Ligand drug conjugate with free load recovery unit

Abstract

The invention relates to a ligand drug conjugate with a free load recovery unit and an application of the ligand drug conjugate in prevention or treatment of diseases, the ligand drug conjugate can remove free loads through the connected recovery unit capable of being combined with the free loads, the medication safety is remarkably improved, and the application range of the ligand drug conjugate is widened. Meanwhile, the load carried by the ligand drug conjugate can be increased on the whole by recovering the free load, and the activity loss caused by load falling is made up. Therefore, by introducing the recovery unit, the recovery of the free load is realized, the safety and effectiveness of the ligand-coupled drug can be remarkably improved, and the therapeutic window is expanded.

Description

Ligand drug conjugate with free load recovery unit TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a ligand drug conjugate with free load recovery unit and application thereof. BACKGROUND

[0002] Antibody-drug conjugates (ADCs) are a new type of biological drugs produced by conjugating monoclonal antibodies and cytotoxic drugs (also known as payload) through a linker, which can effectively penetrate tumor tissues and be phagocytosed into the lysosome of tumor cells to release the effector molecule by targeting specific antigens. It fully combines the characteristics of strong targeting selectivity of monoclonal antibodies and high activity of cytotoxic drugs, and can not only specifically target cytotoxic drugs to tumor cells, but also has the effects of slow release and improved anti-tumor activity, etc., and is one of the key development directions of tumor treatment.

[0003] The development of ADC technology can be divided into four stages: the first generation of ADC belongs to the early exploration stage, which is basically blank in understanding the key issues such as the activity and release mode of the payload, the properties of the linker, etc. The strategy of mouse-derived antibody + high-activity payload + unstable linker + random coupling is adopted, which has high immunogenicity, is a mixture of different DAR values, is metabolically unstable, has a narrow safety window, and is a high challenge for drug development. The representative product is Mylotarg. The second generation of ADC enriches the types of linkers, adopts the strategy of humanized antibody + high-activity payload + stable linker + random coupling, has low immunogenicity, significantly improves metabolic stability, and increases therapeutic index. The representative products are Kadcyla and Adcetris. Compared with the first generation of ADC, the second generation of ADC shows good clinical efficacy and safety. The third generation of ADC learns from the experience of early ADC: heterogeneity and instability are important factors affecting the in vivo efficacy and toxicity of ADC. High DAR value components have poor in vivo stability, which affects the treatment window of ADC. Therefore, site-specific coupling and low DAR value technology are adopted, but there are still problems such as insufficient release of the payload and high drug resistance. The fourth generation of ADC technology adopts high DAR value (4-8) and low-to-moderate activity payload strategy. The payload has high bystander effect. The most important feature is that it has good efficacy on tumors with low and high expression of tumor surface antigens. The representative products are Enhertu and Trodelvy. Since 2009, the first ADC drug-gemtuzumab ozogamicin (Mylotarg) was approved by the US FDA for marketing. As of May 2024, a total of 15 ADC drugs have been approved for marketing worldwide (see Table 1), and a large number of ADC drugs are in clinical research or early research stages. Table 1. Basic information of ADC drugs approved for marketing worldwide

[0004] The activity and physicochemical properties of the payload will directly affect the anti-tumor efficacy of ADC drugs. There are two main types of payloads in existing approved ADCs: tubulin inhibitors and DNA inhibitors, which are further divided into DNA damaging agents and topoisomerase inhibitors. As shown in Table 1, tubulin inhibitors are the most widely used type of payload in ADC drug development. Eight of the 15 ADCs on the market are tubulin inhibitors (MMAE, DM1, DM4). In addition, DNA damaging agents, including calicheamicin, PBD, and camptothecin derivatives (SN38, DXD), are also widely used as payloads in the construction of ADC drugs. Representative examples of currently marketed ADC drugs include Mylotarg, Besponsa, Enhertu, Trodelvy, and Zynlonta (Wang Z, Li H, Gou L, Li W, Wang Y. Antibody-drug conjugates: Recent advances in payloads. Acta Pharm Sin B. 2023 Oct; 13(10): 4025-4059).

[0005] Generally, the presence of lysine and cysteine on the antibody provides a reaction site for coupling the payload. Early ADC drugs were basically coupled through lysine or cysteine residues, but since about 40 amino acid residues of the antibody can generally react, different numbers of DAR (0-8) small molecule payloads can be attached to the antibody, resulting in a wide distribution of DAR values. Since IgG1 antibodies have both interchain disulfide bonds and intrachain disulfide bonds. The interchain disulfide bond is exposed on the outside of the antibody and is easily reduced to expose free cysteine residues, thereby providing available sites for the coupling of the linker payload to the antibody. In addition, due to the limited number of binding sites and the unique reactivity of the thiol group, using cysteine as a connection site helps to reduce the heterogeneity of ADCs, so far the most commonly used coupling method is based on the reaction of cysteine. However, both lysine and cysteine residue coupling methods can lead to premature release of the payload due to insufficient stability, which can cause off-target toxicity (Fu Z, Li S, Han S, Shi C, Zhang Y. Antibody drug conjugate: the "biological missile" for targeted cancer therapy. Signal Transduct Target Ther. 2022 Mar 22; 7(1): 93).

[0006] Despite the unprecedented success in the development of ADC new drugs, there are still many challenges in the development of ADCs, including the complexity of pharmacokinetics, insufficient payload release, drug resistance, and inevitable side effects during treatment. The most common serious side effects (grade 3 or higher) are hematologic toxicities, including neutropenia, thrombocytopenia, leukopenia, and anemia. Hematologic toxicity, as well as hepatotoxicity and gastrointestinal reactions, can be related to the premature release of cytotoxic payloads into the blood circulation. This is consistent with conventional chemotherapy drugs that mainly affect healthy cells that proliferate rapidly.

[0007] In addition, in the drug marketing stage, although some ADCs have proven sufficient effectiveness and safety, almost all ADCs exhibit certain toxicity during clinical application. As the in-depth clinical application of ADC drugs, some drugs exhibit certain toxic side effects, leading to reduced or stopped clinical application doses. It is estimated that only about 0.1% of the amount of ADC drugs entering the human body is delivered to the targeted diseased cell population, and the vast majority of drugs are decomposed “off-site” in non-targeted healthy cells, which can cause unnecessary toxicity. ADC toxicity that does not reach the target diseased or target tissue can be divided into “on-target” or “off-target”, and off-target toxicity is mainly caused by free toxins shed from ADC. The complete ADC enters normal cells, which can be through non-specific endocytosis, or by binding to target antigens or Fc / C-type lectin receptors. The payload dissociated from the ADC molecule in the extracellular fluid or the payload released by other targeted / non-targeted apoptotic cells can enter normal cells through passive diffusion or non-specific endocytosis. Off-target payload delivery is a key driver of ADC resistance and ultimately determines the recommended dose for patients (Nguyen TD, Bordéau BM, Balthasar JP. Mechanisms of ADC Toxicity and Strategies to Increase ADC Tolerability. Cancers (Basel). 2023 Jan 24; 15(3): 713.)

[0008] To improve the tolerability of ADCs, Balthasar et al. designed neutralizing antibody fragments (Fab or sdAb) to bind non-conjugated circulating payload molecules, aiming to reduce the amount of cytotoxin reaching normal tissues or cells, thereby reducing the degree of off-target toxicity in patients receiving ADCs (Bordeau BM, Nguyen TD, Polli JR, Chen P, Balthasar JP. Payload-Binding Fab Fragments Increase the Therapeutic Index of MMAE Antibody-Drug Conjugates. Mol Cancer Ther. 2023 Apr 3;22(4):459-470., Nguyen TD, Bordeau BM, Balthasar JP. Use of Payload Binding Selectivity Enhancers to Improve Therapeutic Index of Maytansinoid-Antibody-Drug Conjugates. Mol Cancer Ther. 2023 Nov 1;22(11):1332-1342.). In this method, the ADC and neutralizing antibody fragments need to be co-administered to the subject, which is costly in production and administration, and in addition, the administration ratio and administration method between the ADC and the neutralizing antibody fragment need to be explored, increasing the complexity of the development of the administration scheme. In addition, WO2021113740A1 and WO2024044709A2 also disclose the above method, and disclose a variety of antibodies targeting small molecule toxins, including single domain antibodies and oligopeptides, etc., such as the antibody mouse antibody MA24E2, humanized antibody ABC3315, ABC3320 (heavy chain: SEQ ID NO: 163; light chain: SEQ ID NO: 164) that only binds free MMAE, not MMAE on MMAE-ADC; anti-MMAE single domain antibody MC7; anti-camptothecin toxin mouse 8C2 antibody, humanized 8C2 Fab, humanized 8C2 VHH; anti-camptothecin toxin single domain antibody TF7 and single domain antibody TA2, anti-calicheamicin oligopeptide C2 and C9, anti-calicheamicin single domain antibody CG4, anti-maytansinoid single domain antibody DF3A2, DF3A4, DF4B12, DMFH1, DMOH9. It was found that the use of some antibodies may increase the cytotoxicity of antibody conjugate drugs for unknown reasons, such as 8C2 antibody, MC7 antibody, etc.

[0009] The application creatively connects a recyclable unit for free load that can be detached from the ligand drug conjugate in the ligand drug conjugate, which can remove free load in the blood, reduce off-target toxicity, and generally increase the load carried by the ligand drug conjugate as a whole by recycling free load and making up for the loss of activity caused by load detachment. Therefore, by introducing the recycling unit, the safety of the ligand conjugate drug can be significantly increased. Further, the dosage of ADC can be further improved to correspondingly improve the effectiveness of ADC treatment and expand the therapeutic window.

[0010] In addition, the risk of unknown antibody drug conjugate cytotoxicity enhancement in the above-mentioned combination therapy (WO2021113740A1) is also effectively avoided. On the other hand, compared with the combination therapy using two macromolecular products, the production cost and clinical development cost can be greatly reduced, the drug use can be simplified, the drug risk can be reduced, and the convenience of the subject using the drug can be improved.

[0011] The recycling unit can use existing technologies to screen antibodies, polypeptides or aptamers that target free load, and related screening technologies are also becoming mature. The main method is to screen antibodies or antigen-binding fragments and aptamers that bind to small molecule targets with high affinity and high specificity through specific methods for treatment, diagnosis or research. The main technologies include hybridoma technology, nanobody screening technology, computer-aided design (AI / structural biology), phage display technology, yeast display technology, single B cell cloning technology, microfluidic technology, surface plasmon resonance assisted screening and nucleic acid aptamer screening technology (SELEX). The hybridoma technology is the most traditional screening method, and the main principle is to fuse B cells of immunized animals (such as mice) with myeloma cells to form hybridoma cells, and screen clones secreting target antibodies. The advantage of phage display technology is that it does not require animal immunization and can screen for toxic small molecules or difficult-to-immune targets. The present application uses the above-mentioned technologies to screen antibodies, polypeptides or aptamers targeting small molecule drugs.

[0012] All references cited herein, including patent applications, patent publications, and UniProtKB / Swish Prot accession numbers, are incorporated herein by reference in their entirety, i.e., each individual reference is specifically and individually indicated to be incorporated by reference herein. SUMMARY

[0013] The application provides a ligand drug conjugate with a free load recycling unit and its application in preventing or treating tumors.

[0014] In a specific aspect, the present application provides a ligand drug conjugate comprising a ligand unit targeting a tumor antigen or a non-tumor antigen, a payload unit conjugated, and a recovery unit capable of binding to free payload shed from the ligand drug conjugate.

[0015] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of IgG, the recovery unit is in the form of VHH, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-VHH; (3) VH-CH1-CH2-CH3-VHH; and (4) VL-CL, the hexagonal star mark represents the conjugated or free payload, the schematic diagram is shown in Figure 1.

[0016] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of IgG, the recovery unit is in the form of scFv, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-scFv; (3) VH-CH1-CH2-CH3-scFv; and (4) VL-CL, the hexagonal star mark represents the conjugated or free payload, the schematic diagram is shown in Figure 2.

[0017] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of IgG, the recovery unit is in the form of peptide, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-peptide; (3) VH-CH1-CH2-CH3-peptide; and (4) VL-CL, the hexagonal star mark represents the conjugated or free payload, the schematic diagram is shown in Figure 3.

[0018] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of IgG, the recovery unit is in the form of aptamer, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-aptamer; (3) VH-CH1-CH2-CH3-aptamer; and (4) VL-CL, the hexagonal star mark represents the conjugated or free payload, the schematic diagram is shown in Figure 4.

[0019] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of VHH+Fc, the recovery unit is in the form of VHH, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VHH-CH2-CH3-VHH; and (2) VHH-CH2-CH3-VHH; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 5. 配体 -CH2-CH3-VHH 回收 ; and (2) VHH 配体 -CH2-CH3-VHH 回收 ; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 5.

[0020] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of VHH+Fc, the recovery unit is in the form of scFv, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VHH-CH2-CH3-scFv; and (2) VHH-CH2-CH3-scFv; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 6.

[0021] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of VHH+Fc, the recovery unit is in the form of polypeptide, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VHH-CH2-CH3-polypeptide; and (2) VHH-CH2-CH3-polypeptide; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 7.

[0022] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of VHH+Fc, the recovery unit is in the form of aptamer, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VHH-CH2-CH3-aptamer; and (2) VHH-CH2-CH3-aptamer; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 8.

[0023] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of polypeptide, the recovery unit is in the form of VHH, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: polypeptide-VHH; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 9.

[0024] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a polypeptide, the recovery unit is in the form of a scFv, the recovery unit is directly or indirectly linked to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: polypeptide-scFv; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 10.

[0025] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a polypeptide, the recovery unit is in the form of a polypeptide, the recovery unit is directly or indirectly linked to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: polypeptide 配体 - polypeptide 回收 ; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 11.

[0026] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a polypeptide, the recovery unit is in the form of an aptamer, the recovery unit is directly or indirectly linked to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: polypeptide-aptamer; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 12.

[0027] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a VHH, the recovery unit is in the form of an aptamer, the recovery unit is directly or indirectly linked to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: VHH-aptamer; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 13.

[0028] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a scFv, the recovery unit is in the form of an aptamer, the recovery unit is directly or indirectly linked to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: scFv-aptamer; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 14.

[0029] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of an aptamer, the recovery unit is in the form of a polypeptide, the recovery unit is directly or indirectly linked to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: aptamer-polypeptide; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 15.

[0030] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of an aptamer, the recovery unit is also in the form of an aptamer, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: aptamer 配体 - aptamer 回收 ; the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 16.

[0031] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a bispecific antibody, the recovery unit is in the form of VHH, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-VHH; (3) VH-CH1-CH2-CH3-VHH; and (4) VL-CL, the VH and VL of (1) and (2) form a binding site that specifically binds to a first antigen or a first antigen epitope, the VH and VL of (3) and (4) form a binding site that specifically binds to a second antigen or a second antigen epitope, the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 17.

[0032] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a bispecific antibody, the recovery unit is in the form of scFv, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-scFv; (3) VH-CH1-CH2-CH3-scFv; and (4) VL-CL, the VH and VL of (1) and (2) form a binding site that specifically binds to a first antigen or a first antigen epitope, the VH and VL of (3) and (4) form a binding site that specifically binds to a second antigen or a second antigen epitope, the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 18.

[0033] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a bispecific antibody, the recovery unit is in the form of a polypeptide, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-polypeptide; (3) VH-CH1-CH2-CH3-polypeptide; and (4) VL-CL, the VH and VL of (1) and (2) form a binding site that specifically binds to a first antigen or a first antigen epitope, the VH and VL of (3) and (4) form a binding site that specifically binds to a second antigen or a second antigen epitope, the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 19.

[0034] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of a bispecific antibody, the recovery unit is in the form of an aptamer, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-aptamer; (3) VH-CH1-CH2-CH3-aptamer; and (4) VL-CL, the VH and VL of (1) and (2) form a binding site that specifically binds to a first antigen or a first antigen epitope, the VH and VL of (3) and (4) form a binding site that specifically binds to a second antigen or a second antigen epitope, the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 20.

[0035] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of IgG, the recovery unit is in the form of VHH, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the load is conjugated on the VHH, the structure of the ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-VHH; (3) VH-CH1-CH2-CH3-VHH; and (4) VL-CL, the hexagonal star mark represents the conjugated or free load, the schematic diagram is shown in Figure 21.

[0036] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of IgG, the recovery unit is in the form of VHH, the recovery unit is connected to the C-terminus of the ligand unit through a linker unit; preferably, the load is coupled on the linker unit between IgG and VHH, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-VHH; (3) VH-CH1-CH2-CH3-VHH; and (4) VL-CL, the hexagonal star mark represents the coupled or free load, the schematic diagram is shown in FIG. 22.

[0037] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of IgG, the recovery unit is in the form of VHH, the recovery unit is directly connected to the C-terminus of the ligand unit; preferably, IgG and VHH are directly connected, i.e. there is no linker unit between the ligand unit and the recovery unit, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) VH-CH1-CH2-CH3-VHH; (3) VH-CH1-CH2-CH3-VHH; and (4) VL-CL, the hexagonal star mark represents the coupled or free load, the schematic diagram is shown in FIG. 23.

[0038] In some embodiments, the ligand drug conjugate comprises: the ligand unit is in the form of scFv, the recovery unit is in the form of IgG, the recovery unit is directly or indirectly connected to the C-terminus of the ligand unit; preferably, the structure of ligand unit-recovery unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) scFv-VH-CH1-CH2-CH3; (3) scFv-VH-CH1-CH2-CH3; and (4) VL-CL, the hexagonal star mark represents the coupled or free load, the schematic diagram is shown in FIG. 54.

[0039] In some embodiments, the ligand drug conjugate comprises: the recovery unit is in the form of scFv, the ligand unit is in the form of IgG, the recovery unit is directly or indirectly connected to the N-terminus of the ligand unit; preferably, the structure of recovery unit-ligand unit from N-terminus to C-terminus is as follows: (1) VL-CL; (2) scFv-VH-CH1-CH2-CH3; (3) scFv-VH-CH1-CH2-CH3; and (4) VL-CL, the hexagonal star mark represents the coupled or free load, the schematic diagram is shown in FIG. 56.

[0040] In some embodiments, the ligand unit and the recovery unit are directly connected or not directly connected.

[0041] In some embodiments, the ligand unit and the recycling unit are connected by a linker unit.

[0042] In some embodiments, the ligand drug conjugate further comprises other functional units.

[0043] In some embodiments, the payload is conjugated to the ligand unit and / or the recycling unit and / or the linker unit and / or the other functional unit by the linker.

[0044] In some embodiments, the ligand unit comprises an antibody or an antigen-binding fragment, a receptor, a polypeptide, an oligopeptide, a peptidomimetic, a fusion protein or an aptamer that specifically binds to a tumor antigen.

[0045] Further, the antibody comprises and is not limited to a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody (HcAb), an immunoglobulin new antigen receptor (IgNAR); the antigen-binding fragment comprises and is not limited to scFv, Fab, Fab', F(ab')2, Fd, Fv, dAb, Fd, sdAb, VHH, Diabody; the receptor comprises and is not limited to a T cell receptor.

[0046] Further, the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an artificially engineered antibody.

[0047] Further, the antibody further comprises a constant region of an immunoglobulin.

[0048] Further, the immunoglobulin is selected from human IgG1, IgG2, IgG3 or IgG4.

[0049] Further, the tumor antigen or non-tumor antigen includes, but is not limited to, CD9, CD19, CD20, CD22, CD24, CD25, CD29, CD30, CD33, CD37, CD44, CD45, CD46, CD47, CD49b, CD51, CD52, CD56, CD73 (NT5E), CD79b, CD123, CD133, CD138, CD157, CD166, DLL-3, CDH6, Tissue factor, EpCAM, PSMA, MUC1, MUC16, FOLR1, GPC3, ROR1, ROR2, PD-L1, ENPP3, TDGF1, MSLN, TIM-1, LRRC15, LIV-1 (ZIP6), Claudin6, Claudin9, Claudin18.2, Mesothelin, HER2 (ErbB2), HER3 (ErbB3), EGFR, c-MET, SLITRK6, KIT (CD117), STEAP1, NaPi2B (SLC34A2), SLC44A4, GPNMB, AXL, CD166, B7-H3 (CD276), B7-H4 (VTCN1), PTK7 (CCK4), EFNA4, 5T4, NOTCH3, Nectin-4, TROP-2, GPR20, EphA2, LYPD3, FGFR2, FGFR3, FRa, CEACAMs, CAIX, P-cadherin (CDH3), CDH17, GD3, Cadherin 6, LAMP1, FLT3, BCMA, SLTRK6, Lewis Y, ASCT2, CA-IX, Cripto, DPEP3, Globo H, Ly6E, RNF43, DR5, CDCP1, CEACAM6, FUT3, GD2, CD26E, CD70, CD74, CD163, PRLR, TNFa, CXCR4, LXR, IL-6, CEACAM5, GPRC5D, HLA-DR, CTLA4 (CD152), FAPa, or IL2R.

[0050] Further, the antibody specifically binding to a tumor antigen or a non-tumor antigen includes, but is not limited to, alemtuzumab, arcitumomab, evatumomab, atezolizumab, inotuzumab, anetumab, avelumab, bectumomab, bermekimab, bevacizumab, belimumab, vibostuzumab, catumaxomab, cintirelimab, cetuximab, clivatuzumab, dalotuzumab, daratumumab, desidustau, daratumumab beta, dostarlimab, durvalumab, edrecolomab, epratuzumab, erdafitinib, genmab, gemtuzumab, girentuximab, ibritumomab, inebilizumab, inotuzumab, ipilimumab, isatuximab, labetuzumab, loncastuximab, mogamulizumab, tiragolumab, mor002701, nimotuzumab, naratuximab, necitumumab, nivolumab, oregovomab, ofatumumab, olaratumab, orantinib, panitumumab, pembrolizumab, patritumab, vedotin, racotumomab, ramucirumab, retifanlimab, rituximab, sacituzumab, siltuximab, tocilizumab, tositumomab, trastuzumab, volociximab, zalutumumab, toripalimab, or zanolimumab; and antigen-binding fragments and derivatives of the above antibodies; the polypeptide, oligopeptide, fusion protein targeting binding to a specific antigen includes, but is not limited to, a bicycle, an angiopep-2, a linear peptide, a pH-sensitive peptide, SOR-C27, A6 polypeptide, SOR13; and variants and derivatives of the above polypeptide, oligopeptide or fusion protein; the aptamer targeting binding to a specific antigen includes AS1411, sgc8, sgc4f, sgd5a, TC01, TD05, A9, A10, APT, Min.2, AIR-3A, E3, E07, Waz, P19, Zy1; and variants and derivatives of the above aptamer. The antibody or antigen-binding fragment, receptor fragment or polypeptide, oligopeptide, peptidomimetic, fusion protein or aptamer targeting binding to a specific antigen can be obtained by existing screening techniques, or artificial design and computer-aided design or artificial intelligence-assisted development according to the targeted tumor antigen or non-tumor antigen and binding requirements.

[0051] Further, the ligand drug conjugate can specifically bind to the free load, and the ligand drug conjugates do not aggregate.

[0052] Further, the recovery unit specifically binds to the free load.

[0053] Further, the recovery unit comprises an antibody or an antigen-binding fragment, a receptor, a fusion protein, a polypeptide, an oligopeptide, a peptidomimetic or an aptamer that specifically binds to the free payload.

[0054] Further, the antibody that specifically binds to the free payload comprises, but is not limited to, a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody (HcAb), an immunoglobulin new antigen receptor (IgNAR); the antigen-binding fragment comprises, but is not limited to, an scFv, an Fab, an Fab', an F(ab')2, an Fd, an Fv, a dAb, an Fd, an sdAb, a VHH, a Diabody; and the receptor comprises, but is not limited to, a T cell receptor.

[0055] Further, the antibody that specifically binds to the free payload is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an artificially engineered antibody.

[0056] Further, the recovery unit is capable of specifically binding to the free payload means that the recovery unit has an equilibrium dissociation constant K D (M) less than 10 -4 , or further less than 10 -5 , or further less than 10 -6 , or further less than 10 -7 , or further less than 10 -8 , or further less than 10 -9 , or further less than 10 -10 , or further less than 10 -11 , or further less than 10 -12 , or further less than 10 -13 .

[0057] Further, the recovery unit does not bind to, or has a lower affinity for, the payload in the conjugated state in the ligand drug conjugate, and the lower affinity can also achieve that no molecular aggregation occurs between the ligand drug conjugate molecules.

[0058] Further, the recovery unit specifically targets a non-exposed epitope on the conjugated payload unit.

[0059] Further, the linker or the payload is connected with a payload epitope masking unit that can prevent the recovery unit from binding to the payload still conjugated to the ligand drug conjugate.

[0060] Further, the load epitope shielding unit does not affect the normal separation of the ligand drug conjugate into the target cell after entering the target cell.

[0061] Still further, the recycling unit is one, two or more.

[0062] Further, the recovery unit comprises: (1) a VHH domain selected from CDR1-3 defined by the amino acid sequences set forth in: CDR1-3 set forth in SEQ ID NO: 103, 104, and 105; or (2) an amino acid sequence selected from: the amino acid sequence set forth in SEQ ID NO: 102; or (3) an amino acid sequence selected from a combination of a VH and a VL comprising: a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 2, 3, and 4, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 6, YAS, and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 9, 10, and 11, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 6, FAS, and SEQ ID NO: 13, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 2, 3, and 4, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 6, YAS, and SEQ ID NO: 16, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 2, 3, and 20, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 6, YAS, and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 22, 23, and 24, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 26, WAS, and SEQ ID NO: 27, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 29, 30, and 31, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 33, KVS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 29, 36, and 37, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 33, KLS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 40, 41, and 42, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 44, KVS, and SEQ ID NO: 45, respectively.a VH comprising HCDR1-3 as set forth in SEQ ID NO: 47, 48, and 42, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 50, KVS, and SEQ ID NO: 51, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 53, 54, and 55, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 57, DTS, and SEQ ID NO: 58, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 62, 63, and 64, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 66, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 69, 70, and 71, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 73, WAS, and SEQ ID NO: 74, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 80, GAS, and SEQ ID NO: 81, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 83, 84, and 85, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 87, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 89, 90, and 91, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 93, HGT, and SEQ ID NO: 94, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 99, 77, and 100, respectively; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 5; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 8, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 14, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 15; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 17, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 18;the VH comprises an amino acid sequence of SEQ ID NO: 19, and the VL comprises an amino acid sequence of SEQ ID NO: 18; the VH comprises an amino acid sequence of SEQ ID NO: 21, and the VL comprises an amino acid sequence of SEQ ID NO: 25; the VH comprises an amino acid sequence of SEQ ID NO: 28, and the VL comprises an amino acid sequence of SEQ ID NO: 32; the VH comprises an amino acid sequence of SEQ ID NO: 35, and the VL comprises an amino acid sequence of SEQ ID NO: 38; the VH comprises an amino acid sequence of SEQ ID NO: 39, and the VL comprises an amino acid sequence of SEQ ID NO: 43; the VH comprises an amino acid sequence of SEQ ID NO: 46, and the VL comprises an amino acid sequence of SEQ ID NO: 49; the VH comprises an amino acid sequence of SEQ ID NO: 52, and the VL comprises an amino acid sequence of SEQ ID NO: 56; the VH comprises an amino acid sequence of SEQ ID NO: 59, and the VL comprises an amino acid sequence of SEQ ID NO: 60; the VH comprises an amino acid sequence of SEQ ID NO: 61, and the VL comprises an amino acid sequence of SEQ ID NO: 65; the VH comprises an amino acid sequence of SEQ ID NO: 68, and the VL comprises an amino acid sequence of SEQ ID NO: 72; the VH comprises an amino acid sequence of SEQ ID NO: 75, and the VL comprises an amino acid sequence of SEQ ID NO: 79; the VH comprises an amino acid sequence of SEQ ID NO: 82, and the VL comprises an amino acid sequence of SEQ ID NO: 86; the VH comprises an amino acid sequence of SEQ ID NO: 88, and the VL comprises an amino acid sequence of SEQ ID NO: 92; the VH comprises an amino acid sequence of SEQ ID NO: 95, and the VL comprises an amino acid sequence of SEQ ID NO: 96; the VH comprises an amino acid sequence of SEQ ID NO: 97, and the VL comprises an amino acid sequence of SEQ ID NO: 98; the CDR1, CDR2, CDR3 sequences, the HCDR1, HCDR2, HCDR3 sequences, and the LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT definition scheme;or (4) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129; or (5) an aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO: 162.

[0063] Further, the recovery unit is obtained by immunizing a non-human animal with a conjugate of a small molecule drug and a carrier protein or a biotin-small molecule drug conjugate; or obtained by screening using phage display technology; or obtained by screening using virtual technology; or obtained by screening using SELEX technology; or obtained by screening using hybridoma technology; or obtained by screening using nanobody technology; or obtained by using yeast display technology; or obtained by using single B cell cloning technology; or obtained by using microfluidic technology; or obtained by using surface plasmon resonance assisted screening technology.

