Trop2 antibody or antigen-binding fragment thereof, preparation method therefor, and use thereof

Abstract

A TROP2 antibody or an antigen-binding fragment thereof, a preparation method therefor, and a use thereof. The TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises HCDR1 having a sequence as shown in any one of SEQ ID NOs:1-10, HCDR2 having a sequence as shown in any one of SEQ ID NOs:11-25, and HCDR3 having a sequence as shown in any one of SEQ ID NOs:26-39. The described antibody exhibits good binding to antigens at both protein and cellular levels: said antibody has superior binding capabilities with CHO-K1-hTROP2 and CHO-K1-cynoTROP2 compared to existing antibodies, and said antibody is also capable of binding to HCC1954 and BxPC-3. In addition, the KD value and specific binding of the antibody can reach a level comparable to that in the existing technology.

Description

A TROP2 antibody or antigen-binding fragment thereof and its preparation method and application

[0001] This application claims priority to Chinese patent application No. 2024102631524, filed on March 7, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to a TROP2 antibody or an antigen-binding fragment thereof, and a preparation method and application thereof. Background Art

[0003] TROP2 belongs to the TACSTD family and is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium signal transducer 2 (TACSTD2). It is a transmembrane protein whose extracellular domain is overexpressed in a variety of tumors, making it a natural candidate for targeted therapy development. The limited tissue expression of TROP2 reduces the toxicity of treatment, which is also an advantage of TROP2-targeted therapy.

[0004] TROP2 is a gene closely associated with tumors. It primarily promotes tumor cell growth, proliferation, and metastasis by regulating calcium signaling pathways, cell cycle protein expression, and reducing fibronectin adhesion. TROP2 can also interact with β-catenin in the Wnt signaling cascade, thereby contributing to nuclear oncogene transcription and cell proliferation. TROP2 signaling involves several pathways: Under the action of protein kinase C (PKC), the intracellular tail serine residue (S303) of the TROP2 protein is phosphorylated, promoting the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 interacts with IP3 receptors on the endoplasmic reticulum surface and promotes the release of stored calcium ions in the endoplasmic reticulum, activating the mitogen-activated protein kinase (MAPK) pathway and promoting cell cycle progression. In addition, increased intracellular calcium ions can also activate cyclin D1 and ERK / MEK pathways to enhance the expression of CREB1, c-Jun, NF-κB, Rb, STAT1 and STAT3, thereby inhibiting tumor cell apoptosis.

[0005] Numerous clinical studies and literature reports have demonstrated that TROP2 is overexpressed in a variety of epithelial cancers, including gastric, lung, colorectal, prostate, ovarian, breast, pancreatic, esophageal, and liver cancers. In contrast, TROP2 is rarely or not expressed in normal adult tissues, with expression limited to epithelial cells at lower levels than in cancer, suggesting a role for TROP2 in tumorigenesis. Overexpression of TROP2 in tumor tissue is closely associated with poor patient prognosis and metastasis, while also impacting overall survival. Therefore, TROP2 has become an attractive target for molecularly targeted cancer therapy.

[0006] Heavy-chain-only antibodies retain excellent antigen recognition capabilities, yet possess a small molecular weight, approximately half that of conventional antibodies. These antibodies can be independently developed as drugs and modified for diverse applications. For example, they can be converted into single-domain antibodies, coexist with standard bispecific antibodies, or even flexibly designed into symmetrical or asymmetrical configurations. While there are reports of TROP2 nanobodies or humanized versions obtained by immunizing camels, there are no reports of fully human TROP2 HCAbs. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a TROP2 antibody or antigen-binding fragment thereof, and its preparation method and application. The antibody of the present invention has good binding to antigens at both protein and cell levels: it has better binding ability to CHO-K1-hTROP2 and CHO-K1-cynoTROP2 than existing antibodies, and can also bind to HCC1954 and BxPC-3. In addition, the antibody K D The value and specific binding can reach a level comparable to the existing technology.

