Antibody labeling method

Abstract

Provided is an antibody labeling method. The method comprises using a direct or indirect labeling method to directly couple a target label to an antibody. A flexible platform method is provided for a site-directed antibody labeling process, an antibody recombination process, and a multi-label coupling process.

Description

A method for labeling antibodies

[0001] This application claims priority to Chinese patent application 2024115937152, filed on November 8, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention pertains to antibody conjugation and labeling technology, and particularly relates to a method for labeling antibodies. Background Technology

[0003] Antibodies are widely used in various fields of biological and medical research due to their specific antigen-binding ability, in vivo targeting function, ability to stimulate or inhibit multiple cell signaling pathways, induce other immune responses, and excellent pharmacokinetic properties. Antibody markers refer to the covalent binding of a molecule with indicator function or biological activity to an antibody molecule to achieve the function of labeling, analysis, or therapy. Antibody markers can take the form of in vitro immunoassay reagents and in vivo immunodiagnostic and targeted therapeutic drugs.

[0004] Indicators in labeled antibodies include detectable molecules or groups such as fluoresceins (e.g., FITC and the Alexa fluorescein series (Alexa Fluor 488, Alexa 647, etc.), fluorescent proteins (e.g., green fluorescent protein (EGFP), yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), red fluorescent protein (mCherry, mStrawberry, dTomato), blue fluorescent protein (EBFP)), enzymes, chemiluminescent reagents, radioactive elements (e.g., I-125, I-131, Tc-99m), and metallic colloids (e.g., colloidal gold and colloidal silver). These reagents are widely used in biomedical research and development experiments, immunoassays, and in vivo immunoassay diagnostics.

[0005] Antibody-drug conjugates (ADCs) are conjugates of antibodies and cytotoxic toxins. ADCs combine the excellent targeting ability, compatibility with the human body, and long half-life in the bloodstream of humanized monoclonal antibodies with the high activity of chemical drugs. The main mechanism of action of ADCs is that the antibody binds to the target protein on the cell membrane surface, causing the cell to internalize both the target protein and the ADC. This leads to the release of the toxin in the ADC component within the target cell, where it exerts its effect. Currently, the toxins used in ADC drugs include microtubule inhibitors, DNA damaging agents, and topoisomerase inhibitors, all of which share the characteristic of inhibiting cell proliferation and amplification, thereby causing apoptosis of the target cells. The binding of antibodies to toxins effectively delivers cytotoxic drugs to target tissues or cells, improving the biological activity of targeted antibody drugs while reducing the toxic side effects of chemical drugs, thus significantly expanding the therapeutic window compared to traditional chemical drugs. Since 2000, at least 15 ADC drugs have been approved for marketing by drug regulatory agencies in various countries, achieving good clinical results. Currently, the most successful application of ADCs is in cancer treatment. The first approved ADC drugs included Pfizer's Mylotarg and Besponsa, which utilize antibodies targeting CD33 and CD22 respectively, bound to kacimycin via cleavable linkers. Roche's Kadcyla utilizes an antibody targeting CD30, bound to MMAE via a cleavable linker. These ADC drugs are indicated for hematologic malignancies such as AML, BCP-ALL, and Hodgkin's lymphoma. Many more ADCs focus on treating solid tumors. For example, ADCs targeting HER2, such as Kadcyla, Enhertu, and Adrenaline, have shown superior clinical efficacy compared to traditional therapies for refractory breast cancer, gastric cancer, and lung cancer. Trodelvy, targeting trop2, shows even better efficacy against triple-negative breast cancer (TNBC).

[0006] Currently, antibody labeling methods include non-site-specific coupling and site-specific coupling methods. These two methods are further subdivided into various coupling chemical methods. Non-site-specific coupling includes coupling via the amino groups of the antibody (including lysine residues, side-chain amino groups, and N-terminal residues) and by reducing and cleaving some disulfide bonds between polypeptide chains within the antibody molecule to generate active thiol groups. Site-specific coupling methods include cleaving all disulfide bonds between antibody chains and coupling all active thiol groups; preparing thiol antibodies (Thiomab) by inserting cysteine ​​residues into a specific sequence of the antibody and labeling its side-chain thiol groups; and several enzyme-catalyzed coupling methods, including coupling methods catalyzed by transglutaminase (MTG), sortase, and glycosyltransferase. Recently adopted amino-based site-specific coupling methods utilize a peptide segment with affinity for a specific sequence in the constant region of the antibody to guide the compound to be labeled to that site and couple it with the side-chain amino group of a lysine residue in the adjacent antibody sequence.

[0007] However, current antibody conjugation methods have certain limitations. First, most non-site-specific conjugation methods easily affect antibody activity, leading to heterogeneous labeled products with insufficient signal intensity in low-degree-of-labeling (DOL) products and reduced antigen-binding activity and molecular instability in high-degree-of-labeling products. Furthermore, site-specific labeling of bispecific antibodies or the need for multiple labels to label the same antibody presents greater challenges. For example, the complexity of bispecific antibody structures makes conventional labeling methods prone to producing products that are difficult to label or unstable. Analyzing the uniformity and degree of labeling with multiple labels presents significant challenges.

[0008] These issues are particularly prominent in ADC drugs. First, ADC drugs need to have an appropriate drug-to-antibody ratio (DAR). ADCs with too low a DAR cannot achieve the desired effect, such as effectively inhibiting the activity of target cells. Too high a DAR can easily lead to ADC molecule aggregation and non-specific binding, causing rapid clearance and increased toxic side effects. For example, most natural antibodies have four interchain disulfide bonds, which, when reduced, produce eight thiol groups, while many ADCs only require DAR4 or DAR2. Most currently marketed ADCs fall into this category. For example, Mylotarg (DAR 2-3), Besponsa (DAR 5-7), Kadcyla (DAR 3.5), and Elahere (DAR 3.5) were prepared through random coupling of amino groups. Zynlonta (DAR 2-3), Acetris (DAR 4), Polivy (DAR 3.5), Padcev (DAR 4), Belnrep (DAR 4), Aidixi (DAR 3.5), and Tivdak (DAR 4) were prepared by controlling the reduction conditions to cleave inter-chain disulfide bonds for coupling. These ADCs are all mixtures of non-uniform DARs. Only Enhertu and Trodelvy, two DAR8 ADCs, achieved high-DAR vertex coupling through saturated coupling of inter-chain disulfide bonds.

[0009] Secondly, monoclonal antibodies targeting a single target often encounter insufficient abundance of target proteins on the target cell surface, preventing adequate binding or insufficient endocytosis to effectively inhibit tumor growth. Therefore, a current trend in ADC development is the use of bispecific antibody-drug conjugates (ADCs). The antibody portion of a bispecific ADC has the function of binding to two antigenic epitopes, which can be different targets or different epitopes of the same target. The characteristics of bispecific ADCs are: stronger ability to induce target internalization through binding to two epitopes or two target proteins; broader targeting capability; and more specific selectivity for target tissues by regulating the antibody's affinity for the target protein. Currently, the commonly used method for preparing bispecific ADCs is to first prepare the bispecific antibody and then conjugate it with a small molecule compound, such as a toxin.

[0010] Another challenge facing ADC drugs is drug tolerance. All chemotherapy drugs used clinically to date have developed resistance issues, and the payload carried by an ADC is essentially the same as that of a chemotherapy drug. Similar to combination chemotherapy, ADCs formed by conjugating two or more toxins with different mechanisms of action or resistance to the same antibody are called bitoxin or multitoxin ADCs, which can largely avoid the development of resistance. However, how to perform multitoxin antibody conjugation is a challenge in terms of conjugation technology and analytical methods. Multitoxin conjugation mainly takes two forms: single-site multitoxin and multi-site multitoxin linkage. Multi-site conjugation methods make it difficult to control the ratio of toxins conjugated to the antibody. Moreover, the DAR value of ADCs conjugated with multitoxins is difficult to determine because the complex structure of multitoxins makes it impossible to analyze the DAR distribution of the conjugation product using hydrophobic chromatography. Another method for analyzing DAR is to utilize the difference between the characteristic UV absorption of the toxin and the characteristic absorption peak wavelength of the antibody protein. However, the characteristic UV absorption of multitoxins is affected by the ratio between the toxins; an uncertain toxin ratio results in an uncertain characteristic extinction coefficient, therefore, UV absorption cannot be used to detect the DAR of such ADCs. LC-MS analysis often exceeds the resolution of molecular weight differences due to the similar molecular sizes of toxin molecules. Currently, the most widely used method for multi-toxin conjugation utilizes branched linkers to bind multiple toxins together in a fixed ratio, which are then attached to an antibody via a single site. This maintains a relatively constant ratio between toxins. However, this results in excessively large toxin molecules, making them difficult to couple to the antibody due to conformational hindrance, and their strong hydrophobicity leads to instability in the ADC. Therefore, a simpler procedure is needed for site-specific conjugation of single or multiple toxins to monoclonal or polyclonal antibodies, forming a homogeneous and stable ADC.

[0011] There are several methods for preparing bispecific antibodies, including recombination (recombination) of the Fc cells of different antibodies to form new antibodies containing the antigen-binding properties of the parent antibody. Recombination requires point mutations in the Fc cells of the antibodies at the DNA level, causing changes in the charge and stereostructure of the sites on the Fc structure that fix the pairing between the two heavy chains, thereby enabling the Fc cells of the two antibodies to cross-pair. This is the duobody method and the knock-in-hole method. Summary of the Invention

[0012] To address the technical problems existing in the prior art, this invention provides a method for labeling antibodies. Specifically, this invention employs a combination of bioconjugation technology and antibody engineering technology to label recombinant antibodies. Specifically, the method of this invention first utilizes engineering modification technology to prepare recombinant parent antibodies (hereinafter referred to as parent antibodies). The mixed parent antibodies, without covalent bonds between their heavy chains, can form a re-pairing between the two antibody heavy chains, forming a stable recombinant antibody containing half of each parent antibody. For ease of description, the two recombinant parent antibodies are referred to as the first parent antibody and the second parent antibody, respectively. "First" and "second" are merely for convenience and do not have a specific relationship in terms of order or position. The recombinant antibody includes a first arm and a second arm; the first arm and the second arm are derived from the first parent antibody and the second parent antibody, respectively, and also have no specific relationship in terms of order or position. Furthermore, the first arm and the second arm are connected at the hinge region and Fc segment by disulfide bonds and non-covalent bonds.

