Multi-specific antibody targeting her3 and trop2 and conjugate thereof

Abstract

Provided in the present application are an HER3 single-domain antibody, a multi-specific antigen-binding protein and protein-drug conjugate comprising the single-domain antibody, and the use thereof in the preparation of a drug for treating / preventing tumors.

Description

5, 6, 7, 8, 9, or 10; ; k is an integer from 1 to 20, e.g., 2, 3, 4, the core GlcNAc is directly linked to P*. In some embodiments, the conjugate has an overall DAR value of 1 to 8. In some embodiments, j = 1-10; for example, j = 1-8, j = 1.2-6, j = 1.5-3, or j = 1.5-2.5. In some embodiments, the antigen-binding protein comprises a first polypeptide set forth in SEQ ID NO: 51 and a second polypeptide set forth in SEQ ID NO: 50. In some embodiments, in the protein-drug conjugate, the oligosaccharide is linked to the Fc fragment of P*, preferably to the CH2 domain of the Fc fragment, and more preferably to Asn297 (numbered according to the EU index of Kabat) of the Fc fragment. As described above, the oligosaccharide may be derived from a natural glycan chain of an antibody. Specifically, a preparation method for a precursor of the oligosaccharide (or referred to herein as a protein derivative) comprises the following step: reacting an antibody whose N-glycoform is mainly G0F / G0 with UDP-GalNAz or a salt thereof, or with other UDP-GalNAc azido derivatives, in the presence of a catalyst to give the precursor of the aforementioned oligosaccharide. UDP-GalNAz has the following structure: Methods for obtaining antibodies of the G0F / G0 glycoform are well-known in the art. For example, antibodies expressed by eukaryotic cells are post-translationally modified, and the glycan can be converted to the G0F / G0 form by treatment with galactosidase, which removes any terminal galactose residue and leaves the terminal N-acetylglucosamine residues. In some other embodiments, the starting glycan chain of the G0F / G0 glycoform described in the present disclosure may also be obtained by expression using a B4GALT1 gene knockout cell line and purification. One example of knocking out the B4GALT1 gene from an expression cell line is by homologous recombination technology, and other examples of knocking out the B4GALT1 gene include the use of zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs). Specific methods are described in reports such as Nature Biotechnology, volume 33, pages 842-844 (2015). Int. J. Mol. Sci. 2015, 16(10), 23849-23866. In some embodiments, the aforementioned catalyst is a galactosyltransferase or a functional variant or fragment thereof. In some embodiments, the catalyst is p-1,4-galactosyltransferase or a functional variant or fragment thereof. In some embodiments, the catalyst is bovine p—1,4-galactosyltransferase or human p—1,4-galactosyltransferase or a functional variant or fragment thereof. In some embodiments, the catalyst is human P-(1,4)-GalT1 having mutation Y285L or bovine P-(1,4)-GalT1 having mutation Y289L. In some embodiments, the catalyst is the p—1,4-acetylgalactosyltransferase disclosed in Patent Application WO2016170186. In some embodiments, the catalyst comprises the sequence set forth in any one of SEQ ID NOs: 52-54. Protein Derivative In another aspect, the present application further relates to a protein derivative having the structure represented by formula VII: Man— GlcNAc—Gal** (VII) wherein P* binds to HER3 and TROP2 and comprises the antigen-binding protein described in the present application, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20; GlcNAc Gal** is a modified galactose selected from the following structures:                     and the oligosaccharide is linked to P* by the core GlcNAc. The protein derivative may be obtained by reacting an antigen-binding protein whose N-glycoform is mainly G0F with UDP-GalNAz or a salt thereof, or with other UDP-GalNAc azido derivatives, in the presence of a catalyst. Nucleic Acid of Present Disclosure and Vector and Host Cell Comprising Same In one aspect, the present disclosure provides a nucleic acid encoding any of the above binding protein, single variable domain, VHH, fusion protein, antibody, and multispecific antigen-binding protein or any fragment (e.g., antigen / target-binding fragment) thereof. The present disclosure also encompasses a nucleic acid that hybridizes with the nucleic acid described above under stringent conditions, a nucleic acid having one or more substitutions (e.g., conservative substitutions), deletions, or insertions compared to the nucleic acid described above, or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity compared to the nucleic acid described above. For example, the nucleic acid of the present disclosure comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-51, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-51. As will be appreciated by those skilled in the art, the amino acid sequence of each binding protein, single variable domain, VHH, fusion protein, antibody, or multispecific antigen-binding protein or any fragment thereof may be encoded by a variety of nucleic acid sequences due to codon degeneracy. Nucleic acid sequences encoding the molecules of the present disclosure may be produced using methods well-known in the art, e.g., by de novo solidphase DNA synthesis, or by PCR amplification. In one aspect, the present disclosure provides a nucleic acid encoding any of the above binding protein, single variable domain, VHH, fusion protein, antibody, and multispecific antigen-binding protein or any peptide chain / fragment thereof. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of showing the ability to bind to human HER3 and / or TROP2. In one embodiment, nucleic acids encoding chains of the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, and the multispecific antigen-binding protein of the present disclosure may be in the same vector or different vectors. In yet another embodiment, nucleic acids encoding chains of the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, and the multispecific antigen-binding protein of the present disclosure may be introduced into the same host cell or different host cells for expression. Thus, in some embodiments, a method for producing the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, or the multispecific antigen-binding protein of the present disclosure comprises a step of: culturing a host cell comprising nucleic acids encoding chains of the molecule under conditions suitable for the expression of the chains to produce the antibody or the bispecific binding molecule or the fusion protein or a fragment thereof or the multispecific binding molecule of the present disclosure. In another aspect, the present disclosure provides a vector comprising the nucleic acid described above. In one preferred embodiment, the vector is an expression vector. It will be fully appreciated by those skilled in the art that vectors commonly used in the art to which the present disclosure pertains can be applied to the present disclosure. In one embodiment, the present disclosure provides a host cell comprising the nucleic acid or the vector. The term “host cell” refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be exactly the same as parental cells in terms of nucleic acid content, and may contain mutations. Mutant progeny having the same function or biological activity that are screened or selected from the initially transformed cells are included herein. Host cells are any type of cell system that can be used to produce the antibody molecules of the present disclosure, including eukaryotic cells, such as mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, and yeast cells, and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, and also include cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues. Composition In another aspect, the present disclosure provides a composition, e.g., preferably a pharmaceutical composition, comprising one of or a combination of the HER3-binding protein, fusion protein, multispecific antigen-binding protein, or protein-drug conjugate of the present disclosure, which is formulated together with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic agents, absorption delaying agents, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). According to the route of administration, the active compound, i.e., the antibody molecule, may be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate the compound. The amount of the active ingredient that can be combined with a carrier material to prepare a single dosage form varies depending on the subject being treated and the particular mode of administration. The amount of the active ingredient that can be combined with a carrier material to prepare a single dosage form is generally an amount of the composition that produces a therapeutic effect. Generally, such amounts range from about 0.01% to about 99% of the active ingredient, for example, from about 0.1% to about 70% or from about 1% to about 30% of the active ingredient, based on 100%, combined with a pharmaceutically acceptable carrier. Actual dosage levels of the active ingredient in the pharmaceutical composition of the present disclosure may be varied so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response 36 for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present disclosure employed or an ester, salt, or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, body weight, condition, general health, and medical history of the patient being treated, and similar factors well-known in the medical arts. In some embodiments, the protein-drug conjugate composition of the present disclosure has a DAR value of about 1.0 to 16.0, preferably about 2.0 to 12.0, more preferably about 3.0 to 6.0, and even more preferably about 3.5 to 4.5. For example, the protein-drug conjugate composition has a DAR value of about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0. The composition of the present disclosure may be administered via one or more routes of administration using one or more methods well-known in the