[0008] On the one hand, the present invention provides a TROP2 antibody or an antigen-binding fragment thereof, wherein the TROP2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are respectively the same as the HCDR1, HCDR2 and HCDR3 of the VH having the amino acid sequence shown in SEQ ID NO:40-56.

[0009] As for the TROP2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the heavy chain variable region comprises a HCDR1 with a sequence as shown in any one of SEQ ID NOs: 1-10, a HCDR2 with a sequence as shown in any one of SEQ ID NOs: 11-25, and a HCDR3 with a sequence as shown in any one of SEQ ID NOs: 26-39.

[0010] The heavy chain variable region of the present invention preferably comprises:

[0011] HCDR1 with the sequence shown in SEQ ID NO: 1, HCDR2 with the sequence shown in SEQ ID NO: 11, and HCDR3 with the sequence shown in SEQ ID NO: 26;

[0012] HCDR1 with the sequence shown in SEQ ID NO: 2, HCDR2 with the sequence shown in SEQ ID NO: 12, and HCDR3 with the sequence shown in SEQ ID NO: 27;

[0013] HCDR1 with the sequence shown in SEQ ID NO: 3, HCDR2 with the sequence shown in SEQ ID NO: 13, and HCDR3 with the sequence shown in SEQ ID NO: 28;

[0014] HCDR1 with the sequence shown in SEQ ID NO: 4, HCDR2 with the sequence shown in SEQ ID NO: 14, and HCDR3 with the sequence shown in SEQ ID NO: 28;

[0015] HCDR1 with the sequence shown in SEQ ID NO: 1, HCDR2 with the sequence shown in SEQ ID NO: 17, and HCDR3 with the sequence shown in SEQ ID NO: 31;

[0016] HCDR1 with the sequence shown in SEQ ID NO: 5, HCDR2 with the sequence shown in SEQ ID NO: 16, and HCDR3 with the sequence shown in SEQ ID NO: 30;

[0017] HCDR1 with the sequence shown in SEQ ID NO: 1, HCDR2 with the sequence shown in SEQ ID NO: 15, and HCDR3 with the sequence shown in SEQ ID NO: 29;

[0018] HCDR1 with the sequence shown in SEQ ID NO: 6, HCDR2 with the sequence shown in SEQ ID NO: 18, and HCDR3 with the sequence shown in SEQ ID NO: 32;

[0019] HCDR1 with the sequence shown in SEQ ID NO:7, HCDR2 with the sequence shown in SEQ ID NO:19, and HCDR3 with the sequence shown in SEQ ID NO:33;

[0020] HCDR1 with the sequence shown in SEQ ID NO: 8, HCDR2 with the sequence shown in SEQ ID NO: 20, and HCDR3 with the sequence shown in SEQ ID NO: 34;

[0021] HCDR1 with the sequence shown in SEQ ID NO:9, HCDR2 with the sequence shown in SEQ ID NO:21, and HCDR3 with the sequence shown in SEQ ID NO:35;

[0022] HCDR1 with the sequence shown in SEQ ID NO: 10, HCDR2 with the sequence shown in SEQ ID NO: 22, and HCDR3 with the sequence shown in SEQ ID NO: 36;

[0023] HCDR1 with the sequence shown in SEQ ID NO: 8, HCDR2 with the sequence shown in SEQ ID NO: 23, and HCDR3 with the sequence shown in SEQ ID NO: 37;

[0024] HCDR1 with the sequence shown in SEQ ID NO: 8, HCDR2 with the sequence shown in SEQ ID NO: 24, and HCDR3 with the sequence shown in SEQ ID NO: 38;

[0025] Or, the HCDR1 sequence shown in SEQ ID NO: 1, the HCDR2 sequence shown in SEQ ID NO: 25, and the HCDR3 sequence shown in SEQ ID NO: 39.

[0026] As for the TROP2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the amino acid sequence of the heavy chain variable region is preferably as shown in any one of SEQ ID NOs: 40-56.

[0027] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:58, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:75, and HFWR4 as shown in SEQ ID NO:86.