[0013] The labeled antibody preparation process of this invention combines the features of site-directed conjugation, bispecific antibody preparation, and multiple labeling. This method offers high flexibility and can be used to prepare monoclonal antibodies, bispecific antibodies, or multispecific antibodies with single, double, or multiple labels, thereby solving the difficulties in site-directed conjugation, bispecific antibody conjugation, multiple labeling conjugation, and the challenge of analyzing DOL in multiple labeled conjugates.

[0014] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0015] A first aspect of the present invention provides a method for labeling an antibody or ADC, the method comprising conjugating a target label to an antibody using a direct or indirect labeling method.

[0016] In some embodiments of the present invention, the antibody includes a monoclonal antibody or a bispecific antibody (biantibody).

[0017] In some embodiments of the present invention, the antibody is a recombinant antibody.

[0018] In some embodiments of the present invention, the functional fragment of the monoclonal antibody or the bispecific antibody or the parent antibody of the recombinant antibody is selected from the group consisting of:

[0019] 1) Antibodies containing mutations that can generate controlled Fab arm exchange (cFAE) with other antibodies;

[0020] 2) Contains point mutations, thus enabling it to react with other antibodies to produce antibodies with a knob-into-hole alteration.

[0021] 3) Fc contains antibodies against IgG CH2-IgACH3 chimera.

[0022] In some embodiments of the present invention, the parent antibody can be a monospecific antibody or monoclonal antibody, a bispecific antibody or bispecific antibody, or a multispecific antibody or polyclonal antibody, depending on its antigen recognition characteristics. The antigen binding specificity of a bispecific antibody can be against different antigens or against different epitopes of the same antigen. The specific forms of the parent antibody are as follows:

[0023] In some embodiments of the present invention, the bispecific antibody includes monoclonal antibodies with different antigen binding specificities. The specificity may be against epitopes of different antigens or against different epitopes of the same antigen molecule.

[0024] In some embodiments of the present invention, the parent antibody comprises a naturally occurring IgG-type structure, namely, containing an Fc domain and two attached Fab domains, each containing a specific antigen recognition domain composed of a heavy chain variable region and a light chain variable region (Figure 1). In other embodiments of the present invention, the parent antibody comprises an engineered non-natural form, such as one or both sides lacking a Fab domain, or replacing the Fab domain with a single-chain variable region (scFv), a single-domain antibody (VHH), or other functional peptides, or fusing additional Fab, scFv, VHH, or other functional peptide structures into one or more chains. These reductions, substitutions, and additions of domains in the parent antibody can also be transferred to the recombinant antibody.

[0025] In some embodiments of the present invention, the recombinant antibody is selected from parent antibodies with the same antigen-binding specificity, including parent antibodies with the same variable region sequence or parent antibodies derived from the same WT antibody that have undergone the above-mentioned recombinability modification.

[0026] In some embodiments of the present invention, the first and second arms of the recombinant antibody target different antigens or the same antigen.

[0027] In some embodiments of the present invention, the antigens targeted by the first arm and the second arm are each independently tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs), such as one or more selected from Trop2, Her 2, tissue factor (TF, also known as thromboplastin factor III or CD142), Nectin-4, FRα, CD33, CD79b, BCMA and CD30.

[0028] In some embodiments of the present invention, the first arm or the second arm includes a variable region present at Fab, scFv, Fv and VHH, Fab' and dAb are optional; it also includes a constant region selected from human IgG constant regions such as IgG1 and IgG2 constant regions.

[0029] In some embodiments of the present invention, the antibody recombination method includes any recombination method between different parent antibodies, selected from cFAE method (including DuoBody preparation method), KnH, and charge pairing (CP).

[0030] In some embodiments of the invention, the recombination conditions include dissociating the heavy-chain dimers of a mixed pair of parent antibodies in the presence of a reducing agent and re-pairing the heavy chains of the first and second parent antibodies. Specifically, for parent antibodies that do not contain inter-chain disulfide bonds, the reducing agent may be reduced or removed.

[0031] In some embodiments of the present invention, the first arm includes a first heavy chain and a first light chain; and / or, the second arm includes a second heavy chain and a second light chain.

[0032] In some embodiments of the present invention, the first heavy chain and / or the second heavy chain are mutated in the heavy chain constant region (preferably the CH3 domain) to enable the first arm and the second arm to specifically pair, thereby enabling the first arm and the second arm to assemble to form the recombinant antibody.

[0033] In some embodiments of the present invention, the mutation is selected from F405L, K409R / E / D, R411T, T370K, T366W / S, L368A, Y407V, K392D, N399K, E356K and D399R corresponding to IgG1, and the position of the mutation is EU number.

[0034] In some embodiments of the present invention, the heavy chain constant region introduced by the first arm is mutated to F405L, and the heavy chain constant region introduced by the second arm is mutated to K409R; or the heavy chain constant region introduced by the first arm is mutated to K409R, and the heavy chain constant region introduced by the second arm is mutated to F405L.

[0035] In some embodiments of the present invention, the first arm and the second arm target the same antigen, which is preferably Trop2 or Her 2.

[0036] In some embodiments of the present invention, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:2; and / or

[0037] The amino acid sequence of the second heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the second light chain is shown in SEQ ID NO:2.

[0038] In some embodiments of the present invention, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:4; and / or

[0039] The amino acid sequence of the second heavy chain is shown in SEQ ID NO:6, and the amino acid sequence of the second light chain is shown in SEQ ID NO:7.

[0040] In some embodiments of the present invention, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:2; and / or

[0041] The amino acid sequence of the second heavy chain is shown in SEQ ID NO:6, and the amino acid sequence of the second light chain is shown in SEQ ID NO:7.

[0042] In some embodiments of the present invention, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:12, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:11; and / or

[0043] The amino acid sequence of the second heavy chain is shown in SEQ ID NO:10, and the amino acid sequence of the second light chain is shown in SEQ ID NO:11.

[0044] In some embodiments of the present invention, the antigen targeted by the first arm is Trop2, and the antigen targeted by the second arm is Her2.

[0045] In some embodiments of the present invention, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:2; and / or

[0046] The amino acid sequence of the second heavy chain is shown in SEQ ID NO:3, and the amino acid sequence of the second light chain is shown in SEQ ID NO:4.

[0047] In some embodiments of the present invention, the ADC is a normal IgG type recombinant antibody ADC. Preferably, the toxic molecules in the ADC are selected from Exatecan derivatives (DX-8951 derivative, abbreviated as Dxd), maytansinoids (DM1, DM4), calic acid, pyrrolobenzodiazepine (PBD) such as SGN-CD33A, SN-38, Pseudomonas exotoxin A (PE) and auristatins (e.g. MMAE, MMAF), polypeptides, small nucleic acids, radionuclides, or other functional compounds.

[0048] The preparation of recombinant antibodies includes transfecting the gene encoding the parent antibody into host cells, expressing and purifying the parent antibody, followed by in vitro recombination, and co-transfecting the gene encoding the parent antibody into the same host cell, co-expressing the parent antibody, allowing it to self-assemble into a recombinant antibody, and then purifying the recombinant antibody from the culture system. In some cases, whether in vitro recombination or co-expression and self-assembly, a component of a certain parent antibody may remain. Excess parent antibody needs to be removed by purification. The purification method varies depending on the situation. If the parent antibody and recombinant antibody have different charge properties, ion-exchange column chromatography can be used to remove the excess target antibody; if the molecular weight difference is significant, molecular sieve column chromatography can be used; if a single parent antibody lacks significant binding ability to protein A or protein G, while the recombinant antibody has significant binding ability, protein A or protein G affinity column chromatography can be used to remove the excess target antibody.

[0049] In some embodiments of the present invention, the method for preparing monoclonal antibodies, first parent antibodies, or second parent antibodies includes:

[0050] (1) The nucleotide sequence of the antibody synthesis gene, and

[0051] (2) Transiently transfer it to host cells to prepare a stable cell pool or cell line that can stably express the target antibody; or transiently transfer it to host cells, perform transient cis-transfer expression and purify the antibody; or choose to prepare a stable cell pool or cell line that can stably express the target antibody and then express and prepare the antibody in large quantities.

[0052] (3) The target antibody is purified from the culture product after the cell culture process.

[0053] In some preferred embodiments of the present invention, the host cell is selected from mammalian cells, such as HEK293 cells and CHO-K1 or CHO-S cells, yeast cells, Escherichia coli cells, or plant cells.

[0054] In some preferred embodiments of the present invention, the antibody purification method includes conventional antibody purification steps such as centrifugation, filtration to remove cells and cell debris, affinity chromatography (such as protein A, protein G affinity chromatography or antigen affinity chromatography) combined with molecular sieve (SEC) ion exchange resin (IEX), dialysis, and ultrafiltration.

[0055] In some embodiments of the present invention, the method yields a monoclonal antibody-monoclotent, biclonal antibody-monoclotent, monoclonal antibody-multi-labeled, or biclonal antibody-multi-labeled product.

[0056] In some embodiments of the present invention, the target marker is selected from cytotoxic compounds / toxin molecules constituting antibody-drug conjugates (ADCs) such as camptothecin (e.g., Camptothecin, Dxd, Exatecan, Topotecan, Belotecan, Irinotecan, SN38), maytasinoids (e.g., DM1, DM4, DM21), auristatin (MMAE, MMAF), Calicheamicin), chelating agents (e.g., Nota, Dota), fluorescein, biotin, peptides, amino acids, and other compounds with active reactive groups.