[0064] Further, the small molecule drug includes but is not limited to MMAE, MMAD, DM1, DM2, DM4, eribulin, Exatecan, SN38, DXD, calicheamicin, pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IBD), duocarmycin, amanitin, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, MMAF, ML022-Dxd, ML022-D633-004, ML026-Dxd, ML031-D633-004, ML037-D633-004, ML042-Dxd, ML047-DX8951, MPA-Dxd, Preferably, the carrier protein includes but is not limited to KLH, BSA or OVA.

[0065] Further, the biotin-small molecule drug conjugate includes but is not limited to Biotin-PEG12-Dxd, Biotin-PEG10-Dxd, Biotin-PEG6-Dxd, Biotin-PEG6-Triazole-Dxd or Biotin-PEG10-Triazole-Dxd:

[0066] Further, the coupled load includes a cytotoxic molecule, a nucleic acid, an immunostimulant and a modulator.

[0067] Further, the number of loads is one, two or more.

[0068] Further, the number of loads is 1, 2, 3, 4, 5, 6, 7, 8 or more.

[0069] Further, when two or more loads are coupled, the loads can be the same load or different loads.

[0070] Further, the load is coupled to the ligand unit and / or the recycling unit and / or the linker unit and / or other functional unit by site-specific or random coupling.

[0071] Further, the cytotoxic molecule includes but is not limited to microtubulin inhibitor, DNA inhibitor or other toxin molecule; the DNA inhibitor further includes DNA damaging agent or topoisomerase I inhibitor.

[0072] Further, the microtubulin inhibitor further includes but is not limited to Auristatin, Maytansinoid Derivative, tubulysin, halichondrin, Cryptophycin, EG5 inhibitor.

[0073] Further, the Auristatin further includes but is not limited to Auristatin F (AF), monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF); the Maytansinoid Derivative further includes but is not limited to DM1, DM2, DM4; the halichondrin further includes but is not limited to eribulin.

[0074] Further, the DNA damaging agent further includes but is not limited to calicheamicin, pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IBD), duocarmycin; the topoisomerase I inhibitor further includes but is not limited to camptothecin derivative.

[0075] Further, the camptothecin derivative further includes but is not limited to Exatecan, SN38, DXD.

[0076] Further, the other toxin molecule includes but is not limited to amanitin, Maaa-1181a, taxol, daunomycin, vinblastine, Doxorubicin, methotrexate or its radioisotope and / or pharmaceutically acceptable salt.

[0077] Further, the nucleic acid used in the conjugated payload includes but is not limited to antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), oligonucleotide (AOC).

[0078] Further, the nucleic acid used in the conjugated payload includes but is not limited to DMPK siRNA, Exon-44-skipping PMO, DUX4 siRNA, Exon-51-skipping PMO, DMPK ASO, DUX4 ASO, CpG.

[0079] Further, the immunostimulant used in the conjugated payload includes but is not limited to TLR7 agonist, TLR8 agonist, TLR9 agonist, TGF-β inhibitor, TNIK inhibitor, Sting agonist.

[0080] Further, the modulator used in the conjugated payload includes but is not limited to glucocorticoid receptor modulator (such as dexamethasone, budesonide, fluticasone propionate, glucocorticoid), antibiotic (such as rifalogue), kinase inhibitor (such as dasatinib), liver X receptor (LXR) agonist, phosphodiesterase (PDE4) inhibitor, bisphosphonate (alendronate).

[0081] Further, the structure after the free payload is selected from the group consisting of the structure shown below or the isomer or deuterated compound of the following structure:

[0082] Further, the connecting unit includes but is not limited to a peptide linker, an Fc domain or a fragment thereof; preferably the peptide linker is a flexible peptide linker; further preferably the peptide linker contains one or more amino acids; further preferably the peptide linker contains at least 5 amino acids; or the amino acid sequence of the peptide linker contains: (GGGGS)n, (VPGXG)n, (SG)n, (EAAAK)n, (G)n, (R)n, A(EAAAK)n ALEA(EAAAK)nA, A(EAAAK)nA, (AP)n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, E DVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, GFLG, LE, wherein n is equal to 1, 2, 3 or 4.

[0083] In another aspect, the present application provides a ligand drug conjugate in a pharmaceutically acceptable salt, deuteride, solvate.

[0084] In another aspect, the present application provides a pharmaceutical composition comprising the ligand drug conjugate provided by the present application or a pharmaceutically acceptable salt, deuteride, solvate thereof and a pharmaceutically acceptable excipient.

[0085] In another aspect, the present application provides a use of a recovery unit capable of binding to a free load detached from the ligand drug conjugate in the preparation of a ligand drug conjugate with a free load recovery unit.

[0086] Further, the recovery unit comprises an antibody or an antigen-binding fragment, a receptor, a fusion protein, a polypeptide, an oligopeptide, a peptidomimetic or an aptamer that specifically binds to the free load.

[0087] Further, the antibody that specifically binds to the free load includes, but is not limited to, a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody (HcAb), an immunoglobulin new antigen receptor (IgNAR); the antigen-binding fragment includes, but is not limited to, scFv, Fab, Fab', F(ab')2, Fd, Fv, dAb, Fd, sdAb, VHH, Diabody; and the receptor includes, but is not limited to, a T cell receptor.

[0088] Further, the antibody that specifically binds to the free load is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an artificially modified antibody.

[0089] Further, the recovery unit capable of specifically binding to the free load means that the equilibrium dissociation constant KD(M) of the recovery unit to the free load is less than 10 -4 , or more than 10 -5 , or more than 10 -6 , or more than 10 -7 , or more than 10 -8 , or more than 10 -9 , or more than 10 -10 , or more than 10 -11 , or more than 10 -12 , or more than 10 -13 .

[0090] Further, the recycling unit does not bind or has a lower affinity to the payload in the conjugated state in the ligand drug conjugate, and the lower affinity can also prevent molecular aggregation between the ligand drug conjugate molecules.

[0091] Further, the recovery unit comprises: (1) a VHH domain selected from CDR1-3 defined by the amino acid sequences set forth in: SEQ ID NO: 103, 104, and 105; or (2) an amino acid sequence selected from SEQ ID NO: 102; or (3) an amino acid sequence selected from a combination of VH and VL comprising: a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 2, 3, and 4, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 6, YAS, and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 9, 10, and 11, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 6, FAS, and SEQ ID NO: 13, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 2, 3, and 4, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 6, YAS, and SEQ ID NO: 16, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 2, 3, and 20, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 6, YAS, and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 22, 23, and 24, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 26, WAS, and SEQ ID NO: 27, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 29, 30, and 31, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 33, KVS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 29, 36, and 37, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 33, KLS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 40, 41, and 42, respectively, and a VL comprising LCDR1-3 having the amino acid sequences set forth in SEQ ID NO: 44, KVS, and SEQ ID NO: 45, respectively.a VH comprising HCDR1-3 as set forth in SEQ ID NO: 47, 48, and 42, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 50, KVS, and SEQ ID NO: 51, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 53, 54, and 55, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 57, DTS, and SEQ ID NO: 58, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 62, 63, and 64, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 66, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 69, 70, and 71, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 73, WAS, and SEQ ID NO: 74, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 80, GAS, and SEQ ID NO: 81, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 83, 84, and 85, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 87, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 89, 90, and 91, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 93, HGT, and SEQ ID NO: 94, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 99, 77, and 100, respectively; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 5; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 8, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 14, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 15; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 17, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 18;the VH comprises an amino acid sequence of SEQ ID NO: 19, and the VL comprises an amino acid sequence of SEQ ID NO: 18; the VH comprises an amino acid sequence of SEQ ID NO: 21, and the VL comprises an amino acid sequence of SEQ ID NO: 25; the VH comprises an amino acid sequence of SEQ ID NO: 28, and the VL comprises an amino acid sequence of SEQ ID NO: 32; the VH comprises an amino acid sequence of SEQ ID NO: 35, and the VL comprises an amino acid sequence of SEQ ID NO: 38; the VH comprises an amino acid sequence of SEQ ID NO: 39, and the VL comprises an amino acid sequence of SEQ ID NO: 43; the VH comprises an amino acid sequence of SEQ ID NO: 46, and the VL comprises an amino acid sequence of SEQ ID NO: 49; the VH comprises an amino acid sequence of SEQ ID NO: 52, and the VL comprises an amino acid sequence of SEQ ID NO: 56; the VH comprises an amino acid sequence of SEQ ID NO: 59, and the VL comprises an amino acid sequence of SEQ ID NO: 60; the VH comprises an amino acid sequence of SEQ ID NO: 61, and the VL comprises an amino acid sequence of SEQ ID NO: 65; the VH comprises an amino acid sequence of SEQ ID NO: 68, and the VL comprises an amino acid sequence of SEQ ID NO: 72; the VH comprises an amino acid sequence of SEQ ID NO: 75, and the VL comprises an amino acid sequence of SEQ ID NO: 79; the VH comprises an amino acid sequence of SEQ ID NO: 82, and the VL comprises an amino acid sequence of SEQ ID NO: 86; the VH comprises an amino acid sequence of SEQ ID NO: 88, and the VL comprises an amino acid sequence of SEQ ID NO: 92; the VH comprises an amino acid sequence of SEQ ID NO: 95, and the VL comprises an amino acid sequence of SEQ ID NO: 96; the VH comprises an amino acid sequence of SEQ ID NO: 97, and the VL comprises an amino acid sequence of SEQ ID NO: 98; the CDR1, CDR2, CDR3 sequences, the HCDR1, HCDR2, HCDR3 sequences, and the LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT definition scheme;or (4) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129; or (5) an aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO: 162.

[0092] Further, the recovery unit is obtained by immunizing a non-human animal with a conjugate of a small molecule drug and a carrier protein or a biotin-small molecule drug conjugate; or obtained by screening using phage display technology; or obtained by screening using virtual technology; or obtained by screening using SELEX technology; or obtained by screening using hybridoma technology; or obtained by screening using nanobody technology; or obtained by using yeast display technology; or obtained by using single B cell cloning technology; or obtained by using microfluidic technology; or obtained by using surface plasmon resonance assisted screening technology.

[0093] Further, the small molecule drug includes but is not limited to MMAE, MMAD, DM1, DM2, DM4, eribulin, Exatecan, SN38, DXD, calicheamicin, pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IBD), duocarmycin, amanitin, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, MMAF, ML022-Dxd, ML022-D633-004, ML026-Dxd, ML031-D633-004, ML037-D633-004, ML042-Dxd, ML047-DX8951, MPA-Dxd, Preferably, the carrier protein includes but is not limited to KLH, BSA or OVA.

[0094] Further, the biotin-small molecule drug conjugate includes but is not limited to Biotin-PEG12-Dxd, Biotin-PEG10-Dxd, Biotin-PEG6-Dxd, Biotin-PEG6-Triazole-Dxd or Biotin-PEG10-Triazole-Dxd:

[0095] Further, the linker or payload is connected with a payload epitope masking unit capable of preventing the recovery unit from binding to the payload still conjugated to the ligand drug conjugate.

[0096] Further, the payload epitope masking unit does not affect the normal separation of the payload after the ligand drug conjugate enters the target cell.

[0097] The present application also provides a method for preparing the ligand drug conjugate, comprising the following steps: 1) screening to obtain an antibody or an antigen-binding fragment, a receptor, a fusion protein, a polypeptide, an oligopeptide, a peptidomimetic or an aptamer which specifically binds to the payload; 2) preparing a ligand drug conjugate using the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer which specifically binds to the payload as a recovery unit; 3) detecting whether the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer binds to the prepared ligand drug conjugate, and if not, obtaining a target ligand drug conjugate; or detecting whether the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer binds to the prepared drug-linker, and if not, obtaining a target ligand drug conjugate. 4) If the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer binds to the prepared ligand drug conjugate or drug-linker, or molecular condensation occurs between the prepared ligand drug conjugates: A. further modifying or replacing the antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer in the recovery unit until no molecular condensation occurs between the ligand drug conjugates and no binding to the target ligand drug conjugate; and / or B. screening a payload epitope shielding unit which can be connected to the linker or payload of the ligand drug conjugate, the connected payload epitope shielding unit will prevent the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer from binding to the prepared antibody drug conjugate and will not affect the normal release of the ligand drug conjugate into the cell after the ligand drug conjugate enters the cell, and after screening a suitable payload epitope shielding unit, a target ligand drug conjugate is prepared.

[0098] The present application also provides a method for treating or preventing a disease, comprising administering a prophylactically or therapeutically effective amount of the ligand drug conjugate or a pharmaceutically acceptable salt, deuteride, solvate thereof or the pharmaceutical composition or a pharmaceutically acceptable salt, deuteride, solvate thereof and a pharmaceutically acceptable adjuvant thereof to a subject in need thereof. The disease is preferably a tumor.

[0099] The present application also provides the use of the ligand drug conjugate or a pharmaceutically acceptable salt, deuteride, solvate thereof or the prepared pharmaceutical composition in the preparation of a medicament for treating or preventing a disease.

[0100] The present application also provides an immunogen for screening a recovery unit.

[0101] Further, the immunogen includes but is not limited to MMAF, ML022-Dxd, ML022-D633-004, ML026-Dxd, ML031-D633-004, ML037-D633-004, ML042-Dxd, ML047-DX8951, MPA-Dxd, Biotin-PEG12-Dxd, Biotin-PEG10-Dxd, Biotin-PEG6-Dxd, Biotin-PEG6-Triazole-Dxd, Biotin-PEG10-Triazole-Dxd and conjugates of the said MMAF, ML022-Dxd, ML022-D633-004, ML026-Dxd, ML031-D633-004, ML037-D633-004, ML042-Dxd, ML047-DX8951, MPA-Dxd with carrier proteins.

[0102] Further, the carrier protein includes but is not limited to KLH, BSA or OVA.

[0103] The application also provides a load recovery unit capable of specifically binding to the free load of the ligand drug conjugate. Further, the load recovery unit comprises: (1) a VHH domain selected from CDR1-3 defined by the following amino acid sequences: CDR1-3 shown as SEQ ID NO: 103, 104 and 105; or (2) selected from the amino acid sequence shown as SEQ ID NO: 102; or (3) selected from the amino acid sequence comprising the combination of VH and VL defined as follows: VH comprising HCDR1-3 with the amino acid sequences shown as SEQ ID NO: 2, 3 and 4 respectively, and VL comprising LCDR1-3 with the amino acid sequences shown as SEQ ID NO: 6, YAS and SEQ ID NO: 7 respectively; VH comprising HCDR1-3 with the amino acid sequences shown as SEQ ID NO: 9, 10 and 11 respectively, and VL comprising LCDR1-3 with the amino acid sequences shown as SEQ ID NO: 6, FAS and SEQ ID NO: 13 respectively; VH comprising HCDR1-3 with the amino acid sequences shown as SEQ ID NO: 2, 3 and 4 respectively, and VL comprising LCDR1-3 with the amino acid sequences shown as SEQ ID NO: 6, YAS and SEQ ID NO: 16 respectively; VH comprising HCDR1-3 with the amino acid sequences shown as SEQ ID NO: 2, 3 and 20 respectively, and VL comprising LCDR1-3 with the amino acid sequences shown as SEQ ID NO: 6, YAS and SEQ ID NO: 7 respectively; VH comprising HCDR1-3 with the amino acid sequences shown as SEQ ID NO: 22, 23 and 24 respectively, and VL comprising LCDR1-3 with the amino acid sequences shown as SEQ ID NO: 26, WAS and SEQ ID NO: 27 respectively; VH comprising HCDR1-3 with the amino acid sequences shown as SEQ ID NO: 29, 30 and 31 respectively, and VL comprising LCDR1-3 with the amino acid sequences shown as SEQ ID NO: 33, KVS and SEQ ID NO: 34 respectively; VH comprising HCDR1-3 with the amino acid sequences shown as SEQ ID NO: 29, 36 and 37 respectively, and VL comprising LCDR1-3 with the amino acid sequences shown as SEQ ID NO: 33, KLS and SEQ ID NO: 34 respectively; VH comprising HCDR1-3 with the amino acid sequences shown as SEQ ID NO: 40, 41 and 42 respectively, and VL comprising LCDR1-3 with the amino acid sequences shown as SEQ ID NO: 44, KVS and SEQ ID NO: 45 respectively.a VH comprising HCDR1-3 as set forth in SEQ ID NO: 47, 48, and 42, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 50, KVS, and SEQ ID NO: 51, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 53, 54, and 55, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 57, DTS, and SEQ ID NO: 58, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 62, 63, and 64, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 66, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 69, 70, and 71, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 73, WAS, and SEQ ID NO: 74, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 80, GAS, and SEQ ID NO: 81, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 83, 84, and 85, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 87, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 89, 90, and 91, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 93, HGT, and SEQ ID NO: 94, respectively; a VH comprising HCDR1-3 as set forth in SEQ ID NO: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 as set forth in SEQ ID NO: 99, 77, and 100, respectively; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 5; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 8, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 14, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 15; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 17, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 18;the VH comprises an amino acid sequence of SEQ ID NO: 19, and the VL comprises an amino acid sequence of SEQ ID NO: 18; the VH comprises an amino acid sequence of SEQ ID NO: 21, and the VL comprises an amino acid sequence of SEQ ID NO: 25; the VH comprises an amino acid sequence of SEQ ID NO: 28, and the VL comprises an amino acid sequence of SEQ ID NO: 32; the VH comprises an amino acid sequence of SEQ ID NO: 35, and the VL comprises an amino acid sequence of SEQ ID NO: 38; the VH comprises an amino acid sequence of SEQ ID NO: 39, and the VL comprises an amino acid sequence of SEQ ID NO: 43; the VH comprises an amino acid sequence of SEQ ID NO: 46, and the VL comprises an amino acid sequence of SEQ ID NO: 49; the VH comprises an amino acid sequence of SEQ ID NO: 52, and the VL comprises an amino acid sequence of SEQ ID NO: 56; the VH comprises an amino acid sequence of SEQ ID NO: 59, and the VL comprises an amino acid sequence of SEQ ID NO: 60; the VH comprises an amino acid sequence of SEQ ID NO: 61, and the VL comprises an amino acid sequence of SEQ ID NO: 65; the VH comprises an amino acid sequence of SEQ ID NO: 68, and the VL comprises an amino acid sequence of SEQ ID NO: 72; the VH comprises an amino acid sequence of SEQ ID NO: 75, and the VL comprises an amino acid sequence of SEQ ID NO: 79; the VH comprises an amino acid sequence of SEQ ID NO: 82, and the VL comprises an amino acid sequence of SEQ ID NO: 86; the VH comprises an amino acid sequence of SEQ ID NO: 88, and the VL comprises an amino acid sequence of SEQ ID NO: 92; the VH comprises an amino acid sequence of SEQ ID NO: 95, and the VL comprises an amino acid sequence of SEQ ID NO: 96; the VH comprises an amino acid sequence of SEQ ID NO: 97, and the VL comprises an amino acid sequence of SEQ ID NO: 98; the CDR1, CDR2, CDR3 sequences, the HCDR1, HCDR2, HCDR3 sequences, and the LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT definition scheme;or (4) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129; or (5) an aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO: 162.

[0104] The present application also provides a method for reducing the toxicity of a ligand drug conjugate, further, in a method for treating a patient with a ligand drug conjugate, administering to the patient an effective amount of a corresponding payload recovery unit capable of binding to a free payload in the ligand drug conjugate.

[0105] Further, the ligand drug conjugate and the payload recovery unit can be administered simultaneously or sequentially.

[0106] The present application also provides a nucleic acid encoding the amino acid sequence of the payload recovery unit.

[0107] The present application also provides a vector, further, the vector comprises the nucleic acid.

[0108] The present application also provides a cell, further, the cell comprises the vector. Still further, the cell is selected from CHO cell, COS cell, HEK-293 cell, NSO cell, Sp2.0 cell. BRIEF DESCRIPTION OF DRAWINGS

[0109] Figure 1 shows a schematic diagram of ligand-drug conjugate (the ligand unit is IgG form, the recovery unit is VHH which specifically binds to free payload, the hexagonal star mark represents conjugated or free payload, the same below)

[0110] Figure 2 shows a schematic diagram of ligand-drug conjugate (the ligand unit is IgG form, the recovery unit is scFv which specifically binds to free payload)

[0111] Figure 3 shows a schematic diagram of ligand-drug conjugate (the ligand unit is IgG form, the recovery unit is polypeptide which specifically binds to free payload)

[0112] Figure 4 shows a schematic diagram of ligand-drug conjugate (the ligand unit is IgG form, the recovery unit is aptamer which specifically binds to free payload)

[0113] Figure 5 shows a schematic diagram of ligand-drug conjugate (the ligand unit is VHH+Fc form, the recovery unit is VHH which specifically binds to free payload)

[0114] Figure 6 shows a schematic diagram of ligand-drug conjugate (the ligand unit is VHH+Fc form, the recovery unit is scFv which specifically binds to free payload)

[0115] Figure 7 shows a schematic diagram of ligand-drug conjugate (the ligand unit is VHH+Fc form, the recovery unit is polypeptide which specifically binds to free payload)

[0116] Figure 8 shows a schematic diagram of ligand-drug conjugate (the ligand unit is VHH+Fc form, the recovery unit is aptamer which specifically binds to free payload)

[0117] Figure 9 shows a schematic diagram of ligand-drug conjugate (the ligand unit is polypeptide form, the recovery unit is VHH which specifically binds to free payload, the schematic diagram does not represent that the payload is conjugated to the first amino acid site of the polypeptide, but can be conjugated to any suitable site of the polypeptide, the same below)

[0118] Figure 10 Ligand drug conjugate schematic (ligand unit is in polypeptide form, recycling unit is a scFv that specifically binds to free payload)

[0119] Figure 11 Ligand drug conjugate schematic (ligand unit is in polypeptide form, recycling unit is a polypeptide that specifically binds to free payload)

[0120] Figure 12 Ligand drug conjugate schematic (ligand unit is in polypeptide form, recycling unit is an aptamer that specifically binds to free payload)

[0121] Figure 13 Ligand drug conjugate schematic (ligand unit is in VHH form, recycling unit is an aptamer that specifically binds to free payload)

[0122] Figure 14 Ligand drug conjugate schematic (ligand unit is in scFv form, recycling unit is an aptamer that specifically binds to free payload)

[0123] Figure 15 Ligand drug conjugate schematic (ligand unit is in aptamer form, recycling unit is a polypeptide that specifically binds to free payload)

[0124] Figure 16 Ligand drug conjugate schematic (ligand unit is in aptamer form, recycling unit is an aptamer that specifically binds to free payload)

[0125] Figure 17 Ligand drug conjugate schematic (ligand unit is in bispecific antibody form, recycling unit is a VHH that specifically binds to free payload)

[0126] Figure 18 Ligand drug conjugate schematic (ligand unit is in bispecific antibody form, recycling unit is a scFv that specifically binds to free payload)

[0127] Figure 19 Ligand drug conjugate schematic (ligand unit is in bispecific antibody form, recycling unit is a polypeptide that specifically binds to free payload)

[0128] Figure 20 Ligand drug conjugate schematic (ligand unit is in bispecific antibody form, recycling unit is an aptamer that specifically binds to free payload)

[0129] Figure 21 Ligand drug conjugate recycling unit specifically binds to free payload schematic (ligand unit is in IgG form, recycling unit is a VHH that specifically binds to free payload, payload is conjugated to VHH)

[0130] Figure 22 Ligand drug conjugate schematic (ligand unit is in IgG form, recycling unit is a VHH that specifically binds to free payload, payload is conjugated to a linker unit between IgG and VHH)

[0131] Figure 23 Schematic diagram of ligand drug conjugate (ligand unit is IgG form, recovery unit is VHH specifically binding free load, IgG and VHH are directly connected, i.e. no connecting unit between ligand unit and recovery unit)

[0132] Figure 24 Results of cell protection experiment of chimeric antibody No. 62, 64

[0133] Figure 25 Cell protection activity of PP-1M-#60 and PP-1M-#66 molecules

[0134] Figure 26 Results of cell protection experiment of chimeric antibody No. J4

[0135] Figure 27 Results of cell protection experiment of antibodies No. DXD1-1, DXD1-2, DXD2-1, DXD2-2, DXD5, DXD8, DXD10

[0136] Figure 28 Results of cell protection experiment of antibodies No. JLD1-JLD27

[0137] Figure 29 Results of cell protection experiment of antibodies No. D2 and the like

[0138] Figure 30 Cell protection activity of PP-1X-#9 and PP-1X-#17 molecules

[0139] Figure 31 Results of experiment of specific binding of antibody No. 86 to DXD

[0140] Figure 32 Results of cell protection experiment of antibody No. 86

[0141] Figure 33 Schematic diagram of structure of anti-MMAE polypeptide + Fc fusion molecule

[0142] Figure 34 Results of experiment of specific binding of each molecule to OVA-MMAF, OVA

[0143] Figure 35 Identification results of free MMAE competitively binding polypeptide molecule

[0144] Figure 36 Schematic diagram of structure of anti-MMAE polypeptide + VHH + Fc fusion molecule

[0145] Figure 37 Verification of specific binding of PP-1A-#600 fusion molecule to MMAE

[0146] Figure 38 Cell protection experiment of PP-1A-#600 fusion molecule

[0147] Figure 39 Schematic diagram of structure of anti-DXD polypeptide + Fc fusion molecule

[0148] Figure 40 Results of dot blot experiment

[0149] Figure 41 Cell protection experiment of PP-1A polypeptide

[0150] Figure 42 Results of experiments on specific binding of fusion molecules to OVA-MMAF and BSA-MMAF

[0151] Figure 43 Results of experiments on competition ELISA of PP-1B-#74 molecules with OVA-MMAF and BSA-MMAF

[0152] Figure 44 Results of experiments on specific binding of PP-1D molecules to BSA-MMAF and OVA-MMAF

[0153] Figure 45 Schematic diagram of antibody-adaptor conjugate (targeting MMAE)

[0154] Figure 46 Results of experiments on specific binding of molecules to OVA-DXD, OVA or BSA-DXD, BSA

[0155] Figure 47 Results of experiments on competition ELISA binding of molecules

[0156] Figure 48 Schematic diagram of antibody-adaptor conjugate (targeting DXD)

[0157] Figure 49 Results of experiments on ELISA binding

[0158] Figure 50 Results of experiments on cell protection effect of molecules (inhibition of DXD toxicity)

[0159] Figure 51 Results of PR-ADC cytotoxicity test

[0160] Figure 52 Results of efficacy study of PR-ADC in a human bladder cancer cell HT-1376 subcutaneous xenograft model

[0161] Figure 53 Efficacy study of PR-ADC in a human colon cancer cell RKO subcutaneous xenograft model

[0162] Figure 54 Schematic diagram of ligand-drug conjugate (ligand unit is in the form of scFv, recovery unit is IgG that specifically binds to free load, and load is conjugated to scFv)

[0163] Figure 55 Schematic diagram of bispecific antibody-linker-drug (MMAE)

[0164] Figure 56 Schematic diagram of ligand-drug conjugate (ligand unit is in the form of IgG, and recovery unit is scFv that specifically binds to free load)

[0165] Figure 57 Schematic diagram of conventional antibody-drug conjugate

[0166] Figure 58 Schematic diagram of bispecific antibody-linker-drug (DXD) DETAILED DESCRIPTION

[0167] In the present application, the term "ligand drug conjugate" refers to a class of drugs that can be used to inhibit the proliferation of tumor or cancer cells and for treating cancer or for treating diseases such as autoimmune or inflammatory diseases in a patient. The ligand drug conjugate is used accordingly in a variety of settings to treat cancer, autoimmune diseases or inflammatory diseases and the like. The ligand drug conjugate is used to deliver a drug to a tumor cell, cancer cell or other cell. Without being bound by theory, in some embodiments, the ligand unit of the ligand drug conjugate will bind or conjugate to a relevant antigen of a tumor or non-tumor target, and the ligand drug conjugate is taken up within the tumor cell, cancer cell or other cell by receptor-mediated endocytosis or other internalization mechanisms. In some embodiments, the antigen is attached to the tumor cell, cancer cell or other cell, or can be an extracellular matrix protein associated with the tumor cell, cancer cell or other cell. Once inside the cell, the payload is released intracellularly via activation of the activation unit.

[0168] In the present application, the term "tumor" generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. In embodiments of the present application, the tumor can include a solid tumor and / or a blood tumor. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive when referred to herein. In some embodiments, a tumor can refer to a mass of tissue containing a plurality of cancer cells, e.g., cells exhibiting the characteristics of any of the cancers described herein. Examples of a tumor can include a primary tumor of any of the above types of cancer or a metastatic tumor at a secondary site derived from any of the above types of cancer.

[0169] In the present application, the term "tumor antigen" generally includes the meaning known in the art, which includes any molecule expressed on (or associated with the development of) a tumor cell, known or believed to have a role in the tumorigenic properties of the tumor cell. Many tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined according to techniques and assays well known to those skilled in the art, such as clonogenic assays, transformation assays, in vitro or in vivo tumor formation assays, gel migration assays, gene knockout analysis, and the like. The term "tumor antigen" can refer to a human transmembrane protein, i.e., a cell membrane protein anchored in the cell lipid bilayer.