[0028] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:59, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:75, and HFWR4 as shown in SEQ ID NO:86.

[0029] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:60, HFWR2 as shown in SEQ ID NO:70, HFWR3 as shown in SEQ ID NO:76, and HFWR4 as shown in SEQ ID NO:86.

[0030] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:61, HFWR2 as shown in SEQ ID NO:71, HFWR3 as shown in SEQ ID NO:77, and HFWR4 as shown in SEQ ID NO:87.

[0031] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:59, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:75, and HFWR4 as shown in SEQ ID NO:86.

[0032] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:62, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:78, and HFWR4 as shown in SEQ ID NO:86.

[0033] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:59, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:79, and HFWR4 as shown in SEQ ID NO:88.

[0034] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:59, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:75, and HFWR4 as shown in SEQ ID NO:89.

[0035] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:63, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:80, and HFWR4 as shown in SEQ ID NO:90.

[0036] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:64, HFWR2 as shown in SEQ ID NO:72, HFWR3 as shown in SEQ ID NO:81, and HFWR4 as shown in SEQ ID NO:86.

[0037] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:65, HFWR2 as shown in SEQ ID NO:73, HFWR3 as shown in SEQ ID NO:82, and HFWR4 as shown in SEQ ID NO:86.

[0038] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:66, HFWR2 as shown in SEQ ID NO:73, HFWR3 as shown in SEQ ID NO:83, and HFWR4 as shown in SEQ ID NO:87.

[0039] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:64, HFWR2 as shown in SEQ ID NO:72, HFWR3 as shown in SEQ ID NO:84, and HFWR4 as shown in SEQ ID NO:86.

[0040] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:64, HFWR2 as shown in SEQ ID NO:72, HFWR3 as shown in SEQ ID NO:85, and HFWR4 as shown in SEQ ID NO:91.

[0041] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:68, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:75, and HFWR4 as shown in SEQ ID NO:86.

[0042] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:59, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:79, and HFWR4 as shown in SEQ ID NO:86.

[0043] In a preferred embodiment, the heavy chain variable region comprises HFWR1 as shown in SEQ ID NO:59, HFWR2 as shown in SEQ ID NO:69, HFWR3 as shown in SEQ ID NO:75, and HFWR4 as shown in SEQ ID NO:86.

[0044] The antigen-binding fragment preferably includes Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv, VHH, heavy chain antibody (HCAb) and / or dAb.

[0045] In a preferred embodiment, the heavy chain antibody comprises a constant region comprising the amino acid sequence shown in SEQ ID NO:57.

[0046] Another aspect of the present invention provides an isolated nucleic acid encoding the TROP2 antibody or antigen-binding fragment thereof according to the present invention.

[0047] Another aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid according to one aspect of the present invention.

[0048] The backbone of the recombinant expression vector is preferably a plasmid, cosmid, phage or viral vector.

[0049] The viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0050] Another aspect of the present invention provides a transformant comprising the recombinant expression vector according to one aspect of the present invention.

[0051] The host cell of the transformant is preferably a prokaryotic cell or a eukaryotic cell.

[0052] The eukaryotic cell is preferably a yeast cell or a mammalian cell.

[0053] The mammalian cells are, for example, 293 cells or CHO cells. Another aspect of the present invention provides a method for preparing a TROP2 antibody or an antigen-binding fragment thereof, comprising culturing the transformant according to one aspect of the present invention and obtaining the TROP2 antibody or the antigen-binding fragment thereof from the culture.

[0054] Another aspect of the present invention provides a multispecific antibody, which comprises the TROP2 antibody or antigen-binding fragment thereof as described in one aspect of the present invention, and other anti-tumor antibodies or fragments or analogs thereof.

[0055] The multispecific antibody is preferably a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0056] Another aspect of the present invention provides an antigen-binding protein derivative, which comprises the TROP2 antibody or antigen-binding fragment thereof as described in one aspect of the present invention, and a detectable labeling molecule.