[0057] Compounds denoted by structures such as "linker-toxin molecule" (e.g., GGFG-Dxd, DBCO-PEG4-VA-PBD, or TCO-vc-PAB-Eribulin), "linker-labeling group" (e.g., Maleimide-PEG7-Biotin), or "linker-reactive group" (e.g., NH2CH2CH2-PEG3-SH) in the following text can also be used as the target designation of this invention. The target designations A, B, and C in the following text are for ease of description only and do not have any specific relationship in order or position.

[0058] In some embodiments of the present invention, the target marker is selected from one or more of GGFG-Dxd, DBCO-PEG4-VA-PBD, TCO-vc-PAB-Eribulin, Maleimide-PEG7-Biotin, and NH2CH2CH2-PEG3-SH.

[0059] In some embodiments of the present invention, the indirect labeling method corresponding to the recombinant antibody-single-labeled or multi-labeled product is as follows:

[0060] Method a involves first attaching one or more reactive groups to a specific coupling site on the parent antibody molecule, which then bind to the target marker after the recombinant antibody is obtained; or

[0061] Method b involves first reversibly protecting specific coupling sites on the parent antibody molecule, then deprotecting them after recombining the antibody and binding them to the target marker.

[0062] In some embodiments of the present invention, the indirect labeling method involves first labeling the thiol site of the antibody with a thiol reactive group via a thiol pyridine compound, which can remove the pyridine to generate an active thiol group after the antibody is recombined, and then coupling the target label to it.

[0063] In some embodiments of the present invention, the indirect labeling method involves first coupling an antibody amide group (e.g., 295Q), thiol group, amino group, or other specific site with a click-chemically active group, such as an azide, alkynyl, DBCO, COT, methyltetraazine, or COT group, using an appropriate method. The latter is then linked to the target label after the recombinant antibody is labeled. The role of the click-chemically coupled active group on the parent antibody is to protect the coupling groups on these antibodies, distinguishing the reactivity of the recombinant antibody from that of the paired parent antibody components. Furthermore, these active groups can be further coupled after recombination or used for bioorthogonal reactions (interacting with paired click-reactive groups in vivo).

[0064] In some embodiments of the present invention, the indirect labeling method involves first reversibly protecting specific coupling sites on the antibody molecule, and then deprotecting the recombinant antibody and binding it to the target label after obtaining the recombinant antibody.

[0065] In some embodiments of the present invention, the indirect labeling method involves using mercaptopyridine, cysteine, glutathione, etc., to protect the thiol group of the parent antibody by forming disulfide bonds, preventing it from forming disulfide bonds with the thiol group of the heavy chain hinge region of the paired parent antibody during or after recombination, and selectively deprotecting it and binding it to the target label.

[0066] In some embodiments of the present invention, in method a, the target marker includes 1 to 4 target markers, preferably including 3 target markers (named target marker A, target marker B and target marker C respectively), each independently selected from one of GGFG-Dxd, DBCO-PEG4-VA-PBD and TCO-vc-PAB-Eribulin.

[0067] In some embodiments of the present invention, method a includes:

[0068] (1) First, attach the first active reactive group to the first parent antibody, and then attach the second active reactive group to the second parent antibody;

[0069] (2) The first and second parent antibodies, which are linked to active reactive groups, are specifically paired to obtain recombinant antibodies;

[0070] (3) The recombinant antibody with the obtained active group is directly linked to the target marker A;

[0071] Optionally, method a further includes:

[0072] (4) Link the recombinant antibody that is linked to the target marker A and the target marker B and / or the target marker C that can bind to the first active reactive group and / or the second reactive group.

[0073] In some embodiments of the present invention, the first reactive group and the second reactive group are the same or different, and are preferably linked to the parent antibody via site-directed coupling technology of transglutaminase (mTG).

[0074] In some embodiments of the present invention, the amino acid sequence of the first heavy chain is shown in SEQ ID NO:12, and the amino acid sequence of the first light chain is shown in SEQ ID NO:11.

[0075] In some embodiments of the present invention, the first reactive group is amino-PEG3-C2-Azido; preferably, the DOL of the first parent antibody connecting the first reactive group is 2.

[0076] In some embodiments of the present invention, the first parent antibody connected to the first active reactive group is subjected to linker ring-opening treatment.

[0077] In some embodiments of the present invention, the amino acid sequence of the second heavy chain is shown in SEQ ID NO:10, and the amino acid sequence of the second light chain is shown in SEQ ID NO:11.

[0078] In some embodiments of the present invention, the second reactive group is Methyltetrazine-PEG4-amine; preferably, the DOL of the first antibody connected to the first reactive group is 2.

[0079] In some embodiments of the present invention, the target marker A is GGFG-DXD, which is preferably linked to the recombinant antibody via a maleimide (mc) closed-loop linker to form an ADC.

[0080] In some embodiments of the present invention, the DAR value of the ADC is 8.0 to 10.0.

[0081] In some embodiments of the present invention, the target marker B that can bind to the first active reactive group is DBCO-PEG4-VA-PBD, with a DAR value of 0.8 to 1.0; and / or

[0082] The target label C that can bind to the second active reactive group is TCO-vc-PAB-Eribulin, with a DAR value of 0.6 to 1.0.

[0083] In some embodiments of the present invention, the reaction molar ratio of the recombinant antibody linking target marker A, target marker B, and target marker C is 1:(2-5):(2-5), for example 1:4:4.

[0084] In some embodiments of the present invention, the recombinant antibody conjugated to target marker A, target marker B, and target marker C are reacted in DMSO solution, preferably in 10% DMSO; the reaction temperature is 25-40°C (e.g., 37°C); the reaction pH is 7.2-7.8 (e.g., 7.4); and / or the reaction time is 20-30 hours (e.g., 24 hours).

[0085] In some embodiments of the present invention, the direct labeling method of the recombinant antibody-single-labeled or multi-labeled product includes:

[0086] Method i involves recombining a pre-labeled first and second parent antibodies into a recombinant antibody; the resulting recombinant antibody contains half of the structure of each parent antibody pair, as well as the label carried by the parent antibodies.

[0087] Method ii involves recombining a pre-labeled first parent antibody with a pre-labeled second parent antibody to form a recombinant antibody; the resulting recombinant antibody contains half of the structure of each parent antibody pair, as well as the label carried by the parent antibodies; or

[0088] Method iii: The first parent antibody and the second parent antibody are recombined into a recombinant antibody; then the resulting recombinant antibody is labeled.

[0089] In some embodiments of the present invention, a thiol reducing agent is used to reduce the interchain disulfide bonds of the first parent antibody, the second parent antibody, or the recombinant antibody in method iii and couple the target label thereto.

[0090] In some embodiments of the present invention, the reducing agent includes one or more of TCEP, DTT (dithiothreitol), and β-mercaptoethanol (β-ME).

[0091] In some embodiments of the present invention, the direct marking method includes:

[0092] (1) The parent antibody or the recombinant antibody from method iii is reduced with TCEP in a certain ratio;

[0093] (2) The target label is added to the reaction system to carry out a coupling reaction to obtain the pre-labeled parent antibody.

[0094] In some embodiments of the present invention, the molar ratio of the parent antibody to TCEP is 1:(6-10), for example, 1:8.

[0095] In some embodiments of the present invention, the molar ratio of recombinant antibody to TCEP in method iii is 1:(6-10), for example, 1:10.

[0096] In some embodiments of the present invention, the conditions for the reduction reaction are: pH 7.2-7.8 (e.g. 7.4), temperature 35-40°C (e.g. 37°C), and / or, reaction time 90-150 minutes (e.g. 120 minutes).

[0097] In some embodiments of the present invention, the conditions for the coupling reaction are: a temperature of 20-28°C (e.g., 25°C) and / or a reaction time of 90-150 minutes (e.g., 120 minutes).

[0098] In some embodiments of the present invention, in method i, the target is designated as GGFG-Dxd, which is preferably linked to the first parent antibody via a maleimide (mc) closed-loop linker.

[0099] In some specific embodiments of the present invention, the amino acid sequence of the first heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is shown in SEQ ID NO:2.

[0100] In some specific embodiments of the present invention, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:4, or

[0101] The amino acid sequence of the second heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the second light chain is shown in SEQ ID NO:2.

[0102] In some embodiments of the present invention, in method i, the target is designated as MMAF, which is preferably linked to the first parent antibody via a maleimide (mc) closed-loop linker.

[0103] In some specific embodiments of the present invention, the amino acid sequence of the first heavy chain is shown in SEQ ID NO:3, and the amino acid sequence of the first light chain is shown in SEQ ID NO:4. In other specific embodiments of the present invention, the amino acid sequence of the second heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the second light chain is shown in SEQ ID NO:2; or, the amino acid sequence of the second heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the second light chain is shown in SEQ ID NO:2.

[0104] In some embodiments of the invention, in method ii, the target label attached to the first parent antibody is Maleimide-PEG7-Biotin, which is preferably attached to the first parent antibody via a maleimide (mc) ring-closed linker; and / or

[0105] The target label attached to the second parent antibody is NH2CH2CH2-PEG3-SH, which is preferably attached to the second parent antibody via site-directed coupling technology of transglutaminase (mTG).

[0106] In some embodiments of the invention, in method ii, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:2; and / or

[0107] The amino acid sequence of the second heavy chain differs from that shown in SEQ ID NO:3 by N297A, and the amino acid sequence of the second light chain is shown in SEQ ID NO:4.

[0108] In other specific embodiments of the present invention, in method iii, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:2; and the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:4; and / or

[0109] The target label is GGFG-Dxd, which is preferably linked to the recombinant antibody via a maleimide (mc) closed-loop linker.