[0170] In the present application, the term "ligand unit" refers to a class of small or large molecule compounds that can specifically bind to biomolecules (such as proteins, nucleic acids, receptors, etc.). In the present application, the ligand unit can be, but is not limited to, an antibody or antigen-binding fragment that specifically binds to a tumor antigen, a receptor, a polypeptide, an oligopeptide, a peptidomimetic, a fusion protein, or an aptamer.

[0171] In the present application, the term "payload" refers to a drug molecule that can cause cytotoxicity, for example, any molecule used for treating tumors or cancers can be used, including compounds, DNA, RNA, polypeptides, immunostimulants, etc., or natural drugs (e.g., drugs that naturally contain one or more functional groups that allow covalent attachment to the conjugate), or drugs that are chemically modified to incorporate a functional group (e.g., a group selected from hydroxyl, carboxyl, amino, and thiol) that allows covalent attachment to an adjacent group or moiety, provided that the modified drug has pharmacological activity.

[0172] In the present application, the term "free payload" refers to the molecular part containing the payload that is detached from the ligand drug conjugate after the ligand drug conjugate enters the human body, especially the molecular structure that can cause damage to normal cells in the human body.

[0173] In the present application, the term "conjugation" refers to the process of connecting two or more molecules through a chemical bond to form a new composite molecule, such as a covalent bond.

[0174] In the present application, the term "antibody" (also synonymously referred to as "immunoglobulin (lg)") covers antibodies in natural form and antibodies artificially screened and configured or mutated in amino acids, including but not limited to monoclonal antibodies, bispecific antibodies, multispecific antibodies, nanobodies (heavy chain antibodies, HcAb), immunoglobulin new antigen receptors (IgNAR). For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0175] In the present application, the term "antigen-binding fragment" refers to a fragment or combination of fragments of an antibody that functionally binds to an antigen, typically does not contain an Fc region, such as scFv, Fab, Fab', F(ab')2, Fd, Fv, dAb, Fd, sdAb, VHH, Diabody, Variable domain of immunoglobulin new antigen receptor (VNAR), which comprises at least a portion of an antibody sufficient to impart specific antigen-binding ability to the fragment. The antigen-binding fragment of an antibody (e.g., the above-mentioned antibody fragments) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragment of an antibody is screened for specificity in the same manner as for the intact antibody.

[0176] Preferably, the antibody is an IgG protein, more preferably an lgG1, lgG2, lgG3 or lgG4 protein. Most preferably, the antibody is an lgG1 protein. The antibody can be human or derived from other species. Preferably, the antibody is a human antibody. The antibody can be a monovalent antibody or a bivalent antibody or a multivalent antibody. Native form of human lgG, IgD is a Y-shaped tetrameric molecule (bivalent) composed of two heavy chains and two light chains, each heavy chain having one variable domain (VH) and three constant domains (CH1, CH2, CH3). Each light chain has a variable region (VL) and a constant region (CL).

[0177] In the present application, the term "specifically binds" refers to the antibody or antigen-binding fragment, etc. binds to its target antigen's corresponding epitope in a highly selective manner without binding to a variety of other antigens. Typically, the antibody or antigen-binding fragment, etc. binds to its target antigen with at least about 1 x 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 M affinity binding.

[0178] In the present application, the term "polypeptide" can refer to an entire protein encoded by an entire amino acid sequence or a portion thereof. The term "oligopeptide" is also known as "small peptide" or "oligopeptide", which is a short chain polypeptide composed of a few amino acids connected by peptide bonds.

[0179] In the present application, the term "fusion protein" refers to a protein that is formed by genetically linking two or more proteins so that they can form a single protein molecule when expressed in a cell. It is usually used to enhance the stability of the protein, improve its solubility, increase the expression efficiency or endow the protein with new functions. The fusion protein in the present application includes but is not limited to tag fusion protein, domain fusion protein and multifunctional fusion protein.

[0180] In the present application, the term "peptidomimetics" refers to a class of compounds that mimic the structure of peptides, which have similar chemical or biological properties as peptides but are not natural peptides. They can mimic the secondary structure of peptides (such as α-helix or β-sheet), or have the ability to interact with specific biological targets. The molecular structure of peptidomimetics can contain non-natural amino acids, non-peptide bond connection methods, and various side chain modifications.

[0181] In the present application, the term "aptamer" is also called "aptamer", which usually refers to a special oligonucleotide fragment that can bind to a variety of target substances with high specificity and selectivity, which can be DNA, RNA or XNA (nucleic acid analog) sequence, and is usually obtained from a nucleic acid molecule library through in vitro screening technology - Systematic evolution of ligands by exponential enrichment (SELEX), and is widely used in biosensors, drug delivery, disease treatment and microbial detection. Compared with traditional protein probes, aptamers have the advantages of easy synthesis, good stability, smaller molecular weight and volume, and better thermal stability, easy modification and other characteristics.

[0182] In the present application, the term "bispecific antibody" is used in its broadest sense to encompass antibodies having two epitope specificities, meaning that the antibody is capable of specifically binding to at least two different antigenic determinants. Typically, bispecific antibodies comprise two antigen binding sites, each specific for a different antigenic determinant. In certain embodiments, the bispecific antibody is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two different cells. The entire contents of Kontermann RE, Brinkmann U. Bispecific antibodies. Drug Discov Today. 2015 Jul;20(7):838-47. and Thakur A, Huang M, Lum LG. Bispecific antibody based therapeutics: Strengths and challenges. Blood Rev. 2018 Jul;32(4):339-347. are incorporated herein by reference.

[0183] In the present application, the term "multispecific antibody" is used in its broadest sense to encompass antibodies having two or more epitope specificities, including but not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has two or more epitope specificities; antibodies having two or more VH and VL regions, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies having two or more single variable regions (e.g. VHH), each single variable region binds to a different target or a different epitope of the same target.

[0184] In the present application, the term "lower affinity" refers to the equilibrium dissociation constant Kd of the recovery unit for the payload in the conjugated state in the ligand drug conjugate D (M) greater than 10 -7 .

[0185] In the present application, the term "pharmaceutically acceptable" ingredient generally refers to a substance that is appropriate for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0186] In the present application, the term "solvate" generally refers to an association or complex of one or more solvent molecules and a compound of embodiments of the present application. Non-limiting examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0187] In the present application, the term "isotope" generally includes atoms having the same atomic number but different mass numbers. Examples of isotopes that can be incorporated into the compounds and pharmaceutically acceptable salts thereof described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as, 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 I.

[0188] In the present application, the term "non-exposed epitope" is also sometimes referred to as a "cryptic epitope" or "hidden epitope." A non-exposed epitope refers to an epitope derived from a cancer-specific antigen that is not necessarily processed / presented by an antigen and is "hidden" from immune recognition. Non-exposed epitopes are typically present on APCs at very low concentrations and are not missing self-reactive T cells. Non-exposed epitopes are not presented for recognition by T cells unless they are produced at abnormally high concentrations, or unless they are stripped of their native antigenic context. Cryptic antigenic epitopes derived from cancer-specific antigens can be used to break T cell tolerance to a tumor and induce an effective immune response against the tumor. Such principles have been described in Pardoll, et al., PNAS, Vol. 96, pp. 5340-5342 (1999), the entire contents of which are incorporated herein by reference.

[0189] In the present application, the term "treatment" refers to clinical intervention designed to alter the natural course of the individual or cell being treated, for the purpose of curing, lessening an associated symptom, or improving a clinical condition. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating a disease state, and remission or improved prognosis. For example, alleviating or eliminating one or more symptoms associated with the disease or disorder being treated, such as cancer, inflammation, or autoimmune disease.

[0190] In the present application, the term "effective amount" means an amount that is sufficient to cure or at least partially arrest the disease and its complications in an individual already suffering from the disease. Determining an appropriate effective amount is well within the capabilities of those skilled in the art, and is affected by, among other factors, the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, body weight, and sex, the mode of administration of the drug, and other therapies being administered concurrently, etc.

[0191] Embodiments of the application will be described in detail below with reference to the drawings, but those skilled in the art will understand that the following examples are only for illustration of the application and should not be regarded as limiting the scope of the application.

[0192]

[0193] The structures of MMAE and MMAF are shown above. According to the reports in the literature (Bordeau BM, Nguyen TD, Polli JR, Chen P, Balthasar JP. Payload-Binding Fab Fragments Increase the Therapeutic Index of MMAE Antibody-Drug Conjugates. Mol Cancer Ther. 2023 Apr 3;22(4):459-470., Nguyen TD, Bordeau BM, Balthasar JP. Use of Payload Binding Selectivity Enhancers to Improve Therapeutic Index of Maytansinoid-Antibody-Drug Conjugates. Mol Cancer Ther. 2023 Nov 1;22(11):1332-1342.), it is expected that the target epitope of the screened anti-MMAE / MMAF antibody is to bind the secondary amine at the left N-terminus, and since the carboxyl group on the structure of MMAF is more easily derivatized and conjugated, the immunogen is generated, and after conjugation, it does not affect the secondary amine at the N-terminus. Therefore, in some examples of the present application, MMAF is used instead of MMAE to construct the immunogen. The experimental results show that the related molecules screened have strong binding activity to MMAF, and it can also be understood that the related molecules can also specifically bind to free MMAE. Specific examples Example 1 Preparation of immunogen Example 1-1 Synthesis of ML022-Dxd

[0194] Take 2-((tert-butyldimethylsilyl)oxy)acetic acid (393.84 mg, 2.07 mmol) into a 25 mL single-mouth bottle, add 10 mL of N,N-dimethylformamide to completely dissolve, stir at room temperature, and then add HATU (CAS# 148893-10-1, 786.86 mg, 2.07 mmol), N,N-diisopropyl ethylamine (364.72 mg, 2.82 mmol), and irinotecan methanesulfonate (500 mg, 0.94 mmol) into the reaction system, and react at room temperature. After the reaction is completed, monitor by LC-MS, and purify by reverse-phase flash preparation. The target product ML022-Dxd-01 430 mg is obtained, with a purity of 91.41% and a yield of 68.76%. ESI-MS (m / z): 608.75 [M+H] + .

[0195] Take ML022-Dxd-01 (300 mg, 0.49 mmol) into a 25 mL single-mouth bottle, add 10 mL of dichloromethane to completely dissolve, stir at 5°C, and then add succinic anhydride (74.1 mg, 0.74 mmol) and DBU (CAS# 6674-22-2, 150.3 mg, 0.98 mmol) into the reaction system, and react at room temperature. After the reaction is completed, monitor by LC-MS, and purify by normal-phase column chromatography. The target product ML022-Dxd-02 196 mg is obtained, with a purity of 88.1% and a yield of 49.36%. ESI-MS (m / z): 708.81 [M+H] + .

[0196] Take ML022-Dxd-02 (500 mg, 0.7 mmol) into a 25 mL single-mouth bottle, add 5 mL of tetrahydrofuran and 5 mL of water to ultrasonically dissolve, stir at room temperature, and then add 3 mL of formic acid dropwise into the system, and react at room temperature. After the reaction is completed, monitor by LC-MS, and purify by reverse-phase high-pressure preparation. The target product ML022-Dxd 256 mg is obtained, with a purity of 97.64% and a yield of 59.62%. ESI-MS (m / z): 594.51 [M+H] + Example 1-2 Synthesis of ML022-D633-004

[0197] According to the synthesis steps of ML022-Dxd, the second-step raw material is replaced with ML022-D633-004-01 to obtain the target product ML022-D633-004 19.94 mg. The purity is 94.25%, and the yield is 44.82%. ESI-MS (m / z): 594.65 [M+H] + Example 1-3 Synthesis of ML026-Dxd

[0198] According to the synthesis steps of ML022-Dxd, the raw material was replaced with diethylene glycol anhydride in the second step. The target product ML026-Dxd 60 mg was obtained, with a purity of 95.89% and a yield of 32.01%. ESI-MS (m / z): 610.22 [M+H] + Synthesis of ML031-D633-004

[0199] According to the synthesis steps of ML022-D633-004, the raw material was replaced with carboxyl-octadecaglycol-tert-butyl propionate in the second step. The target product ML037-D633-004 14.41 mg was obtained, with a purity of 94.52% and a yield of 10.72%. ESI-MS (m / z): 947.37 [M+H] + Synthesis of ML037-D633-004

[0200] According to the synthesis steps of ML022-D633-004, the raw material was replaced with carboxyl-octadecaglycol-tert-butyl propionate in the second step. The target product ML037-D633-004 14.41 mg was obtained, with a purity of 94.52% and a yield of 10.72%. ESI-MS (m / z): 947.37 [M+H] + Synthesis of ML042-Dxd

[0201] ML022-Dxd (30 mg, 0.54 mmol) was weighed into a 25 mL single-mouth bottle, 10 mL of dichloromethane was added to completely dissolve it, and it was stirred at room temperature. HATU (21.14 mg, 0.55 mmol), N,N-diisopropylethylamine (9.8 mg, 0.75 mmol), and methylamine hydrochloride (2.35 mg, 0.75 mmol) were sequentially weighed and added to the reaction system, and the reaction was carried out at room temperature. The solvent was removed by rotary evaporation, and reverse-phase high-pressure preparation purification was performed. The target product ML042-Dxd 14.19 mg was obtained, with a purity of 93.75% and a yield of 43.39%. ESI-MS (m / z): 607.67 [M+H] + Synthesis of ML047-DX8951

[0202] Icinotecan mesylate (500 mg, 0.94 mmol), TEA (285.56 mg, 0.28 mmol), and Boc anhydride (410.59 mg, 1.88 mmol) were weighed sequentially and placed in a 100 mL single-necked flask. 50 mL of dichloromethane was added and the mixture was completely dissolved. The mixture was stirred overnight at room temperature. After the reaction was complete as monitored by LC-MS, the mixture was washed with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. 630 mg of the crude product ML047-DX8951-01 was obtained and directly added to the next step. ESI-MS (m / z): 535.95 [M+H] + .

[0203] 1-(9H-fluorene-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecano-13-acid (820.41 mg, 2.05 mmol) was weighed and placed in a 100 mL single-necked flask. 50 mL of dichloromethane was added and the mixture was completely dissolved. The mixture was stirred at room temperature. Then, EDCI (CAS#25952-53-81.07 g, 5.6 mmol), DMAP (CAS#1122-58-3, 684.3 mg, 5.8 mmol), and ML047-DX8951-01 (1.0 g, 1.87 mmol) were weighed and added to the reaction mixture. The reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by LC-MS, the solvent was removed by rotary evaporation, and the mixture was purified by normal silica gel column chromatography. The target product ML047-DX8951-02 was obtained in 1.51 g, with a purity of 95.75% and a yield of 84.44%. ESI-MS (m / z): 918.47 [M+H] + .

[0204] Weigh 200 mg (0.21 mmol) of ML047-DX8951-02 into a 25 mL single-necked flask, add 10 mL of dichloromethane and sonicate to dissolve. Stir at room temperature, then add 3 mL of diethylamine dropwise to the system and react at room temperature. After the reaction is complete as monitored by LC-MS, remove the solvent by rotary evaporation, dissolve the dichloromethane, and evaporate to dryness. Repeat this process three times. 150 mg of the crude target product ML047-DX8951-03 was obtained and directly added to the next step. ESI-MS (m / z): 695.56 [M+H] + .

[0205] Take ML047-DX8951-03 crude product (30 mg, 0.043 mmol) and place it in a 25 mL single-mouth bottle, add 10 mL of dichloromethane and ultrasonically dissolve it, stir at room temperature, and add 2,5-Pyrrolidinedione, 1-(bromoacetyl)-(9CI) (28.5 mg, 0.12 mmol) to the above system. After monitoring the completion of the reaction by LC-MS, wash with water, wash with saturated brine, combine the organic phases, and spin dry to obtain 35 mg of ML047-DX8951-04 crude product, which is directly used in the next step. ESI-MS (m / z): 815.96 [M+H] + .

[0206] Take ML047-DX8951-04 crude product (30 mg, 0.036 mmol) and place it in a 25 mL single-mouth bottle, add 5 mL of dichloromethane and ultrasonically dissolve it, stir at room temperature, and add 2 mL of TFA (CAS# 76-05-1) dropwise to the system and react at room temperature. After monitoring the completion of the reaction by LC-MS, purify by reverse-phase high-pressure preparation. Obtain 3.13 mg of target product ML047-DX8951 with a purity of 96.40% and a yield of 11.46%. ESI-MS (m / z): 715.75 [M+H] + . Synthesis of MPA-Dxd

[0207] Dissolve compound ML022-Dxd-01 (270.12 mg, 0.34 mmol), 3-(tritylsulfanyl)propanoic acid (324.54 mg, 0.68 mmol), DIC (162 μL, 0.68 mmol), and DMPA (27.41 mg, 0.068 mmol) in 6 mL of dichloromethane. React at room temperature for 4 h. Add 10 mL of purified water to the reaction system, extract, and spin dry the organic phase to obtain 240.14 mg of target product MPA-Dxd-01 as a crude product with a yield of 57.54%. ESI-MS (m / z): 938.36 [M+H] + .

[0208] Dissolve MPA-Dxd-01 crude product in 7 mL of dichloromethane, add 2 mL of TFA and 1 mL of triethylsilane, and react at room temperature for 18 h. After purification by preparative liquid chromatography, obtain 62.14 mg of target product MPA-Dxd with a yield of 41.61%. ESI-MS (m / z): 582.16 [M+H] + . Synthesis of Biotin-PEG12-Dxd

[0209] Biotin-PEG12-Dxd-01 (20 mg, 32.91 μmol) was dissolved in 2 mL of dichloromethane and added dropwise to the above system, and the reaction was carried out at room temperature. After the reaction was completed, the reaction solution was spin-dried, and 50 mg of the obtained Biotin-PEG12-Dxd-01 crude product was directly used in the next step. ESI-MS (m / z): 1434.34 [M+H] + .

[0210] The Biotin-PEG12-Dxd-01 crude product (50 mg, 0.28 mmol) was weighed into a 25 mL single-mouth bottle, 2 mL of tetrahydrofuran and 2 mL of water were added and ultrasonically dissolved, stirred at room temperature, and 1 mL of formic acid was added dropwise to the system and reacted at room temperature. After the reaction was completed, it was purified by reverse-phase high-pressure preparation. The target product Biotin-PEG12-Dxd was obtained in an amount of 7.89 mg, with a purity of 95.88% and a yield of 35.74%. ESI-MS (m / z): 1319.94 [M+H] + Example 1-10 Synthesis of Biotin-PEG10-Dxd

[0211] According to the synthesis route of Biotin-PEG12-Dxd, the raw material was changed to Biotin-PEG10-carboxyl. The target product Biotin-PEG10-Dxd was obtained in an amount of 16.6 mg, with a purity of 95.88% and a yield of 13.18%. ESI-MS (m / z): 1232.41 [M+H] + Example 1-11 Synthesis of Biotin-PEG6-Dxd

[0212] According to the synthesis route of Biotin-PEG12-Dxd, the raw material was changed to Biotin-PEG6-carboxyl. The target product Biotin-PEG6-Dxd was obtained in an amount of 22.14 mg, with a purity of 95.91% and a yield of 24.51%. ESI-MS (m / z): 1056.98 [M+H] + Example 1-12 Synthesis of Biotin-PEG6-Triazole-Dxd

[0213] NaH (31 mg) was weighed into a three-necked flask, which was purged with argon and protected, cooled to 0°C and stirred, and a solution of ML022-Dxd-01 (300 mg, 0.5 mmol) in N,N-dimethylformamide (20 mL) was slowly added dropwise, stirred at low temperature for 30 min, and a solution of bromopropargyl (91 mg) in N,N-dimethylformamide (10 mL) was slowly added dropwise, stirred at low temperature and gradually returned to room temperature. Stirred overnight. The system was purified by reversed-phase high-pressure preparation. The target product Biotin-PEG6-Triazole-Dxd-01 23 mg was obtained as a crude product. ESI-MS (m / z): 646.21 [M+H] + .

[0214] Biotin-PEG6-Triazole-Dxd-01 (8 mg, 12 μmol) was dissolved in 2 mL of acetone and 1 mL of water, and compound N3-PEG6-Biotin (7.5 mg, 12 μmol) and copper sulfate pentahydrate (5 mg) were added at room temperature. The system was stirred in a 10°C low-temperature bath. A solution of sodium ascorbate (4 mg) in water (1 mL) was slowly added dropwise to the reaction. Stirred at 10°C for 1 h. The system was quenched with acetonitrile (1 mL). The system was purified by preparative liquid chromatography to obtain the target product Biotin-PEG6-Triazole-Dxd 3.85 mg. Purity 94.48%, yield 25%. ESI-MS (m / z): 1108.25 [M+H] + Example 1-13 Synthesis of Biotin-PEG10-Triazole-Dxd

[0215] According to the synthesis route of Biotin-PEG6-Triazole-Dxd, the second raw material was replaced with N3-PEG10-Biotin. The target product Biotin-PEG10-Triazole-Dxd 2.77 mg was obtained, with a yield of 15%. ESI-MS (m / z): 1329.09 [M+H] + Example 1-14 Synthesis of immunoconjugates (carrier protein (BSA / OVA / KLH)-MMAF, carrier protein (BSA / OVA / KLH)-ML022-Dxd, carrier protein (BSA / OVA / KLH)-ML022-D633-004, carrier protein (BSA / OVA / KLH)-ML031-D633-004)

[0216] The activation process of the drug: dissolve the drug, EDC, and NHS in DMF respectively, activate the drug with EDC and NHS, and react for 15 min at room temperature with 600 r / min shaking.

[0217] Coupling of drug to carrier protein (BSA / OVA / KLH): Dissolve the protein in PBS, add 10-30 eq of activated drug to the carrier protein (BSA / OVA / KLH) system, adjust the concentration to 5 mg / ml, and react overnight at room temperature.

[0218] Liquid exchange and detection: After the reaction is completed, the reaction system is thoroughly exchanged with PBS buffer to remove excess small molecules, and the sample is subjected to ELISA detection of the coupling condition.

[0219] Reduction of carrier protein (BSA / OVA / KLH): Dissolve the protein in a system of 20 mM boric acid, 5 mM borax, pH 8.5, add 10% DTPA and 5-20 eq TCEP to the reaction system, and react for 2 h at room temperature.

[0220] Coupling of drug to carrier protein (BSA / OVA / KLH): Add 5-20 eq of drug to the above reaction system, and react for 3 h at room temperature at 600 r / min.

[0221] Liquid exchange and detection: After the reaction is completed, the system is exchanged into PBS, and an appropriate amount of sample is subjected to ELISA detection of the coupling condition.

[0222] Table 1. Mouse immunization scheme - immunogen is BSA-MMAF (prepared from Example 1-14)

[0223] Table 2. Mouse immunization scheme - immunogen is KLH-MMAF (prepared from Example 1-14) Example 2-1 Screening of antibodies binding to MMAE (first round)

[0224] Generation of hybridomas

[0225] Mice were immunized with BSA-MMAF, and each mouse (mouse strain: CD1) was injected with 40 μg of immunogen each time. The spleen cells of mice with excellent titer detection results were selected and fused with SP2 / 0 myeloma cells at a ratio of 1:1 under electrofusion conditions. The cells were plated in 96-well cell culture plates using HAT medium containing 20% FBS, and 30 plates were plated for each mouse. After 7 days of culture, all the plates were changed to 10% FBS HT medium, and after 3 days of culture, the supernatant was subjected to ELISA detection.

[0226] Primary screening of hybridomas

[0227] OVA-MMAF concentration of 200 ng / mL to ELISA plate, 37°C for 1 h, 5% skim milk powder blocking, 37°C for 2 h, take 100 μL hybridoma supernatant to the plate, 37°C for 1 h, add 1:5000 dilution of HRP-Goat Anti-mouse IgG △IgM, 37°C for 1 h after TMB color development, detection of OD value at 450 nm. And the positive hybridoma supernatant was subjected to negative screening, and the hybridoma specifically binding to MMAE without binding to MMAE-Linker (structure MC-Val-Cit-PAB-MMAE) was screened: OVA-MMAF concentration of 200 ng / mL to ELISA plate, 37°C for 1 h, 5% skim milk powder blocking, 37°C for 2 h, take 50 μL hybridoma supernatant, add PBS, free MMAE and vedisituximab respectively, 37°C for 1 h, add 1:5000 dilution of HRP-Goat Anti-mouse IgG △IgM, 37°C for 1 h after TMB color development, detection of OD value at 450 nm. According to the results, the hybridoma with the same reading as the PBS group and the vedisituximab group and the lower value of the free MMAE group was selected, which was considered to specifically bind to free MMAE without binding to MMAE-Linker. It was subcloned by limiting dilution method, and a total of more than 500 plates were subcloned. A total of 24 mice were fused and screened, and more than 46000 hybridoma strains were detected in the primary screening, and more than 800 plates were subcloned.

[0228] Monoclonal screening

[0229] According to the method of screening positive clones, the variable region of the mouse-derived antibody was carried on the human-derived constant region, and finally more than 160 chimeric antibodies were obtained for subsequent analysis.

[0230] Chimeric antibody analysis-affinity analysis

[0231] ELASA detection: OVA-MMAF concentration of 200 ng / mL to ELISA plate, 37°C for 1 h, 5% skim milk powder blocking, 37°C for 2 h, gradient dilution of antibody chimeric antibody, concentration of 10000, 5000, 2500, 1250, 625, 125, 25, 5, 1, 0.1, 0.01, 0 ng / mL, take 100 μL to the plate, 37°C for 1 h, add 1:5000 dilution of Goat Anti-Human IgG H&L (HRP) (abcam, ab6858), 37°C for 1 h after TMB color development, detection of OD value at 450 nm.

[0232] The experimental results show (Table 3) that the affinity of No. 62 and No. 64 chimeric antibodies to OVA-MMAF is higher than that of AB3320.

[0233] Table 3. EC50 (ng / mL) value of affinity detection of part of candidate molecules

[0234] Biacore detection (binding affinity with free MMAE)

[0235] Antibody immobilization: 20 μg / mL of antibody was immobilized on the surface of the chip with the probe of ProA; binding: the concentration of antigen MMAE was 50, 16.7, 5.5, 1.85, 0 nM, the flow rate was set to 30 μL / min, and the contact time was 600 s; dissociation: the buffer HEPES was flushed for 300 s. Regeneration: 10 mM Glycine-HCl was used for regeneration.

[0236] The experimental results show (Table 4) that the affinity of No. 62 chimeric antibody to free MMAE is higher than that of ABC3320.

[0237] Table 4. Affinity detection of part of candidate molecules

[0238] Chimeric antibody analysis-specific analysis: In order to verify whether the chimeric antibody specifically binds to free MMAE without binding to MMAE-Linker (i.e. to screen out the antibody that does not bind to the conjugated load in ADC), the competition and blocking experiments of the chimeric antibody were carried out.

[0239] Competition experiment: 1) 200 ng / mL of OVA-MMAF was coated on the ELISA plate, incubated at 37°C for 1 h, and blocked with 5% lipid milk, incubated at 37°C for 2 h; 2) the antibody was diluted to a final concentration of 10000, 5000, 2500, 1250, 625, 125, 25, 5, 1, 0.1, 0.01, 0 ng / mL, and MMAE and MMAE-Linker were diluted to a final concentration of 5 μM; 3) 50 μL of PBS+50 μL of chimeric antibody, 50 μL of MMAE+50 μL of chimeric antibody, and 50 μL of MMAE-Linker+50 μL of chimeric antibody were added to the ELISA plate, respectively, and the total volume of each well was 100 μL, which was incubated at 37°C for 1 h, and then the reaction solution was poured out, and the plate was washed with PBST for 3 times; 4) 1:5000 diluted Goat Anti-Human IgG H&L (HRP) (abcam, ab6858) was added, incubated at 37°C for 1 h, and then TMB was developed, and the OD value was detected at 450 nm.

[0240] The experimental results (Table 5) show that the competition EC50 values of the chimeric antibodies No. 62 and No. 64 are 80.9 ng / mL and 84.52 ng / mL, respectively, and the chimeric antibodies No. 62 and No. 64 can specifically bind to free MMAE and do not bind to MMAE-Linker.

[0241] Table 5. Competition EC50 (ng / mL) values of chimeric antibodies No. 62 and No. 64 and ABC3320

[0242] Note: "NA" represents that the measured OD value is too low, and thus the calculated EC50 value is meaningless.

[0243] Blocking experiment: OVA-MMAF was coated on the ELISA plate at a concentration of 200 ng / mL, incubated at 37°C for 1 h, blocked with 5% skim milk, and incubated at 37°C for 2 h. The antibody was diluted to a final concentration of 100 ng / mL, and MMAE and MMAE-Linker were diluted to concentrations of 600, 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09, 0.03, 0.01 nM. In the ELISA plate, 50 μL of MMAE + 50 μL of chimeric antibody, and 50 μL of MMAE-Linker + 50 μL of chimeric antibody were added, and the total volume per well was 100 μL. After incubation at 37°C for 1 h, Goat Anti-Human IgG H&L (HRP) (abcam, ab6858) diluted 1:5000 was added, and after incubation at 37°C for 1 h, TMB was developed, and the OD value was detected at 450 nm. According to the experimental results (Table 6), the blocking IC50 values of the chimeric antibodies No. 62 and No. 64 are 1.427 nM and 4.762 nM, respectively.

