least one antigen-binding site. A "conventional" or "full-length" antibody generally consists of four polypeptides: two heavy chains (HC) and two light chains (LC). As used herein, the definition of "antibody" includes 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-idiotypic antibodies. "Antibody" also includes members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (e.g., IgG2a and IgG2b), or any derivative thereof.

[0042] As used herein, an "antibody fragment" of an antibody refers to any portion of an antibody that contains fewer amino acid residues than the full-length antibody, such as an antigen-binding fragment that contains at least a portion of the variable domain of the antibody (e.g., one or more CDRs) and specifically binds to the cognate antigen of the full-length antibody, or an Fc fragment that contains the heavy chain constant region of the antibody and binds to an Fc receptor on a cell surface. Antibody fragments can be obtained by various methods, such as chemical or enzymatic treatments, chemical synthesis, or recombinant DNA technology. Examples of antibody fragments include Fv (fragment variable region), scFv (single-chain Fv fragment), dsFv (disulfide bond stabilized variable fragment), scdsFv (single-chain disulfide bond stabilized variable fragment), diabody, Fd fragment (the fragment difficult), Fab (antigen-binding fragment), scFab (single-chain Fab), Fab', F(ab') 2 , Fc (Fragment crystallizable) and any derivatives thereof.

[0043] As used herein, the term "payload" refers to a functional moiety included in a conjugate, for example, linked via a linker. Examples of payloads include small molecule compounds (also called small molecule drugs, e.g., inhibitors and toxins (such as cytotoxins)), radionuclides (e.g., 225 Ac, 211 At, 212 Bi, 213 Bi, 67 Ga, 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, linkers, such as 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 peptidomimetics. As used herein, payloads including linkers (e.g., linkers including ligase substrate recognition motifs) and payloads described above are included in the present invention.

[0044] As used herein, the term "naturally occurring amino acid" refers to an amino acid that is a constituent amino acid of protein, and includes the 20 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) and the less common selenocysteine ​​and pyrrolysine.

[0045] As used herein, the term "unnatural amino acid" refers to an amino acid that is not a proteinogenic amino acid. Specifically, the term refers to an amino acid that is not a natural amino acid as defined above.

[0046] As used herein, the term "peptidomimetic" refers to a compound that mimics the conformation and desirable characteristics of a particular peptide.

[0047] As used herein, "receptor" refers to a structure present within or on the surface of a cell that binds to a specific substance to effect a specific action within the cell. Receptors can include T cell receptors, B cell receptors, and receptors for signaling molecules, cell growth factors and cytokines, as described herein.

[0048] As used herein, a "signal transduction factor" refers to any substance that plays a role in signal transduction events across or through a cell. Signal transduction factors include signal molecules (e.g., steroid hormones, retinoic acid, thyroid hormones, vitamin D 3 These may include, but are not limited to, peptide hormones, neuropeptides, eicosanoids, neurotransmitters and cytokines) and their receptors.

[0049] As used herein, the term "immunomodulator" refers to a biologically active substance that can affect the function of the immune system. Immunomodulators may be immunosuppressive (e.g., immunosuppressants / immunosuppressant drugs) or immunostimulatory (e.g., immunostimulants / immunostimulators). Examples of immunomodulators include cytokines, thymic hormones (e.g., thymulin, thymosin, and thymopoietin), lentinan, β-glucan, inulin, levamisole, isoprinosine, IMPDH inhibitors (e.g., azathioprine, leflunomide, mycophenolic acid, mizoribine, ribavirin, and tiazofurin), calcineurin inhibitors (e.g., cyclosporine, tacrolimus), mTOR inhibitors (e.g., sirolimus, tacro ... These may include, but are not limited to, anti-cancer drugs, such as sirolimus, everolimus, P38 inhibitors, NF-κB inhibitors (e.g., bortezomib), corticosteroids (e.g., prednisone, budesonide, and prednisolone), Janus kinase inhibitors (e.g., tofacitinib, baricitinib), anti-cytokine antibodies, and antibodies against T cell receptors.

[0050] As used herein, the term "cell growth factor" refers to any substance capable of stimulating cell growth, fusion, proliferation, survival and differentiation. Examples of growth factors may include, but are not limited to, epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), teratocarcinoma-derived growth factor (TDGF), insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and erythropoietin (EPO).

[0051] 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, but are not limited to, chemokines, lymphokines, colony stimulating factors (CSFs), monocyte chemoattractant proteins (MCPs), angiogenic factors, interleukins, interferons, tumor necrosis factors (TNFs), growth factors, and other secreted and cell surface molecules that can transmit signals to 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.

[0052] A small molecule compound is a molecule of a size similar to that of organic molecules commonly used in medicine. The term does not include biopolymers (e.g., proteins, nucleic acids, etc.), but includes low molecular weight peptides such as dipeptides, tripeptides, tetrapeptides, and pentapeptides, or derivatives thereof. In general, the molecular weight of a small molecule compound can be, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, or about 200 to about 500 Da. As used herein, a small molecule compound can also be referred to as a drug.

[0053] A cytotoxin is a substance that inhibits or blocks cellular expression activity, cellular function, and / or causes cellular destruction. In some cases, cytotoxins currently used in ADCs can be more toxic than commonly used chemotherapy drugs. Examples of cytotoxins include, but are not limited to, drugs that target targets such as the microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and apoptosis regulation. Drugs that target the microtubule cytoskeleton can be, for example, microtubule stabilizers or microtubule protein polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of microtubule protein polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinblastines, colchicines, and dolastatins. DNA targeting drugs can 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 breakers, DNA alkylating agents, and DNA intercalators. The DNA double strand breaker may be, for example, an enediyne antibiotic, including, but not limited to, dynemicin, esperamicin, neocarzinostatin, uncialamycin, etc. The DNA alkylator may be, for example, a DNA bis-alkylator (i.e., a DNA cross-linking agent) or a DNA mono-alkylator. Examples of DNA alkylators include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, 1-(chloromethyl)-2,3-dihydrogen-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, dihydroindolobenzodiazepine (IGN) dimers, duocarmycin-like compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, camptothecins, anthracyclines.Drugs that target RNA can be, for example, drugs that inhibit splicing, examples of which include, but are not limited to, pladienolide. Drugs that target kinesin-mediated protein transport can be, for example, mitotic kinesin inhibitors, examples of which include, but are not limited to, kinesin spindle protein (KSP) inhibitors.

[0054] A spacer is a structure that is located between different structural modules and can spatially separate the structural modules. The definition of a spacer is not limited by whether it has a specific function or can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures. Among them, non-amino acid structures can be, but are not limited to, amino acid derivatives or analogs. "Spacer sequence" refers to an amino acid sequence that serves as a spacer, examples of which include, but are not limited to, a single amino acid such as Leu, Gln, a sequence containing multiple amino acids, such as a sequence containing two amino acids such as GA, or, for example, GGGS, GGGGSGGGGS, etc. Other examples of spacers include self-immolative spacers such as PAB (p-aminobenzyl).

[0055] The term "alkyl group" means a linear or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, which is linked to the remainder of the molecule by a single bond. An alkyl group can contain from 1 to 20 carbon atoms, i.e., C 1 -C 20 It may be alkyl, for example, C 1 -C 4 Alkyl group, C 1 -C 3 Alkyl group, C 1 -C 2 Alkyl group, C 3 Alkyl group, C 4 Alkyl group, C 3 -C 6It is an alkyl group. Non-limiting examples of alkyl 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 isomers thereof. Divalent radical means a group obtained by removing a hydrogen atom from the carbon atom that bears the free valence electron of the corresponding monovalent radical. A divalent radical has two linking sites that are connected to the remainder of the molecule. For example, an "alkylene group" or an "alkylidene group" refers to a straight-chain or branched, saturated divalent hydrocarbon group. Examples of alkylene groups include the methylene group (-CH 2 -), ethylene group (-C 2 H 4 -), propylene group (-C 3 H 6 -), butylene group (-C 4 H 8 -), pentylene group (-C 5 H 10 -), hexylene group (-C 6 H 12 -), 1-methylethylene group (-CH(CH 3 )CH 2 -), 2-methylethylene group (-CH 2 CH(CH 3 )-), methylpropylene group, ethylpropylene group, and the like, but are not limited to these.

[0056] As used herein, when one group is combined with another group, the linkage of the groups may be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination may be linked to the rest of the molecule by any suitable atom within the structure, preferably by a designated chemical bond. For example, C 1-4 Alkylene group and -CH 2 When describing a combination with one of the groups containing -, -NH-, -(CO)-, -NH(CO)-, or -(CO)NH-, C 1-4 The alkylene group can form linear linkages with the above groups, for example, C 1-4 Alkylene-CH 2 -, C 1-4 Alkylene-NH-, C 1-4 Alkylene-(CO)-, C 1-4 Alkylene-NH(CO)-, C 1-4 Alkylene -(CO)NH-, -CH 2 -C 1-4 Alkylene, -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 linked to other portions of the molecule.

[0057] As used herein, the term "isoelectric point (pI)" is the pH (hydrogen concentration index) value of an aqueous solution of a molecule (e.g., a protein) that has no net surface charge, expressed as pH units. The pI of a protein can be experimentally determined using methods well known in the art, such as, for example, imaged capillary isoelectric focusing (iCIEF) and capillary isoelectric focusing (CIEF). Different biomolecules (proteins, nucleic acids, polysaccharides, etc.) with different pIs can have different charges at a given pH value, which allows them to be separated by methods such as ion exchange chromatography or isoelectric focusing.

[0058] As used herein, a molecule with a "basic pI" refers to a molecule with a pI greater than 7.0. As used herein, a molecule with an "acidic pI" refers to a molecule with a pI less than 7.0.

[0059] As used herein, a "protein tag" refers to a polypeptide that can be introduced into a molecule of interest to facilitate detection, separation, immobilization or capture of the molecule of interest or to improve one or more properties of the molecule of interest (e.g., expression levels, solubility, and stability).

[0060] Ion exchange chromatography (IEX) is a commonly used technique for the purification of biomolecules that separates them based on differences in their net surface charge and their affinity for an ion exchanger (also called the medium, resin or stationary phase). For example, in anion exchange chromatography, a protein with a pI lower than the pH of the buffer has a negative net surface charge and binds to the positively charged anion exchanger, while another protein with a pI higher than the pH of the buffer has a positive net surface charge and does not bind to the positively charged anion exchanger, so passes through the medium with the buffer.

[0061] As used herein, the term "support" refers to a water-insoluble material that can be separated from a reaction mixture in solid or semi-solid form, for example, a surface, gel, polymer, matrix, particle, resin, bead, or membrane.

[0062] The term "clarification" refers to the removal of insoluble impurities from a system containing a biomolecule of interest. The "clarification" process can be monitored by nephelometry and can be measured, for example, in nephelometric turbidity units (NTU).

[0063] The term "polishing purification step" refers to a step that further removes trace contaminants and aggregates present in the mixture. Generally, the process for ADC or antibody preparation can employ one or more polishing purification steps, which can be selected from affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, and hydroxyapatite chromatography.

[0064] As used herein, the terms "impurities" and "contaminants" refer to undesirable substances in a target molecule mixture, such as cells, cell debris, host cell proteins and other proteins, endotoxins, media components, lipids, excess reactants (e.g., unreacted linker-payload intermediates), nucleic acids, and viruses.

[0065] As used herein, the term "ppm (parts per million)" refers to the amount of contaminant (e.g., HCP or Protein A) contained in a target molecule (e.g., a target conjugate) per million units of total weight. The term is used as a measure of the purity of the target molecule.

[0066] The term "ultrafiltration" or "UF" refers to a membrane filtration technique that uses a controllable pore, semi-permeable membrane to concentrate or classify 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.

[0067] The term "diafiltration" or "DF" refers to the technique of completely removing, replacing, or reducing the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules using ultrafiltration membranes. The method selectively uses permeable (porous) membrane filters to separate components of solutions and suspensions based on molecular size. Ultrafiltration and diafiltration can be used in combination and are referred to as UF / DF.

[0068] Viral inactivation is used in many biopharmaceutical purification processes to ensure safety. Various viral inactivation techniques are known in the art, including temperature, pH, radiation, and exposure to certain chemical reagents. Generally, viral inactivation can be performed by low pH treatment. For molecules containing Fc fragments, viral inactivation can be performed, for example, after a chromatographic method step (e.g., protein A affinity chromatography or cation exchange chromatography). In this case, a pool containing the target molecule is adjusted to the pH required for viral inactivation and maintained for a certain time (viral inactivation acidification (VIA) step), a combination of pH and time that has been proven to cause viral inactivation. The VIA pool is adjusted to a near-neutral pH value (viral inactivation neutralization (VIN) step) for use in further downstream processes.

[0069] Virus filtration (also called virus-retentive filtration) is a common step in many biopharmaceutical purification processes. Virus filtration can be performed by UF or nanofiltration. Compared to other dedicated virus removal unit operations such as low pH or heat treatment, virus filtration is in most cases gentler, reducing the potential adverse impact on product quality. Depending on the size of the virus to be removed, commercially available virus filtration products can be used.

[0070] The terms "process step" or "unit operation", which may be used interchangeably herein, refer to the use of one or more methods or equipment to achieve a particular result in a refinery process.

[0071] As used herein, the term "continuous process" refers to a method for purifying a target molecule that includes two or more method steps (or unit operations) such that the output from one method step passes directly to the next method step without interruption and / or without collecting all the output from the previous method step before performing the next method step. As described herein, continuous processes also include methods in which the input or output of fluidic materials at any individual method step is discontinuous or intermittent. Such processes are sometimes referred to as "semi-continuous" processes.

[0072] Ligase fusion protein In one aspect, the invention provides a ligase fusion protein comprising a ligase and a Halo tag.

[0073] Ligase The ligase of the invention can be any targeted ligase, and in particular can specifically catalyze the conjugation between a first portion that includes a ligase donor substrate recognition motif and a second portion that includes a ligase acceptor substrate recognition motif to produce a desired conjugate.

[0074] In some embodiments, the ligase is a transpeptidase. The transpeptidase may be naturally occurring or engineered. In some preferred embodiments, the ligase is a sortase, such as, but not limited to, sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), or sortase F (SrtF). As used herein, "sortase" or "sortase enzyme" refers to an enzyme having sortase activity that catalyzes a transpeptidation reaction, including, but not limited to, class A, class B, class C, class D, class E, and class F sortases of the sortase enzyme superfamily (see, e.g., 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 referred to as, but are not limited to, SrtA, SrtB, SrtC, SrtD, SrtE, or SrtF. Sortases may be naturally occurring or engineered.Naturally occurring sortases are found in a variety of Gram-positive bacteria, including, but not limited to, any strain, species or subspecies of Streptococcus (e.g., Streptococcus pneumoniae and Streptococcus pyogenes), Staphylococcus (e.g., Staphylococcus argenteus and Staphylococcus aureus), Bacillus (e.g., Bacillus anthracis), and Listeria (e.g., Listeria monocytogenes). Engineered sortases, such as sortase variants having substitutions, deletions or insertions of one or more amino acid residues, can be obtained from their natural counterparts by methods known in the art, such as protein engineering and chemical synthesis. Also contemplated are other variants of any wild-type sortase known in the art (e.g., those with one or more active groups or labels) that have the same or similar function as the wild-type sortase. One of skill in the art can readily identify and classify sortases into particular classes based on their sequence and other characteristics. However, the definition of a sortase is not limited to any classification method or nomenclature system.

[0075] In some specific embodiments, the ligase is sortase A (SrtA). SrtA may be naturally occurring or engineered. Examples of SrtA include those described in, for example, U.S. Patent No. 7,238,489 and Malik and Kim, 2019, supra, including, but not limited to, any strain, species or subspecies of Streptococcus (e.g., Streptococcus pneumoniae and Streptococcus pyogenes), Staphylococcus (e.g., Staphylococcus argenteus and Staphylococcus aureus), Streptomyces (e.g., Streptomyces coelicolor), Bacillus (e.g., Bacillus anthracis), Lactobacillus (e.g., Lactobacillus plantarum), and Listeria (e.g., Listeria monocytogenes). Various SrtA amino acid sequences can be found, for example, in U.S. Patent No. 7,238,489 or in public sequence databases (e.g., GenBank and Uniprot), the relevant contents of which are incorporated herein by reference. Naturally occurring SrtA amino acid sequences that can be used in embodiments of the present invention are listed under Uniprot accession numbers Q2FV99, A0A3S0JRJ4, A0A2T4Q430, A0A507SMZ3, A0A1F2JEX6, A0A364UNR7, A0A1J3ZU75, A0A0M2NSU2, A0A432A5V1, A0A1J4HB57, A0A4Q8MXV4, W1W5Z3, A0A The engineered SrtA may be, but is not limited to, the proteins 2T4KDK7, A0A2K4DQX6, A0A2T4KHW3, A0A380FYB6, A0A2K4C0Y9, A0A4Q9WQB8, A0A121AFU6, A0A1Q8DH59, A0A5B2YTH7, A0A533IYI6, Q4L923, A0A1F1M8Z4, A0A2A1KC84, and A0A133Q671. Engineered SrtA has been reported in various publications, such as WO2016 / 014501, the relevant contents of which are incorporated herein by reference.For example, engineered SrtA with one or more substitutions (e.g., Pro94Arg, Aspl60Asn, Aspl65Ala, Lysl90Glu, Lysl96Thr, Glul05Lys, and Glul08Gln) compared to Q2FV99, or truncated SrtA with 59 amino acids deleted at the N-terminus compared to Q2FV99, as described in WO2016 / 014501, can be considered. The amino acid sequence of the SrtA variant 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 sequence described above. Also contemplated are any wild-type SrtA variants known in the art (e.g., with one or more active groups or labels) that have the same or similar function as wild-type SrtA.

[0076] In some embodiments, SrtA comprises an amino acid sequence selected from SEQ ID NOs: 1-26 (WT). In other embodiments, SrtA comprises an amino acid sequence selected from SEQ ID NOs: 1-26 and contains 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), Tyr, Asn, Ala, and Thr (i.e., [Tyr34][Asn100][Ala105][Thr136], YNAT), Trp, Asn, Asp, and Thr (i.e., [Trp34][Asn100][Asp105][Thr136], WNDT), or Val, Asn, Asn, and Ser (i.e., [Val34][Asn100][Asn105][Ser136], VNNS), respectively. In some specific embodiments, sortase A comprises the amino acid sequence of SEQ ID NO:27, which is the SNAT counterpart of SEQ ID NO:1.

[0077] In some embodiments, sortase 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 to an amino acid sequence selected from SEQ ID NOs: 1-26.

[0078] In some embodiments, sortase 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 to an amino acid sequence selected from SEQ ID NOs: 1-26, and comprises SNAT, YNAT, WNDT, or VNNS amino acid substitutions at positions 34, 100, 105, and 136.

[0079] In another aspect, the present invention provides SrtA comprising an amino acid sequence selected from SEQ ID NOs: 1-26 and including SNAT, YNAT, WNDT or VNNS amino acid substitutions at positions 34, 100, 105 and 136, 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 to said amino acid mutants. In another aspect, the present invention provides SrtA 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.

[0080] Halo Tag Halo tags are haloalkyl substrates (e.g., the haloalkyl moiety -(CH 2 ) 2-30-X, where X is a halogen such as F, Cl, Br, I, particularly Cl or Br), and forms a covalent bond with the remainder of the substrate. Mutant haloalkane dehalogenases are described, for example, in WO2006 / 093529 and WO2008 / 054821, the relevant contents of which are incorporated herein by reference. Mutant haloalkane dehalogenases that may be used in the present invention may include, but are not limited to, mutants of a Xanthobacter dehalogenase (e.g., Xanthobacter autotrophicus dehalogenase (DhIA)) or a Rhodococcus dehalogenase (e.g., Rhodococcus rhodochrous dehalogenase (DhaA)), including those that contain one or more substitutions at the catalytic triad residues, such as replacement of His272 with Phe / Ala / Gly / Gln / Asn or replacement of Asp106 with Cys or other substitutions, as described in WO 2008 / 054821. The mutant haloalkane dehalogenase may be capable of forming a covalent bond with a haloalkyl substrate.