art. It will be appreciated by those skilled in the art that the route and / or mode of administration varies depending on the desired result. Preferred routes of administration for the HER3-binding protein, multispecific antigen-binding protein, or protein-drug conjugate of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as injection or infusion. The phrase “parenteral administration” as used herein refers to a mode of administration other than enteral and topical administration, which is generally injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Pharmaceutical Combination The term “pharmaceutical combination” or “combination product” refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit. The term “non-fixed combination” means that the active ingredients (e.g., (i) the HER3-binding molecule of the present disclosure, (ii) the multispecific antigen-binding protein of the present disclosure, and (iii) the protein-drug conjugate of the present disclosure) are administered, either simultaneously or sequentially (without a specific time limitation or at identical or different time intervals), to a patient as separate entities, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient. The term “fixed combination” means that two or more active agents are simultaneously administered in the form of a single entity to a patient. The dose and / or time interval of two or more active agents are / is preferably selected such that the combined use of the components can result in a therapeutic effect on the disease or condition greater than that achieved by the use of any one of the components alone. Each of the components may be in a separate formulation form, and their formulation forms may be identical or different. Thus, in yet another aspect, the present application further provides a pharmaceutical combination or combination product comprising the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, the multispecific antigen-binding protein, and the protein-drug conjugate of the present disclosure, wherein for the definitions regarding the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, the multispecific antigen-binding protein, and the protein-drug conjugate in the combination, reference can be made to the technical features described in the first aspect. In some embodiments, the pharmaceutical combination or combination product may further comprise one or more additional therapeutic agents, e.g., a chemotherapeutic agent. The present application further provides a kit of parts comprising the pharmaceutical combination. For example, the kit of parts comprises, within the same package: - a first container containing the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, the multispecific antigen-binding protein, the nucleic acid, the vector, and the host cell of the present disclosure or a pharmaceutical composition comprising the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, the multispecific antigen-binding protein, the nucleic acid, the vector, and the host cell, and - a second container containing the protein-drug conjugate of the present disclosure or a pharmaceutical composition comprising the protein-drug conjugate, wherein for the definitions regarding the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, the multispecific antigen-binding protein, the nucleic acid, the vector, the host cell, and the proteindrug conjugate, reference can be made to the technical features described in the aforementioned aspects of the present disclosure. In some embodiments, the kit of parts further comprises, within the same package, an additional container containing an additional therapeutic agent or comprising the additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. Treatment Method and Use In yet another aspect, the present application further provides a method for treating a tumor or cancer, e.g., a solid tumor or a non-solid tumor, e.g., a hematological system tumor, comprising administering to a patient in need thereof one of or a combination of more than one of the binding protein, the single variable domain, the VHH, the fusion protein, the antibody, the multispecific antigen-binding protein, the nucleic acid, the vector, the host cell, and the protein-drug conjugate of the present disclosure. The present application further provides a method for treating a tumor or cancer, e.g., an advanced or metastatic solid malignancy, comprising administering to a patient in need thereof the pharmaceutical composition of the present application. The description of “administering...a pharmaceutical combination” or “...in combination with...” in the present application includes both the case where a plurality of drugs are administered simultaneously and the case where a plurality of drugs are administered sequentially. When administered sequentially, the plurality of drugs are administered at intervals of no more than 24 h, e.g., no more than 18 h, no more than 15 h, no more than 12 h, no more than 10 h, no more than 8 h, no more than 5 h, no more than 3 h, no more than 2 h, no more than 1 h, or no more than 0.5 h. In another aspect, the present application further relates to use of the HER3-binding protein, the multispecific antigen-binding protein, the nucleic acid, the vector, the host cell, and / or the protein-drug conjugate in the preparation of a medicament for treating and / or preventing a tumor. In some embodiments, the tumor includes a solid tumor and / or a non-solid tumor, e.g., a hematological system tumor. In some embodiments, the tumor is HER3-positive and / or TROP2-positive. In some embodiments, the tumor is one whose treatment would benefit from inhibiting HER3 and / or TROP2. Examples Instruments and equipment Name              M anufacturer Model No. Flow cytometer Beckman CytoFlex 10000396 Cell counter Count-star ICI1000 10000395 Biosafety cabinet Airtech BSC-1804IIA2         10000388 Cell incubator Thermo Model311 10000385 Multimode Molecular Devices SpectraMax I3x         10000397 microplate reader Reagents and consumables Name Manufacturer Catalog No. RPMI1640 Gibco 11875-093 FBS Hyclone 10091-148 Trypsin-EDTA Hyclone 25200056 Penicillin-Streptomycin (PS) Gibco 15140-122 PBS Hyclone SH30028.02 Matrigel Corning 356230 proA biosensors Sartorius 18-5012 Human HER3 Protein, His Tag ACRO ER3-H5223 SA-HRP Abcam ab7403 APC anti-human IgG Fc Antibody Biolegend 366906 / B343083 CellTrace™ Violet cell proliferation Thermo Fisher C34557 / 2535896 Kit, for flow cytometry CCK8 Dojindo CK04 / SW602 Nunc microplate Nunc 442404 96-well polypropylene microplate, F- Sartorius 655209 bottom, black Example 1: Screening, Preparation, and Characterization of HER3 Single-Domain Antibodies 1.1 Construction of HER3 single-domain antibody immune library A healthy camel was selected and immunized with a human HER3-Fc fusion protein as the antigen via multi-point intramuscular injection in the neck once every two weeks, and a total of six immunizations were performed. At the end of the last immunization, 50 mL of peripheral blood was collected from the camel into a vacuum blood collection tube, and the supernatant was collected as post-immunization serum. Lymphocytes were isolated using density gradient centrifugation, and total RNA was extracted using an RNA extraction kit provided by QIAGEN. The extracted RNA was all reverse-transcribed into cDNA using a Super-Script III FIRST STRANDSUPERMIX kit (ThermoFisher) according to the instructions. Nested PCR was performed to amplify nucleic acid fragments encoding the variable regions of heavy-chain antibodies. A nucleic acid fragment encoding the target heavy-chain single-domain antibody was recovered and cloned into the phage display vector pComb3XSS using the restriction endonuclease SfiI. The product was subsequently electroporated into Escherichia coli electrocompetent cells TG1 to construct an anti-HER3 immune single-domain antibody phage display library, and the library was characterized. Through gradient dilution plating, the library size was calculated to be about 1.0 x 108. 1.2. Panning for HER3 single-domain antibody The previously obtained phage library was subjected to panning. In the first round of panning, HER3-muFc (human HER3-mouse Fc fusion protein) was used as the screening antigen, and PD-L1-Fc was used as the negative screening antigen. In the second and third rounds of panning, HER3-muFc was used as the screening antigen, and SP-Fc (signal peptide-Fc fusion protein) was used as the negative screening antigen. After panning, clones binding to HER3-muFc while not binding to Fc were obtained. The binding-positive phages obtained after panning were used to infect blank Escherichia coli cells, and the cells were plated. Subsequently, colonies were picked, inoculated separately into 2TY-AG (containing 10% glycerol), and left to stand overnight at room temperature. The next day, each culture was inoculated into 200 uL of 2TY-AG, with an inoculation amount of 1%, and cultured with shaking at 250 rpm at 37 °C until OD600 was about 0.5. Helper phage M13KO7 was added for infection (at a multiplicity of infection of 1:20). The cultures were left to stand at 37 °C for 15 min and then incubated at 220 rpm for 45 min. 800 uL of 2TY-AG was added to each well, and the plate was incubated overnight at 30 °C at 220 rpm and centrifuged the next day. The supernatants were collected for ELISA analysis. Plates were coated overnight at 4 °C with HER3-Fc and SP-Fc, respectively, and the obtained supernatants were added. The plates were incubated at room temperature for 2 hours. After washing, the secondary antibody goat anti-HA tag HRP (purchased from Abcam) or goat anti-mouse IgG-HRP (purchased from Thermo) was added, and the plates were incubated at room temperature for 2 hours. After washing, the TMB chromogenic solution was added, and the absorbance values at wavelengths of 450 nm and 650 nm were read. The final absorbance values were obtained by subtracting the absorbance values at the wavelength of 650 nm from the absorbance values at the wavelength of 450 nm. The results are shown in Table 1. Table 1. OD values of positive clones