[0244] Table 6. Blocking IC50 (nM) values of chimeric antibodies No. 62 and No. 64 and ABC3320

[0245] Chimeric antibody analysis - cell protection effect analysis: OVCAR3 cells were plated at 5000 cells / well, and after 24 h, the supernatant was discarded, and the antibody and free MMAE were added. The final concentration of MMAE was 69.5 nM, and the final concentration of the antibody was 60, 30, 15, 10, 7.5, 5, 3.75, 2.5 μg / mL. 50 μL of MMAE + 50 μL of chimeric antibody was mixed and added to the OVCAR3 cells, which were incubated for 72 h. Then, CellTiter-MeIuncell luminescence cell viability assay kit (Meiluncell, PWL111) was used for chemiluminescence reading.

[0246] As shown in FIG. 24, the experimental results show that the chimeric antibodies No. 62 and No. 64 have a good protection effect on the killing of MMAE, which is comparable to ABC3320.

[0247] In summary, among the above-mentioned candidate molecules, No. 62 and No. 64 chimeric antibodies have better performance.

[0248] The No. 62 and No. 64 chimeric antibodies were sequenced respectively, and the amino acid sequences of the heavy chain variable region (62H, 64H) and the light chain variable region (62L, 64L) are as follows:

[0249] > 62H (SEQ ID NO: 1; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0250] > HCDR1 of 62H (SEQ ID NO: 2)

[0251] GYSITNANHW

[0252] > HCDR2 of 62H (SEQ ID NO: 3)

[0253] ISSSGTT

[0254] > HCDR3 of 62H (SEQ ID NO: 4)

[0255] VRTYYDGTYEEN

[0256] > 62L (SEQ ID NO: 5; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0257] > LCDR1 of 62L (SEQ ID NO: 6)

[0258] QSVGNN

[0259] > LCDR2 of 62L

[0260] YAS

[0261] > LCDR3 of 62L (SEQ ID NO: 7)

[0262] QQHYNSPWTFAGG

[0263] > 64H (SEQ ID NO: 8; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0264] > HCDR1 of 64H (SEQ ID NO: 9)

[0265] GNSITNTNNW

[0266] HCDR2 of 64H (SEQ ID NO: 10)

[0267] ISSSGST

[0268] HCDR3 of 64H (SEQ ID NO: 11)

[0269] VRTYYDGSYEEN

[0270] 64L (SEQ ID NO: 12; CDR1-3 (IMGT system) are underlined and bolded, respectively):

[0271] LCDR1 of 64L (SEQ ID NO: 6)

[0272] QSVGNN

[0273] LCDR2 of 64L

[0274] FAS

[0275] LCDR3 of 64L (SEQ ID NO: 13)

[0276] QQHYISPWT

[0277] Humanized sequence design of No. 62 antibody

[0278] By comparing the sequence similarity, the antibody germline with the highest similarity to the murine No. 62 antibody was selected as the humanization template. The CDR of the heavy chain and the light chain were replaced with the CDR region of the template, and then the key amino acids affecting the activity of the antibody were back mutated according to the three-dimensional structure predicted by artificial intelligence. The specific humanization process is as follows:

[0279] (1) IGHV4-30-4*01 and IGHJ6*01 were selected as the heavy chain humanization template of the murine No. 62 antibody, and IGKV1-33*01 and IGKJ4*01 of Kappa were selected as the light chain humanization template of the murine No. 62 antibody. The CDR region of the heavy chain or light chain of the murine No. 62 antibody was replaced with the CDR region of the humanization template, and the heavy chain variable region sequence 5759 (SEQ ID NO: 14) and the light chain variable region sequence 5764 (SEQ ID NO: 15) were obtained, respectively;

[0280] (2) The ESMFold module in WeMol software was used to predict the structure of the variable region of the murine No. 62 antibody, and the three-dimensional structure model was obtained;

[0281] (3) According to the variable region structure of the mouse-derived No. 62 antibody, the key amino acids in the framework region affecting the interaction between the heavy chain and the light chain and the interaction with the CDR were judged, the amino acid sites of the back mutation were determined, and the heavy chain variable region sequence 5954 (SEQ ID NO: 17) and the light chain variable region sequence 6052 (SEQ ID NO: 18) were obtained, which constitute the PP-1M-#60 molecule; at the same time, the potential isomerization site DG in the heavy chain CDR3 was removed by translation modification, and the heavy chain variable region sequence 6265 (SEQ ID NO: 19) was obtained, which constitutes the PP-1M-#66 molecule with the light chain variable region sequence 6052 (SEQ ID NO: 18).

[0282] >5759 (SEQ ID NO: 14; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0283] HCDR1 of 5759 (SEQ ID NO: 2)

[0284] GYSITNANHW

[0285] HCDR2 of 5759 (SEQ ID NO: 3)

[0286] ISSSGTT

[0287] HCDR3 of 5759 (SEQ ID NO: 4)

[0288] VRTYYDGTYEEN

[0289] >5764 (SEQ ID NO: 15; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0290] LCDR1 of 5764 (SEQ ID NO: 6)

[0291] QSVGNN

[0292] LCDR2 of 5764

[0293] YAS

[0294] LCDR3 of 5764 (SEQ ID NO: 16)

[0295] QQHYNSPWT

[0296] >5954 (SEQ ID NO: 17; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0297] HCDR1 of >5954 (SEQ ID NO: 2)

[0298] GYSITNANHW

[0299] HCDR2 of >5954 (SEQ ID NO: 3)

[0300] ISSSGTT

[0301] HCDR3 of >5954 (SEQ ID NO: 4)

[0302] VRTYYDGTYEEN

[0303] >6052 (SEQ ID NO: 18; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0304] LCDR1 of >6052 (SEQ ID NO: 6)

[0305] QSVGNN

[0306] LCDR2 of >6052

[0307] YAS

[0308] LCDR3 of >6052 (SEQ ID NO: 7)

[0309] QQHYNSPWTFAGG

[0310] >6265 (SEQ ID NO: 19; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0311] HCDR1 of >6265 (SEQ ID NO: 2)

[0312] GYSITNANHW

[0313] HCDR2 of >6265 (SEQ ID NO: 3)

[0314] ISSSGTT

[0315] HCDR3 of >6265 (SEQ ID NO: 20)

[0316] VRTYYDSTYEEN

[0317] Humanized antibody construction and expression purification

[0318] The heavy chain sequence and the light chain sequence of the antibody were constructed into expression vectors respectively by primer design, and Expi293 cells were used for transient transfection and expression.

[0319] Cell protection experiment of humanized anti-MMAE antibody

[0320] NCI-H292 cells were digested, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, resuspended with 1640+10% FBS medium for counting, adjusted to the corresponding concentration, and added into the Corning 3610 plate at 60 μL per well, 5000 cells per well, and placed in a 37°C incubator for standby;

[0321] The antibody concentration was diluted (40 μg / mL or 20 μg / mL, 2-fold dilution), and the fixed concentration of free MMAE was 10 ng / mL. 30 μL of MMAE was added to the 3610 plate with cells in a 1:1 ratio, and the total culture system was 120 μL. The plate was incubated in a 37°C incubator for 3 days.

[0322] The plate was taken out and equilibrated at room temperature. 100 μL Luminescent Cell was added to each well, mixed, and incubated at room temperature in the dark for 10 min.

[0323] The luminescence values were detected on the microplate reader, and the calculation formula was % Cytoxicity = (Blank-Sample) / Blank*100%.

[0324] The experimental results are shown in Figure 25, which show that the PP-1M-#60 and PP-1M-#66 molecules have good cell protection activity.

[0325] Generation of hybridoma

[0326] Mice were immunized with KLH-MMAF, and each mouse (mouse strain CD1 and Balb / c) was injected with 40 μg of immunogen each time. The spleen cells of mice with excellent titer detection results were selected and fused with SP2 / 0 myeloma cells at a ratio of 1:1 under the condition of electrofusion. The 96-well cell culture plate was plated with HAT medium containing 20% FBS, 30 plates per mouse, and the medium was changed to 10% FBS HT medium after 7 days of culture. After 3 days of culture, the supernatant was detected by ELISA.

[0327] Hybridoma screening

[0328] Coat OVA-MMAF at a concentration of 200 ng / mL to ELISA plates, incubate at 37°C for 1 h, block with 3% BSA, incubate at 37°C for 2 h, take 100 μL of hybridoma supernatant to the plate, incubate at 37°C for 1 h, add 1:5000 diluted HRP-Goat Anti-mouse IgG △IgM, incubate at 37°C for 1 h, then develop with TMB, and detect OD value at 450 nm. The positive hybridoma supernatant was subjected to negative screening to screen hybridomas that specifically bind to MMAE but not to MMAE-Linker: coat OVA-MMAF at a concentration of 200 ng / mL to ELISA plates, incubate at 37°C for 1 h, block with 3% BSA, incubate at 37°C for 2 h, take 50 μL of hybridoma supernatant, add 300 nM of free MMAE and 300 nM of MMAE-Linker respectively, incubate at 37°C for 1 h, add 1:5000 diluted HRP-Goat Anti-mouse IgG △IgM, incubate at 37°C for 1 h, then develop with TMB, and detect OD value at 450 nm. According to the results, select hybridomas with high readings in the PBS group and the MMAE-Linker group and low values in the free MMAE group, which are considered to be able to specifically bind to free MMAE but not to MMAE-Linker. Subclone them by limiting dilution method, a total of 38 plates, and detect more than 700 strains of hybridomas.

[0329] Monoclonal screening

[0330] Repeat the above screening method for monoclonal screening, and load the variable region of the mouse-derived antibody onto the human-derived constant region to finally obtain 6 chimeric antibodies for subsequent analysis.

[0331] Chimeric antibody analysis-specificity analysis

[0332] Competition experiment: the experimental steps are the same as the above competition experiment, and the experimental results (Table 7) show that the competition EC50 value of the No. J4 chimeric antibody is 27.27 ng / mL, which is significantly better than ABC3320.

[0333] Table 7. Competition EC50 (ng / mL) values of No. J4 chimeric antibody and ABC3320

[0334] Blocking experiment: the experimental steps are the same as the above blocking experiment, and according to the experimental results (Table 8), the blocking IC50 value of the No. J4 chimeric antibody is 1.89 nM.

[0335] Table 8. Blocking IC50 (nM) values of No. J4 chimeric antibody and ABC3320

[0336] Chimeric antibody analysis - cell protection effect analysis: the experimental procedure was the same as the cell protection effect analysis experiment, and the experimental results are shown in Figure 26. The results show that the cell protection effect of the J4 chimeric antibody is equivalent to that of ABC3320.

[0337] In summary, among the above candidate molecules, the J4 chimeric antibody has better performance.

[0338] The J4 chimeric antibody was sequenced, and the amino acid sequences of the heavy chain variable region (J4H) and the light chain variable region (J4L) are as follows:

[0339] > J4H (SEQ ID NO: 21; the CDR1-3 (IMGT system) regions are bolded and underlined, respectively):

[0340] > HCDR1 of J4H (SEQ ID NO: 22)

[0341] GFTFSSSY

[0342] > HCDR2 of J4H (SEQ ID NO: 23)

[0343] IYAGTGNT

[0344] > HCDR3 of J4H (SEQ ID NO: 24)

[0345] SRDGYDGRSYDLDY

[0346] > J4L (SEQ ID NO: 25; the CDR1-3 (IMGT system) regions are bolded and underlined, respectively):

[0347] > LCDR1 of J4L (SEQ ID NO: 26)

[0348] QDVGTS

[0349] > LCDR2 of J4L

[0350] WAS

[0351] > LCDR3 of J4L (SEQ ID NO: 27)

[0352] QQFSTYPLT Example 2-3 Screening of antibodies conjugated with MMAE (third round)

[0353] Generation of hybridomas

[0354] The mice were immunized with KLH-MMAF, each mouse (Balb / c strain) was injected with 40 μg of immunogen, and the spleen cells of mice with excellent titer detection results were selected and fused with SP2 / 0 myeloma cells at a ratio of 1:1 under the condition of electrofusion. The 96-well cell culture plates were plated with HAT medium containing 20% FBS, 30 plates per mouse, and after 7 days of culture, all the HT medium containing 10% FBS was replaced, and after 3 days of culture, the supernatant was detected by ELISA.

[0355] Hybridoma primary screening

[0356] OVA-MMAF was coated on the ELISA plate at a concentration of 200 ng / mL, incubated at 37°C for 1 h, blocked with 3% BSA, incubated at 37°C for 2 h, and 100 μL of hybridoma supernatant was added to the plate, incubated at 37°C for 1 h, 1:5000 diluted HRP-Goat Anti-mouse IgG △ IgM was added, incubated at 37°C for 1 h, and then TMB was developed, and the OD value was detected at 450 nm. The positive hybridoma supernatant was subjected to negative screening to screen hybridomas that specifically bind to MMAE but not to MMAE-linker: OVA-MMAF was coated on the ELISA plate at a concentration of 200 ng / mL, incubated at 37°C for 1 h, blocked with 3% BSA, incubated at 37°C for 2 h, and 50 μL of hybridoma supernatant was added, 300 nM free MMAE and 300 nM MMAE-linker were added respectively, and incubated at 37°C for 1 h, 1:5000 diluted HRP-Goat Anti-mouse IgG △ IgM was added, incubated at 37°C for 1 h, and then TMB was developed, and the OD value was detected at 450 nm. According to the results, hybridomas with the same reading as the PBS group and lower values in the free MMAE group were selected, which were considered to specifically bind to free MMAE but not to MMAE-linker. Subcloning was performed by limiting dilution method, and a total of more than 20 plates were subcloned, and more than 400 hybridomas were detected.

[0357] Monoclonal screening

[0358] The above screening method was repeated for monoclonal screening, and the variable region of the mouse-derived antibody was loaded onto the human-derived constant region, and finally 10 chimeric antibodies were obtained for subsequent analysis.

[0359] Chimeric antibody analysis-specificity analysis

[0360] Competition experiment: The experimental steps were the same as the above competition experiment, and the experimental results (Table 9) showed that the competition EC50 values of chimeric antibodies No. 92 and No. 93 were 30.63 ng / mL and 37.22 ng / mL, respectively, which were significantly better than ABC3320.

[0361] Table 9. Competition EC50 (ng / mL) values of chimeric antibodies No. 92, 93 and ABC3320

[0362] Blocking experiment: The experimental procedure was the same as the above blocking experiment. According to the experimental results (Table 10), the blocking IC50 values of chimeric antibodies No. 92 and 93 were 83.75 nM and 54.69 nM, respectively.

[0363] Table 10. Blocking IC50 (nM) values of chimeric antibodies No. 92, 93 and ABC3320

[0364] In summary, among the above candidate molecules, chimeric antibodies No. 92 and 93 have better performance.

[0365] Chimeric antibodies No. 92 and 93 were sequenced respectively, and the amino acid sequences of the heavy chain variable region (92H, 93H) and the light chain variable region (92L, 93L) are as follows:

[0366] > 92H (SEQ ID NO: 28; the bold and underlined regions are CDR1-3 (IMGT system), respectively):

[0367] > HCDR1 of 92H (SEQ ID NO: 29)

[0368] GYTFTSYV

[0369] > HCDR2 of 92H (SEQ ID NO: 30)

[0370] IIHYNDVT

[0371] > HCDR3 of 92H (SEQ ID NO: 31)

[0372] TRSPYYYDGSFDY

[0373] > 92L (SEQ ID NO: 32; the bold and underlined regions are CDR1-3 (IMGT system), respectively):

[0374] > LCDR1 of 92L (SEQ ID NO: 33)

[0375] QSIVHSNGNTY

[0376] > LCDR2 of 92L

[0377] KVS

[0378] > LCDR3 of 92L (SEQ ID NO: 34)

[0379] FQGSHAPYT

[0380] >93H (SEQ ID NO: 35; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0381] HCDR1 of 93H (SEQ ID NO: 29)

[0382] GYTFTSYV

[0383] HCDR2 of 93H (SEQ ID NO: 36)

[0384] IIHYNDGT

[0385] HCDR3 of 93H (SEQ ID NO: 37)

[0386] ARSPYYSDGSFDY

[0387] >93L (SEQ ID NO: 38; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0388] LCDR1 of 93L (SEQ ID NO: 33)

[0389] QSIVHSNGNTY

[0390] LCDR2 of 93L

[0391] KLS

[0392] LCDR3 of 93L (SEQ ID NO: 34)

[0393] FQGSHAPYT Antibodies screened for conjugation to MMAE (fourth round) Examples 2-4

[0394] Generation of hybridomas

[0395] Mice were immunized with BSA-MMAF, 40 μg of immunogen per injection per mouse (mouse strains were CD1 and Balb / c), and spleen cells from mice with excellent titer detection results were selected and fused with SP2 / 0 myeloma cells at a ratio of 1:1 under electrofusion conditions. The 96-well cell culture plates were plated with HAT medium containing 20% FBS, 30 plates per mouse, and after 7 days of culture, all the plates were changed to 10% FBS HT medium, and after 3 days of culture, the supernatant was detected by ELISA.

[0396] Primary screening of hybridomas

[0397] Coat OVA-MMAF at a concentration of 200 ng / mL to the ELISA plate, incubate at 37°C for 1 h, block with 3% BSA, incubate at 37°C for 2 h, take 100 μL of hybridoma supernatant to the plate, incubate at 37°C for 1 h, add 1:5000 diluted HRP-Goat Anti-mouse IgG △IgM, incubate at 37°C for 1 h, then develop with TMB, and detect OD value at 450 nm. The positive hybridoma supernatant was subjected to negative screening to screen hybridomas that specifically bind to MMAE but not to MMAE-Linker. Coat OVA-MMAF at a concentration of 200 ng / mL to the ELISA plate, incubate at 37°C for 1 h, block with 3% BSA, incubate at 37°C for 2 h, take 50 μL of hybridoma supernatant, add 300 nM of free MMAE and 300 nM of MMAE-linker respectively, incubate at 37°C for 1 h, add 1:5000 diluted HRP-Goat Anti-mouse IgG △IgM, incubate at 37°C for 1 h, then develop with TMB, and detect OD value at 450 nm. According to the results, select hybridomas with high readings in the PBS group and the vedisiran group and low values in the free MMAE group, which are considered to be able to specifically bind to free MMAE but not to MMAE-Linker, and subclone them by limiting dilution. A total of more than 50 plates were subcloned, and more than 1000 hybridomas were detected.

[0398] Monoclonal screening

[0399] Repeat the above screening method for monoclonal screening, and load the variable region of the mouse-derived antibody onto the human-derived constant region to finally obtain 23 chimeric antibodies for subsequent analysis.

[0400] Chimeric antibody analysis-specificity analysis

[0401] Competitive experiment: the experimental steps are the same as the above competitive experiment, and the experimental results (Table 11) show that the competitive EC50 values of chimeric antibodies 132 and 133 are 70.61 ng / mL and 34.84 ng / mL respectively, which are significantly better than ABC3320.

[0402] Table 11. Competitive EC50 (ng / mL) values of chimeric antibodies 132 and 133 and ABC3320

[0403] Blocking experiment: the experimental steps are the same as the above blocking experiment, and according to the experimental results (Table 12), the blocking IC50 values of chimeric antibodies 132 and 133 are 1.318 nM and 1.593 nM respectively.

[0404] Table 12. Blocking IC50 (nM) values of chimeric antibodies 132 and 133 and ABC3320

[0405] In summary, among the above-mentioned candidate molecules, No. 132 and No. 133 chimeric antibodies have better performance.

[0406] The No. 132 and No. 133 chimeric antibodies were sequenced respectively, and the amino acid sequences of the heavy chain variable region (132H, 133H) and the light chain variable region (132L, 133L) are as follows:

[0407] > 132H (SEQ ID NO: 39; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0408] > HCDR1 of 132H (SEQ ID NO: 40)

[0409] GFTFSAYY

[0410] > HCDR2 of 132H (SEQ ID NO: 41)

[0411] ISDGGSNT

[0412] > HCDR3 of 132H (SEQ ID NO: 42)

[0413] ARDGGWLLYYFDY

[0414] > 132L (SEQ ID NO: 43; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0415] > LCDR1 of 132L (SEQ ID NO: 44)

[0416] QAIVQSNGETY

[0417] > LCDR2 of 132L

[0418] KVS

[0419] > LCDR3 of 132L (SEQ ID NO: 45)

[0420] FQGSHVPWT

[0421] > 133H (SEQ ID NO: 46; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0422] > HCDR1 of 133H (SEQ ID NO: 47)

[0423] GFIFSDHY

[0424] HCDR2 of 133H (SEQ ID NO: 48)

[0425] ISDGGTYT

[0426] HCDR3 of 133H (SEQ ID NO: 42)

[0427] ARDGGWLLYYFDY

[0428] >133L (SEQ ID NO: 49; CDR1-3 (IMGT system) are bolded and underlined, respectively):

[0429] LCDR1 of 133L (SEQ ID NO: 50)

[0430] QNIVQSDGNTY

[0431] LCDR2 of 133L

[0432] KVS

[0433] LCDR3 of 133L (SEQ ID NO: 51)

[0434] FQGSHLPWT Example 3-1 Screening of antibodies binding to DXD (first round)

[0435] Generation and primary screening of hybridomas

[0436] 1) Immunize mice with KLH-DXD, 40 μg of immunogen per injection, select 2 mice (Balb / c and CD1 mouse strains, respectively) with excellent titer detection results, fuse the spleen cells of the mice with SP2 / 0 myeloma cells by electrochemical method, and culture the hybridoma cells in HAT medium in 70 96-well plates, replace the culture medium with HT medium after one week, and screen for positive hybridoma cells after the fused cells grow to a certain extent and secrete antibodies.

[0437] 2) Select the cell well with OD450 value greater than 0.2 to carry out the negative screening of ADC (structure as shown in Figure 58, Ab-MC-GGFG-DXD) and free DXD (co-incubate the fermentation supernatant and quantified free DXD or ADC, select the cell well in which the incubation of ADC does not affect the OD450 reading value while the incubation of DXD makes the OD450 reading value smaller.), obtain 20 positive hybridomas for subcloning, carry out monoclonal screening by the method of ELISA, finally obtain 7 hybridomas from the pool for sequence fishing work, and the obtained chimeric antibodies are numbered as DXD1-1, DXD1-2, DXD2-1, DXD2-2, DXD5, DXD8, and DXD10, respectively.

[0438] Characterization of chimeric antibodies

[0439] Chimeric antibody primary screening: the above chimeric antibodies are expressed by using 293F cells, and the fermentation supernatant is collected after 7 days and quantified by OCTET.

[0440] Each well is plated with 5000 A549 cells, and after 24 hours, the fermentation supernatant (diluted to 10 μg / mL and 50 μg / mL, respectively) and DXD (100 nM) are added for co-incubation, and after 72 hours, the cell viability is detected to preliminarily screen the fermentation supernatant with DXD protection effect for purification and affinity verification.

[0441] The experimental results are shown in Figure 27, and the results show that the antibodies with cell protection effect preliminarily screened are DXD5 and DXD8.

[0442] After the chimeric antibodies with protection effect preliminarily screened in the early stage are purified, the binding activity of the antibodies to OVA-DXD is detected by ELISA: 500 ng / mL of OVA-DXD is coated in a 96-well plate at 37°C for 2 hours, after being washed twice with PBST, 5% milk powder is added for blocking at 37°C for 2 hours, after the plate is washed twice with PBST, the antibody concentration is diluted to 20000, 10000, 5000, 2500, 1250, 625, 125, 25, 5, 0.5, and 0.05 ng / mL, the diluted antibody is added to the 96-well plate and incubated at 37°C for 1 hour, after the plate is washed twice with PBST, Anti-mouse IgG HRP is added and incubated at 37°C for 1 hour, after the plate is washed three times with PBST, TMB color developing liquid is added, after the color develops to a certain extent, 2M H2SO4 is added and the reading is taken by an enzyme label instrument. The EC50 value is shown in Table 13, and the chimeric antibodies with better binding activity to OVA-DXD are DXD1-1, DXD5, and DXD8.

[0443] Table 13. EC50 (μg / mL) value of chimeric antibody binding to OVA-DXD Example 3-2 antibody screening (second round)

[0444] Generation and primary screening of hybridomas

[0445] 1) Immunize mice with KLH-DXD, 40 μg of immunogen per injection, select mice with excellent titer test results (Balb / c strain) and fuse their spleen cells with SP2 / 0 myeloma cells by electrochemical method, culture hybridoma cells in 30 96-well plates in HAT medium, replace the medium with HT medium after one week, and screen for positive hybridoma cells after the fused cells grow to a certain extent and secrete antibodies.

[0446] 2) Select cell wells with OD450 values greater than 1.8 for ADC and free DXD negative screening (co-incubate the fermentation supernatant and a certain amount of free DXD or ADC, and select cell wells where the incubation of ADC does not affect the OD450 reading but the incubation of DXD makes the OD450 reading smaller.), obtain 52 positive hybridomas for subcloning, screen monoclonal antibodies by ELISA, and finally obtain 27 hybridomas from pools for sequence fishing, and the obtained chimeric antibodies are numbered as JLD1-JLD27.

[0447] Characterization of chimeric antibodies

[0448] Primary screening of chimeric antibodies: use 293F cells to express the above chimeric antibodies, collect the fermentation supernatant after 7 days and use OCTET for quantification.

[0449] Purify and affinity verify the fermentation supernatant with DXD protection effect by co-incubating 5000 A549 cells per well with the fermentation supernatant (diluted to 10 μg / mL and 50 μg / mL, respectively) and DXD (100 nM) after 24 h, and detecting cell viability after 72 h.

[0450] The experimental results are shown in Figure 28, and the results show that the antibodies with cell protection effect are JLD6, JLD7, JLD8, JLD9, JLD10, JLD12, JLD13, JLD14, JLD16, JLD17, JLD18, JLD19, JLD20, JLD21, JLD23, JLD24, JLD25, and JLD27.

[0451] After purification of the chimeric antibodies with protective effects in the previous preliminary screening, the binding activity of the antibodies and OVA-DXD was detected by ELISA: 500 ng / mL of OVA-DXD was coated in a 96-well plate at 37°C for 2 h, after washing twice with PBST, 5% milk powder was added for blocking at 37°C for 2 h, after washing the plate twice with PBST, the antibody concentration was diluted to 20000, 10000, 5000, 2500, 1250, 625, 125, 25, 5, 0.5, 0.05 ng / mL, the diluted antibody was added to the 96-well plate and incubated at 37°C for 1 h, after washing the plate twice with PBST, Anti-mouse IgG HRP was added and incubated at 37°C for 1 h, after washing the plate three times with PBST, TMB color developing solution was added, and after the color developed to a certain extent, 2M H2SO4 was added for reading by an enzyme marker. The EC50 values are shown in Table 14. The chimeric antibodies with better binding activity to OVA-DXD are JLD6, JLD7, JLD17 and JLD20.

[0452] Table 14. Chimeric antibody binding EC50 (ng / mL) value to OVA-DXD Example 3-3 Screening of antibodies binding to DXD (third round)

[0453] Generation of hybridoma and preliminary screening

[0454] 1) The mice were immunized with KLH-DXD, 40 μg of immunogen was injected each time, and the spleen cells of 2 mice with excellent titer detection results (mouse strains were Balb / c and CD1, respectively) were fused with SP2 / 0 myeloma cells by electrochemical method. The hybridoma cells of Balb / c mice were plated in 30 96-well plates, and the hybridoma cells of CD1 mice were plated in 30 96-well plates and cultured in HAT medium. After one week, the culture medium was replaced with HT medium, and after the fusion cells grew to a certain extent and secreted antibodies, the positive hybridoma cells were screened.

[0455] 2) The cell wells with OD450 value greater than 2 were selected for negative screening of ADC and free DXD (the fermentation supernatant and a certain amount of free DXD or ADC were co-incubated, and the cell wells in which the incubation of ADC did not affect the OD450 reading value but the incubation of DXD made the OD450 reading value smaller were selected). A total of 212 positive hybridomas were obtained and subcloned, and monoclonal screening was performed by ELISA method, finally 65 pools of hybridomas were obtained for sequence fishing, and the obtained chimeric antibodies were numbered as D1-D65.

[0456] Characterization of chimeric antibodies

[0457] Chimeric antibody preliminary screening: The above chimeric antibodies were expressed by 293F cells, and the fermentation supernatant was collected after 7 days and quantified by OCTET.

[0458] Each well was laid 5000 A549 cells, after 24h, the fermentation supernatant (diluted to 20 μg / mL) and DXD (1 μM) were added for co-incubation, after 72h, the cell viability was detected, and the fermentation broth with DXD protective effect was screened for purification and affinity verification.

[0459] The experimental results are shown in Figure 29, and the results show that the antibodies with cell protection effect screened preliminarily are D2, D4, D6, D12, D15-1, D15-2, D23-1, D23-2, D24, D28-2, D31, D34, D41, D42, D47, D43-1, D43-3, D49, D50, D52, D56-2, D61, D62, D63, D64, and D65.