[0110] In some embodiments of the present invention, in method iii, the target label is MMAE, which is preferably linked to the recombinant antibody via Val-Cit-PAB(vc).

[0111] In some specific embodiments of the present invention, the amino acid sequence of the first heavy chain is shown in SEQ ID NO:3, and the amino acid sequence of the first light chain is shown in SEQ ID NO:4; the amino acid sequence of the second heavy chain is shown in SEQ ID NO:6, and the amino acid sequence of the second light chain is shown in SEQ ID NO:7.

[0112] In some specific embodiments of the present invention, the amino acid sequence of the first heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is shown in SEQ ID NO:2; the amino acid sequence of the second heavy chain is shown in SEQ ID NO:6, and the amino acid sequence of the second light chain is shown in SEQ ID NO:7.

[0113] In some embodiments of the present invention, the direct labeling method further includes linker ring-opening treatment of the pre-labeled parent antibody; preferably, when the pre-labeled parent antibody contains a maleimide (mc) closed-ring linker, the linker is hydrolyzed into an open-ring structure by incubation in 0.05-0.2M Tris-HCl buffer (pH 8.5-9.5) at 35-39°C for 18-30 hours.

[0114] In some embodiments of the present invention, in the direct labeling method, the first parent antibody and the second parent antibody are recombined in a 1:1 molar ratio in the presence of cysteine ​​hydrochloride to obtain the recombinant protein.

[0115] In some embodiments of the present invention, the final concentration of cysteine ​​hydrochloride is 60–90 mM, for example 75 mM.

[0116] In some embodiments of the present invention, the reaction conditions for the recombination are: a temperature of 31 to 37°C and a reaction time of 4 to 6 hours.

[0117] Specifically, for parent antibodies that do not contain inter-chain disulfide bonds, the reducing agent can be reduced or removed; for some highly hydrophobic labels, organic solvents such as 5-20% DMSO, DMA, etc., can be added to aid dissolution and promote the occurrence of FAE.

[0118] In some specific embodiments of the present invention, the Duobody recombinant antibody method includes: mutating the parent antibody CH3 by F405L and K409R respectively (unless otherwise specified, the present invention uses the EU antibody numbering system); under the action of neutral pH buffer and cysteine ​​hydrochloride (with a concentration of 10-100mM, for example 75mM), the interchain disulfide bonds of the mixed parent antibody will be dissociated, and the F405L mutated heavy chain will recombine with the K409R mutated heavy chain to form a recombinant antibody.

[0119] In some specific embodiments of the present invention, the KnH recombinant antibody method is employed. There are numerous reports of successfully obtaining recombinant antibodies by mutating the CH3 domain of an antibody to form a knock and hole structure. For example, recombinant antibodies are obtained by performing T366W mutations on the parent antibody CH3 to form a "knob," and by performing combined mutations of T366S, L368A, and Y407V on another antibody; or, high proportions of recombinant antibodies are obtained by performing combined mutations of S354C, T366W, and Y349C, T366S, L368A, and Y407V on the parent antibody.

[0120] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain of one parent antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain of the parent antibody is as shown in SEQ ID NO:2; and / or

[0121] The amino acid sequence of the heavy chain of the other parent antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain of the parent antibody is shown in SEQ ID NO:4.

[0122] A second aspect of the present invention provides a product prepared by the above method.

[0123] A third aspect of the present invention provides the use of the above-described product in the preparation of a medicament for treating tumors.

[0124] A fourth aspect of the present invention provides the above-described product for treating tumors.

[0125] A fifth aspect of the present invention provides a method for treating a tumor, the method comprising administering a therapeutically effective amount of the above-described product to a subject in need.

[0126] In some embodiments of the present invention, the tumor is selected from one or more of breast cancer, gastric cancer, ovarian cancer, endometrial cancer, cholangiocarcinoma, triple-negative breast cancer, non-small cell lung cancer, urothelial carcinoma, and colorectal cancer.

[0127] The schematic diagrams of the labeled antibodies of this invention are shown in Figures 1-9. The specific technical solutions include the following:

[0128] I. Preparation of the parent antibody (which can be used interchangeably with the monoclonal antibody in this article)

[0129] Recombinant parent antibodies were synthesized separately. The recombinant parent antibodies were selected from several engineered parent antibodies as follows.

[0130] 1) Controlled Fab Arm Exchange (cFAE) is a duobody that affects the surface charge of the Fc region of an antibody heavy chain by altering certain amino acid residues in the CH3 domain.

[0131] 2) Knob-into-hole alteration, which involves changing certain amino acids in the CH3 domain of the Fc segment of the antibody heavy chain to form a Knob or Hole structure. Antibodies with Knob and Hole structures can form recombinant antibody pairs.

[0132] 3) Fc contains antibodies against IgG CH2-IgA CH3 chimera (see reference Davis, J He et al. SEEDbodies).

[0133] Engineered maternal antibodies can be recombined into recombinant antibodies under appropriate recombinant reaction conditions. Paired maternal antibodies include maternal antibodies with different antigen-binding specificities and maternal antibodies with the same antigen-binding specificity. One inventive aspect of this invention is the selection of maternal antibodies with different antigen-binding specificities, whose specificity can be against epitopes of different antigens or against different epitopes of the same antigen molecule. Another inventive aspect of this invention is the selection of maternal antibodies with the same antigen-binding specificity, including maternal antibodies with the same variable region sequence or maternal antibodies derived from the same WT antibody that have undergone the aforementioned recombinability modification.

[0134] Methods for preparing the parent antibody include, but are not limited to, synthesizing antibody sequence DNA and transiently transferring it into host cells (such as mammalian cells, such as HEK293 cells and CHO-K1 or CHO-S cells, yeast cells, E. coli cells, or plant cells) or preparing a stable cell pool or cell line capable of stably expressing the target antibody. The target antibody is then purified from the culture product after a routine cell culture process. Antibody purification methods include centrifugation, filtration to remove cells and cell debris, affinity chromatography (such as protein A, protein G affinity chromatography or antigen affinity chromatography) combined with molecular sieve (SEC) ion exchange resin (IEX), dialysis, ultrafiltration, and other routine antibody purification steps. The purified antibody undergoes quality control analysis using methods such as SDS-PAGE, SEC-HPLC, HIC-HPLC, RP-HPLC, N-glycosylation removal, LC-MS molecular weight identification, and endotoxin detection (LAL method).

[0135] II. Mother Antibody Markers

[0136] At least one of the parent antibodies in the recombinant antibody is biolabeled to obtain the labeled parent antibody.

[0137] Methods for labeling parent antibodies include direct labeling and indirect labeling. One inventive aspect of this invention is the use of direct labeling to directly couple the target label to the parent antibody. The aforementioned labeling methods can be used to directly couple the target label to the parent antibody.

[0138] Another important inventive point of this invention is the indirect labeling of the parent antibody, which includes two types of indirect labeling methods. One inventive point of this invention employs a first type of indirect labeling method, in which an active reactive group is first attached to a specific coupling site on the antibody molecule, and the latter binds to the target label after recombining the antibody. For example, a mercaptopyridine compound is first labeled to the thiol site of the antibody via the aforementioned thiol reactive group. After recombining the antibody, the pyridine can be removed to produce an active thiol group, which is then coupled to the target label. Alternatively, a thiol, amino, or 295Q group can be first coupled with an active group capable of click chemistry, such as an azide, alkynyl, DBCO, COT, methyltetraazine, or TCO group, using an appropriate method. The latter then binds to the target label after recombining the antibody. Another inventive point of this invention employs a second type of indirect coupling method of the parent antibody, in which a specific coupling site on the antibody molecule is reversibly protected, and then deprotected and bound to the target label after recombining the antibody. For example, mercaptopyridine, cysteine, and glutathione can be used to protect the thiol group of the parent antibody by forming disulfide bonds, preventing it from forming disulfide bonds with the thiol group of the heavy chain hinge region of the paired parent antibody during or after recombination, and selectively deprotecting and coupling it.

[0139] The target markers coupled in this invention include, but are not limited to, cytotoxic compounds such as camptothecin (e.g., Camptothecin, Dxd, Exatecan, Topotecan, Belotecan, Irinotecan, SN38), maytasinoids (e.g., DM1, DM4, DM21), auristatin (MMAE, MMAF), calicamicin, chelating agents (e.g., Nota, Dota), hormones, immunomodulators, fluorescein, biotin, peptides, amino acids, small nucleic acids, radionuclides, and other compounds with active reactive groups, etc.

[0140] III. Recombination of Labeled Progenitor Antibodies

[0141] One of the main inventive aspects of this invention is the recombination of a pre-labeled parent antibody with another compatible parent antibody to form a recombinant antibody. The resulting recombinant antibody contains half of the structure from each parent antibody pair, as well as the label carried by the parent antibody. Depending on the subclass of the parent antibody and the degree of engineering modification, the parent antibody undergoes a cFAE-like recombination process, resulting in a recombinant antibody containing molecular characteristics from both parent antibodies, including their respective labels. The recombination conditions involve dissociating the heavy chain dimer of the parent antibody in the presence of a reducing agent and re-pairing it with the heavy chain of the paired parent antibody. This recombination step needs to be optimized for different parent antibodies. For parent antibodies that do not contain inter-heavy chain disulfide bonds, the reducing agent can be reduced or removed. For some highly hydrophobic labels, appropriate organic solvents, such as 5-20% DMSO, DMA, etc., can be added to aid dissolution and promote FAE. After allowing sufficient time, the proportion and purity of the recombinant antibody produced in the recombinant product are analyzed.

[0142] Methods for antibody recombination include those between different parent antibodies, including but not limited to cFAE (including duobody preparation methods), KnH, and charge pairing (CP), such as strop, P, etc., as reported below.