[0057] The detectable labeling molecule is preferably an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

[0058] Another aspect of the present invention provides an antibody-drug conjugate, which comprises a cytotoxic agent or a label, and the TROP2 antibody or antigen-binding fragment thereof according to one aspect of the present invention.

[0059] The cytotoxic agent is preferably MMAF, MMAE or Dxd, and the label is a fluorescent agent.

[0060] Another aspect of the present invention provides a chimeric antigen receptor, which comprises the TROP2 antibody or antigen-binding fragment thereof according to the present invention.

[0061] Another aspect of the present invention provides a genetically modified cell, wherein the genetically modified cell comprises the TROP2 antibody or antigen-binding fragment thereof according to one aspect of the present invention.

[0062] The genetically modified cells are preferably eukaryotic cells, more preferably isolated human cells; further preferably immune cells such as T cells or NK cells.

[0063] Another aspect of the present invention provides a pharmaceutical composition, which comprises the TROP2 antibody or its antigen-binding fragment as described in one aspect of the present invention, the multispecific antibody as described in one aspect of the present invention, the antigen-binding protein derivative as described in one aspect of the present invention, the antibody-drug conjugate as described in one aspect of the present invention, the chimeric antigen receptor as described in one aspect of the present invention, or the genetically modified cell as described in one aspect of the present invention, and a pharmaceutically acceptable carrier.

[0064] The pharmaceutical composition preferably further contains one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules and vaccines.

[0065] Another aspect of the present invention provides a kit comprising the TROP2 antibody or antigen-binding fragment thereof as described in one aspect of the present invention, the multispecific antibody as described in one aspect of the present invention, the antigen-binding protein derivative as described in one aspect of the present invention, the antibody-drug conjugate as described in one aspect of the present invention, the chimeric antigen receptor as described in one aspect of the present invention, the genetically modified cell as described in one aspect of the present invention, or the pharmaceutical composition as described in one aspect of the present invention.

[0066] The kit preferably further comprises (i) a device for administering the antibody or antigen-binding fragment thereof, antibody drug conjugate, chimeric antigen receptor, genetically modified cell, or pharmaceutical composition; and / or (ii) instructions for use.

[0067] Another aspect of the present invention provides a medicine kit, comprising medicine kit A and medicine kit B:

[0068] The drug kit A contains the TROP2 antibody or antigen-binding fragment thereof as described in one aspect of the present invention, the multispecific antibody as described in one aspect of the present invention, the antigen-binding protein derivative as described in one aspect of the present invention, the antibody-drug conjugate as described in one aspect of the present invention, the chimeric antigen receptor as described in one aspect of the present invention, the genetically modified cell as described in one aspect of the present invention, or the pharmaceutical composition as described in one aspect of the present invention;

[0069] The drug kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

[0070] Another aspect of the present invention provides the use of the TROP2 antibody or antigen-binding fragment thereof as described in one aspect of the present invention, the multispecific antibody as described in one aspect of the present invention, the antigen-binding protein derivative as described in one aspect of the present invention, the antibody-drug conjugate as described in one aspect of the present invention, the chimeric antigen receptor as described in one aspect of the present invention, the genetically modified cell as described in one aspect of the present invention, the pharmaceutical composition as described in one aspect of the present invention, the kit as described in one aspect of the present invention, or the set of kits as described in one aspect of the present invention in the preparation of products for diagnosing, treating and / or preventing tumors.

[0071] The tumor is preferably a TROP2-positive tumor; more preferably a blood cancer or a solid cancer.

[0072] The blood cancer is, for example, a B-cell leukemia, lymphoma, acute myeloid leukemia, Burkitt's lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, or marginal zone lymphoma.

[0073] The solid cancers include breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, or adrenal cancer.

[0074] Another aspect of the present invention provides a method for diagnosing, treating and / or preventing a TROP2-mediated disease or condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of a TROP2 antibody or antigen-binding fragment thereof as described in one aspect of the present invention, a multispecific antibody as described in one aspect of the present invention, an antigen-binding protein derivative as described in one aspect of the present invention, an antibody-drug conjugate as described in one aspect of the present invention, a chimeric antigen receptor as described in one aspect of the present invention, a genetically modified cell as described in one aspect of the present invention, or a pharmaceutical composition as described in one aspect of the present invention to treat the patient in need.