[0460] After the chimeric antibodies with protective effect screened preliminarily were purified, the binding activity of the antibodies and OVA-DXD was detected by ELISA: 500 ng / mL of OVA-DXD was coated at 37°C for 2h in a 96-well plate, after washing twice with PBST, 5% milk powder was added for blocking at 37°C for 2h, after washing the plate twice with PBST, the antibody concentration was diluted to 20000, 10000, 5000, 2500, 1250, 625, 125, 25, 5, 0.5, 0.05 ng / mL, the diluted antibody was added to the 96-well plate and incubated at 37°C for 1h, after washing the plate twice with PBST, Anti-mouse IgG HRP was added and incubated at 37°C for 1h, after washing the plate three times with PBST, TMB color developing liquid was added, and after the color developed to a certain extent, 2M H2SO4 was added for reading by an enzyme marker. The EC50 values are shown in Table 15. The chimeric antibodies with better binding activity to OVA-DXD are D2, D4, D6, D52, D61, D62, D63, and D64.

[0461] Table 15. EC50 (ng / mL) values of chimeric antibodies binding to OVA-DXD Example 3-4 Cell protection experiment of chimeric antibodies

[0462] Specificity detection of chimeric antibodies

[0463] The binding activity of the antibody to DXD was detected by ELISA. The detection was performed according to the following steps: 200 ng / mL of DXD-OVA was coated at 37 °C for 2 h, 5% milk was used for blocking at 37 °C for 2 h, the gradient-diluted antibody was respectively incubated with PBS, ADC (500 nM) and free DXD (40 μM) at 37 °C for 1 h, Anti-human HRP was added for incubation at 37 °C for 1 h, the plate was washed with PBST for 3 times, TMB color developing liquid was added, and the color was developed to a certain degree, 2M H2SO4 was added for reading by an enzyme label instrument. The IC50(μg / mL) values are shown in Table 16. Except for D28-2, the binding specificity of the other chimeric antibodies is good.

[0464] Table 16. Specificity of the binding of the chimeric antibody to DXD

[0465] Chimeric antibody protection experiment on DXD killing cells

[0466] NCI-H292 is a sensitive cell line to DXD. 5000 cells per well were plated in a white 96-well plate, and after 24 h of incubation in an incubator, the mixture of gradient-diluted antibody and small molecule DXD was added for cell killing and antibody protection experiment. After 72 h, the ATP amount of the living cells in each well was detected by a chemiluminescence detection kit to detect the cell protection efficiency of the antibody. The IC50 values are shown in Table 17. The chimeric antibodies with good protection effect on DXD killing cells are DXD8, JLD6, JLD18, D2, D4, D28-2, D52 and D6.

[0467] Table 17. Inhibition IC50(μg / mL) values of the chimeric antibody on DXD killing cells

[0468] Based on the above experimental results, among the above candidate molecules, the chimeric antibodies D4, D6, D28-2, D52, JLD6, JLD18 and DXD8 have better performance and can effectively reduce the cell killing effect of free DXD toxin.

[0469] The above chimeric antibodies were sequenced, and the amino acid sequences of the heavy chain and light chain variable regions are as follows:

[0470] >D4H (SEQ ID NO: 52; the CDR1-3 (IMGT system) in the bold and underlined region)

[0471] HCDR1 of D4H (SEQ ID NO: 53)

[0472] GFTFSDNY

[0473] HCDR2 of D4H (SEQ ID NO: 54)

[0474] ISAGGSYT

[0475] HCDR3 of D4H (SEQ ID NO: 55)

[0476] AGTAMDY

[0477] D4L (SEQ ID NO: 56; CDR1-3 (IMGT system) are bolded and underlined, respectively):

[0478] LCDR1 of D4L (SEQ ID NO: 57)

[0479] SSVSY

[0480] LCDR2 of D4L

[0481] DTS

[0482] LCDR3 of D4L (SEQ ID NO: 58)

[0483] LQWKSYPPT

[0484] D6H (SEQ ID NO: 59; CDR1-3 (IMGT system) are bolded and underlined, respectively):

[0485] HCDR1 of D6H (SEQ ID NO: 53)

[0486] GFTFSDNY

[0487] HCDR2 of D6H (SEQ ID NO: 54)

[0488] ISAGGSYT

[0489] HCDR3 of D6H (SEQ ID NO: 55)

[0490] AGTAMDY

[0491] D6L (SEQ ID NO: 60; CDR1-3 (IMGT system) are bolded and underlined, respectively):

[0492] LCDR1 of D6L (SEQ ID NO: 57)

[0493] SSVSY

[0494] LCDR2 of D6L

[0495] DTS

[0496] LCDR3 of D6L (SEQ ID NO: 58)

[0497] LQWKSYPPT

[0498] D28H (SEQ ID NO: 61; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0499] HCDR1 of D28H (SEQ ID NO: 62)

[0500] GFTFINYW

[0501] HCDR2 of D28H (SEQ ID NO: 63)

[0502] ISLISDDYAT

[0503] HCDR3 of D28H (SEQ ID NO: 64)

[0504] TEAGYFFDY

[0505] D28L (SEQ ID NO: 65; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0506] LCDR1 of D28L (SEQ ID NO: 66)

[0507] QNVGTN

[0508] LCDR2 of D28L

[0509] SAS

[0510] LCDR3 of D28L (SEQ ID NO: 67)

[0511] QQYNSYPLT

[0512] D52H (SEQ ID NO: 68; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0513] HCDR1 of D52H (SEQ ID NO: 69)

[0514] GFTLSNYW

[0515] HCDR2 of D52H (SEQ ID NO: 70)

[0516] IRPKSYNYAT

[0517] HCDR3 of D52H (SEQ ID NO: 71)

[0518] TGFPFDY

[0519] D52L (SEQ ID NO: 72; bold underlined regions are CDRs 1-3 (IMGT system), respectively):

[0520] LCDR1 of D52L (SEQ ID NO: 73)

[0521] QDVGTA

[0522] LCDR2 of D52L

[0523] WAS

[0524] LCDR3 of D52L (SEQ ID NO: 74)

[0525] QQYSSYPLT

[0526] JLD6H (SEQ ID NO: 75; bold underlined regions are CDRs 1-3 (IMGT system), respectively):

[0527] HCDR1 of JLD6H (SEQ ID NO: 76)

[0528] GISITNGNHW

[0529] HCDR2 of JLD6H (SEQ ID NO: 77)

[0530] IRPSGST

[0531] HCDR3 of JLD6H (SEQ ID NO: 78)

[0532] ARGGLEDGKYYYAMDY

[0533] JLD6L (SEQ ID NO: 79; bold underlined regions are CDRs 1-3 (IMGT system), respectively):

[0534] LCDR1 of JLD6L (SEQ ID NO: 80)

[0535] ENVEYFGTSL

[0536] LCDR2 of JLD6L

[0537] GAS

[0538] LCDR3 of JLD6L (SEQ ID NO: 81)

[0539] QQSRKVPST

[0540] JLD18H (SEQ ID NO: 82; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0541] HCDR1 of JLD18H (SEQ ID NO: 83)

[0542] GFTFTNYW

[0543] HCDR2 of JLD18H (SEQ ID NO: 84)

[0544] IRLMSNNCAT

[0545] HCDR3 of JLD18H (SEQ ID NO: 85)

[0546] TYYDYDYYYSMDY

[0547] JLD18L (SEQ ID NO: 86; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0548] LCDR1 of JLD18L (SEQ ID NO: 87)

[0549] QNVATN

[0550] LCDR2 of JLD18L

[0551] SAS

[0552] LCDR3 of JLD18L (SEQ ID NO: 67)

[0553] QQYNSYPLT

[0554] DXD8H (SEQ ID NO: 88; CDR1-3 (IMGT system) underlined and bolded, respectively):

[0555] HCDR1 of DXD8H (SEQ ID NO: 89)

[0556] GHTFTSYV

[0557] HCDR2 of DXD8H (SEQ ID NO: 90)

[0558] INPYNGGI

[0559] HCDR3 of DXD8H (SEQ ID NO: 91)

[0560] YYYGNSYGY

[0561] DXD8L (SEQ ID NO: 92; CDR1-3 (IMGT system) are underlined and bolded, respectively):

[0562] LCDR1 of DXD8L (SEQ ID NO: 93)

[0563] QGINSN

[0564] LCDR2 of DXD8L

[0565] HGT

[0566] LCDR3 of DXD8L (SEQ ID NO: 94)

[0567] IQYAQFPFT

[0568] Humanization sequence design of JLD6 antibody

[0569] By comparing the sequence similarity, the antibody germline with the highest similarity to the mouse JLD6 antibody was selected as the humanization template, the CDR of the heavy chain and light chain were replaced with the CDR region of the template, and then the key amino acids affecting the activity of the antibody were back mutated according to the three-dimensional structure predicted by artificial intelligence. The specific humanization process is as follows:

[0570] (1) IGHV4-30-4*01 and IGHJ6*01 were selected as the heavy chain humanization template of the mouse JLD6 antibody, and IGKV4-1*01 and IGKJ2*01 of Kappa were selected as the light chain humanization template of the mouse JLD6 antibody, the CDR region of the heavy chain or light chain of the mouse JLD6 antibody was replaced with the CDR region of the humanization template, and the heavy chain variable region sequence 6231 (SEQ ID NO: 95) and the light chain variable region sequence 6235 (SEQ ID NO: 96) were obtained, respectively;

[0571] (2) The ESMFold module in WeMol software was used to predict the structure of the variable region of the mouse JLD6 antibody, and the three-dimensional structure model was obtained;

[0572] (3) According to the variable region structure of the mouse-derived JLD6 antibody, the key amino acids in the framework region affecting the interaction between heavy chain and light chain and the interaction with CDR were judged, the amino acid sites of back mutation were determined, and the heavy chain variable region sequence 6233 (SEQ ID NO: 97) was obtained, which constituted the PP-1X-#9 molecule with the light chain variable region sequence 6235. At the same time, the potential isomerization site DG in the heavy chain CDR3 was removed by post-translational modification, and the heavy chain variable region sequence 6496 (SEQ ID NO: 98) was obtained, which constituted the PP-1X-#17 molecule with the light chain variable region sequence 6235.

[0573] >6231 (SEQ ID NO: 95; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0574] HCDR1 of 6231 (SEQ ID NO: 76)

[0575] GISITNGNHW

[0576] HCDR2 of 6231 (SEQ ID NO: 77)

[0577] IRPSGST

[0578] HCDR3 of 6231 (SEQ ID NO: 78)

[0579] ARGGLEDGKYYYAMDY

[0580] >6235 (SEQ ID NO: 96; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0581] LCDR1 of 6235 (SEQ ID NO: 80)

[0582] ENVEYFGTSL

[0583] LCDR2 of 6235

[0584] GAS

[0585] LCDR3 of 6235 (SEQ ID NO: 81)

[0586] QQSRKVPST

[0587] >6233 (SEQ ID NO: 97; the bold and underlined regions are CDR1-3 (IMGT system) respectively):

[0588] HCDR1 of 6233 (SEQ ID NO: 76)

[0589] GISITNGNHW

[0590] HCDR2 of 6233 (SEQ ID NO: 77)

[0591] IRPSGST

[0592] HCDR3 of 6233 (SEQ ID NO: 78)

[0593] ARGGLEDGKYYYAMDY

[0594] > 6496 (SEQ ID NO: 98; CDR1-3 (IMGT system) are bolded and underlined, respectively):

[0595] HCDR1 of 6496 (SEQ ID NO: 99)

[0596] GISITNANHW

[0597] HCDR2 of 6496 (SEQ ID NO: 77)

[0598] IRPSGST

[0599] HCDR3 of 6496 (SEQ ID NO: 100)

[0600] ARGGLEEGKYYYAMDY

[0601] Humanized antibody construction and expression and purification

[0602] The heavy chain sequence and light chain sequence of the antibody were constructed into the expression vector pcDNA3.4 by primer design, respectively, to obtain the corresponding plasmids, and Expi293 cells were used for transient transfection, and the specific transfection and purification process was as follows:

[0603] HEK293SQ cells were cultured in Expi293 expression medium. ExpiFectamine 293 transfection kit was used and transient transfection was performed according to the manufacturer's instructions, and transfection enhancer was added 20h after transfection, and supernatant was collected 120h after transfection for subsequent protein purification;

[0604] Magnetic beads were activated with 0.1M NaOH and PBST, respectively, and after incubation at room temperature for 1.5h, impurities were washed with PBST and water, respectively, and the target protein was eluted with 1000μL elution buffer, and the purity of the sample was determined by 4-12% SDS-PAGE.

[0605] Cell protection experiment of humanized anti-DXd antibody

[0606] Digest NCI-H292 cells, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend with 1640+10% FBS medium for counting, adjust to the corresponding concentration, and add 60 μL per well into the Corning 3610 plate, 5000 cells per well, 37°C incubator standby;

[0607] Dilute the antibody concentration (40 μg / mL or 20 μg / mL, 2-fold dilution), and the fixed concentration of free Dxd is 10 ng / mL. Add 30 μL of Dxd and 30 μL of antibody 1:1 into the 3610 plate with cells, and the total culture system is 120 μL. Incubate in a 37°C incubator for 3 days.

[0608] Take out the plate, equilibrate at room temperature, and add 100 μL Luminescent Cell, mix well, and stand at room temperature in the dark for 10 min.

[0609] Detect the Luminescence values on the microplate reader, and the calculation formula is: % Cytoxicity = (Blank-Sample) / Blank*100%

[0610] The experimental results are shown in FIG. 30, and the results show that the PP-1X-#9 and PP-1X-#17 molecules have good cell protection activity. Example 4-1 Llama immunization scheme and screening method

[0611] Table 18. Llama immunization scheme

[0612] Llama group 2-1 / 2-3

[0613] Primary immunization: The emulsion prepared by mixing Freund's complete adjuvant (FCA) and antigen at a volume ratio of 1:1, and the immunization dose is 500 μg of antigen per head.

[0614] Boosting immunization: Prepare the emulsion with Freund's incomplete adjuvant (FIA) every 14 days, and adjust the dose to 250 μg per head.

[0615] Llama group 2-2

[0616] Primary immunization: Dose of 500 μg of antigen per head.

[0617] Boosting immunization: Dose of 250 μg of antigen per head, and the immunization interval is the same as that of group 2-1.

[0618] Post-immunization screening method

[0619] Alpaca 2-3: venous blood was collected 14 days after the third immunization, and Alpaca 2-1 / 2-2: venous blood was collected 7 days after the fourth immunization, peripheral blood mononuclear cells (PBMCs) were isolated and RNA was extracted. After reverse transcription of the RNA into cDNA, a nanobody phage library was constructed by PCR, restriction enzyme digestion and ligation reaction.

[0620] After two rounds of panning, ELISA detection was performed to select non-redundant sequences. A total of 1000 antibodies were screened in this batch, and then antibodies in the form of VHH+Fc were constructed for antibody ELISA detection and cell protection experiments.

[0621] 1) Non-redundant sequences were constructed into expression vectors for transient transfection in HEK293F suspension cells. 2) Protein purification: Protein A magnetic bead affinity chromatography. The purified protein was subjected to ELISA detection, and OVA-DXD / ADC (structure as shown in Figure 58, Ab-MC-GGFG-DXD) / OVA 100 ng / well was coated, 4°C incubation overnight. 3) The next day, the liquid in the plate was discarded and placed in a plate washer for washing 3 times. Blocking: the blocking solution was 2%-3% BSA, 200 μL of blocking solution was added per well, and incubated at 37°C for 1 h. 4) Washing the plate: discard the liquid in the plate and place it in a plate washer, wash the plate 3 times. 5) Antibody gradient dilution. Each antibody corresponds to five experimental groups, the first group is OVA-DXD plate + antibody + PBS; the second group is OVA-DXD plate + antibody + DXD (40 μM); the third group is OVA-DXD plate + antibody + ADC (500 nM); the fourth group is OVA plate + antibody + PBS; the fifth group is ADC plate + antibody + PBS. 6) Antibody, PBS, DXD, ADC, each 30 μL, 37°C for 1 h. 7) Discard the liquid in the plate, place it in a plate washer, and wash it 3 times. 8) Secondary antibody: Anti ALFA tag Mab HRP, 1:5000 dilution, 37°C incubation for 1 h. Wash the plate: discard the liquid in the plate, place it in a plate washer, and wash it 3 times. 9) Add color developing solution (TMB 100 μL), avoid light color development. Add 50 μL of stop solution, and measure the OD450nm reading value with a microplate reader.

[0622] The experimental results are shown in Figure 31, which shows that antibody No. 86 performs better in terms of specificity.

[0623] The NCI-H292 cells were recovered and cultured in 1640 medium containing 10% FBS to 80% confluence. The cells were trypsinized, centrifuged and resuspended, and the cell density was adjusted to 3×10 4Cells / mL. The cell suspension was seeded into 96-well plates at 100 μL per well (about 3000-5000 cells per well), and the edge wells were filled with PBS to reduce evaporation interference. Incubate at 37℃, 5% CO2 incubator for 24h to allow cell adhesion. Set the following groups: antibody protection group: cells + antibody pretreatment + DXD toxin + medium; control antibody protection group: cells + control antibody IgG1 pretreatment + DXD toxin + medium. Discard the original culture medium, add 100 μL per well of antibody at a concentration of 200 μg / mL prepared with fresh culture medium. Add culture medium containing DXD (10 nM) toxin, 37℃, 5% CO2 for 48h. Add 50 μL of cell proliferation / killing detection reagent per well, shake gently to mix, and incubate at 37℃ for 5-30 min in the dark. Measure the chemiluminescence value with a microplate reader.

[0624] The experimental results are shown in Figure 32, which show that the No. 86 antibody can produce a cell protection effect compared with the control group.

[0625] The No. 86 antibody was sequenced, and the amino acid sequence is as follows:

[0626] >86 (SEQ ID NO: 101; the bold and underlined regions are CDR1-3 (IMGT system), respectively):

[0627] >86VHH (SEQ ID NO: 102; the bold and underlined regions are CDR1-3 (IMGT system), respectively):

[0628]

[0629] >86CDR1 (SEQ ID NO: 103)

[0630] GFTFSIYY

[0631] >86CDR2 (SEQ ID NO: 104)

[0632] INTGGGST

[0633] >86CDR3 (SEQ ID NO: 105)

[0634] ARVLDPSTWEYDT

[0635] Synthesis of random octa / dodeca-loop peptide oligonucleotide primers

[0636] The design strategy for encoding random octa / duodeca cyclic peptide oligonucleotide fragments is to use the NNK codon encoding method. N is any nucleotide (A / T / C / G), and K is G / T. The synthesized random nucleotide sequence is shown in Table 19.

[0637] Table 19. Nucleotide sequence for polypeptide library construction

[0638] PCR amplification of double-stranded DNA fragments

[0639] A palindrome sequence is designed at the 3' end of the long-chain DNA primer to provide a primer for DNA polymerase, guiding 3' to 5' DNA synthesis to obtain a complete double-stranded DNA sequence. The synthesized oligonucleotide template encoding the octapeptide library was dissolved in ddH2O to a 100 μM solution, 5 μg was mixed with an equal molar of universal extension primer in 50 μL ddH2O, and heat denatured at 95°C for 5 min, and naturally cooled to below 37°C in a water bath to form an annealing mixture. An equal volume of PrimeSTAR Max Premix (TAKARA, CAT#: R045B) was added to prepare the PCR solution, mixed well and centrifuged, then extended at 37°C for 10 min and at 65°C for 15 min to synthesize double-stranded DNA fragments encoding the octa / duodeca cyclic peptide library.

[0640] pComb3XSS phage vector and DNA fragment digestion, ligation and transformation

[0641] The DNA fragments and pComb3XSS prepared by restriction enzyme sfi I (NEB, CAT#: R0123L) I were incubated at 50°C for 18 h. 1% agarose gel electrophoresis was used to separate the pComb3XSS digested overnight, and the purified and recovered according to the operation of the gel recovery kit (MN, CAT# 740609.250), then the linearized pComb3XSS plasmid and the digested DNA fragments were ligated using T4 ligase, the molar ratio of vector to DNA fragment was 1:10, and incubated at 16°C for 19 h. The ligation product was purified and recovered using the purification kit, eluted with ultrapure water (30 μL / tube), and the concentration was measured by Nanodrop. Freshly prepared 350 μL TG1 competent cells were added to each electroporation cup (2 mm, pre-cooled in ice water bath), then 500 ng of ligation product was added, the bottom of the electroporation cup was shaken to mix, and incubated in ice water bath for 10 min. The water on the wall and bottom of the electroporation cup was thoroughly wiped off, the voltage was set to 2.5 KV, and the electroporation transformation was performed.

[0642] Identification of peptide library

[0643] Twelve clones were randomly picked for sequencing to analyze the quality of the polypeptide library. Example 5-2 Enrichment screening of phage display cyclic peptide library for MMAE binding peptides

[0644] Polypeptide phage display library panning

[0645] Coating of the immunotubes was performed before panning. The immunotubes were washed with PBS for 3 times and residual liquid was absorbed. According to the immunization strategy, the immunotubes were coated with negative and positive antigens respectively, using 500 μL coating buffer to dilute to the final coating concentration. After mixing at room temperature for 2 h at 150 rpm, the tubes were placed at 4 °C overnight for about 16 h. The P12P phage display library with 1.35E+13 cfu was used as the input for the first round of panning; the phage display library enriched in the previous round was used as the input for the second to fourth rounds of panning, with about 1.00E+13 cfu. The input was blocked with Casein solution (Thermo scientific, Cat. 37528) at a concentration of 50%, and the volume was made up to 500 μL with 1x PBS. The overnight-activated TG1 E. coli was inoculated into 120 mL of 2YT liquid medium, and incubated at 37 °C, 250 rpm / 2-3 h, until the OD reached 0.6-0.8. The immunotubes coated with positive antigens were washed with 1x PBS, and then 1 mL of Casein solution was added for blocking for 1 h. The immunotubes coated with negative antigens were added with the blocked phage input, and mixed at room temperature for 1 h at 150 rpm to remove the binding of phage to toxin-coupled proteins. After blocking, the immunotubes coated with positive antigens were washed, and the phage subjected to negative selection was transferred into the immunotubes, and mixed at room temperature for 1-2 h at 150 rpm. The immunotubes were washed with 1x PBST for 3 times, and placed on a 150 rpm shaker for 1-2 min during washing. Then, 5 nmol of Truncated MMAE solution was added, and mixed at room temperature for 1 h at 150 rpm for competition elution. The eluate was transferred into the immunotubes coated with the second negative antigen, and mixed at room temperature for 1 h at 150 rpm for further negative selection to remove the binding of phage to toxin-coupled proteins. The eluted phage was added into 40 mL of TG1 bacterial solution with an OD of 0.6-0.8, and a glucose solution was added to a final concentration of 2%. The mixture was incubated at 37 °C for 45-60 min, with gentle shaking every 15 min. After incubation, 100 μL of the bacterial solution was diluted 10 1 ,10 2 ,10 3 ,10 4Four gradients, each take 100 μL to coat the small flat plate (10 cm * 10 cm 2YT-Amp-Glu), the next day to count the number of monoclonal, to monitor the enrichment of phage. And take 1 ml of bacteria liquid to add glycerol, stored at -80℃. The remaining bacteria liquid is added with 1 / 1000 Amp (100 mg / mL) and helper phage M13KO7 (Mol 20:1) (NEB, Cat. N0315S), 37℃ static invasion 1h. Centrifugal collection of bacteria liquid, discard the supernatant, add 40 mL 1 / 1000 Kan (50 mg / mL) and IPTG (50 mg / mL) 2YT-Amp liquid medium, 30℃ 220 rpm overnight culture. The bacteria liquid of overnight culture, 10000 rpm centrifugal 10 min; collect the supernatant, 0.45 μM filter, add 1 / 5 volume of PEG-NaCl precipitation. Ice for 1h, 4℃, 10000 rpm centrifugal 30 min, discard the supernatant, PBS resuspended the precipitate, used for panning. Nanodrop determination of phage OD260 value, according to OD260 value to determine the amount of phage used for panning, 1 OD is about 3.0E+12.

[0646] Monoclonal screening of polypeptide phage display library

[0647] 96 well plate (sterile, round bottom), add 600 μL Amp+2YT culture solution in each well. From P12P P1 Round2-4 plate to pick single clone for culture. 37℃, 220 rpm, 3-6h to OD 0.6. The prepared phage is used for phage ELISA. Take 150 μL bacteria liquid to transfer to a new 96 well plate, add glycerol to save the bacteria at -80℃. Add helper phage M13KO7 (Mol 20:1) in the 96 well plate, 37℃ static infection 1h. Add 1 / 1000 Kan (50 mg / mL) and IPTG (50 mg / mL), 30℃ 220 rpm overnight culture. The phage of 96 well plate overnight culture, 4000 rpm centrifugal 30 min; the supernatant (400 μL) is transferred to a new 96 well plate for Phage ELISA.

[0648] Phage ELSIA experimental method

[0649] Phage ELSIA blocking: 96-well plate coated with antigen, washed with PBST for 1 time, added Casein solution for blocking, 200 μL / well, 37°C for 1 h. Phage ELSIA primary antibody incubation: washed with PBST for 3 times, added 100 μL of phage supernatant cultured overnight to each well. Set two negative control wells: Blank and M13KO7 (1:100). Incubated at 37°C for 1-2 h. Phage ELSIA secondary antibody incubation: washed with PBST for 6 times, added secondary antibody anti-M13KO7-HRP (1:10000) diluted with 3% Milk-PBS (Sino. Biological, Cat. 11973-MM05T-H), incubated at 37°C for 1 h. Color development: washed with PBST for 6 times, added 100 μL TMB color developing solution to each well, developed color in dark for 2-10 min. Stop: added 100 μL 2M H2SO4 to each well, read OD450 value with microplate reader, recorded and saved. Analyzed data, selected specific positive clones for sequencing, obtained sequences by CLC software analysis, and performed next step of construction and expression purification. The form of the constructed molecules is shown in Figure 33. Example 5-3 Verification of specific binding of polypeptide molecules to MMAE

[0650] ELISA detection of the binding specificity of Fc fusion polypeptides and MMAE

[0651] The constructed Fc fusion polypeptides PP-1A-#84 to #95 were used to identify the binding specificity of OVA-MMAF and OVA, and to determine the molecules with better specific binding activity to MMAE. After the first round of primary screening, #85 / #86 / #89 / #93 were further verified to specifically bind to OVA-MMAF, but not to the negative protein OVA; and #84 / #87 / #88 / #90 / #91 / #92 / #94 were repeatedly verified for specific binding to MMAE.

[0652] Table 20. Amino acid sequence list of PP-1A-#85 (polypeptide)-PP-1A-#94 (polypeptide) and PP-1A-#85 (Fc fusion polypeptide)

[0653] Polypeptide ELISA binding experiment method

[0654] 1. Antigen coating: MMAF conjugated protein and negative protein were coated on the ELISA plate using coating buffer at a concentration of 1 pg / mL and placed at 4°C overnight for about 16 h. 2. Blocking: The ELISA plate coated with antigen was washed once with PBST using a plate washer, and Casein solution was added for blocking at a volume of 200 pL / well at room temperature for 1 h. 3. Primary antibody incubation: The plate was washed three times with PBST, and polypeptide samples were added at a concentration of 2000 nM in the first well, and then diluted 5 times with Casein solution. The negative control was Fc.uIgG1 molecule. Incubation was performed at room temperature for 2 h. 4. Secondary antibody incubation: The plate was washed three times with PBST, and secondary antibody Goat pAb to Hu IgG (HRP) (1:10000) (Abcam, Cat. Ab97225) diluted with Casein solution was added, and incubation was performed at room temperature for 1 h. 5. Color development: The plate was washed six times with PBST, 100 ul of TMB color developing solution was added to each well, and color development was performed in the dark for 2-10 min. 6. Termination: 100 pL of 2M H2SO4 was added to each well to terminate the reaction, and the OD450 value was read using an enzyme-labeled instrument, and the data were recorded and saved. Finally, the data were analyzed using GraphPad software.

[0655] ELISA binding results are shown in Figure 34, #85 / #86 / #89 / #93 has good binding activity with OVA-MMAF, and no binding with OVA; #88 / #90 / #91 / #92 molecules have specific binding with OVA-MMAF, but the binding activity is weak.

[0656] Free MMAE competitive binding polypeptide molecule identification

[0657] A competition experiment was used to further analyze whether PP-1A-#85 / #93 binds to free MMAE, but not to MMAE conjugated antibody.