[0143] Strop, P. et al. Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair. J. Mol. Biol. 420, 204-219. https: / / doi.org / 10.1016 / j.jmb.2012.04.020(2012).

[0144] The engineering modification of the mother structure using these methods is shown in the table below.

[0145] IV. DAR Analysis

[0146] One of the key points of this invention is to solve the problem of difficult DOL analysis of multiplexed antibodies. This is mainly because it is difficult to distinguish the quantity of each labeled compound in antibody molecules modified with multiple labeled compounds using conventional methods. This invention employs a stepwise labeling method, first analyzing the DOL of individual labeled compounds, then ensuring the label is stably linked to the antibody molecule during the preparation of the recombinant antibody, and finally calculating the DOL of that labeled compound on the recombinant antibody. For compounds coupled in a specific chemical form, the DOL can also be calculated by measuring the residual amount of that compound at the antibody's linking site. In summary, by combining multiple DOL analysis methods, the DOL of multiplexed compounds can be obtained.

[0147] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0148] The reagents and raw materials used in this invention are all commercially available.

[0149] The positive and progressive effects of this invention are as follows:

[0150] This invention provides a flexible platform method for antibody site labeling, antibody recombination, and multi-label conjugation processes. Attached Figure Description

[0151] Figure 1 shows the structure 100 of a conventional monoclonal antibody, composed of two heavy chains 101 and two light chains 102. The heavy chains contain a variable region (vH) 103, a first constant region (CH1) 104, a hinge region 105, a second constant region (CH2) 106, and a third constant region (CH3) 107. The light chains consist of a light chain variable region (VL) 108 and a light chain constant region (CL) 109. The variable regions of both the light and heavy chains contain antigen-binding sites 110. The variable regions of the light and heavy chains, along with the first constant region, form Fab 111, and the second and third constant regions of the heavy chain form Fc 112.

[0152] Figure 2 shows the antibody marker 203 generated by the binding of antibody 201 and marker 201.

[0153] Figure 3 shows the recombinant synthesis of new antibody 303 from parent antibodies 301 and 302 via heavy chain repairing. If parent antibody 301 carries two markers derived from site-directed conjugation, distributed on opposite sides of the half antibody HL, the resulting labeled recombinant antibody 303 is a single-labeled antibody. If 301 and 302 originate from the same parent antibody and only undergo recombinability alteration, 303 effectively functions as a single-labeled monoclonal antibody. Depending on the chosen parent antibody, this method can generate either a single-labeled monoclonal antibody or a bispecific antibody single-labeled product.

[0154] Figure 4 shows the recombination of parent antibody marker 401 and parent antibody 402 with the same variable region sequence through heavy chain repairing to form a new antibody marker 403 with partial parent antibody marker.

[0155] Figure 5 shows how labeled parent antibodies 501 and 502 are recombined into a new bispecific double-labeled antibody 503 through heavy chain repairing.

[0156] Figure 6 shows the reaction of recombinant labeled antibody conjugate 601 and labeled 602 to produce double-labeled antibody conjugate 603.

[0157] Figure 7 shows the reaction of antibody 701 with linker 702 to produce antibody-linker conjugate 703, which then recombines with antibody 704 to form a new antibody-linker conjugate 705. The linker of the latter then reacts with marker 706 to form the final antibody-linker conjugate 707.

[0158] Figure 8 illustrates the indirect labeling method. First, the parent antibody 801 reacts with the intermediate label 802 in the presence of a reducing agent, linking the intermediate label 802 to eight thiol groups to form labeled parent antibody 803. 803 recombines with parent antibody 804 to form recombinant antibody 805, containing the HL structure of antibody 803 and the intermediate label 802. 805 reacts with label 807 via pathway 806 to form a new recombinant labeled antibody 808 containing both 802 and 807. If 808 replaces the intermediate label 802 with label 810 via pathway 809, a double-labeled recombinant antibody 811 is formed. If 808 reacts via pathway 812 to link label 813 to 802, a double-labeled recombinant antibody 814 is produced. If 805 reacts with label 813 via pathway 815, a labeled recombinant antibody 816 is produced. 816 reacts with 807 to produce 814.

[0159] Figure 9 shows the structures of different engineered antibodies. Some contain only one light chain, or two common light chains, or crossmab structures, so there is no need to worry about light chain mismatch. Among them, crossmab is a technology for functional region interchange of antibody Fab arms, HL is the heavy and light chain, scFv is the single-chain variable region fragment, Fc is the constant region functional fragment generated by antibody hydrolysis, H is the heavy chain, and nb is the nanobody.

[0160] Figures 10 and 11 show the SEC-HPLC results of the anti-Trop2 antibody Datopotamab (Dato) (F405L) and the anti-her2 antibody Trastuzumab (Tras) (K409R), respectively.

[0161] Figure 12 shows the MS results confirming the successful synthesis of the duobody.

[0162] Figures 13 and 14 show the ELISA results, confirming the activity of the bispecific antibody. Among them:

[0163] Figure 13 shows that HER2 ECD and TROP2 ECD were successfully labeled with biotin. These proteins were labeled with NHS-biotin and purified by desalting the NHS-biotin to remove excess NHS-biotin.

[0164] The left image in Figure 14 is a schematic diagram of the double-antigen sandwich assay used to test bispecific antibodies with double antigen binding activity.

[0165] The right panel in Figure 14 shows the binding activity of the bispecific antibody in the Trop2-coated ELISA assay using Her2-biotin and SA-HRP as detection reagents.

[0166] Figure 15 shows the recombinant antibody-Dxd conjugate, confirmed by HIC-HPLC results to exist as a whole ADC.

[0167] Figures 16-18 show the cell binding, cytotoxicity, and endocytosis results of the recombinant antibody ADC.

[0168] Figures 19-22 show the HIC-HPLC and SEC-HPLC results of Dato(F405L)-Dxd combined with Dato-K409R and Tras(K409R), respectively.

[0169] Figures 23-24 show the HIC-HPLC and SEC-HPLC results of the combination of Tras(K409R)-MMAF and Dato(F405L).

[0170] Figures 25-26 show the HIC-HPLC and SEC-HPLC results of the combination of Dato(F405L)-Mal-PEG7-Biotin and Tras(K409R), N297A-NH2CHCH-PEG3-SH.

[0171] Figures 27-28 show the LC-MS analysis results of Tras(K409R), N297A, V205C-N3 and Tras(F405L), N297A, V205C-TZ.

[0172] Figure 29 shows the HIC-HPLC analysis results of the bispecific antibody-labeled product (named BSR25) after combining Tras(K409R, N297A, V205C)-N3 and Tras(F405L, N297A, V205C)-Tz.

[0173] Figure 30 shows the RP-HPLC analysis results of BSR25 after coupling with three different payloads (GGFG-DXD, DBCO-PEG4-VA-PBD and COT-PEG4-VC-PAB-Eribulin).

[0174] Figure 31 shows the SEC-HPLC analysis results of BSR25 after coupling with three different payloads (GGFG-DXD, DBCO-PEG4-VA-PBD and COT-PEG4-VC-PAB-Eribulin). Detailed Implementation

[0175] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0176] The sample analysis methods of the embodiments are listed below:

[0177] SEC monomer content analysis

[0178] All samples were filtered through a 0.22 μm filter membrane before analysis.

[0179] LC-MS

[0180] RP-HPLC

[0181] HIC-HPLC

[0182] Example 1

[0183] The heavy chain of the anti-Trop2 antibody Datopotamab (Dato) was mutated at the F405L site and the K409R site (EU numbering system), respectively, and both parent antibodies were expressed. The anti-Her2 antibody Trastuzumab (Tras) was mutated at the K409R site and expressed. The anti-Trop2 antibody Sacituzumab (Saci) was mutated at the F405L site and expressed. The human IgG (hIgG) reference antibody used was a human IgG1 form of an internal anti-ractopamine monoclonal antibody. Antibody expression was performed in HEK293 cells according to standard procedures, followed by purification by Protein A column chromatography, achieving a purity of >95%. The antibody sequences are shown in Tables 1-4 below, and the purification results are shown in Figures 10-11.

[0184] Table 1 Sequences of Dato mutants

[0185] Table 2. Sequences of Tras(K409R)

[0186] Table 3 Sequences of Sacituzumab govitecan (trade name Trodelvy, hereinafter referred to as Saci) mutants

[0187] Table 4 hIgG Sequences

[0188] Table 5 Sequences of Tras mutants

[0189] 1. Recombinant duobody with unlabeled antibody and coupled via reduced interstrand disulfide bonds.

[0190] a) Preparation of unlabeled duobody

[0191] The Dato-Tras bispecific antibody was generated by processing the Dato and Tras mutant antibodies using a duobody procedure according to standard methods. Specific steps:

[0192] The two parent antibodies were mixed in a 1:1 ratio (mol:mol) and 75 mM (final concentration) of cysteine ​​hydrochloride (Sigma-Aldrich, 30078) was added. The mixture was then incubated at 31°C for 5 hours. The mixture was dialyzed overnight into PBS buffer (KEL, KC311-01), and the dialyzing was repeated three times, with each dialyzing lasting more than 6 hours.

[0193] SEC-HPLC analysis confirmed that the generated antibody was a single monomeric component. MS analysis confirmed the successful synthesis of the duobody, and the results are shown in Figure 12 and Table 6.

[0194] Table 6. Analysis of MS results for bispecific antibody recombinant antibody.