[0081] In some preferred embodiments, the Halo tag comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Halo tag 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 to SEQ ID NO:28.

[0082] Embodiments further comprising additional elements and / or modifications Optionally, the ligase fusion protein may further comprise one or more additional elements, such as additional polypeptides or tags. Preferably, the ligase fusion protein substantially retains the desired properties. Those skilled in the art can select the appropriate elements depending on the desired function or properties of the fusion protein. Methods for introducing such elements are known in the art.

[0083] The additional polypeptide may be a protein tag having 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 (basic phosphatase), and HRP (horseradish peroxidase). A binding tag can be effectively bound to a corresponding binding partner in a covalent or non-covalent manner. An example of a binding tag is a polyhistidine tag (i.e., His tag, e.g., His 6 Or His 8 tag), Fc tag (immunoglobulin heavy chain constant region (structural domains 3 and 4)), calmodulin tag, maltose binding protein (MBP), glutathione-S-transferase (GST), S tag (interacts with ribonuclease S protein), avidin / streptavidin / neutravidin binding peptides (e.g., SBP tag, Strep tag, and Strep tag II), Halo tag, SNAP tag, and CLIP tag (DNA repair protein O 6 Solubility enhancing tags include, but are not limited to, engineered mutants of -alkylguanine-DNA alkyltransferase and variants thereof. Solubility enhancing tags, when expressed as part of a recombinant protein, can generally enhance the expression level and solubility of the recombinant protein. Examples of solubility enhancing tags include, but are not limited to, GB1 tag (B1 structural domain of streptococcal protein G), Z structural domain of staphylococcal protein A, SUMO (small ubiquitin-related modifier), thioredoxin, GST, and MBP. It should be understood that the properties of the protein tag do not constitute any limitation on the embodiment, and that the protein tag can have one or more properties, for example, a reporter protein or a binding tag can also be a solubility enhancing tag.

[0084] The additional polypeptide may be a short peptide that can act as a linker, spacer, or enzyme-cleavable sequence (e.g., a TEV protease recognition motif or a thrombin recognition motif). In some embodiments, a rigid or flexible linker peptide (e.g., a polyglycine stretch, (G 4 S) n where G is glycine, S is serine, and n is an integer from 1 to 6, preferably n is an integer from 2 to 5) is inserted to ensure normal function of the fusion protein. In some embodiments, the linker peptide is (G 4 S) 2 In some embodiments, the ligase fusion protein comprises the amino acid sequence of SEQ ID NO:29.

[0085] In some embodiments, the tag is a tracer molecule such as a fluorophore, a radionuclide, a fluorescent molecule, a fluorescent quantum dot, or a nanogold particle. In some embodiments, the tag is an affinity tag such as biotin. Such tags can be used to monitor reactions catalyzed by the fusion protein or to track or immobilize the fusion protein.

[0086] In some embodiments, the ligase fusion protein contains one or more modifications, where the ligase, Halo tag and additional polypeptide (if applicable) are independently modified, for example, by substitution, deletion, addition, insertion of one or more amino acids, or by introduction of a moiety or active group at one or more appropriate residues, such that the desired biological activity or function of the modified fusion protein is substantially similar to the biological activity or function of the corresponding fusion protein.

[0087] Specific embodiments of ligases with a basic pI In a preferred embodiment, the present invention provides a ligase fusion protein with an altered pI compared to the ligase from which it is derived, the ligase having a basic pI and the Halo tag having an acidic pI. By fusing a ligase with a basic pI to a Halo tag with an acidic pI, a ligase fusion protein with an altered pI is obtained, which produces certain beneficial effects under certain circumstances. For example, the ligase fusion protein may have altered charge characteristics compared to the ligase under certain conditions (e.g., in a specific buffer system at a certain pH value or in an in vivo environment), which may result in, for example, altered solubility, stability, or electrostatic interaction pattern (i.e., ability to form electrostatic interactions with charged substances) compared to the ligase.

[0088] 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 embodiments including one or more additional elements (e.g., additional polypeptides or labels or combinations thereof as defined above) and / or modifications (e.g., amino acid substitutions, deletions, additions, insertions, or moieties or active groups), preferably a desired pI difference between the ligase fusion protein and the ligase is achieved. In some embodiments, the additional polypeptide (if applicable) may have a specific pI that aids in achieving the desired pI of the ligase fusion protein.

[0089] In some embodiments, the pI of the ligase fusion protein is about 2.0 to about 2.5 pH units lower than the pI of the ligase, e.g., about 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 pH units lower than the pI of the ligase. In some embodiments, the pI of the ligase fusion protein is about 2.6 to about 3.0 pH units lower than the pI of the ligase, e.g., about 2.6, 2.7, 2.8, 2.9 or 3.0 pH units lower than the pI of the ligase. In some embodiments, the pI of the ligase fusion protein is about 3.1 to about 3.5 pH units lower than the pI of the ligase, e.g., about 3.1, 3.2, 3.3, 3.4 or 3.5 pH units lower than the pI of the ligase. In some embodiments, the pI of the ligase fusion protein is about 3.6 to about 4.0 pH units lower than the pI of the ligase, e.g., about 3.6, 3.7, 3.8, 3.9, or 4.0 pH units lower than the pI of the ligase. In some embodiments, the pI of the ligase fusion protein is about 4.1 to about 4.5 pH units lower than the pI of the ligase, e.g., about 4.1, 4.2, 4.3, 4.4, 4.5 pH units lower than the pI of the ligase.

[0090] In some embodiments, the pI of the ligase fusion protein is about 4.5 to about 6.5, e.g., 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.

[0091] 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.

[0092] In some specific embodiments, the pI of the fusion protein is about 5.0 to about 6.0, and the pI of the ligase is about 7.6 to about 9.7.

[0093] In some embodiments, the ligase is a sortase. The sortase may be selected from the group consisting of SrtA, SrtB, SrtC, SrtD, SrtE and SrtF.

[0094] In some preferred embodiments, the SrtA comprises an amino acid sequence selected from SEQ ID NOs: 1-12 (WT). In some other embodiments, the SrtA comprises an amino acid sequence selected from SEQ ID NOs: 1-12 and comprises 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 SrtAs are shown in Table 1.

[0095] [Table 1]

[0096] In some embodiments, the sortase 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 to an amino acid sequence selected from SEQ ID NOs: 1-12.

[0097] In some embodiments, the sortase 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 to an amino acid sequence selected from SEQ ID NOs: 1-12, and comprises SNAT, YNAT, WNDT, or VNNS amino acid substitutions at positions 34, 100, 105, and 136.

[0098] In a specific embodiment, the sortase A comprises the amino acid sequence of SEQ ID NO: 27, which is the SNAT counterpart of SEQ ID NO: 1 and has a pI of 8.508.

[0099] In some embodiments, a rigid or flexible linker peptide (e.g., a polyglycine stretch, (G 4 S) n where G is glycine, S is serine, and n is an integer from 1 to 6, preferably n is an integer from 2 to 5) is inserted to ensure normal function of the fusion protein. In some embodiments, the linker peptide is (G 4 S) 2 In some embodiments, the ligase fusion protein comprises the amino acid sequence of SEQ ID NO:29.

[0100] Methods for Obtaining Ligase Fusion Proteins The ligase, Halo tag, and additional polypeptide (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., a polyglycine stretch, (G 4 S) n where G is glycine, S is serine, and n is an integer from 1 to 6, preferably n is an integer from 2 to 5) may be inserted to ensure proper function of the fusion protein. In some embodiments, the linker peptide is (G 4 S) 2 In some embodiments, the ligase fusion protein comprises the amino acid sequence of SEQ ID NO:29.

[0101] The ligase fusion protein can be obtained by various techniques known in the art, such as, for example, expression from a nucleic acid by recombinant DNA technology, chemical synthesis, enzyme-catalyzed coupling methods or chemical coupling methods. In some preferred embodiments, the ligase fusion protein is a recombinant protein encoded by a nucleic acid, said nucleic acid comprising a nucleic acid sequence encoding a ligase and a Halo tag. The recombinant protein can be expressed and purified in a suitable host cell, such as, for example, a mammalian cell, a bacterium, a yeast cell, or an insect cell, preferably a bacterium such as E. coli.

[0102] Nucleic acids and vectors The invention further provides a nucleic acid encoding a ligase fusion protein of the invention, comprising a first polynucleotide encoding a ligase of the invention 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 of the invention further comprises a third polynucleotide encoding an additional polypeptide, wherein the additional polypeptide is operably linked to the ligase and the Halo tag. Examples of additional polypeptides are described above.

[0103] In some embodiments, the first polynucleotide encodes a sortase A and the second polynucleotide encodes a Halo tag. In some embodiments, the first polynucleotide encodes a sortase A, the sortase A having an amino acid sequence selected from SEQ ID NOs: 1-26, and the second polynucleotide encodes a Halo tag, the Halo tag having an amino acid sequence of SEQ ID NO: 28. In a specific embodiment, the first polynucleotide encodes SrtA, the SrtA having an amino acid sequence of SEQ ID NO: 27, and the second polynucleotide encodes a Halo tag, the Halo tag having an amino acid sequence of SEQ ID NO: 28. In another specific embodiment, the nucleic acid encodes a ligase fusion protein, the ligase fusion protein having an amino acid sequence of SEQ ID NO: 29. It will be understood by those skilled in the art that one or more nucleotides in a nucleic acid may be optimized without departing from the spirit of the present invention.

[0104] In some embodiments, the nucleic acid of the present invention is prepared as a recombinant nucleic acid, which may further comprise one or more additional polynucleotides, such as, for example, a regulatory element and a polynucleotide encoding a protein tag. Such regulatory elements can regulate the expression of the fusion protein of the present invention, and include, but are not limited to, enhancers, insulators, and internal ribosome entry sites (IRES). Recombinant nucleic acids comprising the nucleic acid of the present invention can be prepared by molecular cloning techniques known in the art, such as chemical synthesis, site-directed mutagenesis, and polymerase chain reaction (PCR) techniques (see Sambrook, J., EF Fritsch, and T. Maniatis (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0105] In some embodiments, the nucleic acid of the invention is cloned into a vector, preferably an expression vector that can be expressed in a host cell (e.g., bacterial, mammalian, yeast or insect cell). A person skilled in the art can select an appropriate expression vector depending on the nature of the ligase fusion protein and the host cell used. In some embodiments, the vector is a bacterial expression vector that is expressed in bacteria (e.g., E. coli). In some embodiments, the expression vector can further include one or more selection marker genes, such as neomycin or puromycin resistance genes. After expression, the fusion protein can be purified by methods known in the art depending on the protein tag used. A person skilled in the art can select an appropriate expression vector, promoter, regulatory element and protein tag depending on the type of host cell used and the purification strategy.

[0106] Immobilized ligase In another aspect, the invention provides an immobilized ligase comprising a ligase fusion protein of the invention immobilized on a support.

[0107] The support can be in solid or semi-solid form made of any material. Non-limiting examples of supports can include, but are not limited to, resins (e.g., agarose resins, organosilicone resins, polymethylmethacrylate resins, epoxy resins, or cellulose resins), gels (e.g., alginate hydrogels), beads / microspheres / particles (e.g., polystyrene beads, magnetic particles), plates, wells, tubes, thin films, membranes, matrices, and glass (e.g., glass slides).

[0108] In some preferred embodiments, the support is a resin. In some more preferred embodiments, the support is selected from the group consisting of an agarose resin, an organosilicone resin, a polymethylmethacrylate resin, and a cellulose resin. In one specific embodiment, the support is a highly cross-linked agarose resin.

[0109] Methods of enzyme immobilization, such as adsorption, covalent or non-covalent binding, embedding, encapsulation, and crosslinking, are known in the art. It is desirable to retain maximum enzymatic activity of the ligase after immobilization and to have minimal free ligase in the conjugate product after the conjugation reaction. Preferably, the surface of the support is modified to include one or more functional groups so that the ligase fusion protein can be covalently immobilized on the support.

[0110] Preferably, the support comprises one or more chemically active functional groups capable of forming covalent bonds with reactive groups (e.g., amines, thiols, and carboxylates) of the ligase fusion protein or reactive groups in a haloalkyl substrate, or the support comprises one or more binding partners of a corresponding binding tag / affinity label comprised 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 known in the art.

[0111] In some embodiments, the support comprises chemically active functional groups capable of forming covalent bonds with reactive groups (e.g., amines, thiols, and carboxylates) on the ligase fusion protein or reactive groups in a haloalkyl substrate. In some specific embodiments, the functional groups contained on the support are selected from the group consisting of cyanate esters, isothiocyanates, isocyanates, carbodiimides, N-hydroxysuccinimide (NHS) esters, amines, carbonates, epoxides, maleimides, haloacetyls, aziridines, ethyl chloroformates, and aliphatic aldehydes.

[0112] In some embodiments, the support is an epoxy-activated resin, a CNBr (cyanogen 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 an epoxy-activated highly cross-linked agarose resin. In some preferred embodiments, the epoxy-activated resin is pretreated to introduce amino groups prior to reaction with a haloalkyl substrate. In some preferred embodiments, the pretreatment of the epoxy-activated resin is carried out using ammonia. In some preferred embodiments, the pretreatment of the epoxy-activated resin introduces amino groups onto the oxirane ring, and the ring opening of the oxirane ring provides hydroxy groups. Such hydroxy groups are optionally end-capped in a subsequent support preparation step. In a specific embodiment, the pretreatment of the epoxy-activated resin introduces amino groups onto the oxirane ring, and the ring opening of the oxirane ring provides hydroxy groups, which are optionally end-capped in a subsequent support preparation step with an esterification reagent (e.g., Ac 2 Optionally, the resin is esterified with an acetylating agent such as 0.25MgCl2O4. Epoxy-activated resins pretreated in this manner are within the scope of "epoxy-activated resins" as defined above. In some preferred embodiments, the resin is an agarose resin (e.g., a highly cross-linked agarose resin) or a polymethylmethacrylate resin.

[0113] In some other embodiments, the support comprises one or more binding partners of the corresponding binding tags / affinity labels contained in the ligase fusion protein, e.g., additional tags or affinity labels. Correspondence between reactive groups or between binding tags / affinity labels and binding partners is known in the art. An example of a binding tag / affinity label and a corresponding binding partner is a His tag and a Ni tag. 2+ , biotin / SPB tag / Strep tag / Strep tag II and streptavidin / avidin / neutravidin, GST tag and glutathione, Fc tag and protein A, calmodulin tag and Ca 2+, MBP and amylose, S-tag and ribonuclease S-protein, SNAP-tag and benzylguanine (BG) derivative, and CLIP-tag and benzylcytosine (BC) derivative.

[0114] In some preferred embodiments, the support is functionalized to form a covalent interaction with the Halo tag by including a haloalkyl linker. The haloalkyl linker may be introduced to the support by covalent bonding of one or more functional groups contained in the support with one or more reactive groups in the haloalkyl substrate, and the support thus obtained is also referred to as a haloalkyl linker-modified support. The haloalkyl linker-modified support is within the scope of the "support" defined above. Examples of haloalkyl substrates include, but are not limited to, those described, for example, in US20060024808A1 and WO2006093529. Haloalkyl substrates and methods for preparing such substrates are described, for example, in U.S. Patent Nos. 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.

[0115] The haloalkyl group substrate may include a haloalkyl moiety that includes a primary or secondary halo group, preferably a primary halo group. The halo groups in the haloalkyl moiety are selected from F, Cl, Br and I, preferably Cl and Br. In some embodiments, the haloalkyl group substrate has the structure of formula (I): (F1 a -H1 b ) r -Lh-(F2 b -H2 a ) s (I) During the ceremony, F1 and F2 are independently a moiety that contains a reactive group capable of forming a covalent bond with a chemically active functional group contained in the support; H1 and H2 are halo C 2-30 independently selected from alkyl, Lh is a chemical bond or C 3-200an alkylene group, wherein one or more of (-CH 2 -) structure is optionally replaced by -O-, -NH-, -(CO)-, -NH(CO)-, and -(CO)NH-; Lh -OC 1-10 Alkyl group, -NH-C 1-10 Alkyl group, -(CO)-C 1-10 Alkyl group, -NH(CO)-C 1-10 Alkyl groups, and -(CO)NH-C 1-10 optionally substituted by 1, 2 or 3 substituents selected from alkyl groups; a is 0 or 1 and b is 0 or 1, provided that a and b are different; r is an integer from 1 to 100; s is an integer from 1 to 100.

[0116] 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 an amino group, an amine, a thiol group, and an active ester. In some embodiments, the active ester comprises one or more carboxylic acid radicals (e.g., in a carbonate monoester of a suitable alcohol or phenol, such as an electron-deficient phenol, such as 4-nitrophenol, or, for example, in an NHS ester or sulfo-NHS ester), or one or more sulfonic acid radicals (e.g., in a methanesulfonic acid active ester, such as MsO-). In a specific embodiment, F1 or F2 is [ka] It is.

[0117] In some embodiments, H1 and H2 are haloC 2-20 Alkyl group, preferably haloC 2-10 Alkyl groups, especially haloC 6In some specific embodiments, the alkyl group in H1 or H2 is a straight chain alkyl. In one specific embodiment, H1 or H2 is (CH 2 ) 2-30 -X, preferably (CH 2 ) 2-20 -X, more preferably (CH 2 ) 2-10 -X, especially (CH 2 ) 6 -X, where X is a halogen selected from F, Cl, Br and I.

[0118] In some preferred embodiments, the support is HaloLink TM Resin (Promega).

[0119] In some more preferred embodiments, the support is a resin that may include a haloalkyl linker, said haloalkyl linker being -(CH 2 ) 2-30 -X structure, where X is a halogen selected from F, Cl, Br and I. In a specific embodiment, the support is a haloallyl linker modified resin, preferably an agarose resin or a polymethyl methacrylate resin, more preferably a highly cross-linked agarose resin.

[0120] In some specific embodiments, a is 1, b is 0, r is 1, s is 1, and F1 is [ka] Lh is [ka] H2 is (CH 2 ) 2-20 -Cl, and the haloalkyl group substrate is a chloroalkyl group substrate having the structure of formula (I-1).

[0121] [ka] In the formula, u is an integer of 1 to 20, v is an integer of 0 to 20, and w is an integer of 1 to 19. In one specific embodiment, u is 3, v is 2, and w is 5, and the chloroalkyl group substrate has the structure of formula (I-1-1): [ka]

[0122] In some embodiments, the support is a chloroalkyl linker-modified support and has the structure of formula (II): [ka] In the formula, u is an integer of 1 to 20, v is an integer of 0 to 20, and w is an integer of 1 to 19. [ka] represents a support, which may be a resin, bead, membrane, gel, matrix, thin film, plate, well, tube, glass slide or surface, preferably a resin, more preferably an agarose resin, an organosilicone resin, a polymethylmethacrylate resin or a cellulose resin, most preferably a highly cross-linked agarose resin. Note that for clarity, only a single chloroalkyl-linker moiety linked to the support is described, but it should be understood that there are many such chloroalkyl-linker moieties linked to the support.

[0123] In some embodiments, the chloroalkyl linker-modified support shown in formula (II) is [ka] and a chloroalkyl substrate of formula (I-1).

[0124] In some embodiments, the chloroalkyl linker modified support of formula (II) is prepared from a pretreated epoxy-activated resin, which is prepared by introducing an amino group onto the oxirane ring of the epoxy-activated resin, and the opening of the oxirane ring in the pretreatment process provides a hydroxyl group, which is converted to Ac in the subsequent support preparation step. 2 The support shown in formula (II), optionally esterified with O, has the structure of formula (II-1). [ka] During the ceremony, Sub-structure [ka] represents the pretreated epoxy-activated resin, where [ka] The moiety represents an oxirane ring, which reacts with an amino group to open the ring and give a hydroxy group, which is then esterified to form AcO-; [ka] The portion represents another portion of the pretreated epoxy activated resin.