[0658] Polypeptide competition ELISA experimental method

[0659] 1. Antigen coating: BSA-MMAF was coated on the ELISA plate using coating buffer at a concentration of 1 μg / mL and incubated at 4°C overnight for about 16 h. 2. Blocking: The coated ELISA plate was washed once with PBST using a plate washer, and then blocked with Casein solution at a volume of 200 μL / well for 1 h at room temperature. 3. Primary antibody incubation: The plate was washed 3 times with PBST, and then a premix of polypeptide samples and truncated free MMAE / null-ADC diluted with Casein solution was added to the plate. The final concentration of the polypeptide samples was 2000 nM, and the initial concentration of the truncated free MMAE and null-ADC (an IgG1 antibody conjugated with MMAE that does not bind to any target) was 1000 nM, which was diluted by a factor of 10. The positive control was ABC3320 molecules at a final concentration of 1.28 nM. The plate was incubated at room temperature for 2 h. 4. Secondary antibody incubation: The plate was washed 3 times with PBST, and then Goat pAb to Hu IgG (HRP) diluted with Casein solution at a dilution of 1:10000 was added to the plate, which was incubated at room temperature for 1 h. 5. Color development: The plate was washed 6 times with PBST, and then 100 μL of TMB color developing solution was added to each well, and the plate was developed in the dark for 2-10 min. 6. Termination: 100 μL of 2M H2SO4 was added to each well to terminate the reaction, and the OD450 value was read using an ELISA reader, and the data was recorded and saved. Finally, the data was analyzed using GraphPad software.

[0660] The results of the competition ELISA are shown in FIG. 35. The binding of PP-1A-#85 / #93 to BSA-MMAF was blocked by truncated free MMAE, but not by null-ADC, indicating that the two polypeptide molecules of #85 / #93 specifically bind to MMAE, but not to null-ADC.

[0661] Specific binding of antibody fusion peptides and MMAE

[0662] According to the identification of binding activity and competition, the PP-1A-#85 molecule was selected to construct the Anti-MSLN-MMAE antibody fusion peptide, and the form of the molecule is shown in Figure 36. After the vector construction, the BL21 strain was chemically transformed, and a single colony was activated and cultured. After the activated bacterial solution was sequenced and confirmed to be correct, it was inoculated into 2x YT medium (1 / 1000 Kan (50 mg / mL)) at a ratio of 1:100, cultured at 37°C, 220 rpm for 1-2 h, and the OD value of the bacterial solution was 0.6-0.8. The shaking temperature was adjusted to 16°C, and after the temperature reached, IPTG solution (0.3 mM) (Solarbio, Cat. 12.02.11020) was added to the cultured bacterial solution, and the induction culture was carried out at 16°C, 220 rpm for 24 h. The bacterial cells were collected by centrifugation, and the bacterial cells were resuspended with pre-cooled PBS solution. The resuspended bacterial solution was ultrasonically broken, the power was 120W, and the breaking time was about 1.25 h. The ultrasonic breaking process needs to maintain the low temperature state of the bacterial solution. The bacterial solution after ultrasonic breaking was collected by multiple centrifugations to obtain a relatively clear solution, and the next step of protein purification was carried out.

[0663] Table 21. PP-1A-#600 amino acid sequence table Example 5-6 verification of specific binding of antibody fusion peptide and MMAE

[0664] The binding activity of the antibody fusion peptide molecule PP-1A-#600 molecule (amino acid sequence as shown in Table 21) and OVA-MMAF and OVA was detected by ELISA to verify its specific binding with MMAE.

[0665] Polypeptide ELISA binding experiment method

[0666] ①Antigen coating: OVA-MMAF and OVA protein were coated on the enzyme-labeled plate using coating buffer at a concentration of 1 μg / mL, and placed at 4°C overnight for about 16 h. ②Blocking: The enzyme-labeled plate coated with antigen was washed once with a plate washer PBST, and Casein solution was added for blocking at a concentration of 200 μL / well at room temperature for 1 h. ③Primary antibody incubation: PBST was washed for 3 times, and polypeptide samples were added at a concentration of 2000 nM in the first well, and diluted with Casein solution at a gradient of 5 times. The positive control was ABC3320 molecule, and the negative control was Fc.uIgG1 molecule at a concentration of 100 nM. Incubation was performed at room temperature for 2 h. ④Secondary antibody incubation: PBST was washed for 3 times, and the secondary antibody Goat pAb to Hu IgG(HRP) was diluted with Casein solution at a ratio of 1:10000, and incubated at room temperature for 1 h. ⑤Color development: PBST was washed for 6 times, 100 μL of TMB color developing liquid was added to each well, and color development was performed in the dark for 2-10 min. ⑥Termination: 100 μL of 2M H2SO4 was added to each well to terminate the reaction, and the OD450 value was read by an enzyme-labeled instrument, and recorded and saved. Finally, the data was analyzed by using GraphPad software.

[0667] The ELISA binding results are shown in FIG. 37, and PP-1A-#600 and OVA-MMAF have strong binding activity, and do not bind to OVA, showing good binding specificity.

[0668] The NCI-H292 tumor cell line was used to detect the inhibition of MMAE toxicity by PP-1A-#600.

[0669] ①NCI-H292 cells were digested and counted, and plated at a concentration of 5E3 / well, 60 μL / well, and adhered in a 37°C incubator. ②Polypeptide PP-1A#600, control antibody BMK219(ABC3320-Fc) and nonsense control antibody Null IgG were diluted at a gradient of 10 times with 2-fold dilution starting from a concentration of 100 μg / ml. The fixed MMAE concentration was 1 ng / mL, 3 ng / mL, 5 ng / mL and 10 ng / mL. The test substance was incubated with MMAE at room temperature for 2 h. ③The incubated sample was added to the 96-well plate at a concentration of 60 μL / well, and cultured in a 37°C incubator for 3 days. ④100 μL of Cell Counting-Lite2.0 Reagent was added to each well, and shaken for 10 min. ⑤The fluorescence signal was read by an enzyme-labeled instrument.

[0670] The experimental results are shown in Figure 38. The results show that the PP-1A-#600 fusion molecule can exhibit good cell protection when the concentration of MMAE is 1, 3, 5, and 10 ng / mL. Example 6-1 Construction of phage display octa / penta linear peptide library

[0671] Synthesis of random octa / penta linear peptide oligonucleotide primers

[0672] The design strategy for encoding random linear octa- and penta-peptide oligonucleotide fragments is to use NNK or NHC encoding. N is any nucleotide (A / T / C / G), K is G / T, and H is A / C / T. The synthesized random nucleotide sequence is shown in Table 22.

[0673] Table 22. Nucleotide sequence for polypeptide library construction

[0674] PCR amplification of double-stranded DNA fragments, enzyme digestion, ligation, and transformation of pComb3XSS phage vector and DNA fragments refer to the related steps in Example 5-1.

[0675] Identification of the peptide library

[0676] Randomly select 20-30 clones for sequencing, and sequence alignment analysis the quality of the polypeptide library.

[0677] Polypeptide phage display library panning

[0678] Liquid and solid phase panning cross strategy, liquid panning experimental method: using streptavidin magnetic beads (Magnosphere, Cat. MS300 / Streptavidin), mix well before use and protect the liquid, take the required concentration of magnetic beads and wash with PBS for 3 times. Negative screening magnetic beads are used to remove phage and non-specific binding of magnetic beads, and 3% BSA (Bovogen, Cat. BSAS 0.1) solution is used for incubation at room temperature for 30 min. Add 1 mL PBS diluted positive screening antigen biotin-DXD to the positive screening magnetic beads, and incubate at room temperature for 1 h. Take 1.00E+13 cfu of phage display library as the input of the first round of panning; take the enriched phage display library of the last round as the input of the second round of panning, about 1.00E+13 cfu. Add phage input to the blocked negative screening magnetic beads, and the blocking solution is 3% BSA solution, and the volume is made up with 1xPBS. At the same time, after the positive screening magnetic beads are combined with the antigen, they are washed with 1xPBS, then 3% BSA solution is added for incubation at room temperature for 1 h. After blocking, the positive screening magnetic beads are washed with PBS for 3 times, and the phage after removing non-specific binding is added, and incubated at room temperature for 1 h. Wash the magnetic beads with PBST / PBS, and increase the washing intensity appropriately with the number of panning rounds. After washing, 1 mL of 0.1M pH2.2 Gly-HCl is added, and the mixture is rotated for elution at room temperature for 10 min. Collect the eluate, and add 100μL of 1M pH8.0 Tris-HCl to neutralize the acid eluate.

[0679] Solid phase panning method: Immunotube coating before panning, immunotube with PBS washing 3 times, and the residual liquid is absorbed. According to the immunization strategy, the immunotube is coated with negative and positive antigens, and 500 μL of coating buffer is diluted to the final coating concentration. After mixing at room temperature for 2 h at 150 rpm, it is placed at 4°C overnight for about 16 h. The third round of panning takes the enriched phage display library of the previous round as input, about 1.00E+13 cfu. The input is blocked with Casein solution (Thermo scientific, Cat. 37528) at a concentration of 50%, and the volume is made up to 500 μL with 1x PBS. After washing the immunotube coated with positive antigens with 1x PBS, 1 ml of Casein solution is added for blocking for 1 h. The immunotube coated with negative screening protein is added with the blocked phage input, and combined at room temperature for 1 h at 150 rpm to remove the binding of phage to toxin-coupled protein. After blocking the immunotube coated with positive antigens, the immunotube is washed, and the phage after negative screening is transferred to the immunotube, combined at room temperature for 1-2 h at 150 rpm. The immunotube is washed 3 times with 1x PBST, and placed on a 150 rpm shaker for 1-2 min during washing. 5 nmol of DXD solution is added, and eluted at room temperature for 1 h at 150 rpm. The eluted phage is added to 40 ml of OD0.6-0.8 TG1 bacterial solution, and 2% glucose solution is added to a final concentration. Incubate at 37°C for 45-60 min, and gently shake the bacterial solution every 15 min. After incubation, 100 μL of bacterial solution is diluted 10 1 2 3 Three gradients, 100 μL is taken and plated on a small flat plate (10 cm x 10 cm2 YT-Amp-Glu), and the number of single colonies is counted the next day to monitor the enrichment of phage. 1 mL of bacterial solution is added to glycerol and stored at -80°C. The remaining bacterial solution is added with 1 / 1000 Amp (100 mg / mL) and helper phage M13KO7 (Mol 20: 1) (NEB, Cat. N0315S), and incubated at 37°C for 1 h. The bacterial solution is collected by centrifugation, and the supernatant is discarded. 40 mL of 1 / 1000 Kan (50 mg / mL) and IPTG (50 mg / mL) 2YT-Amp liquid medium is added, and incubated at 30°C at 220 rpm overnight. The overnight culture is centrifuged at 10000 rpm for 10 min, and the supernatant is collected, 0.45 μM filtered, and 1 / 5 volume of PEG-NaCl is added for precipitation. Place on ice for 1 h, centrifuge at 4°C at 10000 rpm for 30 min, discard the supernatant, and resuspend the precipitate with PBS for panning. The OD260 value of the phage is determined by Nanodrop, and the input amount of phage for panning is determined according to the OD260 value, about 3.0E+12 for 1 OD.

[0680] ​​Monoclonal screening of polypeptide phage display library

[0681] 96 well plates (sterile, round bottom) were filled with 600 μL Amp+2YT broth per well. Single clones from P8P / P12P P5 Round2 / 3 plates were picked and grown. 37°C, 220 rpm, 3-6h to OD 0.6. Prepared phage was used for phage ELISA. 150 μL of bacterial solution was transferred to a new 96 well plate and the bacteria were stored at -80°C in glycerol. Helper phage M13KO7 (Mol 20:1) was added to the 96 well plate and the bacteria were allowed to be infected for 1h at 37°C without shaking. 1 / 1000 Kan (50mg / mL) and IPTG (50mg / mL) were added and the bacteria were grown overnight at 30°C with 220 rpm. The phage from the 96 well plate was centrifuged at 4000 rpm for 30 min. The supernatant (400 μL) was transferred to a new 96 well plate and used for phage ELISA.

[0682] Phage ELSIA experimental procedure

[0683] The procedure was performed according to the procedure described in Example 5-1. The format of the molecule is shown in Figure 39. Example 6-3 Dot-blot screening for DXD specific binding peptides

[0684] Sample preparation: centrifuge 5 min at 5000 rpm to collect 50 mL of IPTG-induced overnight (16°C, 16 h) bacterial solution, discard the supernatant. Add 7 mL of PBS to resuspend the bacterial pellet, and break the cells with 200 W for 5 min. Centrifuge at 12000 rpm for 20 min to collect the supernatant for use. Spotting: Cut the nylon membrane (Bi Yun Tian, CAT#FFN10) to the appropriate size and draw a line. Use a pipette to spot 3 μL of antigen sample (OVA or OVA-DXD, 20 μg / mL) on the membrane. Pay attention to the consistency of the sample amount and the spacing between spots to avoid interference between samples. After spotting, let the membrane dry naturally at room temperature. Blocking: Place the dried membrane in blocking solution (5% non-fat milk) and incubate on a shaker at room temperature for 1-2 h. The purpose of blocking is to prevent non-specific binding and reduce background signal. Washing: After incubating the primary antibody, wash the membrane thoroughly with washing buffer (PBST) for 3 times, 5 min each time, to remove unbound primary antibody. Secondary antibody incubation: Place the membrane in a solution containing the appropriate dilution of secondary antibody and incubate at room temperature for 1-2 h. The secondary antibody is an antibody against the primary antibody, usually with a detectable label such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), etc. Washing: Wash the membrane again with washing buffer to remove unbound secondary antibody, wash 3 times, 5 min each time. Color development: Take an appropriate amount of ECL color developing substrate solution onto the membrane, react at room temperature in the dark, and observe the color development with a gel imager to take a photo record of the membrane. By observing the presence or absence of spots, color intensity, etc., qualitative or semi-quantitative analysis of the target protein in the sample can be performed.

[0685] The results of the dot blot are shown in Figure 40, which shows that molecules 6553, 6677, 6678, 6683, 6685, 6686, 6689, 6560, 6551, 6561, and 6562 can specifically bind to the polypeptide of DXD.

[0686] Table 23. Amino acid sequence of the above molecules Example 6-4 Cell experiment to detect the inhibitory effect of DXD binding peptides on toxin cytotoxicity

[0687] NCI-H292 tumor cells were used to detect the inhibitory effect of DXD binding polypeptides on DXD cytotoxicity.

[0688] NCI-H292 cells were digested, counted and plated at 5E3 / well in 60 μL / well, and incubated at 37°C in an incubator. The polypeptide molecules and the nonsense control antibody Null IgG were started at a concentration of 100 μg / ml, 2-fold dilution, 7 gradients. The fixed DXD concentration was 1 ng / mL. The test substances were incubated with DXD at room temperature for 2 h. The incubated samples were added to a 96-well plate, 60 μL / well; incubated at 37°C in an incubator for 3 days. 100 μL of Cell Counting-Lite2.0 Reagent was added to each well, and shaken for 10 min. The fluorescence signal was read using a microplate reader.

[0689] The experimental results are shown in Figure 41, and the results show that polypeptides PP-1A#414, PP-1A#340, PP-1A#415, and PP-1A#417 exhibit cell protection, and the activity of PP-1A#414 is the best.

[0690] Generation of virtual polypeptide library

[0691] In the preliminary establishment, a natural tripeptide library was generated by permutation and combination using 20 natural amino acids, containing a total of 8000 tripeptides; after batch structure prediction of the tripeptide library using structure prediction software, batch molecular docking was performed with small molecule compounds MMAE; the top 50 tripeptides were screened through affinity results, and 20 different natural amino acids were inserted at each possible position, and after excluding repeated sequences, a new tetrapeptide was generated; in this way, the same method was applied to generate pentapeptide, hexapeptide, heptapeptide, octapeptide libraries, etc.

[0692] Screening of virtual polypeptide library

[0693] Molecular docking: AutoDock Vina docking software was used to dock the predicted polypeptide and small molecule, MMAE was used for the N-terminal setting of ADC coupling, the num_modes parameter was set to 10, the energy_range parameter was set to 5, and the exhaustiveness parameter was set to 16; flexible docking mode was used, with the N-terminal of MMAE as the binding center, and the polypeptide was flexibly combined; after docking, all docking conformations were sorted, and the lower the binding free energy, the more likely it was the potential best binding mode, so as to virtually screen high-affinity polypeptides.

[0694] Table 24. Affinity data

[0695] Therefore, among the above-mentioned candidate molecules, the polypeptide PP-1B-#74 shows higher affinity, and its sequence is: YYVAWRW (SEQ ID NO: 129). Example 7-2 Construction and expression and purification of polypeptide molecules

[0696] Construction of polypeptide molecules

[0697] By primer design, the polypeptide fragment PP-1B-#74 is connected to the C-terminus of Fc to construct a plasmid for producing Fc fusion polypeptide, and the amino acid sequence information is shown in SEQ ID NO: 130.

[0698] SEQ ID NO: 130:

[0699] Expression and purification of polypeptide molecules

[0700] HEK293SQ cells were cultured in Expi293 expression medium. ExpiFectamine 293 transfection kit was used, and transient transfection was performed according to the manufacturer's instructions. Transfection enhancer was added 20h after transfection, and supernatant was collected 120h after transfection for subsequent protein purification; 0.1M NaOH and PBST were used to activate magnetic beads, and the supernatant was incubated at room temperature for 1.5h, then PBST and water were used to wash the impurities, 1000μL elution buffer was used to elute the target protein, and 4-12% SDS-PAGE was used to determine the purity of the sample. Example 7-3 Specific binding identification of Fc fusion polypeptide and MMAE

[0701] The binding of the constructed Fc fusion polypeptide PP-1B-#70-#74 molecules with OVA-MMAF and OVA was identified, and the molecules with better specific binding with MMAE were screened; after the first round of preliminary screening, the binding specificity of #74 with OVA-MMAF and BSA-MMAF was further verified;

[0702] Polypeptide ELISA binding experiment method

[0703] 1. Antigen coating: MMAF-coupled proteins and negative proteins were coated on an enzyme-labeled plate using coating buffer at a concentration of 1 μg / mL and placed at 4°C overnight for about 16 h; 2. Blocking: The enzyme-labeled plate coated with the antigen was washed once with a plate washer PBST, and Casein solution was added for blocking at a concentration of 200 μL / well at room temperature for 1 h; 3. Primary antibody incubation: The plate was washed 3 times with PBST, and polypeptide samples were added at a concentration of 2000 nM in the first well, and the polypeptide samples were diluted 5 times with Casein solution, and incubated at room temperature for 2 h; 4. Secondary antibody incubation: The plate was washed 3 times with PBST, and the secondary antibody Goat pAb to Hu IgG (HRP) (1:10000) (Abcam, Cat. Ab97225) diluted with Casein solution was added, and incubated at room temperature for 1 h; 5. Color development: The plate was washed 6 times with PBST, 100 μL of TMB color developing solution was added to each well, and color development was performed in the dark for 2-10 min; 6. Termination: 100 μL of 2M H2SO4 was added to each well to terminate the reaction, and the OD450 value was read with an enzyme-labeled instrument, and the data were recorded and saved. Finally, the data were analyzed using GraphPad software.

[0704] The experimental results are shown in FIG. 42, and the results show that the PP-1B-#74 molecule can specifically bind to OVA-MMAF and BSA-MMAF.

[0705] According to the binding activity results of the polypeptide molecule, the specific binding of the PP-1B-#74 molecule to MMAE was further verified, and the verification was performed by a competition experiment.

[0706] Polypeptide competition ELISA experimental method

[0707] ①Antigen coating: BSA-MMAF was coated on the enzyme-labeled plate using coating buffer, at a concentration of 1 μg / mL, and placed at 4°C overnight for about 16 h; ②Blocking: The enzyme-labeled plate coated with the antigen was washed once with the plate washer PBST, and 200 μL / well of Casein solution was added for blocking at room temperature for 1 h; ③Primary antibody incubation: After washing 3 times with PBST, the premixed solution of polypeptide sample and truncated free MMAE / null-ADC diluted with Casein solution was added, the final concentration of the polypeptide sample was 2000 nM, and the first-well concentration of the truncated free MMAE and null-ADC was 1000 nM, which was diluted by 10 times in gradient, and incubated at room temperature for 2 h; ④Secondary antibody incubation: After washing 3 times with PBST, Goat pAb to Hu IgG (HRP) (1:10000) diluted with Casein solution was added, and incubated at room temperature for 1 h; ⑤Color development: After washing 6 times with PBST, 100 μL of TMB color developing solution was added to each well, and color development was carried out in the dark for 2-10 min; termination: 100 μl of 2M H2SO4 was added to each well to terminate the reaction, and the OD450 value was read by an enzyme-labeled instrument, and the data were recorded and saved. ⑥Finally, the data were analyzed using GraphPad software.

[0708] The experimental results are shown in FIG. 43, and the results show that the PP-1B-#74 molecule can specifically bind to MMAE.

[0709] The single-stranded DNA library and primers shown in the following sequences were synthesized

[0710] Table 25. Aptamer library and primers

[0711] Wherein: the library is composed of single-stranded DNA of 66 bases in length, with a random sequence (N) of 30 bases in the middle and fixed primer sequences at both ends, and 5B refers to 5' end with Biotin modification. 1214-F and 1214-R-5B are used for each round of symmetric PCR, and 1214-F and 1214-R are used for amplifying the library to be sequenced. The single-stranded DNA library and primers are synthesized by Shengong Biotech (Shanghai) Co., Ltd.

[0712] Aptamer screening

[0713] After centrifugation of the initial ssDNA library at 12000 rpm for 1 min, 100 μL of Binding buffer was used to dissolve (D-PBS with 5 mM final concentration of MgCl2, i.e. DPBS containing 5 mM Mg 2+DNA total amount is 1-5 nmol. 95 °C metal bath 5 min, immediately put on ice for 5 min. This process is used for library denaturation, to refold the conformation of aptamer, after renaturation, add 100 μL Binding buffer, add hole plate for standby. Use empty enzyme label coated with 3% BSA as negative screen, BSA-MMAF coated immune tube as positive screen, add renatured aptamer library in positive screen hole plate, incubate at room temperature for 120 min, to make conformation folded aptamer fully bind to target MMAF. After incubation, discard supernatant, wash with DPBS or DPBST solution, then add 200 μL eluent (20 mmol / L Tris-HCl, 4 mol / L guanidine isothiocyanate, 1 mmol / L dithiothreitol (pH 8.3)), incubate at 80 °C for 15 min, wash ssDNA bound to monoclonal antibody, collect eluent. Use eluent as template for first round of PCR amplification, system is: 200 μL template, 400 μL PrimeStar Max, 32 μL 1214-F, 32 μL 1214-R, 332 μL ddH2O, total system is 800 μL, each PCR tube is divided into 80 μL. Reaction conditions 95 °C pre-denaturation 2 min; 95 °C denaturation 30 s, 60 °C annealing 5 s, 72 °C extension 5 s, 6 cycles of amplification; finally 72 °C extension 2 min.

[0714] To determine the optimal number of PCR cycles, take 100 μL of the first round of PCR product as template, prepare the reaction system according to the table above, take 80 μL from it and add to 4 PCR tubes, respectively, to amplify 2, 4, 6 and 8 cycles, then perform 3% agarose gel electrophoresis, select the number of bands clear, no non-specific amplification for the third PCR, the third PCR reaction system and cycle number optimization system are consistent, the remaining mixture is amplified according to the optimized cycle number.

[0715] Biotinylated PCR products were mixed with streptavidin magnetic beads and incubated at room temperature for 15 min. After washing twice with lx PBS, 200 mM NaOH was added and incubated for 5 min to make the double-stranded DNA into single-stranded DNA. The magnetic stand was used to make the ssDNA with biotin remain on the streptavidin magnetic beads and adsorbed on the tube wall, while the other ssDNA without biotin was in the supernatant. The obtained ssDNA secondary library was collected and purified by ethanol precipitation (1 / 10 3M NaAC and 2.5 times the volume of absolute ethanol were mixed well and then placed in a -80 °C refrigerator to precipitate overnight. The next day, it was directly taken out and centrifuged at 13000 rpm for 10 min at 4 °C. After the supernatant was discarded, 1 mL of 75% ethanol was added and centrifuged at 13000 rpm for 10 min at 4 °C. The supernatant was discarded, and the precipitate was dissolved in 50 μL Binding buffer and the concentration was detected by Nanodrop. 1 μg of DNA was taken as the secondary library for the next round of screening, and the excess part was stored in a -20 °C refrigerator).

[0716] The screening pressure was changed by adjusting the target amount, incubation time and washing conditions in each round. The positive screening antigen was reduced from 100 μg to 30 μg, and the positive screening time was reduced from 120 min to 30 min. The negative screening antigen was 10 μg to remove non-specific binding sequences, and the incubation time was increased from 20 min to 60 min.

[0717] A total of 6 aptamers were screened in the first round, P-#1-P-#6.

[0718] Table 26. Aptamer sequence Example 8-2 Synthesis and screening of the second round of aptamers

[0719] The single-stranded DNA library and primers shown in the following sequences were synthesized

[0720] Table 27. Aptamer library and primers

[0721] Wherein: the library is composed of single-stranded DNA of 110 bases in length.

[0722] Aptamer screening

[0723] After centrifugation at 12000 rpm for 1 min, the initial ssDNA library was dissolved with 1 mL of ddH2O, and the total amount of DNA was 1-5 nmol. Then, 60 min of incubation was performed at room temperature after adding the Capture oligo (10+40) and rotating. The molar mass ratio of the Capture oligo (10+40) to ssDNA was 1.2:1. After the incubation, streptavidin magnetic beads were added to the sample (the ratio of the amount of biotin carried by the magnetic beads to the amount of Biotin was 1.5:1 according to the amount of biotin carried by the magnetic beads), and incubation was performed for 30 min. Then, the EP tube was placed on a magnetic stand for 30 s, and the supernatant was discarded. The sample was washed with 1xPBS three times. Then, 15 nmol of truncated MMAE was added, and incubation was performed at room temperature for 120 min. The aptamer targeting MMAE would undergo a conformational transition, fall off the Capture oligo, and be converted into a conformation that binds to MMAE. After the incubation, the supernatant was collected, and the first round of PCR amplification was performed using the collected supernatant as a template. The system was as follows: 400 μL of PrimeStar Max, 32 μL of P1 (10+40), 32 μL of P2 (10+40), and 332 μL of ddH2O were used as the total system, and 80 μL was dispensed into each PCR tube. The reaction conditions were as follows: 95 °C for 2 min of pre-denaturation; 95 °C for 30 s of denaturation, 60 °C for 5 s of annealing, and 72 °C for 5 s of extension, for a total of 6 cycles; and finally, 72 °C for 2 min of extension.

[0724] To determine the optimal number of cycles of PCR, 100 μL of the first round of PCR product was used as a template, and the reaction system was prepared according to the following table. Then, 80 μL was added to four PCR tubes, and 2, 4, 6, and 8 cycles of amplification were performed, respectively. Subsequently, 3% agarose gel electrophoresis was performed, and the number of cycles was selected according to the clear band and non-specific amplification. The third PCR reaction system and cycle number were the same as the optimized system, and the remaining mixture was amplified according to the optimized cycle number.

[0725] Biotinylated PCR products were mixed with a certain amount of streptavidin magnetic beads and incubated at room temperature for 15 min. After washing twice with lx PBS, 200 mM NaOH was added and incubated for 5 min to make the double-stranded DNA into single-stranded DNA. The magnetic stand was used to make the ssDNA containing biotin remain on the streptavidin magnetic beads and adsorb on the tube wall, while the other ssDNA without biotin existed in the supernatant. The obtained ssDNA secondary library was collected and purified by ethanol precipitation (1 / 10 3M NaAC and 2.5 times the volume of absolute ethanol were mixed well and then placed in a -80°C refrigerator to precipitate overnight. The next day, it was directly taken out and centrifuged at 13000 rpm for 10 min at 4°C. After the supernatant was discarded, 1 mL of 75% ethanol was added for washing, and centrifuged at 13000 rpm for 10 min at 4°C. The supernatant was discarded, and the precipitate was dissolved with 50 μL Binding buffer and the concentration was detected by Nanodrop. 1 μg of DNA was taken as the secondary library for the next round of screening, and the excess part was stored in a -20°C refrigerator).

[0726] MMAE coupled with vc was used as negative screening antigen, and MMAF coupled with biotin was used as positive screening antigen. The screening pressure was changed by adjusting the amount of target, incubation time and washing conditions in each round. The positive screening antigen was reduced from 15 nmol to 0.12 nmol, and the positive screening time was reduced from 120 min to 60 min. From the third round, the negative screening step was added to remove the sequences that were non-specifically combined with streptavidin magnetic beads, and the incubation time was increased from 30 min to 60 min. After each round of incubation, the supernatant was collected, and the ssDNA combined with MMAE in the supernatant was used as a template for subsequent PCR and single-strandization steps.

[0727] A total of 6 aptamers were obtained from PP-1-#148-PP-1-#153 in the second round of screening.

[0728] Table 28. Aptamer sequences Example 8-3 Antigen binding experiment

[0729] Experimental steps

[0730] Coating: Coat 1 pg / mL of BSA-MMAF and BSA, OVA-MMAF and OVA on 96-well enzyme-labeled plates respectively, 4°C overnight. Blocking: Coat the antigen 96-well plate, wash the plate with PBST 1 time, add 2% casein blocking, 200 pL / well, 37°C blocking for 1 h. First antibody: PBST wash 3 sides, add the aptamer diluted with binding buffer to each well, the first well concentration is 3 pM, 3-fold dilution for 8 gradients, carefully mix well, 37°C incubation for 1-2 h. Second antibody: wash with PBST for 3 times, dilute the second antibody Streptavidin-HRP (1:10000) with binding buffer, 37°C, incubate for 1 h. Color development: wash with DPBST (containing 5 mM Mg 2+ ) for 6 times, add 100 pL TMB color developing liquid to each well, avoid light color development for 2-10 min. Termination: add 100 pL ELISA termination liquid to each well, read OD450 value with a microplate reader, record and save.