[0075] Another aspect of the present invention provides the method according to one aspect of the present invention, wherein the disease or condition is a tumor, preferably a blood cancer or a solid cancer.

[0076] Said hematological cancer is preferably as defined in the above application of the present invention.

[0077] The solid cancer is preferably as defined in the above application of the present invention.

[0078] On the other hand, the present invention provides a method for immunodetection or determination of TROP2, which comprises using the TROP2 antibody or antigen-binding fragment thereof as described in the present invention, the multispecific antibody as described in the present invention, the antigen-binding protein derivative as described in the present invention, the antibody-drug conjugate as described in the present invention, the chimeric antigen receptor as described in the present invention, the genetically modified cell as described in the present invention, the pharmaceutical composition as described in the present invention, or the kit as described in the present invention.

[0079] Said detection is preferably for non-diagnostic, preventive and / or therapeutic purposes.

[0080] Another aspect of the present invention provides a combination therapy, which comprises administering to a patient in need thereof a TROP2 antibody or an antigen-binding fragment thereof as described in one aspect of the present invention, a multispecific antibody as described in one aspect of the present invention, an antigen-binding protein derivative as described in one aspect of the present invention, an antibody-drug conjugate as described in one aspect of the present invention, a chimeric antigen receptor as described in one aspect of the present invention, a genetically modified cell as described in one aspect of the present invention, or a pharmaceutical composition as described in one aspect of the present invention; and a second therapeutic agent, respectively.

[0081] The second therapeutic agent preferably comprises other anti-tumor antibodies or pharmaceutical compositions comprising such other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0082] Another aspect of the present invention provides a drug delivery device, which includes the TROP2 antibody or its antigen-binding fragment as described in one aspect of the present invention, the multispecific antibody as described in one aspect of the present invention, the antigen-binding protein derivative as described in one aspect of the present invention, the antibody-drug conjugate as described in one aspect of the present invention, the chimeric antigen receptor as described in one aspect of the present invention, the genetically modified cell as described in one aspect of the present invention, the pharmaceutical composition as described in one aspect of the present invention, the kit as described in one aspect of the present invention, or the set of medicine kits as described in one aspect of the present invention.

[0083] The drug delivery device preferably also includes a component for containing or administering the TROP2 antibody or its antigen-binding fragment, the multispecific antibody, the antigen-binding protein derivative, the antibody-drug conjugate, the chimeric antigen receptor, the genetically modified cell, the pharmaceutical composition, the kit, or the set of kits to a subject, such as a syringe, an implantable drug delivery device, or an infusion device.

[0084] It should be noted that the CDR region division of the present invention refers to the Combined definition rule, which includes the Combined definition rule of Kabat definition and Chothia definition.

[0085] Bench marker 1 used in the present invention is Sacituzumab govitecan from Immunomedics (now Gilead), whose heavy chain amino acid sequence is shown in SEQ ID NO: 67, and light chain amino acid sequence is shown in SEQ ID NO: 74.

[0086] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0087] The reagents and raw materials used in the present invention are commercially available.

[0088] The positive progress effect of the present invention is:

[0089] The antibody of the present invention has good binding to antigens at both protein and cell levels: it has better binding ability to CHO-K1-hTROP2 and CHO-K1-cynoTROP2 than existing antibodies, and can also bind to HCC1954 and BxPC-3. D The value and specific binding can reach a level comparable to the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] FIG1 shows the ELISA detection of the binding activity of purified antibodies to hTROP2-ECD-his.

[0091] FIG2 shows the ELISA detection of the binding activity of the purified antibody to cynoTROP2-ECD-his.

[0092] FIG3 shows the binding activity of purified antibodies to CHO-K1-huTROP2 detected by FACS.