[0125] In some embodiments, the immobilized ligase has the structure:

[0126] Support----Linker----HaloTag----Ligase Where: Support is a support (e.g., a solid support), e.g., selected from a resin, a bead, a membrane, a gel, a matrix, a thin film, a plate, a well, a tube, a glass slide, or a surface, preferably a resin, more preferably an agarose resin, an organic silicone resin, a polymethyl methacrylate resin, or a cellulose resin, and most preferably a highly cross-linked agarose resin; Linker is a linker moiety that is covalently attached to the support, e.g., a linker moiety comprising a chain of 10 to 60 carbon atoms, optionally comprising one or more ether, ester, carbamate and / or amide bonds, e.g., a linker moiety of formula (II-1') or (II'): [ka] In the formula, u is an integer of 1 to 20, v is an integer of 0 to 20, and w is an integer of 1 to 19. HaloTag is a Halo tag (haloalkane dehalogenase polypeptide) covalently attached to a linker; Ligase is a ligase. wherein one or more "--Linker--HaloTag--Ligase" moieties are attached to the same support.

[0127] In some embodiments, an immobilized ligase comprising a linker moiety of formula (II-1') is obtained by the reaction of 1) and 2) below: 1) one or more chloroalkyl group substrates are reacted with a support to form a chloroalkyl linker-modified support; 2) the chloroalkyl linker-modified support is then reacted with a HaloTag (e.g., a Halo tag contained in a ligase fusion protein) to obtain said immobilized ligase.

[0128] Applications of ligase fusion proteins and immobilized ligase The present invention further provides the use of the ligase fusion protein or immobilized ligase of the present invention in preparing a conjugate.The type of conjugate is not limited.The conjugate can be obtained by contacting the ligase fusion protein or immobilized ligase with a first part and a second part to conjugate, where one of the first part and the second part comprises a ligase donor substrate recognition motif and the other comprises a ligase acceptor substrate recognition motif.

[0129] In some embodiments, the conjugate is a bioconjugate. Examples of bioconjugates may include, but are not limited to, siRNA conjugates, peptide-hormone conjugates, peptide-peptide conjugates, peptide-drug conjugates, antibody-drug conjugates, and multispecific antibodies. In some embodiments, the conjugate comprises a receptor, an antibody, or an antibody fragment. In some embodiments, the conjugate is an antibody-drug conjugate.

[0130] In some embodiments, the pI of the conjugate is about 1.0 to about 4.0 pH units higher than the pI of the ligase fusion protein, e.g., about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 pH units higher than the pI of the ligase fusion protein. In some preferred embodiments, the pI of the conjugate is about 2.0 to about 4.0 pH units higher than the pI of the ligase fusion protein.

[0131] In some embodiments, the pI of the conjugate is about 5.5 to about 10.5, e.g., about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some preferred embodiments, the pI of the conjugate is about 7.5 to about 10.0. In some specific embodiments, the pI of the conjugate is about 8.0 to about 9.0.

[0132] In some embodiments, the pI of the conjugate is about 5.5 to about 10.5 and the pI of the ligase fusion protein is about 4.5 to about 6.5. In some specific embodiments, the pI of the conjugate is about 8.0 to about 9.0 and the pI of the ligase fusion protein is about 5.0 to about 6.0.

[0133] In some embodiments, the ligase fusion protein and the conjugate can be separated from each other using ion exchange chromatography (IEX). The IEX can be anion exchange chromatography (AEX), cation exchange chromatography (CEX), or a combination thereof. In other embodiments, the ligase fusion protein and the conjugate can be separated from each other using isoelectric focusing, such as iCIEF or CIEF.

[0134] In some specific embodiments, the ligase is a sortase, preferably sortase A, and the conjugate is an antibody-drug conjugate.

[0135] Methods of the Invention In another aspect, the present invention provides a method for preparing a conjugate comprising a first moiety and a second moiety, the method comprising: (a) providing a system 1 including a first portion and providing a system 2 including a second portion; (b) contacting system 1 and system 2 of step (a) with a ligase unit to catalyze a conjugation reaction between the first moiety and the second moiety to obtain the conjugate; The ligase unit comprises a ligase, the first and second moieties each independently comprise a biomolecule, a protein, an antibody, an antibody fragment, a receptor, a signal transduction factor, a cell growth factor, a nucleic acid or a nucleic acid analog, a small molecule compound, a glycan, a PEG moiety, a radionuclide, a cytokine, an immunomodulator, a tracer molecule, a fluorophore, a fluorescent molecule, a peptide, a polypeptide, or a peptidomimetic; One of the first and second parts further includes a ligase donor substrate recognition motif, and the other of the first and second parts includes a ligase acceptor substrate recognition motif.

[0136] In some embodiments, the first part and the second part are linked to each other by the coupling of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif.

[0137] In some embodiments, at least one of the first part and the second part includes a linker, preferably, the ligase recognition motif (i.e., the ligase donor substrate recognition motif or the ligase acceptor substrate recognition motif) included in the first part or the second part is part of the linker. In some embodiments, the first part or the second part includes a payload and a linker, and the linker may include a ligase recognition motif and one or more structural moieties linked to the payload. In other embodiments, the first part or the second part includes a biomolecule and a linker, and the linker may include a ligase recognition motif and one or more structural moieties linked to the biomolecule. In further embodiments, the biomolecule and / or the payload are independently modified to include one or more additional moieties such as reactive groups, spacers, and labels.

[0138] The terms "first part" and "second part" of the conjugate are used herein to refer to each part of the conjugate. For example, in the case of a bioconjugate, the first part may be the biomolecule part of the conjugate, and the second part may be another functional part or the remaining part of the conjugate. It should be understood that "first" and "second" are used merely to designate different parts for clarity purposes and do not constitute any limitation.

[0139] The terms "System 1" and "System 2" are simply used to designate different moieties comprising the moiety to be conjugated and do not constitute any limitation. System 1 and System 2 can each independently be in any form, such as aqueous, solid or semi-solid form. Preferably, at least one of System 1 and System 2 is in an aqueous form, such as a (water) solution or fluid. System 1 and System 2 can each independently be selected from cultures (e.g., tissue cultures, mammalian cell cultures, yeast cell cultures, bacterial cell cultures and phage cultures), harvested cell culture fluids, solutions containing antibodies, solutions containing linker-payload intermediates, etc. "System 1" and "System 2" can be the same or different, but are preferably different.

[0140] Ligase Unit The ligase unit can include any ligase without limitation. Specifically, it can recognize the recognition motifs on two moieties and catalyze the conjugation between the two moieties. In some embodiments, the ligase is a transpeptidase. In some embodiments, the ligase is a sortase. The sortase can be selected from the group consisting of SrtA, SrtB, SrtC, SrtD, SrtE, SrtF, and combinations thereof. In some embodiments, the ligase is SrtA as described above. In some embodiments, the ligase is further modified by including one or more additional elements, such as a protein tag or label as described above, or including one or more amino acid substitutions, deletions, or insertions.

[0141] The ligase may be free or immobilized on a support. Preferably, the ligase is immobilized on a support, which can achieve higher operational stability and reusability, lower enzyme contamination, smaller footprint, and continuous production. The support may be in solid or semi-solid form made of any material. Non-limiting examples of supports may include, but are not limited to, resins (e.g., agarose resin, organic 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).

[0142] Methods for enzyme immobilization are known in the art, including adsorption, covalent or non-covalent binding, embedding, encapsulation, and cross-linking. It is desirable to be able to retain maximum enzymatic activity of the ligase after immobilization and to have minimal amounts of free ligase present in the conjugate product after the conjugation reaction.

[0143] More preferably, the ligase is covalently immobilized on the support to reduce the amount of free ligase that falls off the support. Methods for non-specific covalent immobilization of proteins are known in the art. In some embodiments, the support comprises chemically active functional groups capable of forming covalent bonds with reactive groups (e.g., amines, thiol groups, and carboxylates) on the ligase. Such functional groups can be selected from the group consisting of isothiocyanates, isocyanates, carbodiimides, N-hydroxysuccinimide (NHS) esters, carbonates, epoxides, maleimides, haloacetyls, aziridines, ethyl chloroformates, and aliphatic aldehydes.

[0144] Most preferably, the ligase is covalently immobilized on the support by a self-labeling protein tag so that maximum enzymatic activity is retained. The self-labeling protein tag can form a covalent interaction with its substrate. Such protein tags can include, but are not limited to, SNAP tags, CLIP tags, Halo tags and variants thereof. Accordingly, the support can include the corresponding substrate of the protein tag.

[0145] In some specific embodiments, the ligase unit comprises a ligase fusion protein of the invention. In some specific embodiments, the ligase unit comprises an immobilized ligase of the invention.

[0146] Conjugates The method can be used to prepare a variety of conjugates, hi some embodiments, the conjugates are bioconjugates as described above.

[0147] In some embodiments, the conjugate has a structure of Formula (III), where the first moiety comprises T and the second moiety comprises a linker-payload intermediate of Formula (IV): [ka] During the ceremony, T comprises a biomolecule that is optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif; L comprises a linker comprising the other of the ligase donor substrate recognition motif and the ligase acceptor substrate recognition motif; P contains the payload, z is an integer from 1 to 20; t is an integer from 1 to 20.

[0148] t represents the number of payloads that are coupled to a single linker to form a linker-payload intermediate of formula (IV). Z represents the number of compounds of formula (IV) that are coupled to a single T to form a compound of formula (III).

[0149] In one embodiment, z is selected from an integer of 1 to 10, 1 to 8, 1 to 6, or 1 to 4. In another embodiment, z is 1 or 2. In one specific embodiment, z is 2.

[0150] Biomolecules In the present invention, the biomolecule can be selected from the group consisting of proteins, peptides, antibodies, antibody fragments, receptors, signal transduction factors, cell growth factors, and nucleic acids and analogs. In one embodiment, T optionally comprises one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif, or is optionally modified to have one of such motifs.

[0151] In some embodiments, T is a molecule comprising a receptor, an antibody, or an antibody fragment, optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. In other embodiments, T is a receptor, an antibody, or an antibody fragment, 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 other embodiments, T is a soluble receptor, optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif.

[0152] In some embodiments, T is a targeting molecule that is optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. Targets recognized by the targeting molecule (e.g., an antibody or antigen-binding fragment thereof) include CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4, EGFRvIII, SLC44A4 / AGS-5, CEACAM5 , PSMA, TIM1, LY6E, LIV1, Nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin6, FGFR2, FGFR3, CA6, CanAg, integrin αV, TDGF1, Ephrin A4, Trop2, PTK7, NOTCH3, C4.4A, FLT3.

[0153] In some embodiments, the targeting molecule is an anti-human HER2 antibody or antigen-binding fragment thereof, optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. Examples of anti-human HER2 antibodies include, but are not limited to, Pertuzumab and Trastuzumab.

[0154] In one embodiment, the targeting molecule is one or more selected from anti-human TROP2 antibodies or antigen-binding fragments thereof, optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif. In a specific embodiment, the anti-human TROP2 antibody is one or more selected from engineered anti-TROP2 antibodies based on hrS7 (US20140120035). In another specific embodiment, the anti-human TROP2 antibody is one or more selected from engineered anti-TROP2 antibodies based on MAAA1181a (US20160297890).

[0155] In a preferred embodiment, the anti-human HER2 or TROP2 antibody is a recombinant antibody selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetics. In some embodiments, the antibody mimetics are selected from the group consisting of scFvs, mini-antibodies, bi-antibodies, and nano-antibodies. As described below, in order to couple to the compound of formula (IV), the targeting molecule of the present invention may include a modification portion linked to D1 or D2 in the compound of formula (V), i.e., a portion containing a ligase acceptor or donor substrate recognition motif in the linker. The introduction position of such a modification portion is not limited, and for example, when the targeting molecule is an antibody, the introduction position may be, but is not limited to, the C-terminus or N-terminus of the antibody heavy or light chain.

[0156] In some other embodiments, the modifying moiety for coupling to Dl or D2 in the compound of formula (V) may be introduced at a non-terminal position of the antibody heavy or light chain, for example, using chemical modification methods.

[0157] In some embodiments, the targeting molecule of the present invention is an antibody or an antigen-binding fragment thereof that may include a terminal modification. Terminal modification refers to a modification at the C-terminus or N-terminus of the antibody heavy or light chain, which includes, for example, a ligase recognition motif. In some other embodiments, the terminal modification may further include a spacer Sp2 of 2 to 100 amino acids, where the antibody, Sp2 and the ligase recognition motif are linked in sequence. In a preferred embodiment, Sp2 is a spacer sequence comprising 2 to 20 amino acids. In some specific embodiments, the Sp2 spacer sequence is selected from the group consisting of GA, GGGS and GGGGSGGGGS, in particular GA.

[0158] In a preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof is selected from the group consisting of wild type (LC), C-terminal modified light chain (LCCT) modified by directly introducing the ligase recognition motif LPXTG, and C-terminal modified light chain (LCCT) modified by introducing a short peptide spacer and the ligase donor substrate recognition motif LPXTG. L The heavy chain of the antibody or antigen-binding fragment thereof includes three types: wild type (HC), C-terminal modified heavy chain (HCCT) modified by directly introducing the ligase recognition motif LPXTG, and C-terminal modified heavy chain (HCCT) modified by introducing a short peptide spacer and the ligase donor substrate recognition motif LPXTG. L ) X can be any natural or unnatural single amino acid. When z in the compound of formula (IV) is 1 or 2, the combination of the heavy and light chains can form eight preferred antibody molecules, as shown in the amino acid sequence table.

[0159] In some preferred embodiments, the light chain of the antibody or antigen-binding fragment thereof is selected from the group consisting of wild type (LC), N-terminally modified light chain (LCNT) modified by directly introducing the ligase recognition motif GGG, and N-terminally modified light chain (LCNT) modified by introducing a short peptide spacer and the ligase receptor substrate recognition motif GGG. LThe heavy chain of the antibody or antigen-binding fragment thereof includes three types: wild type (HC), N-terminal modified heavy chain (HCNT) modified by directly introducing the ligase recognition motif GGG, and N-terminal modified heavy chain (HCNT) modified by introducing a short peptide spacer and the ligase receptor substrate recognition motif GGG. L ) and three types.

[0160] The conjugate of the present invention may further comprise a payload, said payload being as described herein.

[0161] Linker In some embodiments, the linker, i.e., L in formula (III) and formula (V), is a compound of formula (V): (A1 p -D1 q -Y) t -Lk-(W-A2 q -D2 p ) t (V) During the ceremony, D1 and D2 are independently a moiety that contains a ligase acceptor or donor substrate recognition motif; A1 and A2 independently represent a bond that links to a payload or a moiety that includes a reactive group that can be coupled to a payload; Lk is a chemical bond, L 1 -L 2 -L 3 Or L 1 -L 2 -L 3 -L 4 Or L 4 -L 1 -L 2 -L 3 Or L 4 and L 1 and L 3 are each independently -CH 2 -, -NH-, -(CO)-, -NH(CO)-, -(CO)NH-, and C 1-4 Alkylene group and -CH 2Selected from the group consisting of a combination with one of the groups of —, —NH—, —(CO)—, —NH(CO)—, —(CO)NH— L 2 is absent or is C 7-34 an alkylene group, wherein one or more (—CH 2 ) structures in the alkylene group are optionally replaced by —O— L 1 , L 2 and L 3 are each optionally and independently replaced by one, two or three substituents selected from —OR 1 and —NR 1 R 2 R 1 and R 2 are each independently hydrogen, —C 1-6 alkyl group, —(CO)—C 1-6 alkyl group, and —S(═O) 2 —C 1-6 alkyl group L 4 is a peptide sequence (the amide bond is formed by a condensation reaction of an α-amino group and a carboxyl group), wherein the peptide sequence optionally contains derivatized Lys (lysine) (the number is 1 to 100), or optionally contains derivatized Cys (cysteine) (the number is 1 to 100), Y and W are each independently absent or selected from the group consisting of a cleavable sequence, a spacer Sp1 and combinations thereof The cleavable sequence contains an amino acid sequence cleavable by an enzyme, and the cleavable sequence contains 1 to 10 amino acids Sp1 is selected from the group consisting of a spacer sequence containing 1 to 20 amino acids, PAB and combinations thereof Provided that p and q are different, p is 0 or 1, and q is 0 or 1 t is as defined in formula (III).

[0162] ​In some embodiments, the linker of formula (V) is linked to the payload by A1 or A2 and linked to the biomolecule T by coupling of D1 or D2 with a ligase receptor substrate recognition motif or a ligase donor substrate recognition motif contained in the biomolecule T. Optionally, the ligase recognition motif in the biomolecule T is present in the form of a modified moiety introduced into the biomolecule, for example, by a recombinant method or a chemical modification method.

[0163] In some embodiments, formula (V) is included in the first part of system 1 or in the second part of system 2.

[0164] In some embodiments, L 1 , L 2 and L 3 are independently substituted by one, two or three substituents selected from -OR 1 and -NR 1 R 2 . The substitution occurs, for example, on (-CH 3 ), (-CH 2 -) or

Chemical formula

[0165] In some embodiments, L 2 is a C 7 - 34 alkylene group, where the alkylene group is a straight-chain or branched-chain alkylene group, one or more of the (-CH 2 -) structures in the alkylene group can be optionally replaced by -O-, and the alkylene group is optionally substituted by one, two or three substituents selected from -OR 1 and -NR 1 R 2 . In a further embodiment, L 2 is -OR 1 and -NR 1 R 2wherein the groups are methylene, ethylene, propylene, butylene, pentylene, hexylene, 1-methylethylene, 2-methylethylene, 2-methylpropylene, and 2-ethylpropylene.

[0166] In some other embodiments, L 2 Ha-(C 2 H 4 -O) i -C 1-4 is an alkylene group, and i is an integer from 2 to 10. 2 H 4 -O) i "-" represents the structure formed by polymerization of PEG units, where i represents the number of PEG units. 2 Ha-(C 2 H 4 -O) i -C 1-2 In some specific embodiments, L is an alkylene group. 2 Ha-(C 2 H 4 -O) i -C 2 H 4 In some other embodiments, L 2 is C 1-4 Alkylene group -(OC 2 H 4 ) i In some other embodiments, L 2 is C 1 - 2 Alkylene group -(OC 2 H 4 ) i In some specific embodiments, L 2 -C 2 H 4 -(OC 2 H 4 ) i In some embodiments, i is selected from 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 4 to 6. In one specific embodiment, i is 4.

[0167] L 4 In another embodiment of the present invention, the ε-amino group of a lysine can be used to introduce a maleimide functional group to the A1 or A2 moiety by a suitable bifunctional crosslinker, depending on the number of couplings desired, or can be used to form an amino bond with the α-carboxyl group of another lysine to form a branched chain, after which the α- and ε-amino groups of the lysine in the branched chain can be introduced with a suitable bifunctional crosslinker. Thus, by increasing the number of lysines in the main chain and / or in the branched side chain, it is possible to increase the number of such L 4 The number of A1 or A2 moieties introduced by the moiety can reach 1-1000.

[0168] L 4 In another embodiment of the present invention, the mercapto group of each cysteine ​​can be used to react with a maleimide functional group in A1 or A2, depending on the desired number of couplings. A1 or A2 can then be linked to Lk. A1 and A2 each further comprise a reactive group capable of coupling to a payload. L 4 Medium, for example L 4 By increasing the number of cysteines in the main chain and / or branched side chains of 4 The number of A1 or A2 moieties introduced by the moiety can reach 1-1000.

[0169] In some embodiments, L 4 is an optionally derivatized lysine.