[0731] The experimental results are shown in Figure 44, and the results show that P-#3, P-#4, PP-1-#148, and PP-1-#151 can specifically bind to MMAF.

[0732] The NHS active ester group in the DBCO-PEG8-NHS linker reacts with the primary amine group on the antibody to introduce the DBCO group, and the DBCO group covalently binds to the azido group in the aptamer through alkyne-azide cycloaddition reaction (SPAAC) to obtain an antibody-aptamer conjugate.

[0733] Experimental steps

[0734] Take 1 mg of antibody, add 12 molar equivalents of DBCO-PEG8-NHS (MCE, 2553412-88-5) according to the molar ratio, react at 25°C for 3 h, and after the reaction is completed, use a centrifugal desalting column (ZebaTM, 7K 0.5 mL) to replace it into PBS; add 4 molar equivalents of azido-modified aptamer to the above sample after desalting, and react at 4°C overnight. After the reaction is completed, use an ultrafiltration concentration tube (ThermoFisher, 100K MW, 88503) to replace the above sample into PBS to obtain an antibody-aptamer conjugate, and the schematic diagram of the constructed antibody-aptamer conjugate is shown in Figure 45.

[0735] The single-stranded DNA library and primers shown in the following sequences were synthesized

[0736] Table 29. Aptamer library and primers

[0737] Library consists of single-stranded DNA of 66 bases in length, with a 30-base random sequence (N) in the middle and fixed primer sequences at both ends. 5B refers to 5' end with Biotin modification. 1214-F and 1214-R-5B are used for each round of symmetric PCR, and 1214-F and 1214-R are used for amplifying the library to be sequenced. The single-stranded DNA library and primers are synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0738] Adaptor screening

[0739] After centrifugation at 12000 rpm for 1 min, the initial single-stranded DNA library is dissolved with 100 μL Binding buffer (D-PBS with 5 mM final concentration of MgCl2, i.e. DPBS containing 5 mM Mg 2+ ), and the total amount of DNA is 1-5 nmol. 95°C metal bath for 5 min, immediately cooled on ice for 5 min. This process is used for library denaturation, which refolds the conformation of aptamer. After denaturation, 900 μL Binding buffer is added. 0.5 μg DXD-Biotin is added, and the sample is incubated at room temperature for 120 min to allow the refolded aptamer to bind to the target DXD. Streptavidin magnetic beads are added to the sample after incubation, and the sample is incubated for 15 min. Then, the EP tube is placed in a magnetic stand and left for 30 s, and the supernatant is discarded. The magnetic beads are washed with 1xPBS for three times. The washed magnetic beads are used as templates for the first round of PCR amplification. The system is as follows: all templates, 400 μL PrimeStar Max, 32 μL 1214-F, 32 μL 1214-R, 332 μL ddH2O, total system is 800 μL, and each PCR tube is divided into 80 μL. The reaction conditions are as follows: 95°C pre-denaturation for 2 min; 95°C denaturation for 30 s, 60°C annealing for 5 s, 72°C extension for 5 s, 6 cycles of amplification; and finally 72°C extension for 2 min.

[0740] To determine the optimal number of cycles for PCR, 100 μL of the first round of PCR product is taken as a template, 250 μL of PrimeStar Max, 20 μL of 1214-F, 20 μL of 1214-R-5B, and 110 μL of ddH2O are added to four PCR tubes, and 80 μL of each is taken for amplification for 2, 4, 6, and 8 cycles, respectively. Then, 3% agarose gel electrophoresis is performed, and the number of cycles with clear bands and no non-specific amplification is selected for the third PCR. The reaction system and cycle number of the third PCR are the same as those of the optimized system, and the remaining mixture is amplified according to the optimized cycle number.

[0741] Biotinylated PCR products were mixed with a certain amount of streptavidin magnetic beads and incubated at room temperature for 15 min. After washing twice with lx PBS, 200 mM NaOH was added and incubated for 5 min to make the DNA double strands into single strands in an alkaline environment. The magnetic stand was turned on to make the biotin-containing ssDNA remain on the streptavidin magnetic beads and adsorb on the tube wall, while the other ssDNA without biotin existed in the supernatant. The obtained ssDNA secondary library was collected and purified by ethanol precipitation (1 / 10 3M NaAC and 2.5 times the volume of absolute ethanol were mixed well and then placed in a -80°C refrigerator to precipitate overnight. The next day, it was directly taken out and centrifuged at 13000 rpm for 10 min at 4°C. After the supernatant was discarded, 1 mL of 75% ethanol was added and centrifuged at 13000 rpm for 10 min at 4°C. The supernatant was discarded, and the precipitate was dissolved with 50 μL Binding buffer and the concentration was detected by Nanodrop. 1 μg of DNA was taken as the secondary library for the next round of screening, and the excess part was stored in a -20°C refrigerator.

[0742] Streptavidin magnetic beads were used as negative screening antigens, and DXD coupled with biotin was used as positive screening antigen. The screening pressure was changed by adjusting the amount of target, incubation time and washing conditions in each round. A total of six rounds of screening were performed, and the positive screening antigen was reduced from 0.5 μg to 0.2 μg, and the positive screening time was reduced from 120 min to 60 min. From the third round, the negative screening step was added to remove the sequences that were non-specifically combined with streptavidin magnetic beads, and the incubation time was increased from 30 min to 60 min. After each round of incubation, the supernatant was discarded, washed with PBST solution containing 0.01%-0.05% Tween20, and then washed with lx PBS, and the supernatant was discarded. The ssDNA combined with the magnetic beads was used as a template for subsequent PCR and single-strandization steps.

[0743] A total of 14 aptamers were obtained from D-#3-D-#18.

[0744] Table 30. Aptamer sequences

[0745] To improve the affinity of the aptamer, a complementary sequence was added to both ends of the aptamer for modification:

[0746] Table 31. Modified aptamer sequences Example 9-2 Antigen binding experiment

[0747] Experimental steps

[0748] Coating: Coat 1 μg / mL OVA-DXD and OVA on 96-well ELISA plate, 4°C overnight. Blocking: Block the coated 96-well ELISA plate with 2% casein, 200 μL / well, 37°C for 1 h. Primary antibody: Wash the plate 3 times with PBST, add the diluted aptamer to each well, the first well concentration is 3 μM, 3-fold dilution for 8 gradients, mix well, 37°C for 1-2 h. Secondary antibody: Wash the plate 3 times with PBST, dilute the secondary antibody Streptavidin-HRP (1:10000) with binding buffer, 37°C for 1 h. Color development: Wash the plate 6 times with DPBST (containing 5 mM Mg 2+ ), add 100 μL TMB color developing solution to each well, color development for 2-10 min in the dark. Termination: Add 100 μL ELISA termination solution to each well, read OD450 value with a microplate reader, record and save.

[0749] The experimental results are shown in Figure 46. The results show that D-#5 / D-#14 / D-#17 can specifically bind to DXD, and after modification, the binding affinity of Capture D-#5 / Capture D-#14 / Capture D-#17 to DXD is significantly improved.

[0750] Experimental procedure

[0751] Coating: Coat 1 μg / mL OVA-DXD on 96-well ELISA plate, 4°C overnight. Blocking: Block the coated 96-well ELISA plate with 2% casein, 200 μL / well, 37°C for 1 h. Primary antibody: Wash the plate 3 times with PBST, add the diluted aptamer to each well, the antibody concentration is 2000 nM, the first well concentration of DXD and GGFG-DXD is 20 μM, 3-fold dilution for 8 gradients, mix well, 37°C for 1-2 h. Secondary antibody: Wash the plate 3 times with PBST, dilute the secondary antibody Streptavidin-HRP (1:10000) with binding buffer, 37°C for 1 h. Color development: Wash the plate 6 times with DPBST, add 100 μL TMB color developing solution to each well, color development for 2-10 min in the dark. Termination: Add 100 μL ELISA termination solution to each well, read OD450 value with a microplate reader, record and save.

[0752] The experimental results are shown in Figure 47. The results indicate that both the parental D-#5 / D-#14 / D-#17 molecules and the modified molecules Capture D-#5 / Capture D-#14 / Capture D-#17 can competitively bind to free DXD. Example 9-4: Preparation of Antibody-Amtamanisome Conjugates

[0753] The active ester group of the NHS in the DBCO-NHS or DBCO-PEG8-NHS linker reacts with the primary amine group on the antibody to introduce the DBCO group. The DBCO group then covalently binds to the azide group in the aptamer via an alkyne-azidocycloaddition reaction (SPAAC) to obtain the antibody-aptamer conjugate. The specific experimental steps are as follows:

[0754] Experimental steps

[0755] Take 1 mg of antibody and add 12 molar equivalents of DBCO-NHS (MCE, 1384870-47-6) or DBCO-PEG8-NHS (MCE, 2553412-88-5) at a molar ratio. React at 25°C for 3 h. After the reaction, use a centrifugal desalting column (Zeba™, 7K 0.5 mL) to replace the sample with PBS. Add 4 molar equivalents of the azide-modified aptamer to the desalted sample and incubate overnight at 4°C. After the reaction, use an ultrafiltration concentrator (ThermoFisher, 100K MW, 88503) to replace the sample with PBS to obtain the antibody-aptamer conjugate. A schematic diagram of the constructed antibody-aptamer conjugate is shown in Figure 48. Example 9-5 Antigen binding experiment of antibody-aptamer conjugate

[0756] The experimental results are shown in Figure 49. The results indicate that the constructed antibody-aptamer conjugates PP-1D#501, PP-1D#502, PP-1D#503, and PP-1D#504 can all specifically bind to DXD. Examples 9-6: Cell Protection Experiment

[0757] The experimental results are shown in Figure 50. The results indicate that capture D-#5-RS920-#101, D-#14-RS920-#101, and capture D-#17-RS920-#101 can effectively inhibit DXD toxicity and have varying degrees of protective effect on cells. Example 10: Preparation methods of PR-ADC (Payload Recycling-Antibody Drug Conjugates) and conventional ADCs. Example 10-1: Preparation of PR-ADC (CDCP1A9-DL01-MMAE, CDCP1A12-DL01-MMAE, Nectin4A14-DL01-MMAE, CDCP1A10-DL01-MMAE).

[0758] The configuration schematic of CDCP1A9-DL01-MMAE is shown in Figure 2, in which the ligand unit is an IgG antibody targeting CDCP1 (CDCP1A9), the recovery unit is an scFv against MMAE, and the two constitute an anti-CDCP1-IgG+anti-MMAE-scFv bispecific antibody. The bispecific antibody-linker-drug structure schematic is shown in Figure 55.

[0759] The configuration schematic of CDCP1A12-DL01-MMAE is shown in Figure 54, in which the ligand unit is an scFv targeting CDCP1 (CDCP1A12), the recovery unit is an IgG antibody against MMAE, and the two constitute an anti-CDCP1-scFv+anti-MMAE-IgG bispecific antibody. The bispecific antibody-linker-drug structure schematic is shown in Figure 55.

[0760] The configuration schematic of Nectin4A14-DL01-MMAE is shown in Figure 56, in which the recovery unit is an scFv against MMAE, and the ligand unit is an IgG antibody targeting Nectin-4 (Nectin4A14), and the two constitute an anti-MMAE-scFv+anti-Nectin-4-IgG bispecific antibody. The bispecific antibody-linker-drug structure schematic is shown in Figure 55.

[0761] The configuration schematic of CDCP1A10-DL01-MMAE is shown in Figure 56, in which the recovery unit is an scFv against MMAE, and the ligand unit is an IgG antibody targeting CDCP1 (CDCP1A10), and the two constitute an anti-MMAE-scFv+anti-Nectin-4-IgG bispecific antibody. The bispecific antibody-linker-drug structure schematic is shown in Figure 55.

[0762] Preparation method

[0763] The reducing agent and metal chelating agent were prepared with purified water as follows: 5mM TCEP (Tris-2-carboxyethyl-phosphine), 10mM DTPA (Diethylene triamine pentacetate acid) stock solution, 10mg / mL of the corresponding bispecific antibody described above, 10% DTPA by volume, the final concentration molar ratio of TCEP to antibody was 2-3:1, and the proportion of TCEP was adjusted slightly according to the different antibodies, and the reaction was stirred at 25°C for 1h.

[0764] The reduced antibody can be directly conjugated. A linker-toxin (DL01-MMAE) solution with a certain concentration (5 mM) is prepared in DMSO (dimethyl sulfoxide), and the drug is slowly added according to a molar ratio of drug to thiol of 1.5-2:1, and the reaction is stirred at 25°C for 2 h. After the reaction is completed, the ADC buffer is centrifuged and ultrafiltrated to remove residual unreacted drugs and free small molecules such as DMSO, and the conjugation is detected by hydrophobic high performance liquid chromatography (HIC-HPLC) and liquid chromatography-mass spectrometry (LC-MS) methods, and the purity of the conjugated sample is detected by size exclusion chromatography (SEC).

[0765] The average number of drugs conjugated to each antibody (DAR) in the prepared CDCP1A9-DL01-MMAE (PR-ADC) is 3.86, and the SEC detection purity is 94.38%; the average number of drugs conjugated to each antibody (DAR) in the prepared CDCP1A12-DL01-MMAE (PR-ADC) is 3.92, and the SEC detection purity is 94.38%; the average number of drugs conjugated to each antibody (DAR) in the prepared Nectin4A14-DL01-MMAE (PR-ADC) is 3.89, and the SEC detection purity is 94.94%; the average number of drugs conjugated to each antibody (DAR) in the prepared CDCP1A10-DL01-MMAE (PR-ADC) is 4.10, and the SEC detection purity is 92.7%. Preparation of conventional ADC (CDCP1A6-DL01-MMAE, Enfortumab vedotin (in-house) - referred to as EV) in Example 10-2

[0766] The configuration diagram of CDCP1A6-DL01-MMAE, EV is shown in FIG. 57, wherein the antibody part is an antibody targeting CDCP1 and Nectin-4, respectively, and the antibody-linker-drug structure diagram is shown in FIG. 55.

[0767] Referring to the preparation method of Example 10-1, CDCP1A6-DL01-MMAE (conventional ADC), EV (conventional ADC) are obtained.

[0768] The average number of drugs conjugated to each antibody (DAR) in the prepared CDCP1A6-DL01-MMAE (conventional ADC) is 4.08, and the SEC detection purity is 99.37%; the average number of drugs conjugated to each antibody (DAR) in the prepared EV (conventional ADC) is 3.85, and the SEC detection purity is 98.81%. Cell experiment in Example 11

[0769] The experimental results (Figure 51) show that the PR-ADC (Nectin4A8-DL01-MMAE, Nectin4A9-DL01-MMAE) maintains similar cell killing activity as Enfortumab vedotin (an antibody drug conjugate targeting Nectin-4, EV). Example 11-2 Comparison of binding activity of naked antibody molecules, conventional ADC, PR-ADC to Nectin-4

[0770] Table 32. Comparison of binding activity of various molecules to Nectin-4

[0771] The experimental results (Table 32) show that the Nectin4A7, Nectin4A8 naked antibody molecules and their corresponding conventional ADC (EV) or PR-ADC (Nectin4A8-DL01-MMAE) have comparable binding ability to Nectin-4. And the binding ability of Enfortumab and EV to Nectin-4 is at the same level. Example 11-3 Cell killing activity

[0772] Table 33. Experimental results of cell killing activity

[0773] The experimental results (Table 33) show that the PR-ADC (Nectin4A8-DL01-MMAE) and the conventional ADC (EV) have similar cell killing activity. Example 12 Safety study of PR-ADC (MMAE load) in animal models Example 12-1 Comparative study of toxicity in BALB / c mice 1. Comparison of PR-ADC and conventional ADC targeting CDCP1 (MMAE load)

[0774] Test 1: 18 BALB / c mice, female, body weight at grouping 16-19 g, divided into PR-ADC group (CDCP1A9-DL01-MMAE, 140 mg / kg), conventional ADC group (CDCP1A6-DL01-MMAE, 100 mg / kg), both at equimolar dose, 9 in each group (5 toxicology + 4 TK), a total of 2 groups. Single administration, administration volume 10 mL / kg. The toxicity related to the drugs in BALB / c mice was investigated and compared through death, clinical observation, body weight, toxicokinetic detection, etc., and the test lasted for 14 days.

[0775] Free MMAE was determined by Hybrid LBA LC-MS / MS method. After the ADC / PR-ADC, PR-ADC bound with MMAE and anti-MMAE antibody bound with MMAE in serum were separated by Protein A / G-Beads, the remaining sample was detected by LC-MS / MS method after protein precipitation to detect free MMAE.

[0776] Table 34. Results of toxicity test of animals in each group

[0777] According to the experimental results, the tolerance of BALB / c mice to PR-ADC group (CDCP1A9-DL01-MMAE) is greater than 140 mg / kg, and the tolerance of BALB / c mice to conventional ADC group (CDCP1A6-DL01-MMAE) is less than 100 mg / kg. The comparison results of toxicity at equimolar dose are conventional ADC group (CDCP1A6-DL01-MMAE, 100 mg / kg) > PR-ADC group (CDCP1A9-DL01-MMAE, 140 mg / kg).

[0778] The experimental results show that the increase of the recovery unit of PR-ADC structure compared with the conventional ADC structure effectively reduces the toxicity to animals.

[0779] Table 35. Toxicokinetic parameters of free MMAE in serum of animals in each group in vivo

[0780] The toxicokinetic results show that in the serum of BALB / c mice, the exposure of free MMAE in PR-ADC group (CDCP1A9-DL01-MMAE) is significantly less than that of conventional ADC group (CDCP1A6-DL01-MMAE), and the exposure level of free MMAE is positively correlated with toxicity, indicating that PR-ADC can significantly improve the in vivo toxicity of conventional ADC. max

[0781] Test two: 16 BALB / c mice, female, the body weight was 16-20 g when grouped, divided into PR-ADC group (CDCP1A12-DL01-MMAE, 134 mg / kg) and conventional ADC group (CDCP1A6-DL01-MMAE, 100 mg / kg) dose groups, both at equimolar dose, 8 in each group (4 toxicology + 4 TK), a total of 2 groups. Single administration, administration volume 10 mL / kg. The related toxicity of BALB / c mice to each group of drugs was investigated and compared through death, clinical observation, body weight, toxicokinetic detection, etc., and the test lasted for 14 days.

[0782] ​Free MMAE was also determined by Hybrid LBA LC-MS / MS method.

[0783] Table 36. Results of toxicity test of each group of animals

[0784] According to the experimental results, the tolerance of BALB / c mice to the PR-ADC group (CDCP1A12-DL01-MMAE) is greater than 134 mg / kg, and the tolerance of the conventional ADC group (CDCP1A6-DL01-MMAE) is less than 100 mg / kg. The comparison results of toxicity at equimolar dose are conventional ADC group (CDCP1A6-DL01-MMAE, 100 mg / kg) > PR-ADC group (CDCP1A12-DL01-MMAE, 134 mg / kg).

[0785] The experimental results also show that the increase of the recovery unit of the PR-ADC structure compared with the conventional ADC structure effectively reduces the in vivo toxicity to animals.

[0786] Table 37. Pharmacokinetic parameters of free MMAE in serum of each group of animals in vivo

[0787] The pharmacokinetic results show that in the serum of BALB / c mice, the exposure of free MMAE in the serum of mice in the PR-ADC group (CDCP1A12-DL01-MMAE) is significantly less than that of the conventional ADC group (CDCP1A6-DL01-MMAE), and the exposure level of free MMAE is basically positively correlated with toxicity, indicating that the PR-ADC can significantly improve the in vivo toxicity of the conventional ADC. max 2. Comparison of PR-ADC and conventional ADC targeting Nectin-4 (MMAE load)

[0788] 18 BALB / c mice, female, weighing 16-19 g when grouped, divided into PR-ADC group (Nectin4A14-DL01-MMAE, 136 mg / kg) and conventional ADC group (EV, 100 mg / kg), both at equimolar dose, 9 in each group (5 toxicology + 4 TK), a total of 2 groups.

[0789] Free MMAE was also determined by Hybrid LBA LC-MS / MS method.

[0790] Table 38. Results of toxicity test of each group of animals

[0791] According to the experimental results, the tolerance of BALB / c mice to the PR-ADC group (Nectin4A14-DL01-MMAE) is greater than 136 mg / kg, and the tolerance of the mice to the conventional ADC group (EV) is less than 100 mg / kg. The comparison of toxicity at the equimolar dose is conventional ADC group (EV, 100 mg / kg) > PR-ADC group (Nectin4A14-DL01-MMAE, 136 mg / kg).

[0792] The experimental results also show that, compared with the conventional ADC structure, the increase of the recovery unit of the PR-ADC structure effectively reduces the in vivo toxicity to animals.

[0793] Table 39. Toxicokinetic parameters of free MMAE in serum of animals in each group

[0794] The toxicokinetic results show that, in the serum of BALB / c mice, the exposure of free MMAE in the serum of mice in the PR-ADC group (Nectin4A14-DL01-MMAE) is significantly less than that in the conventional ADC group (EV), and the exposure level of free MMAE is basically positively correlated with the toxicity, indicating that the PR-ADC can significantly improve the in vivo toxicity of the conventional ADC. max Example 12-2 Comparison of toxicity of SD rats 1. Comparison of PR-ADC and conventional ADC targeting CDCP1 (MMAE load)

[0795] Sixteen SD rats, female, weighing 220-270 g at the time of grouping, were divided into a PR-ADC group (CDCP1A9-DL01-MMAE, 42 mg / kg) and a conventional ADC group (CDCP1A6-DL01-MMAE, 30 mg / kg), both at an equimolar dose, 8 rats in each group (4 rats for toxicology + 4 rats for TK), a total of 2 groups. The test was a single intravenous administration at a dose of 10 mL / kg. The toxicity related to each group of drugs was investigated and compared by observing death, clinical observation, body weight, toxicokinetic detection, etc. The test lasted for 14 days.

[0796] Free MMAE was also determined by Hybrid LBA LC-MS / MS method.

[0797] Table 40. Toxicity test results of animals in each group

[0798] According to the experimental results, the tolerance of SD rats to the PR-ADC group (CDCP1A9-DL01-MMAE) is less than 42 mg / kg, and the tolerance of SD rats to the conventional ADC group (CDCP1A6-DL01-MMAE) is less than 30 mg / kg, and the comparison of toxicity results is conventional ADC group (CDCP1A6-DL01-MMAE, 30 mg / kg) > PR-ADC group (CDCP1A9-DL01-MMAE, 42 mg / kg).

[0799] The SD rat toxicity test also shows that the increase of the recovery unit of the PR-ADC structure compared with the conventional ADC structure effectively reduces the in vivo toxicity to animals.

[0800] Table 41. Pharmacokinetic parameters of free MMAE in serum of animals in each group

[0801] The pharmacokinetic results show that, under the condition of equimolar dose, the exposure (AUC) of free MMAE in the PR-ADC group (CDCP1A9-DL01-MMAE) is less than that in the conventional ADC group (CDCP1A6-DL01-MMAE) in the serum of SD rats. max The exposure level of free MMAE is positively correlated with toxicity. It also shows that the PR-ADC can significantly improve the toxicity of the conventional ADC group. Example 13-1 Comparison of the efficacy of PR-ADC and conventional ADC targeting Nectin-4 ligand unit in human bladder cancer cell HT-1376 subcutaneous xenograft model

[0802] Research method

[0803] After the human bladder cancer cell HT-1376 was cultured by amplification, it was inoculated subcutaneously on the back of the right front limb of BALB / c-nu mice (Jingjia Pharmaceutical, female). The growth of the tumor was observed, and when the tumor volume reached about 90-200 mm 3 , the mice were randomly grouped according to the tumor volume and administered.

[0804] The day of administration was recorded as D0. During the experiment, the long diameter, short diameter and body weight of the tumor were measured twice a week, and the clinical symptoms were observed. At the end of the experiment, the experimental mice were euthanized.

[0805] The experimental results (Fig. 52 and Table 42) show that in the HT-1376 subcutaneous xenograft model, both the PR-ADC and the conventional ADC molecules exhibit significant anti-tumor effects (P≤0.05), and under the condition of equimolar dose, the anti-tumor effect of the PR-ADC group (Nectin4A14-DL01-MMAE) is equivalent to that of the conventional ADC group (EV).

[0806] Table 42. TGI results of each group Example 13-2 Comparison of pharmacodynamic study of PR-ADC targeting CDCP1 with conventional ADC in human colon cancer cell RKO subcutaneous tumor model

[0807] Research method

[0808] After the human colon cancer cell RKO was expanded and cultured, it was inoculated subcutaneously on the back of the right forelimb of BALB / c-nu mice (Beijing Huafukang Biosciences Co., Ltd., female). The growth of the tumor was observed, and when the tumor volume reached about 100-200 mm 3 , the mice were randomly grouped according to the tumor volume and administered with drugs.

[0809] The day of administration was recorded as D0. During the experiment, the long diameter, short diameter and body weight of the tumor were measured twice a week, and the clinical symptoms were observed. At the end of the experiment, the experimental mice were euthanized.

[0810] The experimental results (Figure 53 and Table 43) showed that in the RKO subcutaneous tumor model, all molecules showed significant anti-tumor effect (P≤0.01). At the same molar dose, the anti-tumor effect of the PR-ADC group (CDCP1A10-DL01-MMAE, CDCP1A12-DL01-MMAE) was comparable to that of the conventional ADC group (CDCP1A6-DL01-MMAE).

[0811] Table 43. TGI results of each group

[0812] The above only discloses some embodiments of the present application, and does not limit the present application in other forms. Those skilled in the art can understand that the present application is not limited to each specific embodiment. Those skilled in the art can make some improvements and modifications without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A ligand drug conjugate, characterized in that, The ligand drug conjugate comprises a ligand unit targeting a tumor antigen or a non-tumor antigen, a payload unit coupled with the ligand unit, and a recovery unit capable of binding to free payload falling off from the ligand drug conjugate, wherein the payload unit comprises a payload and a linker.

2. The ligand drug conjugate of claim 1, wherein, The ligand unit and the recovery unit are directly connected or not directly connected.

3. The Ligand Drug Conjugate of claim 1 or 2, wherein, The ligand unit and the recovery unit comprise a connecting unit.

4. The ligand drug conjugate according to any one of claims 1-3, wherein, The ligand drug conjugate further comprises other functional units.

5. The ligand drug conjugate according to any one of claims 1-4, wherein, The payload is coupled to the ligand unit and / or the recovery unit and / or the connecting unit and / or the other functional units through the linker.

6. The ligand drug conjugate according to any one of claims 1-5, wherein, The ligand unit comprises an antibody or an antigen-binding fragment, a receptor, a polypeptide, an oligopeptide, a peptidomimetic, a fusion protein, or an aptamer specifically binding to a tumor antigen or a non-tumor antigen.

7. The ligand drug conjugate of claim 6, wherein, The antibody includes, but is not limited to, a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody (HcAb), an immunoglobulin new antigen receptor (IgNAR); the antigen-binding fragment includes, but is not limited to, scFv, Fab, Fab', F(ab')2, Fd, Fv, dAb, Fd, sdAb, VHH, Diabody; the receptor includes, but is not limited to, a T cell receptor.

8. The Ligand Drug Conjugate of claim 6 or 7, wherein, The antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an artificially engineered antibody.

9. The ligand drug conjugate of claim 8, wherein, The antibody further comprises a constant region of an immunoglobulin.

10. The ligand drug conjugate of claim 9, wherein, The immunoglobulin is selected from human IgG1, IgG2, IgG3, or IgG4.

11. The ligand drug conjugate according to any one of claims 1-10, wherein, The tumor or non-tumor antigen includes, but is not limited to, CD9, CD19, CD20, CD22, CD24, CD25, CD29, CD30, CD33, CD37, CD44, CD45, CD46, CD47, CD49b, CD51, CD52, CD56, CD73 (NT5E), CD79b, CD123, CD133, CD138, CD157, CD166, DLL-3, CDH6, Tissue factor, EpCAM, PSMA, MUC1, MUC16, FOLR1, GPC3, ROR1, ROR2, PD-L1, ENPP3, TDGF1, MSLN, TIM-1, LRRC15, LIV-1 (ZIP6), Claudin6, Claudin9, Claudin 18.2, Mesothelin, HER2 (ErbB2), HER3 (ErbB3), EGFR, c-MET, SLITRK6, KIT (CD117), STEAP1, NaPi2B (SLC34A2), SLC44A4, GPNMB, AXL, CD166, B7-H3 (CD276), B7-H4 (VTCN1), PTK7 (CCK4), EFNA4, 5T4, NOTCH3, Nectin-4, TROP-2, GPR20, EphA2, LYPD3, FGFR2, FGFR3, FRa, CEACAMs, CAIX, P-cadherin (CDH3), CDH17, GD3, Cadherin 6, LAMP1, FLT3, BCMA, SLTRK6, Lewis Y, ASCT2, CA-IX, Cripto, DPEP3, Globo H, Ly6E, RNF43, DR5, CDCP1, CEACAM6, FUT3, GD2, CD26E, CD70, CD74, CD163, PRLR, TNFa, CXCR4, LXR, IL-6, CEACAM5, GPRC5D, HLA-DR, CTLA4 (CD152), FAPa, or IL2R.