[0195] The activity of the bispecific antibody was confirmed by sandwich ELISA analysis using Trop2 and Her2 antigens, as shown in Figures 13-14. The specific experimental steps are as follows:

[0196] (1) The antigen was labeled with NHS-biotin, and excess NHS-biotin was removed by dialysis in 1xPBS (pH 7.4). The biotin labeling effect was analyzed by ELISA (the coated plate was blocked after avidin coating (concentration of 1 μg / mL). Biotinylated antigen (concentration of 10 μg / mL, diluted 1:5) was added and incubated at 37°C for 1 hour. After washing, avidin-horseradish peroxidase (diluted 1:5000) was added and incubated at 37°C for 40 minutes. After washing, the plate was detected by a microplate reader). The results showed that HER2 ECD and TROP2 ECD were successfully labeled with biotin.

[0197] (2) The bispecific antibodies were analyzed by a sandwich ELISA using Trop2 and Her2 antigens. Trop2 was coated at a concentration of 1 μg / mL. After blocking, monoclonal antibody and bispecific antibody (concentration 10 μg / mL, 1:5 dilution) were added, and the plate was incubated at 37°C for 1 hour. After washing, biotinylated Her2 (dilution 1:2500) was added, and the plate was incubated at 37°C for 1 hour. After washing, avidin-horseradish peroxidase (dilution 1:5000) was added, and the plate was incubated at 37°C for 1 hour. After washing, the plate was analyzed using a microplate reader. The ELISA experiment confirmed the binding activity of the bispecific antibodies, indicating successful bispecific antibody recombination.

[0198] b) Preparation and characterization of recombinant antibody-drug conjugation ADC

[0199] The recombinant Dato(F405L)-Tras(K409R) bispecific antibody was reduced by TCEP (ALDRICH, SLBN1521V) and coupled with GGFG-Dxd (MCE, HY-13631E) using conventional methods to prepare an ADC. The specific steps are as follows:

[0200] 1. Add 8 times the molar ratio of TCEP to the antibody and reduce it in 1×PBS, pH 7.4 solution at 37°C for 2 hours.

[0201] 2. After reducing the antibody, add 10% DMSO (Damas-beta, 75927P) and mix with GGFG-Dxd at a 10-fold molar ratio, and couple at 25°C for 2 hours.

[0202] 3. Through Zeba TM Desalination column 7K MWCO (Thermo, 89893) removes excess GGFG-Dxd.

[0203] Analysis after coupling showed that the coupling was successful and an ADC with a DAR of ~8.0 was obtained.

[0204] HIC-HPLC (compared to naked antibody) confirmed the presence of the recombinant antibody-Dxd conjugate Dato-Tras-Dxd as a whole ADC, as shown in Figure 15.

[0205] c) The three parent antibodies (Saci, Dato, and Tras) and the recombinant Saci-Tras and Dato-Tras bispecific antibodies, along with their corresponding ADCs (prepared using a method similar to that in Example b), and huIgG-MMAE (negative control) were mixed with Trop2 at a concentration of 133.33 nM. + Her2 +Cells NCI-N87 (ATCC, CRL-5822, 100,000 cells / well) were bound, and the reaction was carried out at 4°C for 1 hour. After washing with FACS buffer (PBS + 2% bovine serum), the plate was tested with goat anti-human IgG-Alexa-488 (Invitrogen, 2428531), and the reaction was carried out at 4°C for 45 minutes. The results (Figure 16 and Table 7) showed that the binding activity of the bispecific antibody was improved compared with that of the monoclonal antibody.

[0206] Table 7 Cell binding results

[0207] Three parent antibodies (Saci, Dato, and Tras) and two recombinant bispecific antibodies (Saci-Tras and Dato-Tras) conjugated with vcMMAE were used to create an ADC (antibody-drug conjugated with huIgG-MMAE) (negative control). A 1:1 molar mixture of MMAE toxin (MCE, HY-15162) and parent antibody-vcMMAE conjugated ADC was also used as a positive control. This mixture was used to study the effects on Trop2. + Her2 + Cytotoxicity assays were performed on NCI-N87 cells with high expression. ADC at an initial concentration of 133.33 nM was serially diluted 5-fold with culture medium, with 9 spots in total. Then, 100 μL / well / ADC was added to each well of a microplate pre-coated with 1000 cells / well. After incubation at 37°C for 144 hours, cells were collected, and cytotoxicity was detected using CTG reagent (PROMEGA, G7573). The results (Figure 17 and Table 8) showed that both the parent antibody and the bispecific antibody-conjugated ADCs exhibited target-specific cell inhibition and considerable cytotoxicity.

[0208] Table 8 Cytotoxicity Results

[0209] The parent antibodies Saci, Dato, and Tras, along with the recombinant Saci-Tras and Dato-Tras bispecific antibodies, were incubated with pHrodo dye (reaction system containing 10% DMSO) at 25°C for 2 hours. After the reaction, the mixture was dialyzed overnight (PBS, pH 7.4) to remove unbound dye. The pHrodo-labeled antibodies were then reacted with Trop2 at a concentration of 66.67 nM. + Her2 + Cells bound to NCI-N87 (100,000 cells) were incubated at 4°C for 0.5 hours. After binding, the cells were removed and incubated at 4°C and 37°C, respectively. Flow cytometry analysis was performed at 0, 6, and 24 hours. The results (Figure 17) showed that after 24 hours, the endocytosis rate of the bispecific antibody was stronger than that of either parent antibody.

[0210] Example 2: Preparation of recombinant bispecific antibody ADC with recombinant parent antibody

[0211] a) Preparation of normal IgG type recombinant antibody ADC

[0212] DAR7.2 was obtained by labeling mc-GGFG-Dxd with the Dato(F405L) mutant antibody. The method used was a standard reductive coupling method: the antibody was treated with 8 eq TCEP in 1×PBS solution at pH 7.4 and 37°C for 2 hours, followed by the addition of 10 eq mc-GGFG-Dxd. The reaction was continued at 25°C for 2 hours, and the product was then desalted to remove excess mc-GGFG-Dxd. 1×PBS, pH 7.4 buffer was used as the elution and storage solution.

[0213] The ADC underwent MC ring-opening treatment (hydrolysis to open the maleimide ring structure to increase the stability of thiosuccinimide): the ADC solution was adjusted to pH 9.0 using 0.1M Tris-HCl (Damas-beta, 829971B) buffer, pH 9.0, and reacted at 37°C for 24 hours. The reaction was then carried out using Zeba... TM The desalting column was replaced with 7K MWCO in PBS buffer to obtain the ADC after ring-opening treatment.

[0214] The ring-opening ADC was then mixed with Dato (K409R) and Tras (K409R) in a 1:1 molar ratio, and 75 mM cysteine ​​hydrochloride was added. After being placed at 37°C for 5 hours, the reaction product was transferred to a dialysis bag and dialyzed with PBS.

[0215] The mixing records of the reactions are shown in the table below.

[0216] Table 9 Recombinant Antibody Feeding Table

[0217] The antibody solution was collected after dialysis for 2 days and analyzed by SEC-HPLC and HIC-HPLC.

[0218] The analysis confirmed the presence of a significant bispecific antibody component in the recombinant product. The results are shown in Figures 19-22. "Mixture" indicates that the two parent antibodies were mixed after MC ring-opening treatment and then directly subjected to SEC-HPLC and HIC-HPLC. "Recombinant" indicates the SEC-HPLC and HIC-HPLC analysis results two days after recombinant dialysis. RT (Retention Time) represents the peak elution time.

[0219] Specifically, the HIC-HPLC chromatogram showed a distinct recombinant antibody peak in the recombinant product. The SEC-HPLC chromatogram showed that most of the half-antibody peaks disappeared in the recombinant product, and all peaks were replaced by full antibody peaks.

[0220] Example 3: Testing the recombinant performance of other mc-small molecule labeled antibodies

[0221] DAR7.1 was obtained by labeling the Tras(K409R) mutant antibody with mc-MMAF (MCE, HY-156377). The method was a conventional reductive coupling method, and the ADC was subjected to mc ring-opening treatment, as in Example 2.

[0222] The ring-opening ADC was then mixed with unlabeled Dato (F405L) in a 1:1 molar ratio, and 75 mM cysteine ​​hydrochloride was added. After incubation at 37°C for 5 hours, the reaction product was transferred to a dialysis bag and dialyzed with PBS.

[0223] The mixing records of the reactions are shown in the table below.

[0224] Table 10 Recombinant Antibody Feeding Table

[0225] The antibody solution was collected after dialysis for 2 days and analyzed by SEC-HPLC and HIC-HPLC.

[0226] The results confirmed the significant production of bispecific antibodies during the recombination process. The results are shown in Figures 23-24. "Mixture" indicates that the two parent antibodies were mixed after MC ring-opening treatment and then directly subjected to SEC-HPLC and HIC-HPLC. "Recombination" indicates the SEC-HPLC and HIC-HPLC analysis results two days after recombination. RT (Retention Time) represents the peak elution time.

[0227] Example 4: Preparation of dual-antibody dual-labeled product

[0228] According to the options in Table 11, either the parent antibody recombination of the payload is labeled separately or the parent antibody recombination of the linker is labeled separately.

[0229] Table 11 Double Antibody and Double Label Combinations

[0230] Specifically, the Dato(F405L) mutant antibody was labeled with Maleimide-PEG7-Biotin (MCE, HY-156377) to obtain DOL6.50. The method was a conventional reductive coupling method, and the conjugate was subjected to mc ring-opening treatment, the same as in Example 2.

[0231] A Tras(K409R, N297A) mutant antibody was labeled with thiol-PEG3-amine (molecular formula SH-PEG3-CH2CH2NH2) (MCE, HY-130871A) to obtain DAR1.78. The method used was site-directed conjugation with transglutaminase (mTG). Specifically, the antibody was treated with 200 eq NH2CH2CH2-PEG3-SH and 1 U / mg mTG in 1×PBS solution, pH 7.4, overnight at 37°C. The product was then concentrated by ultrafiltration to remove excess NH2CH2CH2-PEG3-SH and mTG. 1×PBS, pH 7.4 buffer was used as the replacement and storage solutions.