[0170] In a preferred embodiment, the derivatization of lysine is selected from the group consisting of 1) and 2) below: 1) amidation of a carboxyl group to obtain the amide NH 2 is C 1-6 2) The carboxyl and / or amino groups are linked to an amino acid fragment containing 1-10 amino acids or a nucleotide fragment containing 1-10 nucleotides, wherein the amino acid fragment is preferably Gly.

[0171] In some embodiments, Y and W are each independently absent or selected from the group consisting of a cleavable sequence, a spacer Spl, and combinations thereof. In a specific embodiment, Y is absent. In another specific embodiment, W is absent. In a further specific embodiment, both Y and W are absent. In some embodiments, the cleavable sequence comprises an amino acid sequence that can be recognized as an enzyme substrate and cleaved by an enzyme. In some specific embodiments, the cleavable sequence is enzymatically cleavable in the lysosomes of a cell. In another specific embodiment, the cleavable sequence can be cleaved by a protease, in particular a cathepsin. In a further specific embodiment, the cleavable sequence can be cleaved by a glutaminase. In some embodiments, the cleavable sequence is selected from the group consisting of a cathepsin restriction site, a glutaminase restriction site, and combinations thereof. In some embodiments, the cleavable sequence is selected from Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, and combinations thereof.

[0172] In one embodiment, Y and W are each independently absent or selected from the spacer Spl. In another embodiment, Sp1 is a spacer sequence comprising 1-10, preferably 1-6, more preferably 1-4 amino acids. In a specific embodiment, Sp1 is Leu. In another specific embodiment, Sp1 is Gln. In one embodiment, Sp1 is PAB. In a further embodiment, Y and W are each independently selected from the group consisting of Phe-Lys-PAB, Val-Cit-PAB, and Val-Lys-PAB.

[0173] In some embodiments, the amino acids included in Y and / or W can be natural or unnatural. In some specific embodiments, Y is absent or is amino acid fragment 1. Amino acid fragment 1 comprises 1-30 natural or unnatural amino acids, each of which is independently the same or different. Amino acid fragment 1 is selected from the group consisting of a cleavable sequence comprising 1-10 amino acids, a spacer sequence comprising 1-20 amino acids, and combinations thereof. In another specific embodiment, W is absent or is amino acid fragment 2. Amino acid fragment 2 comprises 1-30 natural or unnatural amino acids, each of which is independently the same or different. Amino acid fragment 2 is selected from the group consisting of a cleavable sequence comprising 1-10 amino acids, a spacer sequence comprising 1-20 amino acids, and combinations thereof.

[0174] In some embodiments, p=0, q=1, and the structure of the compound of formula (V) is as shown in formula (V-1) below.

[0175] D1-Y-Lk-(W-A2) t (V-1) In the formula, A2, D1, Y, Lk and W are each as defined in formula (V).

[0176] In some other embodiments, p=1, q=0, and the structure of the compound of formula (III) is as shown in formula (V-2) below.

[0177] (A1-Y) t -Lk-W-D2(V-2) In the formula, A1, D2, Y, Lk and W are each as defined in formula (V).

[0178] In one embodiment, the suitable linker may be any linker selected from Figures 13 to 16 of WO2014177042A. In a further embodiment, the suitable linker may be any linker selected from Figures 7 to 10 of WO2015165413A.

[0179] In one embodiment, a suitable linker may be any linker selected from Figures 1 to 12 of WO2014177042A. In a further embodiment, a suitable linker may be any linker selected from Figures 3 to 6 of WO2015165413A.

[0180] Moieties containing ligase acceptor or donor substrate recognition motifs In some embodiments, the ligase is a transpeptidase. In some embodiments, the ligase is selected from the group consisting of naturally occurring transpeptidases, non-naturally occurring transpeptidases, variants of both, and combinations thereof. The non-naturally occurring transpeptidase can be, but is not limited to, one obtained by engineering a naturally occurring transpeptidase.

[0181] In some preferred embodiments, the ligase is selected from the group consisting of natural sortases, non-natural sortases, and combinations thereof. Natural sortase types include SrtA, SrtB, SrtC, SrtD, SrtE, SrtF, etc. (see, e.g., US20110321183A1 and EP3647419A1). To achieve specific coupling between different molecules or structural fragments, the ligase types correspond to ligase recognition motifs.

[0182] In some embodiments, the ligase acceptor substrate recognition motif is selected from the group consisting of oligomeric glycine, oligomeric alanine, and oligomeric glycine / alanine mixtures having degrees of polymerization between 3 and 10. In some specific embodiments, the ligase acceptor substrate recognition motif is G n where G is glycine (Gly) and n is an integer from 3 to 10.

[0183] In some embodiments, the ligase is SrtA, and the donor recognition motif can be LPXTG, where X is any natural or non-natural amino acid. In some embodiments, the ligase is SrtB, and the donor recognition motif can be NPXTG, where X is any natural or non-natural amino acid. In some embodiments, the ligase is SrtC, and the donor recognition motif can 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 can be LPXTA, where X is any natural or non-natural amino acid. In some further embodiments, the ligase is SrtE, and the donor recognition motif can be LAXTG, where X is any natural or non-natural amino acid. In some other embodiments, the ligase is SrtF, and the donor recognition motif can be LPXTG, where X is selected from the group consisting of A, R, N, D, Q, I, L, and K.

[0184] In another specific embodiment, the ligase is SrtA derived from Staphylococcus aureus. Accordingly, the ligase recognition motif can be the typical recognition motif LPXTG of the enzyme. In a further specific embodiment, the ligase donor substrate recognition motif is LPXTGJ, and the ligase acceptor substrate recognition motif is G n where X can be any single natural or non-natural amino acid, and J is either absent or an amino acid fragment containing 1 to 10 amino acids optionally labeled. In one embodiment, J is absent. In a further 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 some other embodiments, J is G mwhere m is an integer between 1 and 10. In a further specific embodiment, the ligase donor substrate recognition motif is LPETG. In another specific embodiment, the ligase donor substrate recognition motif is LPETGG. In one embodiment, the ligase is SrtB from Staphylococcus aureus and the corresponding donor substrate recognition motif can be NPQTN. In another embodiment, the ligase is SrtB from Bacillus anthracis and the corresponding donor substrate recognition motif can be NPKTG. In a further embodiment, the ligase is SrtA from Streptococcus pyogenes and the corresponding donor substrate recognition motif can be LPXTGJ, where J is as defined above. In another embodiment, the ligase is SrtE from Streptomyces coelicolor and the corresponding donor substrate recognition motif can be LAXTG. In a further embodiment, the ligase is SrtA from Lactobacillus plantarum and the corresponding donor substrate recognition motif can be LPQTSEQ. The ligase recognition motif may be any other novel recognition sequence for transpeptidases optimized by manual screening.

[0185] LPXTGJ to G n When coupled to , the peptide bond upstream of the glycine in the LPXTGJ sequence is cleaved by sortase A, resulting in an intermediate G n The resulting amino acid sequence is LPXTG n The array G n and LPXTGJ are as defined above.

[0186] In some specific embodiments, the ligase is SrtA from Staphylococcus aureus, the donor recognition motif is LPETGG, and the acceptor recognition motif is GGG.

[0187] Moiety containing reactive groups In some embodiments, A1 and A2 in formula (V) are each independently selected from the group consisting of an amino compound, a maleimide and its derivatives, a thiol compound, a pyridyldithiol, a haloacetylic acid, and an isocyanate. In other embodiments, the reactive groups in A1 and A2 are each independently selected from the group consisting of an amino group, a maleimide group, a thiol group, a pyridyldithio group, a haloacetyl, and an isocyanate.

[0188] In some embodiments, A1 and A2 can each independently be covalently bonded to a Michael acceptor (the acceptor molecule of a Michael addition) via a disulfide bond, a thioether bond, a thioester bond, or a urethane bond, depending on the structure of the reactive group therein. In a specific embodiment, A1 and A2 are each independently selected from optionally derivatized cysteine.

[0189] In some other specific embodiments, A1 and A2 are each independently selected from an optionally derivatized cysteine. In some preferred embodiments, the derivatization of cysteine ​​is selected from the group consisting of 1), 2) and 3) below: 1) amidating the carboxyl group to form the resulting amide NH 2 is C 1-6 Optionally substituted with an alkyl group; 2) acylation of the amino group; 3) linking the carboxyl and / or amino group to an amino acid fragment comprising 1-10 amino acids or a nucleotide fragment comprising 1-10 nucleotides, wherein said amino acid fragment is preferably Gly. In a specific embodiment, derivatization of cysteine ​​refers to amidation of the carboxyl group of cysteine ​​or linkage with glycine.

[0190] In some embodiments, A2 is [ka] where x is hydrogen, OH, NH 2 In some embodiments, A1 is selected from the group consisting of an amino acid fragment comprising 1 to 10 amino acids, and a nucleotide fragment comprising 1 to 10 nucleotides. [ka] where x is selected from the group consisting of hydrogen, an amino acid fragment comprising 1-10 amino acids, and a nucleotide fragment comprising 1-10 nucleotides. In some embodiments, acylation of an amino group refers to acylation of an amino group of a cysteine ​​to a C 1-6 It refers to being substituted by an alkylcarbonyl group.

[0191] In some embodiments, t in the linking unit of (V-1) is 1 and D1 is G. n So, A2 is [ka] and the structure of the compound of formula (V-1) is as shown in the following formula (V-1-1). [ka] x is hydrogen, OH, NH 2 , an amino acid fragment comprising 1 to 10 amino acids, and a nucleotide fragment comprising 1 to 10 nucleotides; Lk is L 1 -L 2 -L 3 and L 1 , L 2 , L 3 , t, Y and W are each defined as in formula (V). In some preferred embodiments, in formula (V-1-1), x is OH, NH 2 and Gly.

[0192] In some specific embodiments, in formula (V-1-1), Y and W are both absent, and Lk is L 1 -L 2 -L 3 So, L 1 is -NH-, L 3 is -(CO)-, L 2 Ha-(C 2 H 4 -O) i -C 2 H 4 -, 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]

[0193] In some specific embodiments, in formula (V-1-1), W is absent, Y is L, L is leucine (Leu), and Lk is L 1 -L 2 -L 3 So, L 1 is -NH-, L 3 is -(CO)-, L 2 Ha-(C 2 H 4 -O) i -C 2 H 4 -, i=4, and the structure of the linking unit is as shown in the following formula (V-1-1-2). [ka]

[0194] In some further specific embodiments, in formula (V-1-1), W is absent, Y is Q, Q is glutamine (Gln), and Lk is L 1 -L 2 -L 3 So, L 1 is -NH-, L 3 is -(CO)-, L 2 Ha-(C 2 H 4 -O) i -C 2 H4 -, i=4, and the structure of the linking unit is as shown in the following formula (V-1-1-3). [ka]

[0195] In some specific embodiments, in formula (V-1-1), Y and W are both absent, and Lk is L 1 -L 2 -L 3 So, L 1 is -NH-, L 3 is -(CO)-, L 2 -C 5 H 10 -, and the structure of the linking unit is as shown in the following formula (V-1-1-4). [ka]

[0196] In some further specific embodiments, in formula (V-1-1), Y and W are both absent, and Lk is L 1 -L 2 -L 3 So, L 1 is -NH-, L 3 is -(CO)-, L 2 is one -NR 1 R 2 -C substituted by group 5 H 10 - group, R 1 is hydrogen, R 2 HA-(CO)CH 3 and the structure of the linking unit is as shown in the following formula (V-1-1-5). [ka]

[0197] In some embodiments of the linking unit of formula (V-2), t is 1, D2 is LPXTG and A1 is [ka] In the case where the compound of formula (V-2) has a structure as shown in formula (V-2-1) below. [ka] wherein x is selected from hydrogen, an amino acid fragment comprising 1 to 10 amino acids, and a nucleotide fragment comprising 1 to 10 nucleotides; Lk is L 1 -L 2 -L 3 and L 1 , L 2 , L 3 , Y and W are each as defined in formula (V).

[0198] In one embodiment, x is hydrogen.

[0199] In some embodiments, A1 and A2 are each independently a maleimide functional group. The maleimide functional group is introduced into the molecule of formula (V) by a suitable bifunctional crosslinker.

[0200] In some preferred embodiments, bifunctional crosslinkers for introducing maleimide functionality include N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), the "long chain" analogue of SMCC N-[alpha-maleimidoacetoxy]succinimide ester (AMAS), N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS), 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), 6-maleimidohexanoic acid N-hydroxysuccinimide ester ( ... ester, EMCS), N-succinimidyl 4-(4-maleimidophenyl) butyrate (SMPB), succinimidyl 6- [(beta-maleimidopropionamido)hexanoate (SMPH), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate, LC-SMCC), N-succinimidyl 11-(maleimido)undecanoate (KMUS), and N-hydroxysuccinimidyl-(polyethylene glycol alcohol). n (N-hydroxy succinimide-(polyethyleneglycol alcohol)n ,SM(PEG) n ), where n represents 2, 4, 6, 8, 12 or 24 polyethylene glycol (PEG) units. Examples of maleimide functional groups that may be introduced into A1 or A2 upon reaction with a bifunctional crosslinker are listed in the following table:

[0201] [Table 2]

[0202] In some embodiments, A1 and A2 are each independently selected from mc and mcc.

[0203] In some embodiments of the linker of formula (V-1), t is 1 and D is G n In this case, G is glycine, A2 is mcc, W is absent, and Lk is L. 4 So, L 4 is an optionally derivatized lysine, and the structure of the compound of formula (V-1) is as shown below in formula (V-1-2). [ka] In the formula, n is an integer of 3 to 10, x is hydrogen, OH, NH 2 , an amino acid fragment comprising 1 to 10 amino acids, and a nucleotide fragment comprising 1 to 10 nucleotides; Y is as defined in formula (V).

[0204] In one specific embodiment, in formula (V-1-2), Y is absent, n=3, x is OH, and the structure of the linker is as shown below (Linker LU104). [ka]

[0205] payload In the present invention, the payload can be selected from the group consisting of hydrogen, small molecule compounds (e.g., inhibitors and toxins (e.g., cytotoxins)), glycans, PEG moieties, radionuclides, cytokines, immunomodulators, nucleic acids and analogs (e.g., interfering RNAs), tracer molecules (e.g., fluorophores and fluorescent molecules), polypeptides (e.g., protein tags, bioactive peptides, protein toxins and enzymes), peptidomimetics, antibodies and antibody fragments.

[0206] In some embodiments, the payload is selected from the group consisting of small molecule compounds, immunomodulators, nucleic acids and analogs, tracer molecules, radionuclides, peptidomimetics, glycans, and PEG moieties.

[0207] In some embodiments, the payload is selected from the group consisting of bioactive peptides, cytokines, antibodies, antibody fragments, and protein receptors.

[0208] In some embodiments, the payload is selected from the group consisting of small molecule compounds, nucleic acid molecules, and tracer molecules. In some preferred embodiments, the payload is selected from small molecule compounds. In more preferred embodiments, the payload is selected from the group consisting of cytotoxins and fragments thereof. In some embodiments, the payload is one or more radionuclides. In some other embodiments, the payload is one or more cytokines. In some embodiments, the payload is one or more immunomodulators.

[0209] In some embodiments, the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton. In some preferred embodiments, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, or the like. A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinblastines such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine, dolastatin 10 and analogs, halichondrin B and eribulin, indole-3-oxamides, podophyllotoxin, 7-diethylamino-3(2'-benzoxazolyl)-coumarin (DBC), discodermolide, and laulimalide. In some alternative embodiments, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors, such as camptothecins and derivatives, mitoxantrone, and mitoguazone.In some preferred embodiments, the cytotoxin is selected from the group consisting of nitrogen mustards, e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenamet, phenesterine, prednimustine, trofosfamide, uracil mustard, etc. In some further preferred embodiments, the cytotoxin is selected from the group consisting of nitrosoureas, such as, for example, carmustine, flubenzuron, formoterol, lomustine, nimustine, and ranimustine. In some embodiments, the cytotoxin is selected from the group consisting of aziridines. In some preferred embodiments, the cytotoxin is selected from the group consisting of benzodopa, carboquone, meturedepa, and uredepa. In some embodiments, the cytotoxin is selected from the group consisting of antitumor antibiotics. In some preferred embodiments, the cytotoxin is selected from the group consisting of enediyne antibiotics. In some more preferred embodiments, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarzinostatin, and aclacinomycin.In some further preferred embodiments, the cytotoxin is actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, carminomycin and cardinophyllin, actinomycin D, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, ferric adriamycin, ferric serotonin ... In further preferred embodiments, the cytotoxin is selected from the group consisting of trichothecenes. In some more preferred embodiments, the cytotoxin is selected from the group consisting of T-2 toxin, verrucarin A, bacillocporin A, and anguidine. In some embodiments, the cytotoxin is an antitumor amino acid derivative. In some preferred embodiments, the cytotoxin is selected from the group consisting of ubenimex, azaserine, and 6-diazo-5-oxo-L-norleucine. In some further embodiments, the cytotoxin is selected from the group consisting of folic acid analogs.In some preferred embodiments, the cytotoxin is selected from the group consisting of dimethylfolate, methotrexate, pteropterin, trimetrexate, and edatrexate. In some embodiments, the cytotoxin is selected from the group consisting of purine analogs. In some preferred embodiments, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine. In further embodiments, the cytotoxin is selected from pyrimidine analogs. In some preferred embodiments, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, and floxuridine. In some embodiments, the cytotoxin is selected from androgens. In some preferred embodiments, the cytotoxin is selected from the group consisting of calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone. In some further embodiments, the cytotoxin is selected from the group consisting of anti-adrenal drugs. In some preferred embodiments, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In some embodiments, the cytotoxin is selected from anti-androgens. In some preferred embodiments, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprorelin acetate, and goserelin. In further embodiments, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors.In some specific embodiments, the cytotoxin is selected from the group consisting of vinblastines, colchicines, taxanes, auristatins, and maytansinoids. In some specific embodiments, the cytotoxin is an auristatin, such as, for example, MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), or MMAD (monomethyl auristatin D). The synthesis and structure of monomethyl auristatin compounds are as described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0210] The payload comprises a reactive group capable of reacting with the reactive group in the compound of formula (V), thereby covalently linking the payload to the compound of formula (V). Compounds that do not contain a reactive group must be appropriately derivatized to obtain the payload. In some embodiments, the reactive group in the payload is a maleimide, or compounds that do not contain a maleimide can be subjected to an appropriate reaction to obtain a maleimide derivative. For example, MMAF is derivatized to obtain mc-MMAF (where mc is maleimidocaproyl). MMAE is derivatized to obtain mc-Val-Cit-PAB-MMAE. The mc in the above structure can be replaced by mcc (4-(maleimidomethyl)cyclohexane-1-carbonyl) or maleimide-R structure, where R is C. 1-20 an alkylene group, optionally including one or more (-CH 2 The -) structure may be replaced by -O-.

[0211] In some embodiments, step (a) comprises reacting a linker of formula (V) with a payload to obtain system 2. Preferably, the compound of formula (V) is independently covalently attached via a reactive group contained in the A1 or A2 moiety to another reactive group contained in the payload to form a linker-payload intermediate of formula (IV).

[0212] The reactive groups contained in the A1 or A2 moieties are as described above.

[0213] In some specific embodiments, the covalent bond formed between the compound of formula (V) and the payload is one or more selected from the group consisting of an amide bond, a disulfide bond, a thioether bond, a thioester bond, a peptide bond, a hydrazone bond, an ester bond, an ether bond, and a urethane bond.

[0214] In some embodiments, the reactive group in A1 or A2 of the compound of formula (V) is a maleimide or a maleimide derivative, and another reactive group in the payload is a Michael acceptor. After reacting with the payload, the maleimide or maleimide derivative is converted to a succinimide or a succinimide derivative.

[0215] In some embodiments, p=0 and q=1, and the intermediate of formula (IV) is as shown in formula (IV-1) below. D1-Y-Lk-(W-A2-P) t (IV-1).