Sample OD (HER3-Fc) OD (SP-Fc) Sample OD (HER3-Fc) OD (SP-Fc) iBT42 2.992 0.062 iBT19 2.423 0.06 iBT47 2.928 0.079 iBT27 2.414 0.046 iBT44 2.843 0.055 iBT17 2.382 0.055 iBT16 2.764 0.051 iBT34 2.318 0.044 iBT6 2.754 0.059 iBT28 1.925 0.052 iBT11 2.741 0.052 iBT40 1.915 0.057 iBT21 2.739 0.059 iBT14 1.799 0.056 iBT4 2.677 0.081 iBT7 1.717 0.195 iBT9 2.64 0.061 iBT22 1.695 0.053 iBT24 2.56 0.051 iBT3 1.683 0.042 iBT10 2.522 0.062 iBT33 1.593 0.047 iBT13 2.515 0.051 iBT46 1.502 0.053 iBT41 2.505 0.064 iBT25 1.448 0.068 iBT23 2.501 0.05 iBT20 1.359 0.049 iBT12 2.486 0.048 iBT26 1.337 0.051 iBT39 2.474 0.049 iBT45 0.859 0.046 iBT32 2.472 0.099 iBT35 0.512 0.052 iBT1 2.47 0.051 iBT15 0.484 0.047 iBT38 2.468 0.047 iBT43 0.324 0.053 iBT18 2.462 0.052 iBT31 0.3 0.115 iBT5 2.459 0.05 iBT8 0.108 0.054 iBT2 2.439 0.046 iBT36 0.101 0.045 iBT29 2.432 0.048 iBT30 0.096 0.04 iBT37 2.431 0.055 The positive clones specifically binding to HER3 obtained by screening were sequenced. The amino acid sequence of iBT11 is shown below: QVQLQESGGGSVQSGGSLRLSCAASGYTTSSVCMAWFRQAPGNEREGVAHITRDGRTMYADSVRGRFTIS QDNAKNTLFLQMNSLKPEDTGMYYCAARVCEWRSTVQAPRSEAYQLWGRGTQVTVSS (SEQ ID NO: 1) 1.3. Preparation of HER3 single-domain antibody and its Fc fusion protein A sequence fragment encoding the HER3 single-domain antibody VHH was amplified by PCR and fused with a DNA fragment encoding human IgG1-Fc, or a His tag (Chis) was fused to its C-terminus. The resulting sequence was cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing an HER3 single-domain antibody-Fc / Chis fusion protein in mammals. The universal primers used for PCR amplification are shown below: Upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC (SEQ ID NO: 57) Downstream primer cccGGATCCTGAGGAGACGGTGACCTGG (SEQ ID NO: 58) The constructed plasmid vectors were transfected into HEK293 cells for transient antibody expression. The recombinant expression plasmids were diluted with Freestyle293 culture medium, and a PEI (polyethylenimine) solution required for transformation was added. Each plasmid / PEI mixture was added to a suspension of HEK293 cells, and suspension cell culture was performed at 37 °C with 5% CO2. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography or using a Ni column to obtain the target HER3 single-domain antibody-Fc / Chis fusion protein. 1.4. Determination of affinity for HER3 The affinity of the HER3 single-domain antibody was determined by ELISA: Coating was performed overnight at 4 °C with 5 ug / mL Her3-muFc. The HER3 single-domain antibody-Chis to be tested was serially diluted 3-fold with 1% BSA + 0.05% PBST20 from a starting concentration of 5 ug / mL to obtain a total of 10 gradients, and incubation was performed at room temperature for 2 h. After plate washing with 1% BSA + 0.05% PBST20, the secondary antibody anti-6*his tag HRP (purchased from abcam, diluted in a 1:10,000 ratio) was added. After plate washing with 1% BSA + 0.05% PBST20, the TMB chromogenic solution was added, and color development was performed at room temperature for 5 min. The absorbance values at wavelengths of 450 nm and 650 nm were read. The final absorbance values were obtained by subtracting the absorbance values at the wavelength of 650 nm from the absorbance values at the wavelength of 450 nm. The results are shown in FIG. 1. The EC50 value of iBT11-Chis was 5.39 ng / mL. The binding kinetics of the HER3 single-domain antibody-Fc fusion protein to HER3-chis was studied using biolayer interferometry (BLI). The iBT11-Fc fusion protein was diluted to 10 ug / mL and then immobilized on an AHC biosensor, and then HER3-chis was diluted to 5 gradients: 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM, and allowed to bind to the immobilized iBT11-Fc fusion protein. The equilibrium dissociation constant (KD) was calculated using Octet K2 data analysis software 9.0. The results are shown in Table 2. Table 2. The affinity of iBT11-Fc (BLI) Sample KD (M) kon (1 / Ms) kdis (1 / s) iBT11-Fc 2.27E-09 1.88E+05 4.26E-04 1.5. Humanization of HER3 single-domain antibody The humanization was accomplished using protein surface amino acid humanization (resurfacing) and VHH humanization universal framework grafting (CDR grafting to a universal framework). First, the universal humanization VHH framework hNbBcII10FGLA (PDB No. 3EAK), designed by Cecile Vincke et al. based on sequence homology, was obtained. The framework design is based on the nanobody NbBcII10 (PDB No. 3DWT). Modeling was performed using Modeller 9, and the relative solvent accessibility of amino acids on the framework was calculated based on the three-dimensional structure of the protein. The specific procedure of VHH humanization universal framework grafting is as follows: A highly homologous human antibody sequence is obtained through IMGT, and the target sequence is humanized with reference to the universal humanization VHH framework hNbBcII10FGLA (PDB No. 3EAK). A highly homologous sequence framework is used as the framework template, and the CDRs are replaced with the CDR regions of the target antibody. Non-surface amino acids on the framework are then back-mutated based on the model to complete the humanization of the target antibody. The antibody iBT11 was humanized to obtain the following humanized variants: huiBT11v1 (SEQ ID NO: 2), huiBT11v3 (SEQ ID NO: 3), huiBT11v4 (SEQ ID NO: 4), huiBT11v5 (SEQ ID NO: 5), huiBT11v6 (SEQ ID NO: 6), and huiBT11v7 (SEQ ID NO: 7). Genes encoding the humanized sequences described above were synthesized and fused with a DNA fragment encoding human IgG1-Fc, and the resulting sequences were cloned into conventional mammalian expression vectors to obtain recombinant plasmids for expressing HER3 single-domain antibody-Fc fusion proteins in mammals. The constructed vectors were transfected into HEK293 cells for transient antibody expression. The recombinant expression plasmids were diluted with Freestyle293 culture medium, and a PEI (polyethylenimine) solution required for transformation was added. Each plasmid / PEI mixture was added to a suspension of HEK293 cells, and the cells were cultured at 37 °C at 130 rpm with 5% CO2. After four hours, EXCELL293 culture medium and 2 mM glutamine were added, and the cells were cultured at 130 rpm. After 24 hours, 3.8 mM VPA was added. After 72 hours, 4 g / L glucose was added. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target huiBT11 single-domain antibody-Fc fusion proteins. 1.6. Determination of affinity of humanized antibodies The affinity of humanized antibodies for HER3 was determined by ELISA: Coating was performed overnight at 4 °C with 5 ug / mL Her3-muFc. The antibodies were serially diluted 4-fold with 1% BSA + 0.05% PBST20 from a starting concentration of 100 ug / mL to obtain a total of 10 gradients, and incubation was performed at room temperature for 2 h. After plate washing with 1% BSA + 0.05% PBST20, the detection secondary antibody goat anti-human IgG (Fc specific)-HRP (purchased from Sigma, diluted in a 1:8000 ratio) was added, and incubation was performed at room temperature for 2 h. After plate washing with 1% BSA + 0.05% PBST20, the TMB chromogenic solution was added, and color development was performed at room temperature for 5 min. The absorbance values at wavelengths of 450 nm and 650 nm were read. The final absorbance values were obtained by subtracting the absorbance values at the wavelength of 650 nm from the absorbance values at the wavelength of 450 nm. The results are shown in Table 3. For the sequences of the positive control antibody AV203, reference was made to Patent WO2011136911: Genes encoding AV203HC (SEQ ID NO: 55) and AV203LC (SEQ ID NO: 56) were synthesized and cloned into conventional mammalian expression vectors, and the constructed vectors were transfected into HEK293 cells for transient antibody expression. Table 3. The affinity of humanized antibodies (ELISA method) Conc(ng / mL) huiBT11v1-Ld-Fc huiBT11v4-Ld-Fc huiBT11v3-Ld-Fc huiBT11v5-Ld-Fc huiBT11v6-Ld-Fc huiBT11v7-Ld-Fc Av203 100000 2.311 2.3 2.346 2.313 2.322 2.381 2.345 25000 2.243 2.259 2.28 2.267 2.28 2.283 2.263 6250 2.347 2.257 2.262 2.315 2.272 2.29 2.24 1562.5 2.251 2.254 2.292 2.264 2.279 2.275 2.231 390.625 2.232 2.291 2.257 2.261 2.301 2.274 2.226 97.656 1.777 1.763 1.759 1.758 1.79 1.782 1.481 24.414 0.763 0.741 0.765 0.738 0.772 0.794 0.332 6.104 0.243 0.244 0.245 0.239 0.253 0.268 0.096 1.526 0.089 0.089 0.092 0.086 0.092 0.092 0.052 0.381 0.051 0.05 0.053 0.051 0.053 0.051 0.044 0.095 0.042 0.04 0.042 0.042 0.042 0.043 0.041 EC50(ng / mL) 41.7 42.4 42.4 43.3 41.2 41 70.7 1.7. Determination of blocking activity of humanized antibodies The blocking activity of humanized antibodies against the binding of HER3-muFc to NRG1b1 was determined by ELISA. Coating was performed overnight at 4 °C with 1 ug / mL NRG1 beta 1 (purchased from Sino) and SP-NRGlb-Chis. The antibodies to be tested were serially diluted 4-fold with 1% BSA + 0.05% PBST20 + 200 ng / mL Her3-muFc from a starting concentration of 20 ug / mL to obtain a total of 6 gradients, and incubation was performed at room temperature for 2 h. After plate washing with 1% BSA + 0.05% PBST20, the detection secondary antibody goat anti-mouse IgG1-HRP (purchased from Thermo, diluted in a 1:3000 ratio) was added, and incubation was performed at room temperature for 2 h. After plate washing with 1% BSA + 0.05% PBST20, the TMB chromogenic solution was added, and color development was performed at room temperature for 7.5 min. The absorbance values at wavelengths of 450 nm and 650 nm were read. The final absorbance values were obtained by subtracting the absorbance values at the wavelength of 650 nm from the absorbance values at the wavelength of 450 nm. The results are shown in Table 4. Table 4. The blocking activity of humanized antibodies (ELISA method) Conc (ng / mL) huiBT11v 1-Ld- Fc(Her3-muFc)-NRG1b1 huiBT11v 4-Ld- Fc(Her3-muFc)-NRG1b1 huiBT11v 3-Ld- Fc(Her3-muFc)-NRG1b1 huiBT11v 5-Ld- Fc(Her3-muFc)-NRG1b1 huiBT11v 6-Ld- Fc(Her3-muFc)-NRG1b1 huiBT11v 7-Ld- Fc(Her3-muFc)-NRG1b1 Av203(Her 3-muFc)-NRG1b1 Av203(Her 3-muFc)-SP-NRG1b-Chis 20000 0.251 0.232 