[0232] Then, the ring-opening Dato(F405L)-Maleimide-PEG7-Biotin and Tras(K409R, N297A)-NHCH2CH2-PEG3-SH were mixed in a 1:1 molar ratio, and 75 mM cysteine ​​hydrochloride was added. After incubation at 37°C for 5 hours, the reaction product was transferred to a dialysis bag and dialyzed with PBS.

[0233] The mixing records of the reactions are shown in the table below.

[0234] Table 12 Recombinant Antibody Feeding Table

[0235] Two days after dialysis, the antibody solution was collected and processed by SEC-HPLC and HIC-HPLC.

[0236] The analysis confirmed the significant production of bispecific antibodies during the recombination process. The results are shown in Figures 25-26. "Mixture" indicates that the two parent antibodies were mixed after MC ring-opening treatment and then directly subjected to SEC-HPLC and HIC-HPLC. "Recombination" indicates the SEC-HPLC and HIC-HPLC analysis results two days after recombination. RT (Retention Time) represents the peak elution time.

[0237] Example 5: Preparation of dual-antibody triple-load coupling product

[0238] First, Tras(K409R, N297A, V205C) mutant antibody was labeled with the linker amino-PEG3-C2-Azido(N3) (MCE, HY-W021401) under the action of mTG to obtain DOL 2.00 (Figure 27). Tras(F405L, N297A, V205C) mutant antibody was labeled with the linker Methyltetrazine-PEG4-amine(TZ) (MCE, HY-141261) to obtain DOL 2.00 (Figure 28, Table 13). The method used was site-directed conjugation of transglutaminase (mTG). Specifically, the antibody was treated overnight in 1×PBS solution (pH 7.4) at 37°C with 200 eq of linker and 1 U / mg mTG. The product was then concentrated by ultrafiltration to remove excess linker and mTG. 1×PBS, pH 7.4 buffer was used as the replacement and preservation solutions.

[0239] Table 13 MS-HPLC results of Tras(K409R, N297A, V205C)-N3 and Tras(F405L, N297A, V205C)-TZ

[0240] Antibody recombination: Tras(K409R, N297A, V205C)-N3 and Tras(F405L, N297A, V205C)-Tz were mixed in a 1:1 molar ratio, and 75 mM cysteine ​​hydrochloride was added. After incubation at 37°C for 5 hours, the reaction product was transferred to a dialysis bag and dialyzed with PBS. The recombinant bispecific antibody-labeled product was named BSR25. The recombination rate was detected by HIC-HPLC. The recombination rate was 100% (Figure 29).

[0241] Subsequently, it was coupled with mc-GGFG-DXD to obtain BSR25-DXD10, as described in Example 2. RP-HPLC analysis showed that the coupling DAR value was 10.00 (Figure 30).

[0242] A double "CLICK" reaction was then performed, involving the simultaneous addition of DBCO-PEG4-VA-PBD (MCE, HY-133433) and TCO-vc-PAB-Eribulin (Chempatner). The mixture contained 3 mg / mL BSR25-DXD10, 4 eq DBCO-PEG4-VA-PBD, and 4 eq TCO-vc-PAB-Eribulin, 10% DMSO, and PBS (pH 7.4). The mixture was reacted at 37°C for 24 hours, followed by Zeba... TM Desalination column 7K MWCO and Purified using an ultracentrifuge, 30kDa MWCO (Zhejiang Zhongzai Medical, UT0650030), and the coupling results were analyzed (Figures 30-31). RP-HPLC analysis, using a single DBCO-PEG4-VA-PBD reaction as a control, showed that the heavy chain of BSR25-DXD10 migrated to the right to a retention time of approximately 12.1 minutes. Therefore, in the double peaks of the rightward migration of the heavy chain DAR3(Dxd) in the double "CLICK" reaction, the peak with a retention time of 12.11 minutes represents the PBD coupling peak, accounting for 46.87%, with a DAR value of 0.94. The peak with a retention time of 12.46 minutes represents the Eribulin coupling peak, accounting for 33.16%, with a DAR value of 0.66. Therefore, the final results showed that the coupling DAR values ​​were 10.00×Dxd, 0.94×PBD, and 0.66×Eribulin (Figures 30-31, Table 14). SEC-HPLC analysis showed that the monomer content of BSR25-DXD / PBD / Eribulin was 84.99%.

[0243] Table 14 RP-HPLC Analysis Results of BSR25-PBD1 / Eribulin1 / Dxd10 Coupling Products

[0244] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for labeling antibodies or ADCs, characterized in that, The method includes directly or indirectly labeling a target marker to an antibody.

2. The method as described in claim 1, characterized in that, The antibody includes monoclonal antibodies or bispecific antibodies, preferably recombinant antibodies; Preferably, the functional fragment of the monoclonal antibody or the bispecific antibody or the parent antibody of the recombinant antibody is selected from the group consisting of: 1) Antibodies containing Fc mutations that can form controlled Fab arm exchanges (cFAEs); 2) Antibodies containing Fc mutations that can form a knock-in-hole alteration; 3) Fc contains antibodies against IgG CH2-IgA CH3 chimera; And / or, the recombinant antibody includes monoclonal antibodies with different antigen binding specificities, the specificity of which may be against epitopes of different antigens or against different epitopes of the same antigen molecule; And / or, the recombinant antibody is selected from monoclonal antibodies with the same antigen binding specificity, including parent antibodies with the same variable region sequence or monoclonal antibodies derived from the same WT antibody that have undergone the above-mentioned recombinability modification; And / or, the recombinant antibody includes a first arm and a second arm; the first arm and the second arm target different antigens or the same antigen; and the hinge regions and Fc of the first arm and the second arm are bound together by disulfide bonds and non-covalent forces. And / or, methods for preparing monoclonal antibodies or parental antibodies include: (1) The nucleotide sequence of the antibody synthesis gene, and (2) Transfer it to host cells to prepare a stable cell pool or cell line that can stably express the target antibody; The target antibody is purified from the culture product after the cell culture process.

3. The method as described in claim 2, characterized in that, The antigens targeted by the first arm and the second arm are each independently tumor-associated antigens or tumor-specific antigens, such as one or more selected from Trop2, Her 2, tissue factor, Nectin-4, FRα, CD33, CD79b, BCMA and CD30; And / or, the first arm or the second arm includes a variable region selected from Fab, scFv, Fv, VHH and dAb; optionally, it also includes a constant region selected from human IgG constant regions such as IgG1 and IgG2 constant regions; And / or, the method of antibody recombination includes recombination between different parent antibodies, selected from cFAE method (e.g., DuoBody preparation method), KnH, charge pairing; preferably, the recombination conditions include dissociating the heavy chain dimer of the mixed parent antibodies in the presence of a reducing agent to re-pair the heavy chain of the first parent antibody with the heavy chain of the second parent antibody.

4. The method as described in claim 3, characterized in that, The first arm includes a first heavy chain and a first light chain; and / or, the second arm includes a second heavy chain and a second light chain; And / or, the first heavy chain and / or the second heavy chain introduces a mutation in the heavy chain constant region (preferably the CH3 domain) to enable the first arm and the second arm to specifically pair, thereby enabling the first arm and the second arm to assemble to form the recombinant antibody; And / or, the first arm and the second arm target the same antigen, which is preferably Trop2 or Her 2; Preferably, the mutation is selected from F405L, K409R / E / D, R411T, T370K, T366W / S, L368A, Y407V, K392D, N399K, E356K and D399R corresponding to IgG1, and the position of the mutation is EU number; More preferably, the heavy chain constant region mutation introduced by the first arm is F405L, and the heavy chain constant region mutation introduced by the second arm is K409R; or the heavy chain constant region mutation introduced by the first arm is K409R, and the heavy chain constant region mutation introduced by the second arm is F405L. More preferably, the recombinant antibody is selected from one of the following: (1) The antigen targeted by both the first and second arms is Trop2; preferably: The amino acid sequence of the first heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is shown in SEQ ID NO:2; and / or The amino acid sequence of the second heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the second light chain is shown in SEQ ID NO:2; (2) The antigen targeted by the first arm is Trop2, and the antigen targeted by the second arm is Her2; preferably: i. The amino acid sequence of the first heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is shown in SEQ ID NO:2; And / or, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:4; or ii. The amino acid sequence of the first heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:2; and / or The amino acid sequence of the second heavy chain is shown in SEQ ID NO:6, and the amino acid sequence of the second light chain is shown in SEQ ID NO:7; (3) The antigen targeted by both the first and second arms is Her2; preferably: i. The amino acid sequence of the first heavy chain differs from that of the amino acid described in SEQ ID NO:3 by N297A / V205C; and the amino acid sequence of the first light chain is shown in SEQ ID NO:4; And / or, the amino acid sequence of the second heavy chain differs from that of the amino acid described in SEQ ID NO:6 by N297A / V205C; and the amino acid sequence of the second light chain is as shown in SEQ ID NO:7; or ii. The amino acid sequence of the first heavy chain is as shown in SEQ ID NO:12, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:11; and / or The amino acid sequence of the second heavy chain is shown in SEQ ID NO:10, and the amino acid sequence of the second light chain is shown in SEQ ID NO:

11.

5. The method according to any one of claims 1-4, characterized in that, The ADC is a normal IgG type recombinant antibody ADC; preferably, the toxic molecules in the ADC are selected from Dxd, MMAF maytansine, DM1, DM4, kazimidic acid, PBD such as SGN-CD33A, SN-38, PE and Auristatins (e.g. MMAE, MMAF), peptides, small nucleic acids, radionuclides, or other functional compounds.

6. The method as described in claim 2, characterized in that, The host cells are selected from mammalian cells, such as HEK293 cells and CHO-K1 or CHO-S cells, yeast cells, Escherichia coli cells, or plant cells; And / or, antibody purification methods include centrifugation, filtration to remove cells and cell debris, affinity chromatography (such as protein A, protein G affinity chromatography or antigen affinity chromatography) combined with molecular sieve ion exchange resin, dialysis, and ultrafiltration.