[0216] In some other embodiments, p=1 and q=0, and the structure of the compound of formula (IV) is as shown in formula (IV-2) below. (P-A1-Y) t -Lk-W-D2(IV-2).

[0217] In some embodiments, a linker-payload intermediate of formula (IV) comprising a succinimide or succinimide derivative may be subjected to a ring-opening reaction to obtain a "ring-opened" intermediate. The ring-opening reaction may be carried out in a manner similar to that described in WO2015165413A. The ring-opened intermediate is also included within the scope of formula (IV).

[0218] In some embodiments, the ring-opening reaction of the succinimide in the linker-payload intermediate of formula (IV) forms a ring-open intermediate as shown in formula (IV-1-2) below, which is included within the scope of formula (IV-1). D1-Y-Lk-(W-A2open-P) t (IV-1-2).

[0219] In some other embodiments, the succinimide ring-opening reaction forms a ring-open intermediate shown in formula (IV-2-2) in the linker-payload intermediate of formula (IV), where formula (IV-2-2) is included within the scope of formula (IV-2). (P-A1open-Y) t -Lk-W-D2(IV-2-2) In the formula, the structures of "-A1open-" and "-A2open-" are [ka] is selected from.

[0220] Specific Embodiments of the Linker-Payload Intermediate In some embodiments, A1 and A2 are each independently selected from mc and mcc, and the maleimide functional group contained therein is linked to a thiol group in the payload (P), whereby the linker and the payload are linked to each other via a thiosuccinimide linkage. The succinimide ring in the thiosuccinimide linkage may be subjected to the ring-opening reaction described above to obtain the ring-opened thiosuccinimide structure shown below. [ka]

[0221] In some embodiments, the structure of L is as defined in formula (V-1-1) and the linker-payload intermediate of formula (IV-1) has the structure of formula (IV-1-1). The linker-payload intermediate of formula (IV-1-1) may be prepared by reaction of the corresponding linker of formula (V-1-1) with a payload (P).

[0222] In some specific embodiments, in formula (IV-1), D is G n So, G is glycine and A2 is [ka] In some embodiments, Lk is L 4 And L 4 is an optionally derivatized lysine. In some embodiments, D2 in formula (IV-2) is G n and G is glycine. In some embodiments, the linker-payload intermediate has the structure shown in formula (IV-1-1): [ka]

[0223] In some preferred embodiments, in formula (IV-1-1), both Y and W are absent, the payload is mc(open-ring)-Toxin, and the linker-payload intermediate has the structure shown in formula (IV-1-2-1) or formula (IV-1-2'-1) below.

[0224] [ka] In the formula, Toxin represents a cytotoxin defined by formula (III), and n, Lk, and x are each defined as in formula (IV-1-1).

[0225] In some more preferred embodiments, in formula (IV-1-2-1) and formula (IV-1-2'-1), the cytotoxin is MMAF, i.e., the payload is mc(open ring)-MMAF, and the linker-payload intermediate has the structure shown in formula (IV-1-3) or formula (IV-1-3') below.

[0226] [ka] Formulae (IV-1-3) and (IV-1-3') are isomers, T, n, Lk and x are each as defined in formula (IV-1-1).

[0227] In some other specific embodiments, in formulas (IV-1-3) and (IV-1-3′), Lk is L 1 -L 2 -L 3 So, L 1 is -NH-, L 3 is -(CO)-, L 2 Ha-(C 2 H 4 -O) i -C 2 H 4 -, where i=4, and the linker-payload intermediate has the structure shown in formulas (IV-1-4) and (IV-1-4') below.

[0228] [ka] Formulae (IV-1-4) and (IV-1-4') are isomers, n and x are each as defined in formula (IV-1-1).

[0229] In some specific embodiments, in formula (IV-1), W is absent, t is 1, the payload is Toxin, A2 is mcc, and Lk is L 4 So, L 4 is an optionally derivatized lysine and D1 is G nwherein G is glycine, and the linker-payload intermediate has the structure shown in formula (IV-1-5-1) below. [ka] In the formula, Toxin, n, Y and x are as defined above.

[0230] In some embodiments, the linker-payload intermediate has the structure shown in formula (IV-1-5) or (IV-1-5'):

[0231] [ka] In the formula, Toxin, n, Y and x are as defined above.

[0232] Linker-payload intermediates of formula (IV-1-5) and (IV-1-5') may be prepared by a ring-opening reaction of formula (IV-1-5-1).

[0233] In some preferred embodiments, the Toxin is a maytansinoid, preferably DM1.

[0234] In some specific embodiments, in formulas (IV-1-5) and (IV-1-5'), the cytotoxin is DM1, Y is absent, and the linker-payload intermediate has the structure shown in formula (IV-1-6) or (IV-1-6') below. Formula (IV-1-6) is within the scope of formula (IV-1-5), and formula (IV-1-6') is within the scope of formula (IV-1-5').

[0235] [ka] where n and x are as defined above.

[0236] Specific embodiments of the conjugates In some embodiments, t is 1 and the conjugate has a structure selected from formulas (1), (2), (2'), (3), (3'), (4), (4'), (5), (5'), (6), and (6'):

[0237] In some embodiments, the structure of LP is as defined in formula (V-1-1), and the conjugate of formula (III) has the structure of formula (1). The conjugate of formula (1) may be prepared by a conjugation reaction between the corresponding linker-payload intermediate of formula (V-1-1) and a biomolecule (T). [ka] In the formula, n is an integer of 3 to 10, x is OH, NH 2 or Gly, Lk is L 1 -L 2 -L 3 and T, Payload and z are as defined in formula (III), and L 1 , L 2 , L 3 , Y and W are each as defined in formula (V).

[0238] In some embodiments, the structure of LP is as defined by formula (IV-1-2-1) or (IV-1-2'-1), and the conjugate of formula (III) has a structure selected from the following formulas (2) and (2'). In some specific embodiments, the structure of LP is as defined by formula (IV-1-3) or (IV-1-3'), and the conjugate of formula (III) has a structure selected from the following formulas (3) and (3'). In some more specific embodiments, the structure of LP is as defined by formula (IV-1-4) or (IV-1-4'), and the conjugate of formula (III) has a structure selected from the following formulas (4) and (4'). Formula (4) is within the scope of formula (3), formula (3) is within the scope of formula (2), and formula (2) is within the scope of formula (1). Formula (4') is included in the scope of formula (3'), formula (3') is included in the scope of formula (2'), and formula (2') is included in the scope of formula (1).

[0239] In some preferred embodiments, Y and W are both absent, the payload in formula (1) is an mc(open ring)-toxin, and the structure of the conjugate is as shown in formula (2) or formula (2') below.

[0240] [ka] In the formula, Toxin represents a cytotoxin defined by formula (III), and T, n, Lk, x and z are each as defined in formula (1).

[0241] In some more preferred embodiments, the cytotoxin in formula (2) and formula (2') is MMAF, i.e., the payload is mc(open-ring)-MMAF, and the structure of the conjugate is as shown in formula (3) or formula (3') below.

[0242] [ka] Formulae (3) and (3') are isomers, wherein: T, n, Lk, x and z are each defined as in formula (1).

[0243] In some other specific embodiments, Lk is L 1 -L 2 -L 3 where L 1 is -NH-, L 3 is -(CO)-, L 2 is -(C 2 H 4 -O) i -C 2 H 4 -, and i = 4. The structure of the conjugate is as shown in the following formulas (4) and (4’).

[0244]

Chemical Formula

[0245] In some specific embodiments, the targeting molecule is the antibody pertuzumab, hrS7, or MAAA1181a.

[0246] In some embodiments, t is 1, the structure of L-P is as defined by formula (IV-1-5) or (IV-1-5’), and the conjugate of formula (III) has a structure selected from the following formulas (5) and (5’). The conjugate of (5) or (5’) may be prepared by a conjugation reaction of the corresponding linker-payload intermediate of formula (IV-1-5) or (IV-1-5’) with the biomolecule (T). In some specific embodiments, the conjugate of formula (III) has a structure selected from the following formulas (6) and (6’). The conjugate of (6) or (6’) may be prepared by a conjugation reaction of the corresponding linker-payload intermediate of formula (IV-1-6) or (IV-1-6’) with the biomolecule (T). Formula (6) is included in the scope of formula (5), and formula (6’) is included in the scope of formula (5’).

[0247] [ka]

[0248] In some specific embodiments, Y is absent. The structure of the conjugate is as shown in formulas (6) and (6') below.

[0249] [ka] Formulae (6) and (6') are isomers. In some embodiments, T is an anti-human HER2 antibody. In other embodiments, T is a modified trastuzumab.

[0250] Specific embodiments of the method of the present invention The method of the present invention differs from conventional chemical coupling methods in the art in that the conjugation step is site-specifically catalyzed by a ligase, which specifically recognizes a recognition motif on the moiety to be conjugated, whereas in conventional chemical coupling reactions, it is desirable to purify the moiety to be conjugated prior to the coupling reaction to avoid undesired by-products due to non-specific coupling.

[0251] Thus, in one aspect, the methods of the present invention do not require purification of the moiety to be conjugated prior to the conjugation step, reducing overall operation time and steps and increasing the final yield. Accordingly, in some embodiments, system 1 and / or system 2 of step (a) further comprise one or more impurities.

[0252] In another embodiment, the method is particularly advantageous for conjugates that include chemically labile molecules such as biomolecules (e.g., proteins). In some specific embodiments, the method is suitable for preparing bioconjugates, in which at least one of the first and second moieties includes a biomolecule.

[0253] Generally, biomolecule production methods involve cell culture methods to produce target proteins, such as antibodies or antibody fragments, using, for example, mammalian or bacterial host cell systems. In most cases, the resulting harvest is clarified to remove cells and cell debris, resulting in a harvested clarified cell culture fluid (HCCF) containing impurities, such as host cell proteins (HCPs), medium components, and nucleic acids. In typical chemical coupling-based methods, the HCCF is subjected to a series of further purification steps to obtain a high purity biomolecule for use in downstream conjugation reactions. In some cases, the harvested harvest undergoes extraction, clarification, and concentration to precipitate the target protein, and then the precipitate is redissolved and purified. Since the ligase in the method of the present invention can specifically catalyze the conjugation between the first and second moieties, impurities in system 1 and / or system 2 have little effect on the conjugation efficiency and / or specificity of step (b), and high purity of system 1 and system 2 is no longer a prerequisite for step (b). In some embodiments, at least one of system 1 and system 2 in step (a) is harvested clarified cell culture fluid (HCCF). HCCF can be obtained from tissue culture, mammalian cell culture, yeast cell culture, bacterial cell culture, phage culture, etc. In some embodiments, other samples besides HCCF can be used.

[0254] In another preferred embodiment, the method of the present invention can be flexibly integrated with the production process of a biomolecule to obtain a bioconjugate comprising said biomolecule. For example, the method can be easily integrated with the production process of a monoclonal antibody or antibody fragment to produce a bioconjugate comprising a monoclonal antibody or antibody fragment. The bioconjugate can be produced in the same manufacturing facility, has the same product cycle and similar overall yield as the biomolecule involved, and existing production facilities and pipelines can remain largely unchanged.

[0255] In some further embodiments, the method further comprises: (1) prior to step (b), subjecting the system 1 of step (a) to one or more chromatographic steps to remove one or more impurities; and / or (2) prior to step (b), subjecting system 2 of step (a) to one or more chromatography steps to remove one or more impurities; and / or (3) reacting the conjugate obtained in step (b) with and performing one or more chromatography steps to remove one or more impurities.

[0256] The chromatography steps can be independently selected from the group consisting of affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, mixed mode chromatography, hydroxyapatite chromatography, and combinations thereof. The ion exchange chromatography can be selected from the group consisting of anion exchange chromatography, cation exchange chromatography, mixed mode ion exchange chromatography, and combinations thereof. In some preferred embodiments, the ion exchange chromatography is a combination of anion exchange chromatography and cation exchange chromatography. In some embodiments, the chromatography step in step (3) is also referred to as a purification step. The purification step can include affinity chromatography, hydrophobic interaction chromatography, anion exchange chromatography, cation exchange chromatography, mixed mode chromatography, and hydroxyapatite chromatography.

[0257] In some embodiments, at least one of the first and second portions comprises an antibody or an antibody fragment, and at least one of steps (1)-(3) comprises affinity chromatography. The antibody can be a conventional antibody, a recombinant antibody, a multispecific antibody, a fully human antibody, a non-human antibody, a humanized antibody, a chimeric antibody, an intrabody, or a nanobody. The antibody can be of any type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b), or any active derivative thereof. The antibody fragment can be an Fv fragment, a scFv fragment, a dsFv fragment, a scdsFv fragment, a Fd fragment, a Fab fragment, a scFab fragment, a Fab' fragment, a F(ab') 2 Depending on the nature of the antibody or antibody fragment, affinity chromatography can be performed using Protein A affinity chromatography, Protein G affinity chromatography, Protein L affinity chromatography (e.g. Capto TM L affinity chromatography), KappaSelect affinity chromatography, LambdaFabSelect affinity chromatography, or Mabselect TMThe affinity chromatography may be a protein A affinity chromatography. In some embodiments, at least one of the first and second moieties comprises an Fc fragment, and the affinity chromatography is a protein A affinity chromatography. In some embodiments, the first moiety comprises an Fc fragment. Preferably, the Fc fragment is an Fc fragment of an IgG type antibody, for example selected from IgG1, IgG2, IgG3 and IgG4. In some specific embodiments, T is an antibody, and the affinity chromatography is a protein A affinity chromatography. A person skilled in the art can select an appropriate affinity chromatography method based on the nature of the moiety to be conjugated.

[0258] In some preferred embodiments, the chromatography steps in steps (1), (2) and (3) are selected from affinity chromatography, ion exchange chromatography, and combinations thereof.

[0259] In some embodiments, steps (1) and (2) are absent, and step (3) is a combination of affinity chromatography and ion exchange chromatography (Process 1). In some other embodiments, at least one of steps (1) and (2) comprises affinity chromatography, and step (3) comprises ion exchange chromatography (Process 2). In some further embodiments, at least one of steps (1) and (2) comprises affinity chromatography, and step (3) comprises a combination of affinity chromatography and ion exchange chromatography (Process 3). In some further embodiments, at least one of steps (1) and (2) comprises affinity chromatography and / or ion exchange chromatography, and step (3) comprises a combination of affinity chromatography, hydrophobic interaction chromatography, and ion exchange chromatography (Process 4).

[0260] In some specific embodiments, steps (1) and (2) are absent and step (3) is: (3a-1) Protein A affinity chromatography; (3a-2) Anion exchange chromatography; (3a-3) cation exchange chromatography, in any order.

[0261] In some specific embodiments, at least one of steps (1) and (2) comprises Protein A affinity chromatography, and step (3) comprises: (3b-1) anion exchange chromatography; (3b-2) cation exchange chromatography, in any order.

[0262] In some further specific embodiments, at least one of steps (1) and (2) comprises Protein A affinity chromatography, and step (3) comprises: (3c-1) Protein A affinity chromatography; (3c-2) anion exchange chromatography; (3c-3) cation exchange chromatography, in any order.

[0263] In some specific embodiments, the first portion and / or the second portion comprises an Fc fragment, steps (1) and (2) are absent, and step (3) comprises: (3a-1) Protein A affinity chromatography; (3a-2) Anion exchange chromatography; (3a-3) cation exchange chromatography, in any order.

[0264] In some specific embodiments, the first portion comprises an Fc fragment, step (1) comprises Protein A affinity chromatography, and step (3) comprises: (3b-1) anion exchange chromatography; (3b-2) cation exchange chromatography, in any order.

[0265] In some further specific embodiments, the first portion comprises an Fc fragment, step (1) comprises Protein A affinity chromatography, and step (3) comprises: (3c-1) Protein A affinity chromatography; (3c-2) anion exchange chromatography; (3c-3) cation exchange chromatography, in any order.

[0266] In some further specific embodiments, the second portion comprises an Fc fragment, step (2) comprises Protein A affinity chromatography, and step (3) comprises: (3b-1) anion exchange chromatography; (3b-2) cation exchange chromatography, in any order.

[0267] In some further specific embodiments, the second portion comprises an Fc fragment, step (2) comprises Protein A affinity chromatography, and step (3) comprises: (3c-1) Protein A affinity chromatography; (3c-2) anion exchange chromatography; (3c-3) cation exchange chromatography, in any order.

[0268] In some further specific embodiments, step (1) or step (2) comprises Protein A affinity chromatography and anion exchange chromatography, and step (3) comprises: (3d-1) Protein A affinity chromatography; (3d-2) Cation exchange chromatography; (3d-3) hydrophobic interaction chromatography, in any order.

[0269] In some embodiments, affinity chromatography is performed in bind-elute mode, hi other embodiments, ion exchange chromatography is performed in bind-and-elute mode or in flow-through mode.

[0270] In some preferred embodiments, anion exchange chromatography is performed in flow-through mode. In some embodiments, the sample obtained in the flow-through purification method step flows continuously to the next method step. In other preferred embodiments, cation exchange chromatography is performed in bind-elute mode. In some embodiments, the sample obtained in the bind-elute purification method step flows continuously to the next method step.

[0271] In some embodiments, step (b) is performed in a batch mode, a semi-continuous mode, or a continuous mode. In other embodiments, at least one of steps (a), (b), and (1)-(3) is performed in a semi-continuous mode or a continuous mode, and preferably steps (a), (b), and (1)-(3) are performed in a continuous mode.

[0272] In some specific embodiments, the method of the present invention is carried out in a continuous mode, said method comprising: (a') providing a system 1 in a fluid and providing a system 2 in a fluid; (b') subjecting System 1 and / or System 2 independently to a chromatography step to obtain a System 1 eluate and / or a System 2 eluate, wherein the System 1 eluate and / or the System 2 eluate have reduced impurity levels; (c') mixing system 1 and system 2 in step (a') or (b') to form a reaction fluid, and applying a ligase unit to the reaction fluid to catalyze a conjugation reaction between T and a linker-payload intermediate of formula (IV) to obtain a crude conjugate mixture, wherein the crude conjugate mixture comprises the target conjugate and one or more impurities; (d') subjecting the crude conjugate mixture of step (c') to a chromatography step to remove impurities and obtain the target conjugate having a desired purity; Where: Steps (a')-(d') are connected to maintain fluid interconnection so that the sample can flow continuously from one method step to the next.

[0273] The method of the present invention can be adapted for the preparation of a particular conjugate, for example by further comprising additional steps selected from the group consisting of fermentation, clarification, chromatography, pH adjustment, viral inactivation, viral filtration, ultrafiltration, diafiltration, sterile filtration, formulation, and combinations thereof. A person skilled in the art can combine the additional steps with the method of the present invention and arrange the sequence of steps to prepare a particular conjugate.

[0274] In some embodiments, the method of the invention further comprises steps of viral inactivation, viral filtration, UF / DF and / or formulation. In some embodiments, viral inactivation is completed by low pH treatment, for example after protein A affinity chromatography. Viral filtration can be performed after or before the conjugation step, i.e., step (b). Preferably, viral filtration is performed after at least one chromatography step. In some embodiments, viral filtration is performed after the conjugation step, for example after a purification step (e.g., ADC processes 1-3). In some other embodiments, viral filtration is performed before the conjugation step, for example after ion exchange chromatography (e.g., ADC process 4). In some embodiments, UF / DF is performed after the conjugation step and before step (3) (e.g., ADC processes 3-4).

[0275] In some specific embodiments, step (1) or step (2) comprises Protein A affinity chromatography, and UF / DF is performed after the conjugation step and before step (3), where step (3) comprises: (3e-1) anion exchange chromatography; (3e-2) cation exchange chromatography, in any order.

[0276] In some other specific embodiments, step (1) or step (2) comprises Protein A affinity chromatography and anion exchange chromatography, and UF / DF is performed after the conjugation step and before step (3), where step (3) comprises cation exchange chromatography or hydrophobic interaction chromatography.