0.249 0.255 0.245 0.254 0.354 0.083 5000 0.234 0.214 0.218 0.229 0.23 0.23 0.388 0.076 1250 0.24 0.224 0.233 0.235 0.228 0.241 0.419 0.076 312.5 0.269 0.256 0.27 0.269 0.265 0.274 0.538 0.102 78.125 1.791 1.817 1.837 1.864 1.823 1.8 2.14 1.981 19.531 2.254 2.23 2.256 2.265 2.246 2.23 2.252 2.205 4.883 2.333 2.289 2.281 2.305 2.303 2.26 2.292 2.208 IC50 (ng / mL) 105 109 110 110 108 108 160 122 Example 2: Preparation of Antibody-Drug Conjugate 2.1. Preparation of antibody A Genes encoding the sequences of the first polypeptide (SEQ ID NO: 51) and the second polypeptide (SEQ ID NO: 50) of the target bispecific antibody (antibody A) were synthesized, amplified by PCR using primers, and cloned into conventional pcDNA3.4 mammalian expression vectors to obtain pcDNA3.4-A recombinant plasmids. HEK293 cells were cultured in 293M culture medium at 37 °C at 130 ± 10 rpm with 5% CO2. Before transient transfection, the HEK293 cell density was adjusted to 6 x 106 cells / mL. The pcDNA3.4-A plasmids and the transfection reagent PEI were well mixed in a 1:6 ratio, and the well-mixed transfection reagent and plasmid complex was added to about 2 mL of expression culture medium OPM-293-CD05. The mixture was left to stand for 5 min. The above solution was added to 100 mL of expression culture medium, and the mixture was incubated at 37 °C at 130 ± 10 rpm with 5% CO2. On day 7, the mixture was centrifuged at 1500 rpm for 10 min, and the supernatant was collected. The collected supernatant was subjected to Protein A affinity purification to obtain the target protein. The size of the target protein was confirmed to be correct by SDS PAGE. 2.2. Preparation of linker-toxin The following linker-toxin (LP1) was prepared according to the process described in Patent Application CN113264983A (the content of which is incorporated herein by reference): Analysis showed LP1 MS m / z (ESI): 1375.77. 2.3. Preparation of antibody A-ADC Step 1: A stock solution of antibody A (mainly G0F) was desalted into an HEPES buffer, and the resulting solution was set aside for later use. Ultrapure water (210 ^L), MnCl2 (1 M, 5 ^L), UDP-GalNAz (100 mM, 25 ^L), Tris-HCl (1 M, 5 ^L), antibody A (25.5 mg / mL, 196 ^L), and GalTl (5.2 mg / mL, 12 ^L, SEQ ID NO: 52) were sequentially added to a 1.5 mL centrifuge tube. The mixture was left to react at 30 °C at 400 rpm for 12 h. After the reaction was complete, the reaction mixture was purified by Protein A and concentrated by ultrafiltration into DPBS to give antibody A-(N3)4 for later use. Step 2: LP1 (16 ^L, 50 mM), DMSO (25 ^L), and the antibody A-(Nj)4 obtained in step 1 (10 mg / mL, 800 ^L) were sequentially added to a 1.5 mL centrifuge tube. The mixture was left to react at 30 °C at 400 rpm for 12 h. After the reaction was complete, the reaction mixture was desalted into DPBS to obtain antibody A-ADC. The molecular weight was measured by mass spectrometry. The results are shown in FIG. 2. Comparison with the antibody A before conjugation confirmed that the target product had been obtained. RP-HPLC analysis confirmed that the product had a DAR value of about 3.8. The overall reaction scheme is shown in FIG. 3. Example 3: Characterization of Biological Activity of Antibody A-ADC 3.1. Determination of TROP2-binding activity of antibody A-ADC (BLI method) The affinity of antibody A-ADC for human and monkey TROP2 was determined based on bio-layer interferometry (BLI). First, antibody A-ADC was diluted to 10 ^g / mL and immobilized on a proA biosensor. Then, the TROP2 protein was diluted to obtain 7 concentrations and allowed to bind to antibody A-ADC. Binding signals with different intensities could be detected. Data were analyzed using Data Analysis HT 12.0 software, fitting was performed using a 1:1 model, and the equilibrium dissociation constant (KD) values of the samples were calculated. The results are shown in Table 5. The ability of antibody A-ADC to bind to the human TROP2 protein and the monkey TROP2 protein was comparable to that of the naked antibody. Table 5. The affinity of samples for the human and monkey TROP2 proteins Sample ID Loading Sample ID KD (M) ka (1 / Ms) kdis (1 / s) Antibody A-ADC 2.854E-09 7.19E+04 2.052E-04 hTROP2-His hRS7-IgG1 3.008E-09 1.446E+05 4.351E-04 Antibody A 3.376E-09 7.479E+04 2.525E-04 Antibody A-ADC 4.443E-09 9.969E+04 4.429E-04 cynoTROP2-His hRS7-IgG1 3.801E-09 1.795E+05 6.9824E-04 Antibody A 5.099E-09 9.059E+04 4.619E-04 3.2. Determination of HER3-binding activity of antibody A-ADC (BLI method) The affinity of antibody A-ADC for the human and monkey HER3 proteins was determined using a method similar to that described above. The results are shown in Table 6. The ability of antibody A-ADC to bind to the human HER3 protein and the monkey HER3 protein was comparable to that of the naked antibody. Table 6. The affinity of samples for the human and monkey HER3 proteins Sample ID Loading Sample ID KD (M) ka (1 / Ms) kdis (1 / s) Antibody A-ADC 1.233E-09 5.944E+04 7.331E-05 hHER3-His iBT11V7-Fc 1.442E-09 1.052E+05 1.516E-04 Antibody A 3.438E-09 5.933E+04 2.04E-04 Antibody A-ADC 5.035E-09 6.058E+04 3.05E-04 cynoHER3-His iBT11V7-Fc 3.292E-09 1.078E+05 3.547E-04 Antibody A 4.46E-09 6.183E+04 2.758E-04 3.3. Determination of simultaneous binding activity of antibody A-ADC to HER3 and TROP2 The ability of antibody A-ADC to simultaneously bind to the HER3 and TROP2 proteins was assessed by ELISA. The test sample (multiple concentration gradients) was added to a microplate pre-coated with antigen 1 (HER3-His), and the plate was incubated. Then, biotinylated antigen 2 (TROP2-Biotin, prepared in-house) was added, and the plate was incubated. Finally, a horseradish peroxidase-labeled detection antibody (SA-HRP) was added to form a solid-phase antigen 1-antibody A-ADC sample-antigen 2-enzyme-labeled detection antibody complex. Absorbance values were read at 450 nm using a microplate reader, and data were analyzed by four-parameter fit curve analysis. The results are shown in Table 7. Both antibody A-ADC and antibody A could bind to the human HER3 and TROP2 proteins simultaneously, with the binding EC50 values being 31.42 nM and 29.95 nM, respectively, indicating that the two were comparable in binding ability. iBT11V7-Fc and hRS7-IgG1 could not bind to the two proteins simultaneously. Table 7. The binding activity of antibody A-ADC and antibody A to the human HER3 and TROP2 proteins Sample EC50(nM) Antibody A-ADC Antibody A 31.42 29.95 3.4. Determination of binding activity of antibody A-ADC to tumor cells The triple-negative breast cancer cell line MDA-MB-468, the human gastric cancer cell line NCI-N87, and the human pancreatic cancer cell line BxPC-3 (all purchased from Beina Chuanglian Biotech Co., Ltd.) (1.0 x 105 / well) were separately co-incubated with antibody A-ADC and antibody A (the highest final concentration: 200 nM, 3-fold serial dilution, 10 concentrations in total) on ice for 30 min. After 2 washes, the APC anti-human IgG Fc fluorescent antibody was added, and incubation was performed on ice in a dark place for 30 min. After 2 washes, the fluorescence intensity on the cell surface was measured using a flow cytometer. The flow cytometry results were analyzed using CytExpert software, the mean fluorescence intensity (MFI) of each test sample was calculated, and four-parameter fitting was performed for the experimental results using GraphPad Prism 7.0. The results are shown in Table 8. Antibody A-ADC and the naked antibody, antibody A, exhibited similar binding activity to tumor cells. Table 8. The binding activity of antibody A-ADC to tumor cells Sample EC50 (nM) MDA-MB-468 NCI-N87 BxPC-3 Antibody A-ADC 4.959 2.93 4.18 Antibody A 4.732 2.927 3.538 3.5. Determination of bridging effect of antibody A-ADC on 293T-TROP2 and 293T-HER3 cells 293T-TROP2-GFP cells (1.0 x 105 / well, HEK293T cells expressing TROP2 on the cell surface and expressing green fluorescent protein intracellularly, prepared in-house) and 293T-HER3-CellTrace Violet cells (1.0 x 105 / well, expressing HER3 on the cell surface, labeled with CellTrace™ Violet fluorescent dye, purchased from Kyinno Biotechnology Co., Ltd.) were mixed in a 1:1 ratio. After the cell mixture was well mixed with antibody A-ADC, antibody A, and a mixture of iBT11V7-FC and hRS7-IgG1 (concentration range: 0.016-50 nM), the resulting mixtures were incubated on ice in a dark place for 30 min. After 2 washes, fluorescence signals of 293T-HER3-CellTrace Violet cells and fluorescence signals of 293T-TROP2-GFP cells were collected by the PB450 / FITC channels of a CytoFelx flow cytometer, respectively. The dual fluorescence signal events in the two-dimensional scatter plots of the PB450 channel and the FITC channel reflected the bridging effect of antibody A-ADC on the two types of target cells. The results are shown in Table 9. As the concentration of antibody A-ADC increased, the percentage of dual fluorescence signal events among the total events in the test sample gradually increased from 1.82% to 22.46%, showing significant concentration dependence; however, as the concentration of the drug continued to increase, the percentage of dual fluorescence signal events among the total events gradually decreased, and the bridging effect of antibody A-ADC on the two types of cells decreased, showing a “Hook-effect”. The ability of antibody A to bridge the two types of cells was comparable to that of antibody A-ADC. The results show that antibody A, as an anti-HER3 and TROP2 bispecific antibody-drug conjugate, could bind to TROP2- and HER3-positive cells simultaneously and had the ability to bridge the two types of cells. In contrast, the mixture of the anti-TROP2 monoclonal antibody and the anti-HER3 single-domain antibody-Fc fusion protein showed no significant bridging effect. Table 9. The bridging effect of antibody A-ADC on TROP2- and HER3-positive cells Double positive events % Conc. nM 50 10 2 0.4 0.08 0.016 Antibody A-ADC 1.34% 14.65% 22.46% 17.47% 10.41% 1.82% Antibody A 1.93% 18.85% 21.99% 17.65% 7.00% 1.39% iBT11V7-FC+hRS7-IgG1 0.75% 0.49% 0.77% 0.81% 0.72% 0.69% 3.6. Determination of killing effect of antibody A-ADC on