[0093] FIG4 shows the binding activity of the purified antibody to CHO-K1 detected by FACS.

[0094] FIG5 shows the binding activity of purified antibodies to CHO-K1-cynoTROP2 detected by FACS.

[0095] FIG6 shows the binding of purified antibodies to HCC1954 cells detected by FACS.

[0096] FIG7 shows the binding of purified antibodies to BxPC-3 cells detected by FACS.

[0097] FIG8 shows the binding of purified antibodies to A431 determined by FACS.

[0098] FIG9 shows that the purified antibody Trop2 is internalized in A431 cells.

[0099] Figures 10A-10D show the killing activity of Trop2-ADC on A431 cells in vitro.

[0100] Figures 11A-11B show the PK experiments of Trop2 heavy chain-only antibody in rats. DETAILED DESCRIPTION

[0101] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0102] In this application, the term "antibody" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or backbone portion that allows the portion that binds to the antigen to adopt a conformation that promotes antibody binding to the antigen. It may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. For example, a "heavy chain antibody" in this application does not contain a VL region, but only a VH region. The VH or VL region can be further divided into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH or VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. Examples of antibodies include, but are not limited to, full-length antibodies, heavy-chain antibodies (HCAbs), antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and / or dAbs), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

[0103] In this application, the term "variable" generally refers to the fact that some parts of the sequence of the variable domain of an antibody vary strongly, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the entire variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, called CDRs or hypervariable regions (HVRs), and FRs are more highly conserved parts of the variable domains. The variable domains of native heavy and light chains each comprise four FR regions, most of which adopt a β-sheet configuration, are connected by three CDRs, form loops connected, and in some cases form a part of a β-sheet structure. The CDRs in each chain are closely together through the FR region and form the antigen binding site of the antibody together with the CDRs from another chain. The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's cytotoxicity that depends on the antibody.

[0104] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain. The CH2 domain may comprise one or more mutations that reduce or eliminate binding of the CH2 domain to one or more Fc domain receptors (e.g., Fc domain binds α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α, α Alternatively, it is known that conserved N-linked glycosylation sites can be mutated to produce α-glycosylated antibodies by mutating the asparagine (N) at position 84.4 of the IMGT site in the CH2 domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q) to reduce IgG1 effector function (Wang et al., 2018). Alternatively, it is known that complement activation (C1q binding) and ADCC can be reduced by mutating the proline at position 114 of the IMGT site in the CH2 domain to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations can be combined to generate antibody molecules with further reduced or no ADCC or CDC activity.

[0105] In this application, the term "Fab" generally refers to the antigen-binding portion of a conventional antibody (e.g., IgG), comprising the antibody's heavy chain variable region VH, light chain variable region VL, heavy chain constant region domain CHI, and light chain constant region CL. In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CHI to form the heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form the light chain, and the C-terminus of CHI is further linked to the hinge region and other constant region domains of the heavy chain to form the heavy chain. In some embodiments, "Fab" also refers to variant structures of Fab. For example, in certain embodiments, the C-terminus of VH is linked to the N-terminus of CL to form one polypeptide chain, and the C-terminus of VL is linked to the N-terminus of CHI to form another polypeptide chain, forming a Fab (cross VH / VL) structure. In certain embodiments, the CHI of Fab is not linked to the hinge region, but the C-terminus of CL is linked to the hinge region of the heavy chain, forming a Fab (cross Fd / LC) structure.

[0106] In this application, the term "VH" generally refers to the heavy chain variable region VH domain of an antibody, that is, it can be the heavy chain variable region VH of a conventional antibody (H2L2 structure) of humans or other animals, or the heavy chain variable region VHH of a heavy chain antibody (HCAb structure) of animals such as camelids, or the heavy chain variable region VH of a fully human heavy chain antibody (HCAb structure) produced using Harbour HCAb transgenic mice.

[0107] The term "antigen-binding fragment" generally refers to any functional region of a protein that can specifically bind to an antigen, which can be "Fab", "VH", or other antigen-binding forms (such as lipocalins, neural cell adhesion molecules (NCAM), fibronectin, ankyrin repeat proteins (DARPins) and other derived protein structures).