[0277] In some specific embodiments, the conjugate is an ADC, and the method includes ADC process 1, ADC process 2, ADC process 3, and ADC process 4, as shown in Figure 15. Compared with the conventional ADC process, the method of the present invention has fewer steps, which reduces the operation time, materials, and space required for the method. In addition, in the conventional ADC process, conjugation is usually performed after virus filtration, whereas in the method of the present invention, conjugation can be performed before (processes 1-3) or after (process 4) virus filtration, allowing for more flexible production.

[0278] In some embodiments, the present invention provides a ligase fusion protein comprising a ligase and a Halo tag (the ligase fusion protein according to the present invention). For example, the present invention provides the ligase fusion proteins described in 1.1 to 1.10 below.

[0279] 1.1. A ligase fusion protein according to the invention, wherein said ligase is a transpeptidase, preferably a sortase.

[0280] 1.2. A ligase fusion protein according to the invention, wherein said ligase is sortase A.

[0281] 1.3. The ligase is a sortase, preferably sortase A, and / or the ligase donor substrate recognition motif is LPXTGJ, preferably LPXTG or LPETGG, and / or the ligase acceptor substrate recognition motif is G n wherein G is glycine (Gly), n is an integer from 3 to 10, X is any natural or unnatural amino acid, and J is absent or an amino acid fragment comprising 1 to 10 amino acids, each amino acid being independently any natural or unnatural amino acid, preferably J is absent or G. m Any of the preceding ligase fusion proteins, wherein m is an integer from 1 to 10.

[0282] 1.4. Any of the ligase fusion proteins wherein the ligase is capable of catalyzing the conjugation between a first portion that includes a ligase donor substrate recognition motif (e.g., includes a terminal sequence LPXTG or LPXTGG, where X is any naturally occurring amino acid) and a second portion that includes a ligase acceptor substrate recognition motif (e.g., includes a terminal polyglycine sequence such as GGG) to generate a conjugate of the first portion and the second portion.

[0283] 1.5. Any of the ligase fusion proteins, wherein the ligase comprises an amino acid sequence selected from a. to c. below: An amino acid sequence of any one of SEQ ID NOs: 1 to 26. b. The amino acid sequence of any of SEQ ID NOs: 1-26, wherein the amino acid residues at positions 34, 100, 105, and 136 are optionally substituted with Ser, Asn, Ala, and Thr (i.e., [Ser34][Asn100][Ala105][Thr136], SNAT), Tyr, Asn, Ala, and Thr (i.e., [Tyr34][Asn100][Ala105][Thr136], YNAT), Trp, Asn, Asp, and Thr (i.e., [Trp34][Asn100][Asp105][Thr136], WNDT), or Val, Asn, Asn, and Ser (i.e., [Val34][Asn100][Asn105][Ser136], VNNS), respectively. For example, the ligase comprises the amino acid sequence of SEQ ID NO: 27 (i.e., the SNAT counterpart of SEQ ID NO: 1). c. An amino acid sequence having sortase activity and having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to any one of (a) or (b).

[0284] 1.6. Any of the above ligase fusion proteins wherein the Halo tag is a polypeptide that catalyzes the removal of a halogen from a haloalkyl moiety and forms a covalent bond with the dehalogenated alkyl moiety.

[0285] 1.7. Any of the ligase fusion proteins wherein the Halo tag is derived from a bacterial haloalkane dehalogenase that catalyzes the removal of a halogen from a haloalkyl moiety, forms a covalent bond with the dehalogenated alkyl moiety, and is mutated to prevent hydrolysis of the covalent bond formed, e.g., a Xanthobacter autotrophicus or Rhodococcus rhodochrous haloalkane dehalogenase mutated at a residue involved in hydrolysis, e.g., a histidine residue corresponding to amino acid residue 272 of Rhodococcus rhodochrous dehalogenase.

[0286] 1.8. Any of the ligase fusion proteins wherein the Halo tag comprises the amino acid sequence of SEQ ID NO:28, or an amino acid sequence having dehalogenase activity and at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO:28.

[0287] 1.9. Any of the ligase fusion proteins, wherein the ligase has an isoelectric point (pI) of about 7.5 to about 10.0, the Halo tag has an isoelectric point of about 4.5 to about 5.0, and the isoelectric point (pI) of the ligase fusion protein is about 2.0 to about 4.5 pH units lower than the isoelectric point (pI) of the ligase.

[0288] 1.10. Any of the above ligase fusion proteins comprising the sequence of SEQ ID NO:29, or an amino acid sequence having dehalogenase activity, sortase activity and having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO:29.

[0289] In some further embodiments, the present invention provides an immobilized ligase (an immobilized ligase as described herein) comprising a ligase linked to a support via a Halo tag. For example, the immobilized ligase is immobilized by reaction of a ligase fusion protein (e.g., any ligase fusion protein as described herein) comprising a ligase and a Halo tag with a support comprising a haloalkyl linker (preferably a chloroalkyl linker) on its surface, whereby the ligase fusion protein is immobilized to the support via covalent interaction between the haloalkyl linker and the Halo tag. The immobilized ligase may be, for example, any of the following 1.1 to 1.13.

[0290] 1.1. An immobilized ligase according to the present invention, which is immobilized by reaction of a ligase fusion protein comprising a ligase and a Halo tag with a support comprising a haloalkyl linker, said ligase fusion protein being any of the ligase fusion proteins according to the present invention.

[0291] 1.2. Immobilization by reaction of a ligase fusion protein comprising a ligase and a Halo tag with a support comprising a haloalkyl linker, the haloalkyl linker being generated from a haloalkyl substrate having the structure of formula (I-1-1) or (I-1); [ka] In the formula, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19.

[0292] 1.3. Immobilization by reaction of a ligase fusion protein comprising a ligase and a Halo tag with a support comprising a haloalkyl linker, for example, the support having the structure of formula (II-1) or (II): [ka] During the ceremony, [ka] is a support, e.g. selected from a resin, bead, membrane, gel, matrix, thin film, plate, well, tube, glass slide or surface, preferably a resin, more preferably an agarose resin, an organosilicone resin, a polymethylmethacrylate resin or a cellulose resin, most preferably a highly cross-linked agarose resin; In the formula, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19.

[0293] [Note that for clarity, only a single chloroalkyl-linker moiety linked to the support is described, but it should be understood that there are many such chloroalkyl-linker moieties linked to the support.]

[0294] 1.4. Having the structure Support----Linker----HaloTag----Ligase Where: Support is a support (e.g., a solid support), e.g., selected from a resin, a bead, a membrane, a gel, a matrix, a thin film, a plate, a well, a tube, a glass slide, or a surface, preferably a resin, more preferably an agarose resin, an organosilicone resin, a polymethylmethacrylate resin, or a cellulose resin, most preferably a highly cross-linked agarose resin; Linker is a linker moiety that is covalently attached to the support, e.g., a linker moiety comprising a chain of 10 to 60 carbon atoms, optionally comprising one or more ether, ester, carbamate and / or amide bonds, e.g., a linker moiety of formula (II-1') or (II'): [ka] In the formula, u is an integer of 1 to 20, v is an integer of 0 to 20, and w is an integer of 1 to 19. HaloTag is a Halo tag (haloalkane dehalogenase polypeptide) covalently attached to a linker; Ligase is a ligase polypeptide, Any of the above immobilized ligases, wherein one or more "----Linker----HaloTag----Ligase" moieties are attached to the same support.

[0295] 1.5. A sortase, preferably sortase A, and / or a ligase donor substrate recognition motif is LPXTGJ, preferably LPXTG or LPETGG, and / or a ligase acceptor substrate recognition motif is G n wherein G is glycine (Gly), n is an integer from 3 to 10, X is any natural or unnatural amino acid, and J is absent or an amino acid fragment comprising 1 to 10 amino acids, each amino acid being independently any natural or unnatural amino acid, preferably J is absent or G. m Any of the above immobilized ligases, wherein m is an integer from 1 to 10.

[0296] 1.6. Any of the above immobilized ligases that are capable of catalyzing conjugation between a first portion that includes a ligase donor substrate recognition motif (e.g., includes a terminal sequence LPXTG or LPXTGG, where X is any naturally occurring amino acid) and a second portion that includes a ligase acceptor substrate recognition motif (e.g., includes a terminal polyglycine sequence such as GGG), to generate a conjugate of said first portion and second portion.

[0297] 1.7. Any of the above immobilized ligases, comprising an amino acid sequence selected from a. to c. below. An amino acid sequence of any one of SEQ ID NOs: 1 to 26. b. The amino acid sequence of any of SEQ ID NOs: 1-26, wherein the amino acid residues at positions 34, 100, 105, and 136 are optionally substituted with Ser, Asn, Ala, and Thr (i.e., [Ser34][Asn100][Ala105][Thr136], SNAT), Tyr, Asn, Ala, and Thr (i.e., [Tyr34][Asn100][Ala105][Thr136], YNAT), Trp, Asn, Asp, and Thr (i.e., [Trp34][Asn100][Asp105][Thr136], WNDT), or Val, Asn, Asn, and Ser (i.e., [Val34][Asn100][Asn105][Ser136], VNNS), respectively. For example, the ligase comprises the amino acid sequence of SEQ ID NO: 27 (i.e., the SNAT counterpart of SEQ ID NO: 1). c. An amino acid sequence having sortase activity and having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to any one of (a) or (b).

[0298] 1.8. Any of the above ligase fusion proteins wherein the Halo tag is a polypeptide that catalyzes the removal of a halogen from a haloalkyl moiety and forms a covalent bond with the dehalogenated alkyl moiety.

[0299] 1.9. Any of the ligase fusion proteins wherein the Halo tag is derived from a bacterial haloalkane dehalogenase that catalyzes the removal of a halogen from a haloalkyl moiety, forms a covalent bond with the dehalogenated alkyl moiety, and is mutated to prevent hydrolysis of the covalent bond formed, e.g., a Xanthobacter autotrophicus or Rhodococcus rhodochrous haloalkane dehalogenase mutated at a residue involved in hydrolysis, e.g., a histidine residue corresponding to amino acid residue 272 of Rhodococcus rhodochrous dehalogenase.

[0300] 1.10. Any of the ligase fusion proteins wherein the Halo tag comprises the amino acid sequence of SEQ ID NO:28, or an amino acid sequence having dehalogenase activity and at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO:28.

[0301] 1.11. Any of the ligase fusion proteins having an isoelectric point (pI) of about 7.5 to about 10.0, wherein said Halo tag has an isoelectric point of about 4.5 to about 5.0, and wherein the isoelectric point (pI) of said ligase fusion protein is about 2.0 to about 4.5 pH units lower than the isoelectric point (pI) of said ligase.

[0302] 1.12. Any of the above ligase fusion proteins comprising the sequence of SEQ ID NO:29, or an amino acid sequence having dehalogenase activity, sortase activity and having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO:29.

[0303] 1.13. Any of the above immobilized ligases comprising the sequence of SEQ ID NO:29, or an amino acid sequence having dehalogenase activity, sortase activity and at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO:29, and attached to a support via a linker.

[0304] In some further embodiments, the present invention provides a method (method according to the present invention) for preparing a conjugate (e.g., a drug-antibody conjugate) comprising a first portion and a second portion, wherein one of the first portion and the second portion comprises a ligase donor substrate recognition motif and the other of the first portion and the second portion comprises a ligase acceptor substrate recognition motif, the method comprising contacting the first portion and the second portion in the presence of a ligase unit, wherein the ligase unit is an immobilized ligase, or a ligase fusion protein comprising a ligase and a Halo tag. The method is, for example, as described in 1.1 to 1.11 below.

[0305] 1.1. The method of the present invention, wherein the ligase unit is an immobilized ligase comprising a ligase linked to a support via a Halo tag, e.g., the immobilized ligase is any of the immobilized ligases of the present invention.

[0306] 1.2. The method according to the invention, wherein said ligase unit is any of the ligase fusion proteins according to the invention.

[0307] 1.3. (a) providing a system 1 including a first portion and providing a system 2 including a second portion; (b) contacting system 1 and system 2 of step (a) with a ligase unit to catalyze a conjugation reaction between the first moiety and the second moiety to obtain a conjugate; the first and second moieties each independently comprise a biomolecule, a protein, an antibody, an antibody fragment, a receptor, a signal transduction factor, a cell growth factor, a nucleic acid or a nucleic acid analog, a small molecule compound, a glycan, a PEG moiety, a radionuclide, a cytokine, an immunomodulator, a tracer molecule, a fluorophore, a fluorescent molecule, a peptide, a polypeptide, or a peptidomimetic; one of the first and second portions further comprises a ligase donor substrate recognition motif, and the other of the first and second portions comprises a ligase acceptor substrate recognition motif, whereby the ligase unit catalyzes conjugation between the ligase donor substrate recognition motif and the ligase acceptor substrate recognition motif; For example, any of the preceding methods, wherein step (b) is carried out in batch mode, semi-continuous mode, or continuous mode.

[0308] 1.4. (1) prior to step (b), subjecting the system 1 of step (a) to one or more chromatographic steps to remove one or more impurities; and / or (2) prior to step (b), subjecting system 2 of step (a) to one or more chromatography steps to remove one or more impurities; and / or (3) reacting the conjugate obtained in step (b) with performing one or more chromatography steps to remove one or more impurities; For example, the chromatography steps are independently selected from affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, and combinations thereof, and the ion exchange chromatography is selected from anion exchange chromatography, cation exchange chromatography, and combinations thereof; Preferably, the chromatography step is selected from affinity chromatography, ion exchange chromatography and combinations thereof; For example, at least one of steps (a), (b) and (1)-(3) is performed in a semi-continuous mode or a continuous mode, e.g., steps (a), (b) and (1)-(3) are performed in a continuous mode; For example, at least one of the first and second portions comprises an antibody or an antibody fragment, and at least one of steps (1)-(3) comprises affinity chromatography, e.g., where the antibody or antibody fragment comprises an Fc fragment, and the affinity chromatography is Protein A affinity chromatography; For example, the first or second portion comprises an Fc fragment, Steps (1) and (2) do not exist, and step (3) is (3a-1) Protein A affinity chromatography; (3a-2) Anion exchange chromatography; (3a-3) cation exchange chromatography, in any order; Step (1) or step (2) comprises protein A affinity chromatography, and step (3) comprises: (3b-1) anion exchange chromatography; (3b-2) cation exchange chromatography, in any order; or Step (1) or step (2) comprises protein A affinity chromatography, and step (3) comprises: (3c-1) Protein A affinity chromatography; (3c-2) anion exchange chromatography; (3c-3) cation exchange chromatography, in any order; or Step (1) or step (2) comprises protein A affinity chromatography and anion exchange chromatography, and step (3) comprises: (3d-1) Protein A affinity chromatography; (3d-2) Cation exchange chromatography; (3d-3) hydrophobic interaction chromatography, in any order; For example, the affinity chromatography is performed in bind-elute mode and / or the ion exchange chromatography is performed in bind-elute mode or flow-through mode, e.g., the anion exchange chromatography is performed in flow-through mode and / or the cation exchange chromatography is performed in bind-elute mode.

[0309] 1.5. Any of the preceding methods, wherein one or more impurities are present in the reaction between the first and second portions.

[0310] 1.6. Any of the foregoing methods, wherein the first or second portion is contained in harvested clarified cell culture fluid (HCCF).

[0311] 1.7. Any of the foregoing methods, wherein the ligase unit is a ligase fusion protein comprising a ligase and a Halo tag, and further comprising the steps of reacting the ligase unit with a support comprising a haloalkyl linker, and removing the formed immobilized ligase, such as any immobilized ligase described in the present invention, after the reaction between the first and second parts is substantially complete.

[0312] 1.8. Any of the foregoing methods, wherein the ligase unit is an immobilized ligase, such as any of the immobilized ligases described herein, and further comprising the step of removing the immobilized ligase after the reaction between the first and second parts is substantially complete.

[0313] 1.9. the conjugate has a structure of formula (III), the first moiety comprises T, and the second moiety comprises a linker-payload intermediate of formula (IV), [ka] During the ceremony, T comprises a biomolecule that is optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif; L comprises a linker comprising the other of the ligase donor substrate recognition motif and the ligase acceptor substrate recognition motif; P contains the payload, z is an integer from 1 to 20; Any of the preceding methods, wherein t is an integer from 1 to 20.

[0314] 1.10. T includes a protein, a peptide, an antibody, an antibody fragment, a receptor, a signal transduction factor, a cell growth factor, and a nucleic acid or analogue; and / or The method, wherein P comprises hydrogen, a small molecule compound (preferably a toxin), a glycan, a PEG moiety, a radionuclide, a cytokine, an immunomodulator, a nucleic acid or analogue, a tracer molecule, a fluorophore, a fluorescent molecule, a peptide, a polypeptide, a peptidomimetic, an antibody, an antibody fragment, or a protein.

[0315] 1.11. The ligase is a sortase, preferably sortase A, and / or the ligase donor substrate recognition motif is LPXTGJ, preferably LPXTG or LPETGG, and / or the ligase acceptor substrate recognition motif is G n where G is glycine (Gly) and n is an integer from 3 to 10; X is any natural or unnatural amino acid; J is absent or an amino acid fragment comprising 1 to 10 amino acids, where each amino acid is independently any natural or unnatural amino acid, preferably J is absent or G. m Any of the preceding methods, wherein m is an integer from 1 to 10.

[0316] Beneficial effects In one aspect, the present invention provides a ligase fusion protein, which has at least one of the following advantageous features (1) to (3): (1) The ligase fusion protein is highly expressible and soluble, reducing the cost of enzyme purification. (2) The ligase fusion protein can be easily purified in large quantities, has high purity and activity, and is particularly suitable for industrial applications. (3) The ligase fusion protein is easily immobilized under physiological conditions, which not only facilitates storage and transportation of the ligase, but also helps maintain maximal enzyme activity and improve production scalability.

[0317] In another aspect, the present invention provides an immobilized ligase comprising the ligase fusion protein, the immobilized ligase having at least one of the following advantageous characteristics (1) to (4): (1)High stability. (2) It is highly reusable. After the reaction is completed, the immobilized ligase can be easily recovered and separated from the reaction system with almost no loss of enzymatic activity. (3) Its excellent base resistance makes it possible to wash the immobilized ligase with alkali. (4) The enzyme loading and enzyme activity are high, and therefore, compared with free ligase, the immobilized ligase can catalyze conjugation reactions with high concentrations of enzyme activity in a confined space, thereby reducing working and storage space and reagent costs.

[0318] Thus, the immobilized ligase of the present invention is controllable, reusable, cost-effective and easy to scale up, and therefore particularly advantageous for industrial applications.

[0319] In some specific embodiments, the ligase fusion protein has a changed isoelectric point compared to the ligase from which it is derived, allowing for effective removal of residual enzyme contaminants from the final conjugate product. This feature is particularly important for efficient affinity purification of conjugates that do not contain an affinity moiety. For example, for a conjugate with a pI of about 8 to about 9, a ligase fusion protein with a pI of about 5 to about 6 can be used, and the conjugate and ligase fusion protein can be separated by anion exchange chromatography, where the ligase fusion protein binds to the chromatographic medium while the conjugate flows through. Alternatively, they can be separated by cation exchange chromatography, where the conjugate binds to the chromatographic medium while the ligase fusion protein flows through. More importantly, because the pI of the ligase fusion protein has been changed, traces of free enzyme that may be present (e.g., enzymes nonspecifically adsorbed to the support that may be shed during the catalysis process) can be easily removed.

[0320] In another aspect, the invention provides methods for preparing conjugates using the ligase fusion proteins or immobilized ligases of the invention.