tumor cells Human pancreatic cancer BxPC-3 cells were plated on 96-well plates at 5 x 103 / well and cultured overnight, and then antibody A-ADC, antibody A, and iso-ADC (an isotype control ADC drug, the antibody of which was a human IgG1 isotype control, and the toxin-linker of which was the same as that of antibody A-ADC, obtained by the same preparation process as antibody A-ADC) were separately added, with the final concentrations being 0.006-25 nM. The cells were cultured at 37 °C for 120 h. The OD values at 450 / 650 nm were determined using the CCK-8 method and a microplate reader, the viability of the experimental cells was calculated, and thus, the inhibition rate of each experimental group was calculated. Then, the logarithmic values of the standard curve sample concentration (x) and the calculated inhibition rates (%) (y) were fitted using the 4-parameter logistic fit in GraphPad Prism 7.0 software to calculate the IC50 of the test samples. The experimental results are shown in FIG. 4. Antibody A-ADC exhibited a significant killing effect on BxPC-3 cells, with an IC50 of 0.141 nM, while antibody A and iso-ADC exhibited no significant killing effects on BxPC-3 cells. The killing effects of antibody A-ADC on the human epidermal carcinoma cell line A431 (purchased from Beina Chuanglian Biotech Co., Ltd., the final concentrations of the sample: 0.003-50 nM), the human pancreatic cancer cell line CAPAN-2 (purchased from Beina Chuanglian Biotech Co., Ltd., the final concentrations of the sample: 0.006-100 nM), and the human lung adenocarcinoma cell line HCC827 (purchased from Nanjing Cobioer Biosciences Co., Ltd., the final concentrations of the sample: 0.012-50 nM) were tested using a similar method. The results are shown in FIGs. 5-7. Antibody A-ADC exhibited significant killing effects on all these tumor cells, while antibody A and iso-ADC exhibited no significant killing effect. 3.7. Determination of bystander killing effect of antibody A-ADC on TROP2- and HER3-negative cells To investigate the bystander effect of antibody A-ADC, a BxPC-3 (TROP2- and HER3-positive) mono-culture system, a 293T-GFP (TROP2- and HER3-negative) mono-culture system, and a BxPC-3 and 293T-GFP (1:1) coculture system were treated with antibody A-ADC and iso-ADC. After 96 h, the cells in all sample wells were counted and assayed using a flow cytometer: Mono-culture plating: The density of both cell types was adjusted to 3 x 104 / mL using a culture medium (RPMI1640 + 10% FBS), and each type of cells was plated on a 96-well plate at 100 ^L / well (3 x 103 cells / well). The cells were cultured overnight in an incubator at 37 °C with 5% CO2. Co-culture plating: The two types of cells were mixed in a culture medium (RPMI-1640 + 10% FBS), with the density of BxPC-3 cells being 3.0 x 104 / mL and the density of 293T-GFP cells being 3.0 x 104 / mL. The cells were plated on a 96-well plate at 100 ^L / well and cultured overnight at 37 °C with 5% CO2. Antibody A-ADC and iso-ADC (working concentration: 10 nM) were added at 100 ^L / well, and a drug-free control group (negative control) was set. Three replicates were set for each sample. Then, the cells were cultured at 37 °C with 5% CO2 for 96 h. Subsequently, 20 liL of CCK-8 was added to the mono-culture sample wells, and after 3 h of standing at 37 °C, the OD values at 450 / 650 nm were determined using a microplate reader. The cells in the coculture sample wells were digested with trypsin and counted, and the remaining cells were washed once with DPBS and then assayed using a flow cytometer. The number of cells N was determined using a cell counter, and the proportion of TROP2- and HER3-positive cells (V1L% parental group) and the proportion of TROP2- and HER3-negative cells (V1R% parental group) in each concentration group of the co-culture group were determined using a flow cytometer. For the mono-culture system: The inhibition rate (%) of the test sample at each concentration =  1 - Experimental group OD value Control group OD value * 100% For the co-culture system: The inhibition rate (%) of the test sample at each concentration against BxPC-3 = Number of cells in experimental group N * Number of cells in experimental group V1L% parental group 1--,  ‘     --------4----------------;-----------, „  ---—------------------ * 100% Number of cells in control group N * Control cell group V1L% parental group The inhibition rate (%) of the test sample at each concentration against 293T-GFP = Number of cells in experimental group N * Number of cells in experimental group V1R% parental group 1--* 100% Number of cells in control group N * Control cell group V1R% parental group The results are shown in FIG. 8. In the mono-culture system, the average inhibition rate of 10 nM antibody A-ADC against BxPC-3 cells was 65.31%, and its killing effect on 293T-GFP cells was not significant; in the co-culture system, the average inhibition rate of 10 nM antibody A-ADC against BxPC-3 cells was 43.86%, and its average inhibition rate against 293T-GFP cells was 78.8%. It can be seen that antibody A-ADC exhibited a significant bystander killing effect on TROP2- and HER3-negative cells compared to iso-ADC. 3.8. Evaluation of anti-tumor effect of antibody A-ADC in human lung cancer CDX model HCC827 subcutaneous xenograft mouse animal model BALB / c Nude mice (purchased from Vital River (Beijing) Laboratory Animal Technology Co., Ltd.) were subcutaneously injected with human HCC827 cells to establish an animal model: 0.2 mL (10 x 106 cells) of HCC827 cells (mixed with Matrigel in a 1:1 volume ratio) was subcutaneously inoculated into the right dorsal side of each mouse. When the mean tumor volume reached about 123 mm3, animals were randomly divided into 5 groups of 6 according to body weight and tumor volume: a vehicle control group (PBS), an iso-ADC group (10 mg / kg), an antibody A-ADC low-dose group (1 mg / kg), an antibody A-ADC medium-dose group (3 mg / kg), and an antibody A-ADC high-dose group (10 mg / kg). The day of grouping was defined as day 0. The test articles were intraperitoneally injected on days 1, 5, 8, and 12 (4 injections in total). During the experiment, tumor volume and mouse body weight were measured twice weekly. The results showed that during the experiment, there was no significant difference in animal body weight between the PBS group and the antibody A-ADC treatment groups, and there were no significant abnormalities in the status of the mice. As shown in FIG. 9, 32 days after grouping and administration, the mean tumor volume of the vehicle control group was 1552 mm3, and the tumor volumes of the antibody A-ADC (1 mg / kg, 3 mg / kg, and 10 mg / kg) treatment groups were 1073 mm3, 743 mm3, and 120 mm3, respectively, with TGI% (relative tumor growth inhibition rate, in percentage terms) of 33.4%, 56.5%, and 100.2%, respectively (with p values of 0.0103, 0.0001, and 0.0001, respectively). It can be seen that in the CDX model of human lung cancer HCC827, antibody A-ADC, administered 4 times at doses ranging from 1 to 10 mg / kg, exhibited a significant dose-dependent tumor-inhibiting effect. In addition, the body weight change results showed that antibody A-ADC was well tolerated at each dose. 3.9. Evaluation of anti-tumor effect of antibody A-ADC in human gastric cancer CDX model NCI-N87 subcutaneous xenograft mouse animal model 0.2 mL (10 x 106 cells) of NCI-N87 cells (mixed with Matrigel in a 1:1 volume ratio) was subcutaneously inoculated into the right dorsal side of each BALB / c Nude mouse. When the mean tumor volume reached about 169 mm3, animals were randomly divided into 3 groups of 6 according to body weight and tumor volume: a vehicle control group (PBS), an antibody A-ADC low-dose group (3 mg / kg), and an antibody A-ADC high-dose group (10 mg / kg). Administration was performed once weekly for a total of 2 weeks. The results showed that the single-drug test group of each test article showed no significant influence on the body weight of the mice, and there were no significant abnormalities in the status of the mice. As shown in FIG. 10, 20 days after the start of administration, the mean tumor volume of the tumor-bearing mice in the solvent control group reached 826 mm3. In the treatment groups of the test article antibody A-ADC (10 mg / kg and 3 mg / kg), the tumor volumes were 209 mm3 and 591 mm3 (T / C% = 27.22% and 72.64%; TGI% = 94.07% and 35.65%; p < 0.0001 and p = 0.0807), respectively. The treatment group of the test article antibody A-ADC (10 mg / kg) showed a significant anti-tumor effect compared to the control group. 3.10. Evaluation of anti-tumor effect and tolerability of antibody A-ADC in osimertinib-resistant human lung cancer PDX model LU-01-1377 subcutaneous xenograft NOD / SCID mouse animal model The establishment of human-derived tumor PDX models initially comes from clinical samples obtained through surgical resection, which are defined as the P0 generation after being implanted into experimental mice. After implantation of tumor tissue from the P0 generation into the next generation, the P1 generation is obtained. Similarly, this process of implanting tumor tissue into experimental mice can continue. FP3 tumors are obtained by re-thawing the P2 generation. The next generation resulting from the FP3 generation is defined as FP4, and so on. Tumor tissue from the FP8 generation was used in this pharmacodynamic experiment with the LU-01-1377 model. A 20-30 mm3 LU-01-1377 tumor tissue block was inoculated into the right dorsal side of each NOD / SCID mouse (purchased from Vital River (Beijing) Laboratory Animal Technology Co., Ltd.), and tumors were allowed to grow. The in vivo pharmacodynamic experiment was conducted on day 33 after the inoculation of the LU-01-1377 tumor blocks. When the mean tumor volume reached 141 mm3, random grouping and administration were started. The day of grouping was defined as day 1, and administration was started immediately after grouping. The experiment included antibody A-ADC treatment groups (1 mg / kg, 3 mg / kg, and 10 mg / kg) and a vehicle PBS negative control group, with 6 animals in each