7. The method as described in claim 1, characterized in that, The method yields monoclonal antibody-single labeling, bispecific antibody-single labeling, monoclonal antibody-multi-labeling, or bispecific antibody-multi-labeling products.

8. The method as described in claim 1, characterized in that, The target markers are selected from cytotoxic compounds (such as camptothecin, Dxd, Exatecan, Topotecan, Belotecan, Irinotecan, SN38, maytasinoids (such as DM1, DM4, DM21, Auristatin (MMAE, MMAF), Calicheamicin), chelating agents (such as Nota, Dota), fluorescein, biotin, peptides, amino acids, and other compounds with active reactive groups; Alternatively, the target marker may be selected from one or more of GGFG-Dxd, DBCO-PEG4-VA-PBD, TCO-vc-PAB-Eribulin, Maleimide-PEG7-Biotin, and NH2CH2CH2-PEG3-SH.

9. The method according to any one of claims 1-8, characterized in that, The indirect labeling methods corresponding to bispecific antibody-single-labeled or bispecific antibody-multi-labeled products are as follows: Method a involves first attaching one or more reactive groups to a specific coupling site on the parent antibody molecule, which then bind to the target marker after the recombinant antibody is obtained; or Method b involves first reversibly protecting specific coupling sites on the parent antibody molecule, then deprotecting them after recombining the antibody and binding them to the target marker. Preferably, the indirect labeling method involves first reversibly protecting specific coupling sites on the antibody molecule, then deprotecting the recombinant antibody and binding it to the target label; more preferably, the indirect labeling method involves first labeling the thiol site of the antibody with a thiol reactive group via a thiol pyridine compound, which can remove the pyridine to produce an active thiol group after the recombinant antibody is obtained, and then coupling the target label to it. Alternatively, the indirect labeling method involves first coupling the antibody's amide, thiol, or amino groups with an active group capable of click chemistry, such as azide, alkynyl, DBCO, COT, methyltetraazine, or COT groups, using an appropriate method. The latter is then linked to the target label after the antibody is recombined.

10. The method as described in claim 9, characterized in that, In method a, the target marker includes 1 to 4 target markers, preferably 3 target markers, each independently selected from one of GGFG-Dxd, DBCO-PEG4-VA-PBD and TCO-vc-PAB-Eribulin; Preferably, method a includes: (1) First, attach the first active reactive group to the first parent antibody, and then attach the second active reactive group to the second parent antibody; (2) The first and second parent antibodies, which are linked to active reactive groups, are specifically paired to obtain recombinant antibodies; (3) The recombinant antibody with the obtained active group is directly linked to the target marker A; Optionally, method a further includes: (4) Link the recombinant antibody to target label A and target label B and / or target label C that can bind to the first active reactive group and / or the second reactive group; More preferably, the first reactive group and the second reactive group are the same or different, and they are preferably linked to the parent antibody via site-directed coupling technology of glutamine transaminase; More preferably, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:12, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:11; And / or, the first reactive group is amino-PEG3-C2-Azido; preferably, the DOL of the first parent antibody connected to the first reactive group is 2; more preferably, the first parent antibody connected to the first reactive group is subjected to linker ring-opening treatment; And / or, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:10, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:11; And / or, the second reactive group is Methyltetrazine-PEG4-amine; preferably, the DOL of the second antibody connected to the second reactive group is 2; And / or, the target marker A is GGFG-DXD, which is preferably linked to the recombinant antibody via a maleimide closed-loop linker to form an ADC; the DAR value of the ADC is 8.0 to 10.0; And / or, the target marker B that can bind to the first active reactive group is DBCO-PEG4-VA-PBD, with a DAR value of 0.8 to 1.0; And / or, the target label C that can bind to the second active reactive group is TCO-vc-PAB-Eribulin, with a DAR value of 0.6 to 1.0; And / or, the reaction molar ratio of the recombinant antibody linked to target label A, target label B, and target label C is 1:(2-5):(2-5), for example, 1:4:4; And / or, the recombinant antibody conjugated to target marker A, target marker B, and target marker C are reacted in DMSO, preferably in 10% DMSO; the reaction temperature is 25-40°C (e.g., 37°C); the reaction pH is 7.2-7.8 (e.g., 7.4); and / or, the reaction time is 20-30 hours (e.g., 24 hours).

11. The method according to any one of claims 1-8, characterized in that, Direct labeling methods for recombinant antibody-single-labeled or multi-labeled products include: Method i involves recombining a pre-labeled first parent antibody with a pairable second parent antibody to form a recombinant antibody; the resulting recombinant antibody contains half of the structure of each parent antibody pair, as well as the label carried by the parent antibody. Method ii involves recombining a pre-labeled first parent antibody with a pairable pre-labeled second parent antibody to form a recombinant antibody; the resulting recombinant antibody contains half of the structure of each parent antibody pair, as well as the label carried by the parent antibody; or Method iii involves recombining the first parent antibody and a pairable second parent antibody to form a recombinant antibody; then labeling the resulting recombinant antibody. Preferably, the interchain disulfide bonds of the first parent antibody, the second parent antibody, or the recombinant antibody in method iii are reduced and coupled with the target label using a thiol reducing agent; the reducing agent preferably includes one or more of TCEP, DTT, and β-mercaptoethanol; More preferably, the direct labeling method includes: (1) The parent antibody or the recombinant antibody from method iii is reduced with TCEP in a certain ratio; (2) The target marker is added to the reaction system to carry out a coupling reaction to obtain the pre-labeled parent antibody; More preferably, the direct labeling method includes one or more of the following conditions: 1) The molar ratio of the parent antibody to TCEP is 1:(6-10), for example, 1:8; 2) The molar ratio of recombinant antibody to TCEP in method iii is 1:(6-10), for example, 1:10; 3) The conditions for the reduction reaction are: pH 7.2-7.8 (e.g. 7.4), temperature 35-40℃ (e.g. 37℃), and / or, reaction time 90-150 minutes (e.g. 120 minutes).

12. The method as described in claim 11, characterized in that, The method includes one of the following: (1) In method i: The target label is GGFG-Dxd, which is preferably linked to the first parent antibody via a maleimide (mc) closed-loop linker; And / or, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:2; And / or, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:4, or, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:5, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:2; (2) In method i: The target is labeled MMAF, which is preferably linked to the first parent antibody via a maleimide (mc) closed-loop linker; And / or, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:4; And / or, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:2; or, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:5, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:2; (3) In method ii: The target marker attached to the first parent antibody is Maleimide-PEG7-Biotin, which is preferably attached to the first parent antibody via a maleimide (mc) ring-closed linker; And / or, the target label attached to the second parent antibody is NH2CHCH-PEG3-SH, which is preferably attached to the second parent antibody via site-directed coupling technology with glutamine transaminase; And / or, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:2; And / or, the amino acid sequence of the second heavy chain differs from that of the amino acid sequence shown in SEQ ID NO:3 by N297A, and the amino acid sequence of the second light chain is shown in SEQ ID NO:4; (4) In method iii: 1) The amino acid sequence of the first heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is shown in SEQ ID NO:2; and the amino acid sequence of the second heavy chain is shown in SEQ ID NO:3, and the amino acid sequence of the second light chain is shown in SEQ ID NO:4; And / or, the target label is GGFG-Dxd, which is preferably linked to the recombinant antibody via a maleimide (mc) closed-loop linker; or 2) The target marker is MMAE, which is preferably linked to the recombinant antibody via Val-Cit-PAB(vc); Preferably, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the first light chain is as shown in SEQ ID NO:4; and the amino acid sequence of the second heavy chain is as shown in SEQ ID NO:6, and the amino acid sequence of the second light chain is as shown in SEQ ID NO:7; or The amino acid sequence of the first heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the first light chain is shown in SEQ ID NO:2; the amino acid sequence of the second heavy chain is shown in SEQ ID NO:6, and the amino acid sequence of the second light chain is shown in SEQ ID NO:

7. More preferably, the direct labeling method further includes linker ring-opening treatment of the pre-labeled parent antibody; preferably, when the pre-labeled parent antibody contains maleimide (mc) closed-ring linkers, the linkers are hydrolyzed into open-ring structures by incubation in 0.05-0.2M Tris-HCl buffer (pH 8.5-9.5) at 35-39°C for 18-30 hours.

13. The method as described in claim 11 or 12, characterized in that, In the direct labeling method, the first parent antibody and the second parent antibody are recombined in a 1:1 molar ratio in the presence of cysteine ​​hydrochloride to obtain the recombinant protein; Preferably, the final concentration of cysteine ​​hydrochloride is 60–90 mM, for example 75 mM; And / or, the reaction conditions for the recombination are: a temperature of 31–37°C and a reaction time of 4–6 hours.

14. The method according to any one of claims 9-13, characterized in that, For parent antibodies that do not contain inter-chain disulfide bonds, the reducing agent can be reduced or removed; for some highly hydrophobic labels, organic solvents such as 5-20% DMSO, DMA, etc., can be added to aid dissolution and promote the formation of FAE.

15. The method according to any one of claims 3-5 and 9-14, characterized in that, The Duobody recombinant antibody method involves: mutating the parent antibody CH3 with F405L and K409R respectively; and then recombining the mixed parent antibody in a neutral pH buffer and mercaptoethanol (at a concentration of, for example, 75 mM).

16. The product prepared by the method according to any one of claims 1-15.

17. The use of the product as described in claim 16 in the preparation of a medicament for treating tumors; Preferably, the tumor is selected from one or more of breast cancer, gastric cancer, ovarian cancer, endometrial cancer, bile duct cancer, triple-negative breast cancer, non-small cell lung cancer, urothelial carcinoma, and colorectal cancer.

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