[0321] Conventional methods for preparing conjugates containing protein moieties include at least two sets of purification processes: upstream and downstream purification of the protein before the conjugation step, and downstream purification of the conjugation product after the conjugation step, each set including multiple chromatography steps. For the preparation of ADCs (antibody-drug conjugates), the current mainstream conjugation technology is based on chemistry, where drugs are chemically conjugated to lysine or cysteine ​​residues in the antibody via a linker. Before the conjugation step, high purity antibodies are prepared by upstream and downstream processes, because low purity antibodies can lead to unpredictable results. For example, if the antibody feed contains impurities, the conjugation reaction of the payload with lysine / cysteine ​​can produce by-products. Impurities in the antibody feed reduce the yield of the conjugation step, which puts pressure on the productivity of the process, requires additional inputs, and further increases the overall process complexity. After the conjugation step, another downstream purification process is required to remove aggregates, solvents, by-products, and impurities in the ADC. The two downstream steps in the conventional method not only significantly increase the cost and time, but also reduce the yield.In addition, the conjugation reaction needs to be carried out in a chemical isolator from the viewpoint of safety, which makes the method difficult to scale up.In short, the conventional method that includes multiple upstream and downstream purification steps is time-consuming, uneconomical, inflexible, and lacks scalability.

[0322] The method of the present invention achieves at least one of the following technical effects:

[0323] (1) Due to the substrate specificity of the ligase, the method can be performed without prior purification of the moieties to be conjugated from the raw materials, thereby reducing overall operating time and steps, reducing costs (e.g., pure water or water for injection, various buffer reagents, chromatography media, etc.), and increasing yields. (2) The method can be flexibly integrated with the conjugation step of the biomolecular moiety, such as an antibody, to be conjugated, i.e., the targeted conjugate can be produced in the same production facility and with the same product cycle as the biomolecular moiety involved, the overall yields will be similar, and existing production facilities and pipelines can remain largely unchanged. (3) The method can be easily scaled up to meet industrial needs, especially when immobilized ligase is used. (4) Simplification of in-process analysis of product quality is achieved. (5) Effective removal of impurities from upstream catalytic reactions, such as excess reactants and residual enzyme contaminants, is achieved. (6) Improved economy in terms of time and space will be achieved.

[0324] In addition to the above advantages, the method of the present invention has at least the following special advantages (1) and (2) over conventional methods in preparing bioconjugates. (1) The formation of aggregates (e.g., antibody and ADC aggregates) is minimized, improving final yields and reducing the effort required for aggregate removal. (2) The DAR ratio and conjugation sites in bioconjugates can be easily controlled, resulting in bioconjugates with higher homogeneity and defined physicochemical properties.

[0325] Working Example In order to more clearly explain the objectives and technical solutions of the present invention, the present invention will be further described below by specific examples. It should be understood that these examples are not intended to limit the scope of the present invention. Any specific experimental methods not described in the following examples are carried out according to conventional experimental methods.

[0326] Apparatus, materials and reagents Unless otherwise stated, the above-mentioned equipment and reagents may be commercially available or may be prepared according to conventional methods in the art.

[0327] MabSelect Sure ProA is from GE and Q Sepharose FF / Capto S ImpAct is from GE. CHO cells for antibody expression are from Thermo Fisher Scientific. pcDNA3.3 is from Life Technology.

[0328] The HIC-HPLC was Butyl-HIC. The mobile phase A was 25 mM PB, 2M (NH 4 ) 2 SO 4 , pH 7.0. Mobile phase B is 25 mM PB, pH 7.0. Flow rate is 0.8 ml / min. Collection time is 25 min. Sample injection amount is 20 μg. Column temperature is 25° C. Detection wavelength is 280 nm. Sample chamber temperature is 8° C.

[0329] Common methods Common methods for producing antibodies For antibody preparation methods, see, for example, US20170112944A1, the entire contents of which are incorporated herein by reference. Briefly, a plasmid construct encoding the anti-human HER2 antibody trastuzumab is transfected into CHO cells, and the C-terminus of the antibody light chain is modified by including the donor recognition motif LPETGG to produce T-LCCT L -HC is obtained. From the transfected CHO cells, a high expression cell population is screened, which is cultured in a 5-10L reactor by referring to the culture method of Trastuzumab. The cell culture is centrifuged to obtain harvested clarified cell culture fluid (HCCF).

[0330] The HCCF can be used as the antibody feed for the conjugation reaction (Process 1) or can undergo further downstream processing to provide purified antibody for the conjugation reaction (Processes 2 and 3).

[0331] Common methods for antibody purification For antibody purification methods, see, for example, US20170112944A1, the entire contents of which are incorporated herein by reference. L Purification of -HC is performed by standard methods using a combination of protein A affinity chromatography (MabSelect Sure ProA) and Sepharose S cation exchange chromatography, and the purified product is dissolved in the original trastuzumab drug buffer (5 mM histidine-HCl, 2% trehalose, 0.009% polysorbate 20, pH 6.0).

[0332] General method for preparation of linker-payload intermediates For the production method of the linker-payload intermediate, see, for example, US20170112944A1, the entire contents of which are incorporated herein by reference. Briefly, the method is as follows.

[0333] The linker solution having the structure of formula (V-1-2) is incubated with the payload solution to form a linker-payload intermediate having the structure of formula (IV-1-5-1), which is further subjected to a ring-opening reaction to obtain an open linker-payload intermediate having the structure of formula (IV-1-5) or (IV-1-5'). In the embodiment, the payload is DM1, and the linker-payload intermediate has the structure of formula (IV-1-6) or (IV-1-6'). The open linker-payload intermediate is purified and analyzed by HPLC.

[0334] General methods for conjugation reactions For the method of producing ADC using purified antibodies from Protein A affinity chromatography, see, for example, US20170112944A1, the entire contents of which are incorporated herein by reference. Briefly, the method is as follows.

[0335] As described above, a linker-payload intermediate (0.1 to 50 mg / ml) having the structure of formula (IV-1-6) or (IV-1-6') and an antibody T-LCCTL Prepare buffer solutions containing -HC (1 to 100 mg / ml).

[0336] The immobilized Halo-sortase is loaded into a vessel in the desired amount. The immobilized Halo-sortase is treated with 20 mM Tris-HCl, 1-3 M NaCl (pH 6.0-10.0), 0.1-1.0 M NaOH. The immobilized Halo-sortase is preheated in an air bath or water bath at 10-40°C for about 30 minutes or more. The antibody solution and the linker-payload intermediate solution are mixed in a predetermined ratio (antibody:linker-payload intermediate=1:1-1:100) to obtain a mixture, and then the mixture is added to the vessel containing the treated immobilized Halo-sortase (pull-down mode) or to the Halo-sortase column (flow-through mode). The conjugation reaction is started, and the reaction time is from 5 minutes to 24 hours.

[0337] After the conjugation reaction is completed, the reaction solution or the flow-through of the immobilized Halo-sortase column is collected to obtain a crude conjugate mixture containing the target conjugate having the structure of formula (6) or (6'). The crude conjugate mixture is analyzed by HIC-HPLC to determine the DAR of the ADC to determine the conjugation efficiency of the reaction.

[0338] General methodology for Protein A affinity chromatography The column is equilibrated with 20 mM Tris, 150 mM NaCl, pH 7.5 and loaded with the crude conjugate mixture. Subsequent flushes with 20 mM Tris, 150 mM NaCl, pH 7.5 until the desired offset (baseline) is reached. Optionally, impurities are removed by washing with citric acid-sodium citrate buffer, pH 5.0 (wash step). The desired ADC is eluted with citric acid-sodium citrate buffer, pH 3.3-3.7. The eluate containing the desired ADC is collected. The pH of the eluate is adjusted to pH 5.0-6.0 with 1 M Tris-HCl, pH 9.0. Residual impurities in the eluate, such as HCP, DNA and Protein A, are analyzed by ELISA or qPCR.

[0339] General methodology for anion exchange chromatography Pack the chromatography column with Q Sepharose FF media. Equilibrate the column with 20-100 mM Tris-HCl pH 6.5-8.0 and load the combined eluate collected from Protein A affinity chromatography. Collect the flow-through containing the target ADC. Continue flushing with 20 mM Tris-HCl pH 6.5-8.0 until the desired offset (baseline) is reached. Regenerate the chromatography column with 20-100 mM Tris-HCl, 1 M NaCl pH 6.5-8.0. Clean in place (CIP) with 1 M NaOH for 30 min. Analyze the eluate for residual impurities such as HCP, DNA and Protein A.

[0340] General methodology for cation exchange chromatography Pack the column with Capto S ImpAct media. Equilibrate the column with citric acid-sodium citrate buffer, pH 5.0-6.0 and load with the eluate from Protein A affinity chromatography. Wash successively with citric acid-sodium citrate buffer, pH 5.0-6.0 until the desired offset (baseline) is reached. Elute the target ADC with citrate-sodium citrate buffer, 100-500 mM NaCl, pH 5.0-6.0. Collect the eluate containing the target ADC. Regenerate the chromatography column with citrate-sodium citrate buffer, 1 M NaCl, pH 6.0. Clean in place (CIP) with 1 M NaOH for 30 min. Analyze the eluate for residual impurities such as HCP, DNA and Protein A.

[0341] Example 1 Preparation of ligase fusion proteins 1.1 Cloning and purification of SrtAs Nucleic acids encoding SrtA (SrtA having an amino acid sequence selected from SEQ ID NOs: 1-26 and variants thereof ([Ser34][Asn100][Ala105][Thr136], SNAT. [Tyr34][Asn100][Ala105][Thr136], YNAT. [Trp34][Asn100][Asp105][Thr136], WNDT. [Val34][Asn100][Asn105][Ser136], VNNS) are subcloned into NdeI and EcoRI in the pET-21a(+) expression vector by standard methods of gene synthesis and Gibson assembly techniques. 6 The tag is inserted at the N-terminus of the SrtA open reading frame.

[0342] Transform the SrtA expression plasmid into E. coli BL21(DE3). Culture in LB with 50 μg / ml ampicillin at 37 °C until OD600 = 0.5-0.8, then add IPTG to a final concentration of 0.2 mM and induce sortase expression at 25 °C for 12 h. Target cells are collected by centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl), then lysed by sonication, and the supernatant is purified on Ni-NTA agarose according to the manufacturer's instructions. Sortase purity is judged to be >90% by SDS-PAGE. The concentration of SrtA is calculated from A280 measured by the extinction coefficient method.

[0343] 1.2 Assessment of sortase activity The sortase activity of the recombinant SrtA prepared in Example 1.1 is measured by fluorescence spectrophotometry. Reactions in 96-well plates (total volume 100 μL, 0.085 mM Abz-LPETGK-Dnp, 18 mM triglycine in Buffer A (Buffer A: 5 mM CaCl)) are incubated at 37 °C by adding 0.625 μM purified SrtA (or mutants). 2, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) is started. Abz-LPETGK-Dnp is an internally quenched peptide with 2-aminobenzimidazole (Abz) as the fluorophore and 2,4-dinitrophenyl (Dnp) as the quencher. When sortase cleaves peptide LPETGK, Dnp and Abz are separated, and the fluorescent signal of Abz can be detected to indicate the activity of sortase. After 1 h, the increase in the fluorescent signal is continuously collected (λ exc / λ em = 320 nm / 420 nm, gain = 85, Biotek Cytation3 plate reader).

[0344] As an illustrative example, the activity of SrtA from Staphylococcus warneri (SEQ ID NO: 3) and its mutants ([Ser34][Asn100][Ala105][Thr136], SNAT) is shown in a bar graph (Figure 1). The results show that the SNAT mutant SrtA is approximately 1-fold more active than its wild-type SrtA (SEQ ID NO: 3), and both have sortase activity.

[0345] 1.3 Cloning of the Ligase Fusion Protein (Halo-Sortase) of the Invention Nucleic acids encoding the ligase fusion proteins (Halo-sortase) of the invention are cloned into bacterial expression vectors pET21a or pET24d, and each of the ligase fusion proteins comprises SrtA (SrtA having an amino acid sequence selected from SEQ ID NOs: 1 to 26 and its variants ([Ser34][Asn100][Ala105][Thr136], SNAT; [Tyr34][Asn100][Ala105][Thr136], YNAT; [Trp34][Asn100][Asp105][Thr136], WNDT; [Val34][Asn100][Asn105][Ser136], VNNS) and a Halo tag having the amino acid sequence of SEQ ID NO:28.

[0346] The Halo-sortase having the amino acid sequence of SEQ ID NO: 29 was used in the following examples. The Halo-sortase contains an SrtA variant (SEQ ID NO: 27) derived from Staphylococcus aureus and a Halo tag (SEQ ID NO: 28).

[0347] 1.4 Purification of Halo-sortase Halo-sortase is expressed in Escherichia coli BL21(DE3), purified, and stored at -80°C in 5% - 10% glycerol. His 6 -sortase with a tag and GB1-sortase with a GB1 tag are prepared in a similar manner for comparison.

[0348] 1.5 Activity of Halo-sortase The method is as follows (1) - (4). (1) Mix the purified antibody T-LCCT L -HC and a linker-payload intermediate having the structure of formula (IV-1-6) or (IV-1-6’) in conjugation buffer at an optimal molar ratio (Ab: linker-payload intermediate = 1:1 - 1:100). (2) Incubate the Halo-sortase, His-sortase, or GB1-sortase prepared in Example 1.4 with the mixture from step (1) at 4 - 40°C for 0.5 - 20 hours respectively. (3) Store the product obtained in step (2) at 4°C or -80°C. (4) Perform 12% SDS-PAGE electrophoresis on the product to measure the conjugation efficiency.

[0349] As shown in Figure 2, the conjugation efficiencies of Halo-sortase, GB1-sortase, and His-sortase are over 90%.

[0350] Example 2 Preparation of Immobilized Halo-sortase 2.1 Preparation of Chloroalkyl-linker Modified Resin (Chloro Resin) For the preparation of chlororesins, see, for example, U.S. Patent Nos. 7,429,472, 7,888,086 and 8,202,700, the entire contents of which are incorporated herein by reference. The resins used to prepare the chlororesins are shown in Table 2.

[0351] [Table 3]

[0352] The methods are as follows (1) to (3). (1) Pretreatment About NHS activated resin (Bestchrom) and CNBr activated resin (Bestchrom), Filter the resin with isopropyl alcohol, wash the filter cake once with DMF, then suction dry. Transfer the filter cake to a flask using DMF and stir. Then add ethylenediamine to the mixture and stir for 10-15 hours. Filter and wash the filter cake with DMF. Then drain the liquid. About epoxy activated resin Filter the resin with isopropyl alcohol and hydrate the filter cake. 2 The mixture is washed once with 25-28% concentrated aqueous ammonia and then dried by suction. The filter cake is transferred to a flask and stirred. The mixture is then gradually heated to 40-50°C and reacted at 40-50°C with stirring. The temperature of the mixture is lowered to 20-30°C and the mixture is filtered. H2O is added until the pH of the filtrate reaches about 7-8. 2 Wash the filter cake with O. Then wash the filter cake with DMF and drain. (2) The filter cake from step (1) is transferred to a flask and stirred. Then, the chloroalkyl group-containing DMF and triethylamine having the structure of the above formula (I-1-1) are added to the system in order. The system is reacted with stirring. Then, the system is filtered, the filter cake is washed with DMF, and finally the liquid is drained. (3) The filter cake from step (2) is transferred to a flask and stirred. The mixture is then sequentially added with Ac 2Add O and triethylamine. React with stirring. Then, filter the mixture and wash the filter cake with DMF. Then, wash the filter cake with H 2 O to cut off the liquid. Finally, transfer the mixture to a container using 20% ethanol and store it.

[0353] As a result, a chloro resin having the structure of formula (II-1) is obtained.

Chemical formula

Chemical formula

[0354] 2.2 Immobilization of Halo-sortase on the chloro resin The method is as follows (1) to (5). (1) Incubate the purified Halo-sortase prepared in Example 1.4 and the chloro resin prepared in Example 2.1 at room temperature for 10 minutes to 24 hours. (2) Wash the resin three times with 20 mM Tris-HCl, 150 mM NaCl (pH 6.0 to 10.0). (3) Measure the enzyme activity of the immobilized Halo-sortase. (4) Optionally, fill a column with the immobilized Halo-sortase to obtain a Halo-sortase column. (5) Wash the immobilized Halo-sortase in step (3) or the Halo-sortase column in step (4) with 20 mM Tris-HCl, 1 to 3 M NaCl (pH 6.0 to 10.0), 0.1 to 1.0 M NaOH and store at 4°C.

[0355] Example 3 Characterization of the chloro resin The method is as follows (1) to (3). (1) Take 250 μl of each chloro-resin to be tested, add an excess of Halo-sortase, place the tube on a rotator and incubate at room temperature for 2 hours. (2) At different time points of the immobilization reaction (15 min, 30 min, 1 h and 2 h, respectively), the tubes are centrifuged at 3000 g for 3 min at room temperature, one drop of the supernatant of each chloro-resin is taken, and the concentration of Halo-sortase in the supernatant is measured using a Nanodrop spectrophotometer. (3) The concentration of Halo-sortase at each time point is calculated and subtracted from the initial concentration of Halo-sortase to obtain the amount of immobilized Halo-sortase at each time point, and a curve is plotted of the amount of Halo-sortase immobilized on the chloro resin as a function of conjugation time.

[0356] The results are shown in Figure 3, where the amount of Halo-sortase immobilized on the chloro-resins reached a plateau at 2 hours, indicating the maximum loading of each chloro-resin.

[0357] Example 4 Characterization of immobilized Halo-sortase The methods are as follows (1) to (5). (1) A certain amount of Halo-sortase is immobilized on chloro resin as described in Example 2.2. (2) Wash the immobilized enzyme three times with 5-10 times the resin volume of 1x storage buffer, centrifuging each time at 3000g for 3 minutes at room temperature to precipitate the immobilized enzyme resin, and resuspend the immobilized enzyme in conjugation buffer. (3) 25 μl of each immobilized enzyme resin is taken, and 200 μl of GFP protein containing the donor recognition motif LPETGG and a small molecule reaction buffer containing a linker-payload intermediate (containing the receptor recognition motif GGG coupled to a small molecule compound) are added to initiate the conjugation reaction. (4) At different time points during the conjugation reaction (15 min, 30 min, 1 h, and 2 h, respectively), supernatant samples are taken for HIC-HPLC analysis. (5) The conjugation efficiency is measured by HIC-HPLC, and the results are shown in Figure 4 (the conjugation activity is expressed as DAR over time).

[0358] Example 5 Solubility of ADC Products Catalyzed by Halo-Sortase at Low Temperature The methods are as follows (1) to (5). (1) Prepare ADC samples using recombinant sortase proteins GB1-sortase, His-sortase or Halo-sortase as described in "General Methods." (2) The ADC sample is placed on ice for 10 minutes. (3) Centrifuge the ADC sample at 12,000 g for 5 minutes and transfer the supernatant to a new tube. (4) Add 20 μl of 1×SDS loading buffer to the precipitate from step (3) to dissolve the precipitate, and take 5 μl for sample loading. (5) Boil all samples at 95°C for 10 min and load the samples onto a 12% SDS-PAGE gel for analysis.

[0359] The results are shown in Figure 5 and show that when placed on ice, most ADCs catalyzed by GB1-sortase, but not by Halo-sortase or His-sortase, precipitate. As a result, ADCs catalyzed by Halo-sortase or His-sortase are more stable under cold conditions and are likely to survive the conjugation process (common in the production of bioconjugates) with relatively long retention times compared to ADCs catalyzed by GB1-sortase. Thus, His-sortase and Halo-sortase are superior to GB1-sortase in terms of product solubility.

[0360] Example 6 Separation of Halo-sortase and ADC 6.1 Separation of Halo-sortase and ADC by anion exchange chromatography (AEX) The methods are as follows (1) to (5). (1) Pack a column with Q Sepharose FF media. (2) The column is equilibrated with 20 mM Tris-HCl (pH 7.5), and the samples (ADC (A), Halo-sortase (B), and a mixture of ADC and Halo-sortase (C) (the mass ratio of ADC:Halo-sortase is approximately 100:1, respectively)) are loaded. (3) Flush the column with 20 mM Tris-HCl (pH 7.5) until the baseline, eluate pH, and conductivity are stable. (4) Regenerate the column with 20 mM Tris-HCl, 1 M NaCl (pH 7.5). (5) Clean in place (CIP) with 1M NaOH.