group. After grouping, administration was performed by tail vein injection twice weekly. The efficacy and tolerability were evaluated based on tumor growth inhibition (TGI) rates and body weight changes. The body weight change results showed that on day 27 after the first dose, all the animals in the control group and the test drug groups survived without significant body weight loss. The anti-tumor efficacy results are shown in FIG. 11. The mean tumor volume of the mice in the PBS control group on day 27 after the first dose following grouping was 1244 ± 216 mm3. The mean tumor volume of the antibody A-ADC high-dose treatment group (10 mg / kg) on day 27 after the first dose following grouping was 0 ± 0 mm3 (10 mg / kg), with a relative tumor growth inhibition (TGI) rate (%) of 112.78%; there was a statistically significant difference (p < 0.0001) between this treatment group and the PBS control group. The mean tumor volume of the antibody A-ADC medium-dose treatment group (3 mg / kg) on day 27 after the first dose following grouping was 81 ± 43 mm3, with a relative tumor growth inhibition (TGI) rate (%) of 105.43%; there was a statistically significant difference (p < 0.0001) between this treatment group and the PBS control group. The mean tumor volume of the antibody A-ADC low-dose treatment group (1 mg / kg) on day 27 after the first dose following grouping was 1085 ± 171 mm3, with a relative tumor growth inhibition (TGI) rate (%) of 14.35%; there was no statistically significant difference (p = 0.7601) between this treatment group and the PBS control group. 3.11. Evaluation of anti-tumor effect of antibody A-ADC in HCC827 cell strain subcutaneous graft BALB / c nude female mouse model 100 nL (1 x 106 cells) of a suspension of HCC827 cells in a mixture of PBS and Matrigel (1:1) was subcutaneously inoculated into the right dorsal side of each BALB / c nude mouse (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.). After inoculation, the growth of tumors was observed regularly. When the tumors grew to a mean volume of about 93 mm3, animals were randomly grouped according to tumor size and dosed. The day of grouping was defined as day 0. The experiment included an antibody A-ADC treatment group (11.5 mg / kg), sacituzumab govitecan positive control groups (10 mg / kg and 20 mg / kg, purchased from Gilead Sciences, hereinafter referred to as SG), and a PBS blank control group, with 6 mice in each group. Administration was performed by tail vein injection once weekly, and a total of four doses were administered. The experiment was ended on day 49 after the first dose. The efficacy was evaluated based on tumor growth inhibition rates, and the safety was evaluated based on animal body weight changes and mortality. At the end of the experiment, no animal deaths were observed in any group except for the SG 10 mg / kg group, in which one mouse died, and no abnormal weight loss was observed in the mice. The anti-tumor efficacy results are shown in FIG. 12. The mean tumor volume of the mice in the PBS group at the end of the experiment was 580 mm3. The tumor volume of the antibody A-ADC treatment group (11.5 mg / kg) was 43 mm3, with TGI (%) being 92.6% (p < 0.001) compared to the PBS group. The tumor volumes of the positive control SG 10 mg / kg group and 20 mg / kg group were 244 mm3 and 121 mm3, respectively, with TGI (%) being 58.0% (p < 0.05) and 79.2% (p < 0.001) compared to the PBS group. 3.12. Study of stability of antibody A-ADC in serum of different species Antibody A-ADC was diluted with human, cynomolgus monkey, rat, and mouse serum to prepare 10 ng / mL and 100 ng / mL solutions, and then the solutions were aliquoted at 100 nL per tube, with one aliquot prepared for each time point. The aliquots were incubated at 37 °C for 0, 3, 7, 14, and 21 days, respectively, and then taken out and cryopreserved. The DXd content in serum was determined by LC-MS / MS: DXd in the test samples was extracted by protein precipitation, and the treated samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results showed that by day 21, the DXd release rates of antibody A-ADC (100 ug / mL) in human, cynomolgus monkey, rat, and mouse serum were 0.637%, 0.698%, 0.355%, and 0.439%, respectively. The DXd release rates of antibody A-ADC (10 ug / mL) in human, cynomolgus monkey, rat, and mouse serum were 0.696%, 0.702%, 0.368%, and 0.492%, respectively. This indicates that antibody A-ADC could remain stable in the serum of various species, with relatively low payload toxin (DXd) release rates. SEQUENCE LISTING SEQ ID NO: 1 (iBT11) QVQLQESGGGSVQSGGSLRLSCAASGYTTSSVCMAWFRQAPGNEREGVAHITRDGRTMYADSVRGRFTIS QDNAKNTLFLQMNSLKPEDTGMYYCAARVCEWRSTVQAPRSEAYQLWGRGTQVTVSS SEQ ID NO: 2 (huiBT11v1) QVQLVESGGGSVQSGGSLRLSCAASGYTTSSVCMAWFRQAPGNEREGVAHITRDGRTMYADSVRGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAARVCEWRSTVQAPRSEAYQLWGQGTLVTVSS SEQ ID NO: 3 (huiBT11v3) QVQLVESGGGSVQSGGSLRLSCAASGYTTSSVCMAWFRQAPGNGLEGVAHITRDGRTMYADSVRGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAARVCEWRSTVQAPRSEAYQLWGQGTLVTVSS SEQ ID NO: 4 (huiBT11v4) QVQLVESGGGLVQPGGSLRLSCAASGYTTSSVCMAWFRQAPGKGLEGVAHITRDGRTMYADSVRGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAARVCEWRSTVQAPRSEAYQLWGQGTLVTVSS SEQ ID NO: 5 (huiBT11v5) QVQLVESGGGSVQSGGSLRLSCAASGYTTSSVCMAWFRQAPGKGLEGVAHITRDGRTMYADSVRGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAARVCEWRSTVQAPRSEAYQLWGQGTLVTVSS SEQ ID NO: 6 (huiBT11v6) QVQLVESGGGLVQPGGSLRLSCAASGYTTSSVCMAWFRQAPGNGLEGVAHITRDGRTMYADSVRGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAARVCEWRSTVQAPRSEAYQLWGQGTLVTVSS SEQ ID NO: 7 (huiBT11v7) QVQLVESGGGLVQPGGSLRLSCAASGYTTSSVCMAWFRQAPGNEREGVAHITRDGRTMYADSVRGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAARVCEWRSTVQAPRSEAYQLWGQGTLVTVSS SEQ ID NO: 8 (CDR1 Kabat) SVCMA SEQ ID NO: 9 (CDR2 Kabat) HITRDGRTMYADSVRG SEQ ID NO: 10 (CDR3 Kabat) RVCEWRSTVQAPRSEAYQL SEQ ID NO: 11 (CDR1 AbM) GYTTSSVCMA SEQ ID NO: 12 (CDR2 AbM) HITRDGRTM SEQ ID NO: 13 (CDR3 AbM) RVCEWRSTVQAPRSEAYQL SEQ ID NO: 14 (CDR1 Chothia) GYTTSSV SEQ ID NO: 15 (CDR2 Chothia) TRDGR SEQ ID NO: 16 (CDR3 Chothia) RVCEWRSTVQAPRSEAYQL SEQ ID NO: 17 (CDR1 IMGT) GYTTSSVC SEQ ID NO: 18 (CDR2 IMGT) ITRDGRT SEQ ID NO: 19 (CDR3 IMGT) AARVCEWRSTVQAPRSEAYQL SEQ ID NO: 20 (IgG1 Fc C220S) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO: 21 (hRS7 VH) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGR FAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS SEQ ID NO: 22 (hRS7 VH CDR1 Kabat) NYGMN SEQ ID NO: 23 (hRS7 VH CDR2 Kabat) WINTYTGEPTYTDDFKG SEQ ID NO: 24 (hRS7 VH CDR3 Kabat) GGFGSSYWYFDV SEQ ID NO: 25 (hRS7 VH CDR1 AbM) GYTFTNYGMN SEQ ID NO: 26 (hRS7 VH CDR2 AbM) WINTYTGEPT SEQ ID NO: 27 (hRS7 VH CDR3 AbM) GGFGSSYWYFDV SEQ ID NO: 28 (hRS7 VH CDR1 Chothia) GYTFTNY SEQ ID NO: 29 (hRS7 VH CDR2 Chothia) NTYTGE SEQ ID NO: 30 (hRS7 VH CDR3 Chothia) GGFGSSYWYFDV SEQ ID NO: 31 (hRS7 VH CDR1 IMGT) GYTFTNYG SEQ ID NO: 32 (hRS7 VH CDR2 IMGT) INTYTGEP SEQ ID NO: 33 (hRS7 VH CDR3 IMGT) ARGGFGSSYWYFDV SEQ ID NO: 34 (hRS7 VL) DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFT LTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK SEQ ID NO: 35 (hRS7 VL CDR1 Kabat) KASQDVSIAVA SEQ ID NO: 36 (hRS7 VL CDR2 Kabat) SASYRYT SEQ ID NO: 37 (hRS7 VL CDR3 Kabat) QQHYITPLT SEQ ID NO: 38 (hRS7 VL CDR1 AbM) KASQDVSIAVA SEQ ID NO: 39 (hRS7 VL CDR2 AbM) SASYRYT SEQ ID NO: 40 (hRS7 VL CDR3 AbM) QQHYITPLT SEQ ID NO: 41 (hRS7 VL CDR1 Chothia) KASQDVSIAVA SEQ ID NO: 42 (hRS7 VL CDR2 Chothia) SASYRYT SEQ ID NO: 43 (hRS7 VL CDR3 Chothia) QQHYITPLT SEQ ID NO: 44 (hRS7 VL CDR1 IMGT) SAS SEQ ID NO: 45 (hRS7 VL CDR2 IMGT) QDVSIA SEQ ID NO: 46 (hRS7 VL CDR3 IMGT) QQHYITPLT SEQ ID NO: 47 (IgG1 constant region) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 48 (IgK constant region) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 49 (hRS7 HC) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGR FAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 50 (hRS7 LC) DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFT LTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 51 (antibody A HC) QVQLVESGGGLVQPGGSLRLSCAASGYTTSSVCMAWFRQAPGNEREGVAHITRDGRTMYADSVRGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAARVCEWRSTVQAPRSEAYQLWGQGTLVTVSSGAPGGGGGSQV QLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFA FSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 52 (Mutant GalT1) GSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESP LLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQ QLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDK FGFSLPYVQLFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSR DKKNEPN PQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS SEQ ID NO: 53 (human GalT1 Y285L) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAI GQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLI EFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYV INQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYV QLFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNP QRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS SEQ ID NO: 54 (bovine GalT1 Y289L) MKFREPLLGGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLRRLPQLVGVHPPLQGSSHGAAAI GQPSGELRLRGVAPPPPLQNSSKPRSRAPSNLDAYSHPGPGPGPGSNLTSAPVPSTTTRSLTACPEESPLLVGP MLIEFNIPVDLKLVEQQNPKVKLGGRYTPMDCISPHKVAIIIPFRNRQEHLKYWLYYLHPILQRQQLDYGIY VINQAGESMFNRAKLLNVGFKEALKDYDYNCFVFSDVDLIPMNDHNTYRCFSQPRHISVAMDKFGFSLPY VQLFGGVSALSKQQFLSINGFPNNYWGWGGEDDDIYNRLAFRGMSVSRPNAVIGKCRMIRHSRDKKNEP NPQRFDRIAHTKETMLSDGLNSLTYMVLEVQRYPLYTKITVDIGTPS SEQ ID NO: 55 (AV203HC) MGWSLILLFLVAVATRVLSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYAMSWIRQAPGKGLEWVSTIS DGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREWGDYDGFDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 56 (AV203LC) MDFQVQIISFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWYQQKPGKAPKRLIYAA STLDSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC SEQ ID NO: 57 (common upstream primer for sequence encoding single-domain antibody VHH) cccACCGGTCAGGTGCAGCTGCAGGAGTC SEQ ID NO: 58 (common downstream primer for sequence encoding single-domain antibody VHH) cccGGATCCTGAGGAGACGGTGACCTGG