12. The ligand drug conjugate of any one of claims 1-11, wherein, The antibodies that specifically bind to a tumor antigen or a non-tumor antigen include, but are not limited to, alemtuzumab, arcitumomab, evatumomab, atezolizumab, inotuzumab, anetumab, avelumab, bebtuximab, bermekimab, bevacizumab, belimumab, vibostuzumab, catumaxomab, cibisatamab, cetuximab, clivatuzumab, dalotuzumab, daratumumab, desidustuzumab, daratumumab beta, dostarlimab, durvalumab, edrecolomab, epratuzumab, eculizumab, edaravone, epcoritamab, epratuzumab, edaravone, genmab, gireultuzumab, ibritumomab, inebilizumab, inotuzumab, ipilimumab, isatuximab, labetuzumab, loncastuximab, mogamulizumab, tiragolumab, moxetumomab, nimotuzumab, naratuximab, necitumumab, nivolumab, oregovomab, oritumab, orlaratumab, panitumumab, pembrolizumab, patritumab, vedotin, racotumomab, ramucirumab, rilotumumab, rituximab, rovalpituzumab, secolimumab, tositumomab, tocilizumab, trastuzumab, volociximab, zalutumumab, toripalimab, or zamab; and antigen-binding fragments and derivatives of the above antibodies; the polypeptides, oligopeptides, fusion proteins that target binding to a specific antigen include, but are not limited to, a bicycle, a vasculotide-2, a linear peptide, a pH-sensitive peptide, SOR-C27, an A6 polypeptide, SOR13; and variants and derivatives of the above polypeptides, oligopeptides or fusion proteins; the aptamers that target binding to a specific antigen include AS1411, sgc8, sgc4f, sgd5a, TC01, TD05, A9, A10, APT, Min.2, AIR-3A, E3, E07, Waz, P19, Zy1; and variants and derivatives of the above aptamers.

13. The ligand drug conjugate of any one of claims 1-12, wherein, The ligand drug conjugate can specifically bind to the free load, and the ligand drug conjugates do not aggregate.

14. The ligand drug conjugate of claim 13, wherein, The recovery unit specifically binds to the free load.

15. The ligand drug conjugate of claim 14, wherein, The recovery unit includes an antibody or antigen-binding fragment, a receptor, a fusion protein, a polypeptide, an oligopeptide, a peptidomimetic or an aptamer (aptamer) that specifically binds to the free load.

16. The ligand drug conjugate of claim 15, wherein, The antibody specifically binding to the free load includes, but is not limited to, a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody (HcAb), an immunoglobulin new antigen receptor (IgNAR); the antigen binding fragment includes, but is not limited to, scFv, Fab, Fab', F(ab')2, Fd, Fv, dAb, Fd, sdAb, VHH, Diabody; and the receptor includes, but is not limited to, a T cell receptor.

17. The ligand drug conjugate of claim 16, wherein, The antibody specifically binding to the free load is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an artificially engineered antibody.

18. The ligand drug conjugate of any one of claims 13-17, wherein, The recovery unit is capable of specifically binding to the free load, which means that the equilibrium dissociation constant K D (M) less than 10 -4 , or more specifically, less than 10 -5 , or more specifically, less than 10 -6 , or more specifically, less than 10 -7 , or more specifically, less than 10 -8 , or more specifically, less than 10 - 9 .

19. The ligand drug conjugate of claim 18, wherein, The recovery unit is capable of specifically binding to the free load refers to the equilibrium dissociation constant K D (M) less than 10 -7 Or further less than 10 -8 Or further less than 10 -9 .

20. The ligand drug conjugate of claim 19, wherein, The recovery unit is capable of specifically binding to the free load, which means that the equilibrium dissociation constant K D (M) less than 10 -9 .

21. The ligand drug conjugate of claim 18, wherein, The recovery unit does not bind or has a low affinity for the load in the conjugated state in the ligand drug conjugate, and the low affinity can also prevent molecular aggregation between the ligand drug conjugate molecules.

22. The ligand drug conjugate of claim 21, wherein, The recovery unit specifically targets a non-exposed epitope on the conjugated load unit.

23. The ligand drug conjugate of any one of claims 13-22, wherein, The linker or the load is connected to a load epitope masking unit capable of preventing the recovery unit from binding to the load still conjugated to the ligand drug conjugate.

24. The ligand drug conjugate of claim 23, wherein, The load epitope masking unit does not affect the normal separation of the load after the ligand drug conjugate enters the target cell.

25. The ligand drug conjugate of any one of claims 1-24, wherein, The recovery unit is one, two or more.

26. The ligand drug conjugate of claim 15, wherein, The recovery unit comprises: (1) a VHH domain selected from CDR1-3 defined by the following amino acid sequences: CDR1-3 as shown in SEQ ID NO: 103, 104 and 105; or (2) selected from the amino acid sequence as shown in SEQ ID NO: 102; or (3) selected from the amino acid sequence comprising the following defined VH and VL combinations: a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 2, 3 and 4, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 6, YAS and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 9, 10 and 11, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 6, FAS and SEQ ID NO: 13, respectively; a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 2, 3 and 4, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 6, YAS and SEQ ID NO: 16, respectively; a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 2, 3 and 20, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 6, YAS and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 26, WAS, and SEQ ID NO: 27, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 29, 30, and 31, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 33, KVS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 29, 36, and 37, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 33, KLS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 44, KVS, and SEQ ID NO: 45, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 42, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 50, KVS, and SEQ ID NO: 51, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 53, 54, and 55, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 57, DTS, and SEQ ID NO: 58, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 66, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 69, 70, and 71, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 73, WAS, and SEQ ID NO: 74, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 80, GAS, and SEQ ID NO: 81, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 83, 84, and 85, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 87, SAS, and SEQ ID NO: 67, respectively; VH containing HCDR1-3 as shown in SEQ ID NO:89, 90 and 91 respectively, and VL containing LCDR1-3 as shown in SEQ ID NO:93, HGT and SEQ ID NO:94 respectively; VH containing HCDR1-3 as shown in SEQ ID NO:76, 77 and 78 respectively, and VL containing LCDR1-3 as shown in SEQ ID NO:99, 77 and 100 respectively; VH, containing the amino acid sequence shown in SEQ ID NO:1, and VL, containing the amino acid sequence shown in SEQ ID NO:5; VH, which contains the amino acid sequence shown in SEQ ID NO:8, and VL, which contains the amino acid sequence shown in SEQ ID NO:12; VH, which contains the amino acid sequence shown in SEQ ID NO:14, and VL, which contains the amino acid sequence shown in SEQ ID NO:15; VH, containing the amino acid sequence shown in SEQ ID NO:17, and VL, containing the amino acid sequence shown in SEQ ID NO:18; VH, which contains the amino acid sequence shown in SEQ ID NO:19, and VL, which contains the amino acid sequence shown in SEQ ID NO:18; VH, which contains the amino acid sequence shown in SEQ ID NO:21, and VL, which contains the amino acid sequence shown in SEQ ID NO:25; VH, which contains the amino acid sequence shown in SEQ ID NO:28, and VL, which contains the amino acid sequence shown in SEQ ID NO:32; VH, which contains the amino acid sequence shown in SEQ ID NO:35, and VL, which contains the amino acid sequence shown in SEQ ID NO:38; VH, which contains the amino acid sequence shown in SEQ ID NO:39, and VL, which contains the amino acid sequence shown in SEQ ID NO:43; VH contains the amino acid sequence shown in SEQ ID NO:46, and VL contains the amino acid sequence shown in SEQ ID NO:49; VH, which contains the amino acid sequence shown in SEQ ID NO:52, and VL, which contains the amino acid sequence shown in SEQ ID NO:56; VH, which contains the amino acid sequence shown in SEQ ID NO:59, and VL, which contains the amino acid sequence shown in SEQ ID NO:60; VH, which contains the amino acid sequence shown in SEQ ID NO:61, and VL, which contains the amino acid sequence shown in SEQ ID NO:65; VH, which contains the amino acid sequence shown in SEQ ID NO:68, and VL, which contains the amino acid sequence shown in SEQ ID NO:72; VH, which contains the amino acid sequence shown in SEQ ID NO:75, and VL, which contains the amino acid sequence shown in SEQ ID NO:79; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 82, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 86; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 88, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 92; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 95, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 96; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 97, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 98; the CDR1, CDR2, CDR3 sequences, the HCDR1, HCDR2, HCDR3 sequences, and the LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT definition scheme; or (4) a polypeptide comprising an amino acid sequence as defined in SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 82, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 86; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 88, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 92; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 95, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 96; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 97, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 98; the CDR1, CDR2, CDR3 sequences, the HCDR1, HCDR2, HCDR3 sequences, and the LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT definition scheme; or (4) a polypeptide comprising an amino acid sequence as defined in SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: or (5) an aptamer comprising a nucleotide sequence as defined in SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO:

162.

27. The ligand drug conjugate of any one of claims 1-26, wherein, The recovery unit is obtained by immunizing a non-human animal with a conjugate of a small molecule drug and a carrier protein or a biotin-small molecule drug conjugate; or obtained by screening using phage display technology; or obtained by screening using virtual technology; or obtained by screening using SELEX technology; or obtained by screening using hybridoma technology; or obtained by screening using nanobody technology; or obtained by using yeast display technology; Or obtained by using single B cell cloning technology; or obtained by using microfluidic technology; or obtained by using surface plasmon resonance assisted screening technology.

28. The ligand drug conjugate of claim 27, wherein, The small molecule drugs include, but are not limited to, MMAE, MMAD, DM1, DM2, DM4, eribulin, Exatecan, SN38, DXD, calicheamicin, pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IBD), duocarmycin, amanitin, Maaa-1181a, taxol, daunomycin, vinblastine, Doxorubicin, methotrexate, MMAF, ML022-Dxd, ML022-D633-004, ML026-Dxd, ML031-D633-004, ML037-D633-004, ML042-Dxd, ML047-DX8951, MPA-Dxd, Preferably, the carrier protein includes but is not limited to KLH, BSA or OVA.

29. The ligand drug conjugate of claim 28, wherein, The biotin-small molecule drug conjugates include, but are not limited to, Biotin-PEG12-Dxd, Biotin-PEG10-Dxd, Biotin-PEG6-Dxd, Biotin-PEG6-Triazole-Dxd, or Biotin-PEG10-Triazole-Dxd:

30. The ligand drug conjugate of any one of claims 1-29, wherein, The coupled load includes cytotoxic molecules, nucleic acids, immunostimulants and modulators.

31. The ligand drug conjugate of claim 30, wherein, The number of loads is one, two or more.

32. The ligand drug conjugate of claim 31, wherein, The number of loads is 1, 2, 3, 4, 5, 6, 7, 8 or more.

33. The ligand drug conjugate of any one of claims 30-32, wherein, When two or more loads are coupled, the loads can be the same load or different loads.

34. The ligand drug conjugate of any one of claims 30-33, wherein, The load is coupled to the ligand unit and / or the recovery unit and / or the connecting unit and / or other functional units by site-specific or random coupling.

35. The ligand drug conjugate of any one of claims 30-34, wherein, The cytotoxic molecule includes but is not limited to a tubulin inhibitor, a DNA inhibitor or other toxin molecule; the DNA inhibitor further includes a DNA damaging agent or a topoisomerase I inhibitor.

36. The ligand drug conjugate of claim 35, wherein, The tubulin inhibitors further include, but are not limited to, Auristatins, Maytansinoid Derivatives, tubulysin, halichondrin, Cryptophycins, EG5 inhibitors.

37. The ligand drug conjugate of claim 35, wherein, The Auristatins further include, but are not limited to, Auristatin F (AF), Monomethyl Auristatin D (MMAD), Monomethyl Auristatin E (MMAE), Monomethyl Auristatin F (MMAF); the Maytansinoid Derivatives further include, but are not limited to, DM1, DM2, DM4; the halichondrin further includes, but is not limited to, eribulin.

38. The ligand drug conjugate of claim 35, wherein, The DNA damaging agents further include, but are not limited to, calicheamicin, pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IBD), duocarmycin; the topoisomerase I inhibitors further include, but are not limited to, camptothecin derivatives.

39. The ligand drug conjugate of claim 35, wherein, The camptothecin derivatives further include, but are not limited to, Exatecan, SN38, DXD.

40. The ligand drug conjugate of claim 35, wherein, The other toxin molecules include, but are not limited to, amanitin, Maaa-1181a, taxol, daunomycin, vinblastine, Doxorubicin, methotrexate or its radioisotopes and / or pharmaceutically acceptable salts.

41. The ligand drug conjugate of claim 30, wherein, The nucleic acids used by the conjugated payloads include, but are not limited to, antisense nucleic acids (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), oligonucleotides (AOC).

42. The ligand drug conjugate of claim 30, wherein, The nucleic acids used by the conjugated payloads include, but are not limited to, DMPK siRNA, Exon-44-skipping PMO, DUX4 siRNA, Exon-51-skipping PMO, DMPK ASO, DUX4 ASO, CpG.

43. The ligand drug conjugate of claim 30, wherein, The immunostimulants used by the conjugated payloads include, but are not limited to, TLR7 agonists, TLR8 agonists, TLR9 agonists, TGF-β inhibitors, TNIK inhibitors, Sting agonists.

44. The ligand drug conjugate of claim 30, wherein, The modulators used by the conjugated payloads include, but are not limited to, glucocorticoid receptor modulators, antibiotics, kinase inhibitors, liver X receptor (LXR) agonists, phosphodiesterase (PDE4) inhibitors, bisphosphonates.

45. The ligand drug conjugate of claim 30, wherein, The structure of the free load is selected from the group comprising the following structure or an isomer or deuterated version of the following structure:

46. The ligand drug conjugate of any one of claims 1-45, wherein, The connecting unit includes, but is not limited to, a peptide linker, an Fc domain or a fragment thereof; preferably, the peptide linker is a flexible peptide linker; further preferably, the peptide linker comprises one or more amino acids; further preferably, the peptide linker comprises at least 5 amino acids; or the amino acid sequence of the peptide linker comprises: (GGGGS)n, (VPGXG)n, (SG)n, (EAAAK)n, (G)n, (R)n, A(EAAAK)n ALEA(EAAAK)nA, A(EAAAK)nA, (AP)n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, E DVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, GFLG, LE, wherein n is equal to 1, 2, 3 or 4.

47. The ligand drug conjugate of any one of claims 1-46, wherein the ligand drug conjugate is a pharmaceutically acceptable salt, deuterated form, solvate.

48. A pharmaceutical composition comprising the ligand drug conjugate of any one of claims 1-46 or the salt, deuterated form, solvate of claim 47 and a pharmaceutically acceptable excipient.

49. Use of a recovery unit capable of binding a free payload released from a ligand drug conjugate in the preparation of a ligand drug conjugate with a free payload recovery unit.

50. The use of claim 49, wherein, The recovery unit comprises an antibody or an antigen-binding fragment, a receptor, a fusion protein, a polypeptide, an oligopeptide, a peptidomimetic or an aptamer that specifically binds to the free payload.

51. The use of claim 50, wherein, The antibody that specifically binds to the free payload includes, but is not limited to, a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody (HcAb), an immunoglobulin new antigen receptor (IgNAR); the antigen-binding fragment includes, but is not limited to, an scFv, an Fab, an Fab', an F(ab')2, an Fd, an Fv, a dAb, an Fd, an sdAb, a VHH, a Diabody; the receptor includes, but is not limited to, a T cell receptor.

52. The use of claim 51, wherein, The antibody that specifically binds to the free payload is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an artificially engineered antibody.

53. The use according to any one of claims 49-52, wherein the compound is administered in combination with a second compound. The recovery unit is capable of specifically binding to the free load, which means that the equilibrium dissociation constant K D (M) less than 10 -4 , or more specifically, less than 10 -5 , or more specifically, less than 10 -6 , or more specifically, less than 10 -7 , or more specifically, less than 10 -8 , or more specifically, less than 10 -9 .

54. The use according to any one of claims 49-53, wherein, The recovery unit is capable of specifically binding to the free load refers to the equilibrium dissociation constant K D (M) less than 10 -7 Or further, less than 10 -8 Or further, less than 10 -9 .

55. The use according to any one of claims 49-54, wherein the compound is administered in combination with a second compound. The recovery unit is capable of specifically binding to the free load, which means that the equilibrium dissociation constant K D (M) less than 10 -9 .

56. The use according to any one of claims 49-55, wherein the compound is administered in combination with a second compound. The recovery unit does not bind or has a lower affinity for the payload in a conjugated state in the ligand drug conjugate, and the lower affinity can also achieve that no molecular aggregation occurs between the molecules of the ligand drug conjugate.

57. The use of claim 49, wherein, The recovery unit comprises: (1) a VHH domain selected from CDR1-3 defined by the following amino acid sequences: CDR1-3 as shown in SEQ ID NO: 103, 104 and 105; or (2) selected from the amino acid sequence as shown in SEQ ID NO: 102; or (3) selected from the amino acid sequence comprising a combination of VH and VL defined as follows: a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 6, YAS, and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 6, FAS, and SEQ ID NO: 13, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 6, YAS, and SEQ ID NO: 16, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 20, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 6, YAS, and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 26, WAS, and SEQ ID NO: 27, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 29, 30, and 31, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 33, KVS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 29, 36, and 37, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 33, KLS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 44, KVS, and SEQ ID NO: 45, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 42, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 50, KVS, and SEQ ID NO: 51, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 53, 54, and 55, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 57, DTS, and SEQ ID NO: 58, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 66, SAS, and 67, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 69, 70, and 71, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 73, WAS, and 74, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 80, GAS, and 81, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 83, 84, and 85, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 87, SAS, and 67, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 89, 90, and 91, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 93, HGT, and 94, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 99, 77, and 100, respectively; a VH comprising an amino acid sequence of SEQ ID NO: 1, and a VL comprising an amino acid sequence of SEQ ID NO: 5; a VH comprising an amino acid sequence of SEQ ID NO: 8, and a VL comprising an amino acid sequence of SEQ ID NO: 12; a VH comprising an amino acid sequence of SEQ ID NO: 14, and a VL comprising an amino acid sequence of SEQ ID NO: 15; a VH comprising an amino acid sequence of SEQ ID NO: 17, and a VL comprising an amino acid sequence of SEQ ID NO: 18; a VH comprising an amino acid sequence of SEQ ID NO: 19, and a VL comprising an amino acid sequence of SEQ ID NO: 18; a VH comprising an amino acid sequence of SEQ ID NO: 21, and a VL comprising an amino acid sequence of SEQ ID NO: 25; a VH comprising an amino acid sequence of SEQ ID NO: 28, and a VL comprising an amino acid sequence of SEQ ID NO: 32; a VH comprising an amino acid sequence of SEQ ID NO: 35, and a VL comprising an amino acid sequence of SEQ ID NO: 38; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 39, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 43; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 46, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 49; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 52, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 56; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 59, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 60; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 61, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 65; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 68, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 72; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 75, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 79; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 82, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 86; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 88, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 92; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 95, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 96; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 97, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 98; the CDR1, CDR2, CDR3 sequences, the HCDR1, HCDR2, HCDR3 sequences, and the LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT definition scheme; or (4) a polypeptide comprising an amino acid sequence as defined by SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129; or (5) an aptamer comprising a nucleotide sequence as defined in SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO:

162.

58. The use according to any one of claims 49-57, wherein, The recovery unit is obtained by immunizing a non-human animal with a conjugate of a small molecule drug and a carrier protein or a biotin-small molecule drug conjugate; or obtained by screening using phage display technology; or obtained by screening using virtual technology; or obtained by screening using SELEX technology; or obtained by screening using hybridoma technology; or obtained by screening using nanobody technology; or obtained by using yeast display technology; or obtained by using single B cell cloning technology; or obtained by using microfluidic technology; or obtained by using surface plasmon resonance assisted screening technology.

59. The use according to claim 58, wherein The small molecule drugs include, but are not limited to, MMAE, MMAD, DM1, DM2, DM4, eribulin, Exatecan, SN38, DXD, calicheamicin, pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IBD), duocarmycin, amanitin, Maaa-1181a, taxol, daunomycin, vinblastine, Doxorubicin, methotrexate, MMAF, ML022-Dxd, ML022-D633-004, ML026-Dxd, ML031-D633-004, ML037-D633-004, ML042-Dxd, ML047-DX8951, MPA-Dxd, PEG12-Dxd, Preferably, the carrier protein includes but is not limited to KLH, BSA or OVA.

60. The use of claim 59, wherein, The biotin-small molecule drug conjugates include, but are not limited to, Biotin-PEG12-Dxd, Biotin-PEG10-Dxd, Biotin-PEG6-Dxd, Biotin-PEG6-Triazole-Dxd, or Biotin-PEG10-Triazole-Dxd:

61. The use of claim 49 or 50, wherein, The linker or payload is connected with a payload epitope masking unit capable of preventing the recovery unit from binding to the payload still conjugated to the ligand drug conjugate.

62. The use of claim 61, wherein, The payload epitope masking unit does not affect the normal separation of the payload after the ligand drug conjugate enters the target cell.

63. A method for preparing the ligand drug conjugate of any one of claims 1-46, comprising the following steps: 1) screening to obtain an antibody or antigen binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer that specifically binds to the payload; 2) using the screened antibody or antigen binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer that specifically binds to the payload as a recovery unit to prepare a ligand drug conjugate. 3) detecting whether the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer binds to the prepared ligand drug conjugate, and if not, obtaining the target ligand drug conjugate; or detecting whether the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer binds to the prepared drug-linker, and if not, obtaining the target ligand drug conjugate. 4) if the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer binds to the prepared ligand drug conjugate or drug-linker, or molecular condensation occurs between the prepared ligand drug conjugate or drug-linker: A. further modifying or replacing the antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer in the recovered unit until no molecular condensation occurs between the ligand drug conjugate or drug-linker and no binding to the target ligand drug conjugate or drug-linker; and / or B. screening a payload epitope shielding unit capable of being connected to the linker or payload of the ligand drug conjugate, and the connected payload epitope shielding unit will prevent the screened antibody or antigen-binding fragment, receptor, fusion protein, polypeptide, oligopeptide, peptidomimetic or aptamer from binding to the prepared antibody drug conjugate and will not affect the normal release of the ligand drug conjugate into the cell after entering the cell, and after screening the appropriate payload epitope shielding unit, the target ligand drug conjugate is prepared.

64. A method for treating or preventing a disease, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the ligand drug conjugate of any one of claims 1-46, or a pharmaceutically acceptable salt, deuterated form, solvate thereof, or the pharmaceutical composition of claim 48, or a pharmaceutically acceptable salt, deuterated form, solvate thereof, and a pharmaceutically acceptable adjuvant thereof.

65. The method of claim 64, wherein, The disease is a tumor.

66. Use of the ligand drug conjugate of any one of claims 1-46, or a pharmaceutically acceptable salt, deuterated form, solvate thereof, or the pharmaceutical composition of claim 43, in the preparation of a medicament for treating or preventing a disease.

67. An immunogen for screening the recovered unit of claim 1.

68. The immunogen of claim 67, wherein, The immunogen includes, but is not limited to, MMAF, ML022-Dxd, ML022-D633-004, ML026-Dxd, ML031-D633-004, ML037-D633-004, ML042-Dxd, ML047-DX8951, MPA-Dxd, Biotin-PEG12-Dxd, Biotin-PEG10-Dxd, Biotin-PEG6-Dxd, Biotin-PEG6-Triazole-Dxd, Biotin-PEG10-Triazole-Dxd, and conjugates of a carrier protein coupled to the MMAF, ML022-Dxd, ML022-D633-004, ML026-Dxd, ML031-D633-004, ML037-D633-004, ML042-Dxd, ML047-DX8951, MPA-Dxd.

69. The immunogen of claim 67 or 68, wherein, The carrier protein includes, but is not limited to, KLH, BSA, or OVA.

70. A payload recovery unit capable of specifically binding a free payload of a ligand drug conjugate, wherein the payload recovery unit comprises a moiety capable of specifically binding a moiety of the ligand drug conjugate. The payload recovery unit comprises: (1) a VHH domain selected from CDR1-3 defined by the following amino acid sequences: CDR1-3 as shown in SEQ ID NO: 103, 104, and 105; or (2) selected from the amino acid sequence as shown in SEQ ID NO: 102; or (3) selected from the amino acid sequence comprising the following defined VH and VL combinations: a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 2, 3, and 4, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 6, YAS, and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 9, 10, and 11, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 6, FAS, and SEQ ID NO: 13, respectively; a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 2, 3, and 4, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 6, YAS, and SEQ ID NO: 16, respectively; a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 2, 3, and 20, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 6, YAS, and SEQ ID NO: 7, respectively; a VH comprising HCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 22, 23, and 24, respectively, and a VL comprising LCDR1-3 of the amino acid sequences as shown in SEQ ID NO: 26, WAS, and SEQ ID NO: 27, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 29, 30, and 31, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 33, KVS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 29, 36, and 37, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 33, KLS, and SEQ ID NO: 34, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 44, KVS, and SEQ ID NO: 45, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 42, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 50, KVS, and SEQ ID NO: 51, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 53, 54, and 55, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 57, DTS, and SEQ ID NO: 58, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 66, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 69, 70, and 71, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 73, WAS, and SEQ ID NO: 74, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 80, GAS, and SEQ ID NO: 81, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 83, 84, and 85, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 87, SAS, and SEQ ID NO: 67, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 89, 90, and 91, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 93, HGT, and SEQ ID NO: 94, respectively; a VH comprising HCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and a VL comprising LCDR1-3 of the amino acid sequences set forth in SEQ ID NOs: 99, 77, and 100, respectively; a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 5; a VH comprising the amino acid sequence of SEQ ID NO: 8, and a VL comprising the amino acid sequence of SEQ ID NO: 12; a VH comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising the amino acid sequence of SEQ ID NO: 15; a VH comprising the amino acid sequence of SEQ ID NO: 17, and a VL comprising the amino acid sequence of SEQ ID NO: 18; a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 18; a VH comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 25; a VH comprising the amino acid sequence of SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 32; a VH comprising the amino acid sequence of SEQ ID NO: 35, and a VL comprising the amino acid sequence of SEQ ID NO: 38; a VH comprising the amino acid sequence of SEQ ID NO: 39, and a VL comprising the amino acid sequence of SEQ ID NO: 43; a VH comprising the amino acid sequence of SEQ ID NO: 46, and a VL comprising the amino acid sequence of SEQ ID NO: 49; a VH comprising the amino acid sequence of SEQ ID NO: 52, and a VL comprising the amino acid sequence of SEQ ID NO: 56; a VH comprising the amino acid sequence of SEQ ID NO: 59, and a VL comprising the amino acid sequence of SEQ ID NO: 60; a VH comprising the amino acid sequence of SEQ ID NO: 61, and a VL comprising the amino acid sequence of SEQ ID NO: 65; a VH comprising the amino acid sequence of SEQ ID NO: 68, and a VL comprising the amino acid sequence of SEQ ID NO: 72; a VH comprising the amino acid sequence of SEQ ID NO: 75, and a VL comprising the amino acid sequence of SEQ ID NO: 79; a VH comprising the amino acid sequence of SEQ ID NO: 82, and a VL comprising the amino acid sequence of SEQ ID NO: 86; a VH comprising the amino acid sequence of SEQ ID NO: 88, and a VL comprising the amino acid sequence of SEQ ID NO: 92; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 95, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 96; a VH comprising an amino acid sequence as set forth in SEQ ID NO: 97, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 98; the CDR1, CDR2, CDR3 sequences, the HCDR1, HCDR2, HCDR3 sequences, and the LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT definition scheme; or (4) a polypeptide comprising an amino acid sequence as defined by SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129; or (5) an aptamer comprising a nucleotide sequence as defined by SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO:

162.

71. A method for reducing the toxicity of a ligand drug conjugate, comprising, In a method of treating a patient with a ligand drug conjugate, the patient is administered an effective amount of a corresponding payload recovery unit as defined in claim 70 that is capable of binding to a free payload of the ligand drug conjugate.

72. The method of claim 71 wherein, The ligand drug conjugate and the payload recovery unit can be administered simultaneously or sequentially.

73. A nucleic acid encoding an amino acid sequence of a payload recovery unit of claim 70.

74. A vector comprising the nucleic acid of claim 73. The vector comprises the nucleic acid of claim 73.

75. The vector of claim 74, wherein The vector is an expression vector.

76. A cell, comprising: The cell comprises the vector of claim 74 or claim 75. The cell comprises the vector of claim 74 or claim 75.

77. The cell of claim 76, wherein, The cells are selected from the group consisting of CHO cells, COS cells, HEK-293 cells, NSO cells, cells or Sp2.0 cells.

Images