[0361] The results are shown in Figure 6, where ADC passes through the QFF chromatography column (flow-through [FT] mode) at pH 7.5, which is consistent with conventional ADC & Ab purification methods. Halo-sortase binds to the QFF chromatography column (bind / elute [B / E] mode) and is eluted with 20 mM Tris-HCl, 1 M NaCl pH 7.5. For a mixture of ADC and Halo-sortase, ADC passes through the QFF column, while Halo-sortase binds to the QFF column, resulting in good separation of the two.

[0362] 6.2 Separation of Halo-sortase and ADC by cation exchange chromatography (CEX) The methods are as follows (1) to (5). (1) Pack the column with Capto S ImpAct media. (2) The column is equilibrated with 20 mM citric acid-sodium citrate (pH 6.2) and the samples are loaded (A: ADC, B: Halo-sortase, C: a mixture of ADC and Halo-sortase). (3) Equilibrate the column with 20 mM citric acid / sodium citrate (pH 6.2) until the baseline, eluate pH, and conductivity are stable. (4) Elute the sample with 20 mM citric acid / sodium citrate, 160 mM NaCl, pH 6.2. (5) Regenerate the column with 20 mM citric acid / sodium citrate, 1 M NaCl, pH 6.2, and clean in place (CIP) with 1 M NaOH.

[0363] The results are shown in Figure 7, which shows that at pH 6.2, ADC binds to the Capto S ImpAct column (bind / elute [B / E] mode), and at pH 7.5, Halo-sortase passes through the Capto S ImpAct chromatography column (flow-through [FT] mode). For a mixture of ADC and Halo-sortase, ADC binds to the Capto S ImpAct column, but Halo-sortase passes through, resulting in good separation of the two.

[0364] 6.3 His from reaction products 6 Comparison of removal of -sortase or Halo-sortase His 6 The isoelectric points of His-sortase and Halo-sortase, and 6 The chromatographic modes (AEX and CEX) for separating His-sortase or Halo-sortase and the reaction product ADC are shown in Table 3. 6 -Sortase has an isoelectric point of 8.92, which is close to that of ADC (8-9). 6 Since both Halo-sortase and ADC bind to the cation exchanger in CEX and both pass through the anion exchanger in AEX, it is difficult to separate them. The isoelectric point of Halo-sortase is 5.7, so Halo-sortase and ADC can be easily separated by AEX or CEX.

[0365] [Table 4]

[0366] Example 7 ADC Preparation Using Antibody Purified by Protein A Affinity Chromatography (Process 2) 7.1 Conjugation reactions According to the method described in "General Methods", the monoclonal antibody (mAb) obtained from Protein A affinity chromatography of HCCF (i.e., mAb eluate of Protein A affinity chromatography) is used to prepare the target ADC. The content of HCP in the antibody feed is 1000-2000 ppm. The vessel used for the conjugation reaction is the Halo-sortase column prepared in Example 2.2. The crude conjugate mixture is collected as the flow-through of the Halo-sortase column. According to the HIC-HPLC analysis (see Figure 10), the DAR of the prepared ADC is 1.83. The conjugation efficiency is 91.6% (Table 4).

[0367] [Table 5]

[0368] 7.2 Removal of impurities The crude conjugate mixture collected in 7.1 is subjected to AEX and CEX in sequence, and samples containing the target ADC from each step (ADC flow-through from AEX and ADC eluate from CEX, respectively) are collected. The mAb eluate from Protein A affinity chromatography, the ADC flow-through from AEX, and the ADC eluate from CEX are analyzed by ELISA and qPCR to determine the amount of remaining impurities.

[0369] After AEX chromatography, an approximately 1 log (90%) reduction in HCP content was observed. After a series of chromatographic purifications, HCP, DNA and Protein A levels are all reduced to below 1 ppm (see Figure 9).

[0370] Example 8 Preparation of ADC using antibodies obtained from HCCF (Process 1) 8.1 Conjugation reactions Prepare targeted ADCs using HCCF as the antibody feed according to the method described in "General Methods". The content of CHO HCP in the antibody feed is 100,000-1,000,000 ppm.

[0371] The vessel used for the conjugation reaction is the Halo-sortase column prepared in Example 2.2. The crude conjugate mixture is collected as the flow-through of the Halo-sortase column. According to HIC-HPLC analysis (see Figure 11), the DAR of the prepared ADC is 1.81, and the conjugation efficiency is 90.5% (see Table 5).

[0372] [Table 6]

[0373] 8.2 Detection and Removal of Impurities The crude conjugate mixture collected in 8.1 is subjected to Protein A affinity chromatography, AEX, and CEX in sequence, and the ADC-containing solutions from each step (ADC eluate from Protein A affinity chromatography, ADC flow-through from AEX, and ADC eluate from CEX, respectively) are collected and analyzed by ELISA and qPCR to measure the amount of remaining impurities.

[0374] After AEX chromatography, an 80% reduction in HCP content and an approximately 3 log (99.9%) reduction in DNA content was observed. After CEX chromatography, an approximately 93% reduction in HCP content was observed. After a series of chromatographic purifications, DNA and Protein A content levels were reduced to below 5 ppm, and HCP levels were reduced to below 40 ppm (see FIG. 11).

[0375] The processes of the invention are suitable for rapid preparation of ADCs, particularly for small scale, e.g., laboratory, or high throughput preparation of ADCs for purposes such as bioactivity studies.

[0376] Example 9 ADC Preparation Using Antibody Purified by Protein A Affinity Chromatography (Process 3) 9.1 Conjugation reactions The targeted ADC is prepared using monoclonal antibodies obtained from Protein A affinity chromatography of HCCF (i.e., mAb eluate of Protein A affinity chromatography) according to the method described in "General Methods". The vessel used for the conjugation reaction is a Halo-sortase column prepared in Example 2.2. The crude conjugate mixture is collected as the flow-through of the Halo-sortase column.

[0377] 9.2 Removal of impurities In a similar manner to 8.2, the crude conjugate mixture collected in 9.1 is subjected to Protein A affinity chromatography, AEX and CEX in sequence. The mAb eluate of Protein A affinity chromatography, the ADC eluate of Protein A affinity chromatography, the ADC flow-through of AEX and the ADC eluate of CEX are analyzed by ELISA and qPCR to measure the amount of remaining impurities. The results show that after a series of chromatographic purifications, the levels of HCP and Protein A are reduced to below 2 ppm (see Figure 12). Therefore, the use of two Protein A affinity chromatography steps does not affect the product quality in terms of possible leaching of Protein A. The low content of impurities in the eluate of the second Protein A affinity chromatography step indicates that the method can tolerate higher concentration / more complex loading samples. Therefore, this example may be understood as a proof of concept for a more complex purification process than those described in Examples 7 and 8.

[0378] 9.3 Removal of residual enzyme contaminants (i.e., enzyme that falls off the Halo-sortase column after conjugation) As described in Example 9.1, the crude conjugate mixture was collected as the flow-through of the Halo-sortase column, and then protein A affinity chromatography, AEX, and CEX were performed in sequence. ELISA analysis was performed on the crude conjugate mixture, the ADC eluate of protein A affinity chromatography, the ADC flow-through of AEX, and the ADC eluate of CEX to measure the amount of residual enzyme contaminants.

[0379] As shown in Figure 13, after a series of chromatographic purifications, the residual enzyme contaminants decreased by 3 logarithms (99.9%). In particular, compared to one-step purification using only protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography further reduced the amount of residual enzyme in the final product by approximately 78%.

[0380] Example 10 Separation of Linker-Payload Intermediate and ADC Conventional methods for removing linker-payload intermediates from crude conjugate mixtures containing the target ADC usually involve ultrafiltration (UF). However, the shear force generated during ultrafiltration increases the risk of protein molecule aggregation. This is of particular concern when removing small molecules (e.g., linker-payload intermediates) from the product ADC because the ultrafiltration time of the ADC is longer and its hydrophobicity is enhanced compared to unconjugated protein molecules.

[0381] The present invention provides several options for unit operations (method steps) for removing linker-payload intermediates. In the following, examples of chromatographic steps are provided. In these examples, the ADC sample is prepared according to the method described in the "General Method" using the antibody obtained from protein A affinity chromatography of HCCF or using HCCF as the antibody feed. The chromatographic steps are performed according to the method described in the "General Method".

[0382] 10.1 Protein A affinity chromatography The targeted ADC is prepared in a similar manner as in 9.1. The crude conjugate mixture is collected as the flow-through of the Halo-sortase column, followed by Protein A affinity chromatography using Protein A chromatography media provided by different suppliers (Biomax and GE). The method using GE Protein A includes a wash step, whereas the method using Biomax Protein A does not. The results are shown in Figure 14.

[0383] 10.2 CEX Prepare the targeted ADC in a similar manner as in 9.1. Collect the crude conjugate mixture as the flow-through of the Halo-sortase column, followed by CEX chromatography using CEX media provided by GE.

[0384] The results are shown in Figure 14.

[0385] 10.3 Protein A affinity chromatography, AEX and CEX The targeted ADC is prepared in the same manner as in 7.1. The crude conjugate mixture is collected as the flow-through of the Halo-sortase column, followed by Protein A affinity chromatography, AEX, and CEX, respectively. Samples containing the targeted ADC from each step are analyzed by RP-HPLC to measure the residual linker-payload intermediate (Linker-Toxin).

[0386] A greater than 4 log (>99.99%) reduction in the content of the linker-payload intermediate was observed after Protein A affinity chromatography.

[0387] The present invention provides several methods for removing the linker-payload intermediate, such as protein A affinity chromatography, AEX, CEX, and combinations thereof. After the purification step, further purification can be achieved by additional steps such as ultrafiltration and / or diafiltration. Thus, the linker-payload intermediate can be completely removed.

[0388] Sequence Listing

[0389] [ka] [ka] [ka] [ka]

Claims

1. A ligase fusion protein comprising a ligase and a Halo tag, the ligase is a sortase, the ligase has an isoelectric point (pI) of 7.5 to 10.0 and the Halo tag has an isoelectric point of 4.5 to 5.0; The ligase fusion protein, wherein the pI of the ligase fusion protein is 2.0 to 4.5 pH units lower than the pI of the ligase.

2. The ligase fusion protein of claim 1 , wherein the ligase is sortase A.

3. 3. The ligase fusion protein of claim 2, wherein the sortase A comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-26, or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity thereto.

4. 4. The ligase fusion protein of claim 3, wherein the sortase A comprises SNAT, YNAT, WNDT or VNNS amino acid substitutions at positions 34, 100, 105 and 136.

5. 5. The ligase fusion protein of claim 4, wherein the sortase A comprises the amino acid sequence of SEQ ID NO:27, or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity thereto.

6. 6. The ligase fusion protein of claim 1, wherein the Halo tag comprises an amino acid sequence of SEQ ID NO:28, or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity thereto.

7. 3. The ligase fusion protein of claim 2, wherein the sortase A comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity thereto.

8. 8. The ligase fusion protein of claim 7, wherein the sortase A comprises SNAT, YNAT, WNDT or VNNS amino acid substitutions at positions 34, 100, 105 and 136.

9. An immobilized ligase comprising a ligase fusion protein according to any one of claims 1 to 8 immobilized on a support.

10. 10. The immobilized ligase of claim 9, wherein the support comprises a haloalkyl linker such that the ligase fusion protein is immobilized to the support by a covalent interaction between the haloalkyl linker and a Halo tag.

11. The immobilized ligase of claim 10 , wherein the haloalkyl linker is a chloroalkyl linker.

12. The chloroalkyl linker is generated from a chloroalkyl group substrate having the structure of formula (I-1): 【Chemistry 1】 In the formula, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19. The immobilized ligase according to claim 10 or 11.

13. The support has a structure of formula (II): 【Chemistry 2】 In the formula, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19. 【Chemistry 3】 The immobilized ligase of claim 12, wherein the immobilized ligase is a resin, a bead, a membrane, a gel, a matrix, a thin film, a plate, a well, a tube, a glass slide, or a surface.

14. 1. A method for preparing a conjugate comprising a first moiety and a second moiety, comprising: (a) providing a system 1 including a first portion and providing a system 2 including a second portion; (b) contacting system 1 and system 2 of step (a) with a ligase unit to catalyze a conjugation reaction between the first moiety and the second moiety to obtain the conjugate; The ligase unit comprises a ligase, the first and second moieties each independently comprise a biomolecule, a protein, an antibody, an antibody fragment, a receptor, a signal transduction factor, a cell growth factor, a nucleic acid or a nucleic acid analog, a small molecule compound, a glycan, a PEG moiety, a radionuclide, a cytokine, an immunomodulator, a tracer molecule, a fluorophore, a fluorescent molecule, a peptide, a polypeptide, or a peptidomimetic; one of the first and second portions further comprises a ligase donor substrate recognition motif, and the other of the first and second portions comprises a ligase acceptor substrate recognition motif; The ligase unit comprises a ligase fusion protein according to any one of claims 1 to 8. The method.

15. 15. The method of claim 14, wherein at least one of system 1 and system 2 in step (a) contains one or more impurities.

16. 16. The method of claim 14 or 15, wherein at least one of system 1 and system 2 in step (a) is harvested clarified cell culture fluid (HCCF).

17. The method of any one of claims 14 to 16, wherein the ligase unit comprises a ligase immobilized on a support.

18. The method of claim 17, wherein the ligase is covalently immobilized to a support.

19. 19. The method of claim 17 or 18, wherein the ligase is sortase A.

20. The method according to any one of claims 17 to 19, wherein the ligase unit comprises an immobilized ligase according to any one of claims 9 to 13.

21. (1) prior to step (b), subjecting the system 1 of step (a) to one or more chromatographic steps to remove one or more impurities; and / or (2) prior to step (b), subjecting system 2 of step (a) to one or more chromatographic steps to remove one or more impurities; and / or 21. The method of any one of claims 14 to 20, further comprising: (3) subjecting the conjugate obtained in step (b) to one or more chromatography steps to remove one or more impurities.

22. 22. The method of claim 21, wherein the chromatography steps are independently selected from affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, and combinations thereof, and the ion exchange chromatography is selected from anion exchange chromatography, cation exchange chromatography, and combinations thereof.

23. 23. The method of claim 22, wherein the chromatography step is selected from affinity chromatography, ion exchange chromatography, and combinations thereof.

24. 24. The method of claim 22 or 23, wherein at least one of the first and second portions comprises an antibody or an antibody fragment, and at least one of steps (1) to (3) comprises affinity chromatography.

25. the first or second portion comprises an Fc fragment; Steps (1) and (2) are not present, and step (3) is (3a-1) Protein A affinity chromatography; and (3a-2) anion exchange chromatography; (3a-3) cation exchange chromatography, in any order; Or, Step (1) or step (2) comprises Protein A affinity chromatography, and step (3) comprises: (3b-1) anion exchange chromatography; (3b-2) cation exchange chromatography, in any order; Or, Step (1) or step (2) comprises Protein A affinity chromatography, and step (3) comprises: (3c-1) Protein A affinity chromatography; and (3c-2) anion exchange chromatography; (3c-3) cation exchange chromatography, in any order; Or, Step (1) or step (2) comprises protein A affinity chromatography and anion exchange chromatography, and step (3) comprises: (3d-1) Protein A affinity chromatography; and (3d-2) cation exchange chromatography; (3d-3) hydrophobic interaction chromatography, in any order; Or, Step (1) or step (2) comprises Protein A affinity chromatography, and step (b) is followed by UF / DF prior to step (3), and step (3) comprises: (3e-1) anion exchange chromatography; (3e-2) cation exchange chromatography, in any order; Or, 25. The method of any one of claims 22 to 24, wherein step (1) or step (2) comprises Protein A affinity chromatography and anion exchange chromatography, and step (b) is followed by UF / DF before step (3), and step (3) comprises cation exchange chromatography or hydrophobic interaction chromatography.

26. The affinity chromatography is carried out in bind-elute mode, and / or The method according to any one of claims 22 to 25, wherein the ion exchange chromatography is carried out in bind-elute mode or in flow-through mode.

27. 27. The method of any one of claims 14 to 26, wherein step (b) is carried out in batch mode, semi-continuous mode or continuous mode.

28. 28. The method of any one of claims 21 to 27, wherein at least one of steps (a), (b) and (1) to (3) is carried out in a semi-continuous or continuous mode.

29. The conjugate has the structure of formula (III): the first portion comprises T; The second portion comprises a linker-payload intermediate of formula (IV): 【Chemistry 4】 During the ceremony, T comprises a biomolecule optionally modified to have one of a ligase donor substrate recognition motif and a ligase acceptor substrate recognition motif; L comprises a linker comprising the other of the ligase donor substrate recognition motif and the ligase acceptor substrate recognition motif; P contains the payload, z is an integer from 1 to 20; 29. The method of any one of claims 14 to 28, wherein t is an integer from 1 to 20.

30. T includes a protein, a peptide, an antibody, an antibody fragment, a receptor, a signal transduction factor, a cell growth factor, and a nucleic acid or analogue; and / or 30. The method of claim 29, wherein P comprises hydrogen, a small molecule compound, a glycan, a PEG moiety, a radionuclide, a cytokine, an immunomodulator, a nucleic acid or analogue, a tracer molecule, a fluorophore, a fluorescent molecule, a peptide, a polypeptide, a peptidomimetic, an antibody, an antibody fragment, or a protein.

31. the ligase donor substrate recognition motif is LPXTGJ; and / or The ligase receptor substrate recognition motif is G n where G is glycine (Gly) and n is an integer from 3 to 10; X is any natural or unnatural amino acid; 31. The method of any one of claims 14 to 30, wherein J is absent or is an amino acid fragment comprising 1 to 10 amino acids, where each amino acid is independently any natural or unnatural amino acid, and where m is an integer from 1 to 10.

32. The conjugate has a structure selected from the following formulas (3), (3′), (5), and (5′): 【Chemistry 5-1】 【Chemistry 5-2】 In the formula, n is an integer from 3 to 10; x is OH, NH 2 or Gly, Toxin represents a cytotoxin. Lk is L 1 -L 2 -L 3 and L 1 and L 3 are each independently -CH 2 -, -NH-, -(CO)-, -NH(CO)-, -(CO)NH-, and C 1-4 Alkylene group and —CH 2 in combination with one of the groups -, -NH-, -(CO)-, -NH(CO)-, -(CO)NH-; L 2 does not exist or C 7 - 34 an alkylene group, wherein one or more (—CH 2 -) structure is optionally replaced by -O-; L 1 , L 2 and L 3 each optionally and independently represents -OR 1 and -NR 1 R 2 Substituted by 1, 2 or 3 substituents selected from R 1 and R 2 are each independently hydrogen, -C 1 - 6 Alkyl group, -(CO)-C 1 - 6 Alkyl groups, and -S(=O) 2 -C 1 - 6 alkyl groups, Y is absent or is selected from the group consisting of a cleavable sequence, a spacer Sp1, and combinations thereof; the cleavable sequence comprises an amino acid sequence cleavable by an enzyme, the cleavable sequence comprising 1 to 10 amino acids; 32. The method of any one of claims 14 to 31, wherein Sp1 is selected from the group consisting of a spacer sequence comprising 1 to 20 amino acids, PAB, and combinations thereof, and T and z are each as defined in claim 27.

33. 33. The method of claim 32, wherein Y is absent and the conjugate has a structure selected from formulas (4), (4'), (6) and (6'): 【Chemistry 6-1】 【Chemistry 6-2】

34. Use of a ligase fusion protein according to any one of claims 1 to 8 or an immobilized ligase according to any one of claims 9 to 13 in the preparation of a conjugate.

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