Claims

1. A human epidermal growth factor receptor 3 (HER3)-binding protein, comprising at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises a CDR1, a CDR2, and a CDR3 from the VHH set forth in SEQ ID NO: 1.

2. The HER3-binding protein according to claim 1, wherein the CDR1, the CDR2, and the CDR3 are defined according to the following definition system: Kabat, AbM, Chothia, or IMGT.

3. The HER3-binding protein according to claim 1 or 2, wherein the immunoglobulin single variable domain is of the family Camelidae, humanized, or chimeric.

4. The HER3-binding protein according to any one of claims 1-3, wherein the CDR1, the CDR2, and the CDR3 from the VHH set forth in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NOs: 8-10, SEQ ID NOs: 11-13, SEQ ID NOs: 14-16, and SEQ ID NOs: 17-19.

5. The HER3-binding protein according to any one of claims 1-4, wherein the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 1-7.

6. The HER3-binding protein according to claim 5, wherein the at least one immunoglobulin single variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 1-7.

7. The HER3-binding protein according to any one of claims 1-6, wherein the HER3-binding protein comprises one said immunoglobulin single variable domain.

8. The HER3-binding protein according to any one of claims 1-6, wherein the HER3-binding protein comprises 2, 3, 4, 5, or more said immunoglobulin single variable domains; optionally, the 2, 3, 4, 5, or more immunoglobulin single variable domains have or do not have identical sequences.

9. The HER3-binding protein according to any one of claims 1-8, further comprising an immunoglobulin Fc region, preferably a human immunoglobulin Fc region, and more preferably a human IgG1, IgG2, IgG3, or IgG4 Fc region.

10. The HER3-binding protein according to claim 9, wherein the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO: 20.

11. The HER3-binding protein according to claim 9 or 10, wherein the immunoglobulin Fc region is linked directly or linked indirectly by a linker to the at least one immunoglobulin single variable domain.

12. A multispecific antigen-binding protein, comprising the HER3-binding protein according to claims 1-11 or an antigen-binding fragment thereof, which provides a first binding specificity.

13. The multispecific antigen-binding protein according to claim 12, further comprising a second binding specificity provided by an antibody or an antigen-binding fragment thereof that binds to trophoblast cell surface antigen 2 (TROP2), and optionally more binding specificities.

14. A multispecific antigen-binding protein, comprising an antibody or an antigen-binding fragment thereof that binds to trophoblast cell surface antigen 2 (TROP2), and at least one immunoglobulin single variable domain that binds to human epidermal growth factor receptor 3 (HER3), wherein:preferably,the antibody or the antigen-binding fragment thereof that binds to TROP2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a VH CDR1, a VH CDR2, and a VH CDR3 selected from any one of the following groups: SEQ ID NOs: 22-24, SEQ ID NOs: 25-27, SEQ ID NOs: 6128-30, and SEQ ID NOs: 31-33, andthe light chain variable region comprises a VL CDR1, a VL CDR2, and a VL CDR3 selected from any one of the following groups: SEQ ID NOs: 35-37, SEQ ID NOs: 38-40, SEQ ID NOs: 41-43, and SEQ ID NOs: 44-46;and / orpreferably, the immunoglobulin single variable domain that binds to HER3 comprises a VHH CDR1, a VHH CDR2, and a VHH CDR3 selected from any one of the following groups: SEQ ID NOs: 8-10, SEQ ID NOs: 11-13, SEQ ID NOs: 14-16, and SEQ ID NOs: 17-19.

15. The multispecific antigen-binding protein according to claim 14, wherein the heavy chain variable region of the antibody or the antigen-binding fragment thereof that binds to TROP2 comprises the amino acid sequence set forth in SEQ ID NO: 21.

16. The multispecific antigen-binding protein according to claim 14, wherein the light chain variable region of the antibody or the antigen-binding fragment thereof that binds to TROP2 comprises the amino acid sequence set forth in SEQ ID NO: 34.

17. The multispecific antigen-binding protein according to any one of claims 14-16, wherein the antibody or the antigen-binding fragment thereof that binds to TROP2 comprises a heavy chain that further comprises a human IgG1 constant region or a variant thereof; for example, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 47.

18. The multispecific antigen-binding protein according to any one of claims 14-17, wherein the antibody or the antigen-binding fragment thereof that binds to TROP2 comprises a light chain that further comprises a human IgK constant region or a variant thereof; for example, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 48.

19. The multispecific antigen-binding protein according to any one of claims 14-18, wherein the antibody that binds to TROP2 comprises a heavy chain that comprises the amino acid sequence set forth in SEQ ID NO: 49.

20. The multispecific antigen-binding protein according to any one of claims 14-19, wherein the antibody that binds to TROP2 comprises a light chain that comprises the amino acid sequence set forth in SEQ ID NO: 50.

21. The multispecific antigen-binding protein according to any one of claims 14-20, wherein the at least one immunoglobulin single variable domain that binds to HER3 comprises the amino acid sequence set forth in one of SEQ ID NOs: 1-7.

22. The multispecific antigen-binding protein according to claim 21, comprising one immunoglobulin single variable domain.

23. The multispecific antigen-binding protein according to any one of claims 14-22, wherein the at least one immunoglobulin single variable domain that binds to HER3 is linked directly or linked indirectly by a linker to the antibody or the antigen-binding fragment that binds to TROP2.

24. The multispecific antigen-binding protein according to any one of claims 14-23, wherein the C-terminus of the at least one immunoglobulin single variable domain that binds to HER3 is linked to the N-terminus of a heavy chain of the antibody or the antigen-binding fragment that binds to TROP2.

25. The multispecific antigen-binding protein according to any one of claims 14-23, wherein the N-terminus of the at least one immunoglobulin single variable domain that binds to HER3 is linked to the C-terminus of a heavy chain of the antibody or the antigen-binding fragment that binds to TROP2.

26. The multispecific antigen-binding protein according to any one of claims 14-23, wherein the C-terminus of the at least one immunoglobulin single variable domain that binds to HER3 is linked to the N-terminus of a light chain of the antibody or the antigen-binding fragment that binds to TROP2.

27. The multispecific antigen-binding protein according to any one of claims 14-23, wherein the N-terminus of the at least one immunoglobulin single variable domain that binds to HER3 is linked to the C-terminus of a light chain of the antibody or the antigen-binding fragment that binds to TROP2.

28. The multispecific antigen-binding protein according to any one of claims 14-27, comprising a first polypeptide set forth in SEQ ID NO: 51 and a second polypeptide set forth in SEQ ID NO: 50.

29. A protein-drug conjugate, having the structure of formula I:P-(L1-sp1-L2-sp2-D)n      (I),wherein protein P comprises the multispecific antigen-binding protein according to any one of claims 12-28, D is a substance having biological activity, L1 is a linker for linking to P, sp1 is a first spacing unit, L2 is a cleavable linker, sp2 is a second spacing unit and is linked to D, and n = 1-20.

30. The protein-drug conjugate according to claim 29, wherein L1 is selected from:, andwherein Ar represents C6-10 arylene optionally substituted with halogen or C1-6 alkyl; R1 is selected from hydrogen, halogen, and C1-6 alkyl; Z is selected from a direct bond, C2-6 alkynylene, C2-6 alkenylene, C6-10 arylene, 5-10 membered heteroarylene, amido, sulfonamido, imino, and CF2.

31. The protein-drug conjugate according to claim 29 or 30, wherein sp1 has the structure represented by formula II:(II),wherein a1 = 0 or 1, a2 = an integer from 0 to 6, b1 = 0 or 1, b2 = an integer from 0 to 16, b3 = an integer from 0 to 16, and c = an integer from 0 to 6, and at least one of b2 and b3 is 0.

32. The protein-drug conjugate according to claim 31, wherein:(1) a1 = 0, a2 = 2, 3, 4, 5, or 6, b1 = 0, b2 = 0, b3 = 0, and c = 0;(2) a1 = 0, a2 = 0, b1 = 0, b2 = 0, b3 = 0, and c = 2, 3, 4, 5, or 6;(3) a1 = 1, a2 = 2, 3, 4, 5, or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7, or 8, b3 = 0, and c = 0;(4) a1 = 0, a2 = 2, 3, 4, 5, or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7, or 8, b3 = 0, and c = 0; or(5) a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 2, 3, 4, 5, 6, 7, or 8, and c = 2, 3, 4, 5, or 6.

33. The protein-drug conjugate according to any one of claims 29-32, wherein L2 is a dipeptide, tripeptide, or tetrapeptide amino acid residue.

34. The protein-drug conjugate according to claim 33, wherein L2 is selected from the following dipeptide amino acid residues: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Val-Cit-, -Ala-Lys-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, -Trp-Cit-, -Gly-Gly-, -Ala-Ala-, -Gly-Val-, and -Gly-Glu-; the left side of the dipeptide amino acid residue is linked to sp1, and the right side thereof is linked to sp2.

35. The protein-drug conjugate according to claim 33, wherein L2 is selected from the following tripeptide amino acid residues: -Glu-Val-Ala-, -Glu-Val-Cit-, -aGlu-Val-Ala-, -aGlu-Val-Cit-, -Val-Lys-Gly, and -Val-Cit-Gly-; the left side of the tripeptide amino acid residue is linked to sp1, and the right side thereof is linked to sp2.

36. The protein-drug conjugate according to claim 33, wherein L2 is selected from the following tetrapeptide amino acid residues: -Gly-Gly-Phe-Gly- and -Gly-Phe-Gly-Gly-; the left side of the tetrapeptide amino acid residue is linked to sp1, and the right side thereof is linked to sp2.

37. The protein-drug conjugate according to any one of claims 29-36, wherein sp2 is absent, or sp2 is selected from:R2 is independently selected from hydrogen, C1-6 alkyl, hydroxy, amino, halogen, nitro, cyano,0                             0   ।        0n'LI / '|o^             --NfJ1 d 1 0 1          . .                                 . ., and                     , d is an integer from 1 to 20, and e is an integer from 1 to 20; R3 andR4 are each independently selected from hydrogen and C1-6 alkyl;the alkyl may be optionally substituted with hydroxy, amino, halogen, nitro, and cyano.

38. The protein-drug conjugate according to any one of claims 29-37, wherein -L1-sp1-L2-sp2- is selected from the following structures:0h2n o, andk is an integer from 1 to 20, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10.

39. The protein-drug conjugate according to any one of claims 29-38, wherein D is selected from a cytotoxin, a protein kinase inhibitor, an immune agonist, a glucocorticoid, an oligonucleotide, a radioisotope, a polypeptide, and any combination thereof.

40. The protein-drug conjugate according to claim 39, wherein D is a cytotoxin selected from a DNA alkylating agent, a DNA deconstructing agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a microtubule inhibitor, a ribosome inhibitor, and any combination thereof.

41. The protein-drug conjugate according to claim 40, wherein D is selected from an auristatin derivative, a maitansine derivative, an eribulin derivative, a tubulysin derivative, a pyrrolobenzodiazepine (PDB) derivative, a duocarmycin derivative, a calicheamicin derivative, PNU-159682 and a derivative thereof, a camptothecin derivative, an amatoxin derivative, and any combination thereof.

42. The protein-drug conjugate according to claim 40, wherein D has the structure represented by formula III:(III),wherein X is selected from CH2, NH, O, S, and SO2;Y is absent, or Y has the structure represented by(IV-a) or(IV-b);W1 and W3 are each independently selected from O, S, and NH, and W2 is selected from CH and N;Ra and Rb are each independently selected from hydrogen, deuterium, halogen, C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy, amino, cyano, and nitro; or, Ra and Rb, together with the carbon atom to which they are attached, form 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, or carbonyl; or, Ra is linked to the N atom of the amide moiety to form 3-6 membered heterocycloalkyl, and Rb is hydrogen;ring A is selected from the following groups: 5-10 membered cycloalkylene, 5-10 membered heterocycloalkylene, 6-10 membered arylene, and 5-10 membered heteroarylene;the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, cycloalkylene, heterocycloalkylene, arylene, and heteroarylene are each independently, optionally, and further substituted with a group selected from deuterium, halogen, hydroxy, amino, cyano, carbonyl, and nitro;d and e are each independently selected from integers from 0 to 5.

43. The protein-drug conjugate according to claim 42, wherein D has the structure represented by formula III-a:(III-a),wherein W1 is O or NH;Ra and Rb are each independently selected from hydrogen, deuterium, halogen, C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy, amino, cyano, and nitro; or, Ra and Rb, together with the carbon atom to which they are attached, form 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, or carbonyl; or, Ra is linked to the N atom of the amide moiety to form 3-6 membered heterocycloalkyl, and Rb is hydrogen;the alkyl, alkoxy, cycloalkyl, and heterocycloalkyl are each independently, optionally, and further substituted with a group selected from deuterium, halogen, hydroxy, amino, cyano, and nitro;d is an integer from 0 to 5.

44. The protein-drug conjugate according to claim 43, wherein W1 is O.

45. The protein-drug conjugate according to claim 43 or 44, wherein d is 0, 1, or 2.

46. The protein-drug conjugate according to any one of claims 43-45, wherein Ra is selected from hydrogen, deuterium, halogen, C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy, and amino, and Rb is hydrogen.

47. The protein-drug conjugate according to any one of claims 43-45, wherein Ra and Rb, together with the carbon atom to which they are attached, form 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl.

48. The protein-drug conjugate according to claim 43, wherein D is selected from the following groups:

49. The protein-drug conjugate according to claim 42, wherein D has the structure represented by formula III-b:(III-b),wherein W3 is O or NH, and W2 is CH and N;ring A is selected from the following groups: 5-10 membered cycloalkylene, 5-10 membered heterocycloalkylene, 6-10 membered arylene, and 5-10 membered heteroarylene;the cycloalkylene, heterocycloalkylene, arylene, and heteroarylene are each independently, optionally, and further substituted with a group selected from deuterium, halogen, hydroxy, amino, cyano, carbonyl, and nitro;e is an integer from 0 to 5.

50. The protein-drug conjugate according to claim 49, wherein W3 is O.

51. The protein-drug conjugate according to claim 49 or 50, wherein W2 is CH.

52. The protein-drug conjugate according to any one of claims 49-51, wherein e is 0, 1, or 2.

53. The protein-drug conjugate according to any one of claims 49-52, wherein ring A is 5-10 membered cycloalkylene.

54. The protein-drug conjugate according to claim 49, wherein D is selected from the following groups:, and55. The protein-drug conjugate according to claim 41, wherein D is a camptothecin derivative and is preferably selected from the following structures:and56. The protein-drug conjugate according to claim 29, wherein -L1-sp1-L2-sp2-D is selected from the following structures:, wherein k is an integer from 1 to 20.

57. The protein-drug conjugate according to any one of claims 29-56, wherein L1 is linked to the multispecific antigen-binding protein P by sulfhydryl, and the sulfhydryl is obtained by reducing a disulfide bond between heavy chains and / or a disulfide bond between a heavy chain and a light chain.

58. The protein-drug conjugate according to any one of claims 29-56, wherein L1 is linked to the multispecific antigen-binding protein P by an oligosaccharide.

59. The protein-drug conjugate according to claim 58, wherein the oligosaccharide has the structure represented by formula V-a or formula V-b:Man—GlcNAc—Gal*--|dcNAc— GlcNAc—ManMan— GlcNAc—Gal*—|(V-a) or(V-b),wherein P* binds to HER3 and TROP2 and comprises the antigen-binding protein according to any one of claims 12-28, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20;Gal* is a modified galactose selected from the following structures:andthe oligosaccharide is linked to P* by the core GlcNAc.

60. The protein-drug conjugate according to claim 59, wherein the modified galactose is linked to GlcNAc by a 0-1,4-glycosidic bond.

61. The protein-drug conjugate according to any one of claims 58-60, wherein the oligosaccharide is linked to the Fc fragment of P*, preferably to the CH2 domain of the Fc fragment, and more preferably to Asn297 (numbered according to the EU index of Kabat) of the Fc fragment.

62. The protein-drug conjugate according to any one of claims 29-61, having the structure represented by formulaVI:(VI),wherein P* binds to HER3 and TROP2; for example, P* comprises the antigen-binding protein according to any one of claims 12-28; GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20;Gal* is a modified galactose selected from the following structures:andthe oligosaccharide is linked to P* by the core GlcNAc;LP is selected from the following structures:(a)and / or(b)and / or(c)(d)and / orandand / or;k is an integer from 1 to 20, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10;the core GlcNAc is directly linked to P*.

63. The protein-drug conjugate according to any one of claims 59-62, wherein the antigen-binding protein comprises a first polypeptide set forth in SEQ ID NO: 51 and a second polypeptide set forth in SEQ ID NO: 50.

64. The protein-drug conjugate according to claim 62 or 63, wherein the oligosaccharide is linked to the Fc fragment of P*, preferably to the CH2 domain of the Fc fragment, and more preferably to Asn297 (numbered according to the EU index of Kabat) of the Fc fragment.

65. A protein derivative, having the structure represented by formula VII:Man— GlcNAc—Gal**(VII)wherein P* binds to HER3 and TROP2 and comprises the antigen-binding protein according to any one of claims 12-28, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20;Gal** is a modified galactose selected from the following structures:andOH,N3 lL-o0= / the oligosaccharide is linked to P* by the core GlcNAc.

66. The protein derivative according to claim 65, wherein the core GlcNAc is linked to the Fc fragment of P*, preferably to the CH2 domain of the Fc fragment, and more preferably to Asn297 (numbered according to the EU index of Kabat) of the Fc fragment.

67. An isolated polynucleotide, encoding the HER3-binding protein according to any one of claims 1-11 or an antigen-binding fragment thereof or the multispecific antigen-binding protein according to any one of claims 12-28.

68. A vector, comprising the polynucleotide according to claim 67, wherein preferably, the vector is an expression vector.

69. A host cell, comprising the polynucleotide according to claim 67 or the vector according to claim 68, wherein preferably, the host cell is prokaryotic or eukaryotic; more preferably, the host cell is selected from yeast cells, mammalian cells (for example, the host cell is a CHO cell, such as a CHO-K1 cell or an expiCHO cell, or the host cell is a 293 cell, such as an HEK293 cell), and other cells suitable for preparing an antibody or an antigen-binding fragment thereof.

70. A method for preparing a multispecific antigen-binding protein that binds to an HER3-binding protein or an antigen-binding fragment thereof or comprises the HER3-binding protein or the antigen-binding fragment thereof, comprising culturing a host cell comprising a nucleic acid encoding the HER3-binding protein according to any one of claims 1-11 or an antigen-binding fragment thereof or the multispecific antigen-binding protein according to any one of claims 12-28 under conditions suitable for the expression of the protein, wherein optionally, the method further comprises recovering the binding protein or the multispecific antigen-binding protein or an antigen-binding fragment thereof or a fusion protein thereof from the host cell.

71. Use of the HER3-binding protein according to any one of claims 1-11, the multispecific antigen-binding protein according to any one of claims 12-28, or the protein-drug conjugate according to any one of claims 29-64 in the preparation of a medicament for treating and / or preventing a tumor.

72. The use according to claim 71, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

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