[0108] "Optionally," "either," "any," or "any one" means that the subsequently described event or circumstance may, but need not, occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specific sequence may, but need not, be present. As used herein, "a" and "an" are used herein to refer to one or more than one of the grammatical objects. Unless the context clearly indicates otherwise, the term "or" is used herein to refer to the term "and / or" and is used interchangeably therewith. "About" and "approximately" should generally mean an acceptable degree of error in the amount measured, given the nature or precision of the measurement. Exemplary degrees of error are generally within 10% and more generally within 5%. The methods and compositions disclosed herein encompass polypeptides and nucleic acids having a specified sequence, a variant sequence, or a sequence substantially identical or similar thereto, for example, a sequence at least 85%, 90%, 95%, 99% or more identical to the sequence specified. In the case of amino acid sequences, the term "substantially identical" is used herein to refer to the first amino acid sequence.

[0109] The terms "single-domain antibody" and "nanoantibody" of the present invention have the same meaning, referring to a single-domain antibody consisting only of the variable region of an antibody heavy chain, which is the smallest antigen-binding fragment with complete function.

[0110] The term "heavy chain antibody" of the present invention is also called HCAb antibody, which refers to an antibody that lacks the antibody light chain and only contains the heavy chain, specifically the heavy chain variable domain and the Fc constant domain, relative to the two-chain antibody (immunoglobulin).

[0111] A "humanized antibody" comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises at least one, typically two, variable domains in which all or substantially all HVRs (e.g., CDRs) correspond to HVRs of a non-human antibody, and all or substantially all FRs correspond to FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0112] A "human antibody" has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0113] The terms "polynucleotide," "nucleic acid," or "nucleotide sequence" herein refer to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0114] The term "isolated" nucleic acid molecule or polynucleotide of the present invention refers to a nucleic acid molecule, DNA or RNA, that has been separated from its natural environment. In the present invention, a recombinant polynucleotide encoding a polypeptide contained in a vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified from solution. Isolated polynucleotides include polynucleotide molecules that are normally contained in cells containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, in positive and negative strand forms, and in double-stranded form. Isolated polynucleotides or nucleic acids of the present invention further include such molecules that are synthetically generated. In addition, the polynucleotide or nucleic acid can be or can include regulatory elements, such as a promoter, a ribosome binding site, or a transcription terminator.

[0115] The terms "vector" or "expression vector" and "expression construct" of the present invention are used interchangeably to refer to a DNA molecule that is operably linked to a specific gene and is introduced into a target cell and directs expression. The vector includes a vector that is a self-replicating nucleic acid structure and a vector that is incorporated into the genome of the host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can perform large amounts of stable mRNA transcription. Once the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is generated by the cell's transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0116] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, and also include the progeny of such cells. Host cells include "transformants / transformants" and "transformed cells," including primary transformed cells and progeny derived therefrom. The nucleic acid of the progeny may not be completely identical to that of the parent cell and may contain mutations. Host cells are any type of cell that can be used to generate the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, for example, cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, and also include transgenic animals, transgenic plants, or cells contained within cultured plant or animal tissues.

[0117] "Specific binding" refers to binding selectivity for an antigen and can be distinguished from unwanted or non-specific binding. The ability of an antigen binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) technology and traditional binding assays. In one embodiment, the degree of binding of the antigen binding molecule to an unrelated protein, as measured, for example, by SPR, is less than about 10% of the degree of binding of the antigen binding molecule to the antigen. In certain embodiments, the dissociation constant (Kd) of the molecule that binds to the antigen is ≤1M, ≤100nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM or ≤0.001nM (e.g., 10 -7 M or lower, such as 10 -7 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0118] "Affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Binding affinity can generally be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).

[0119] The term "high affinity" in the present invention refers to an antibody with a Kd of 10 -9 M or lower, even 10 -10M or lower. The term "low affinity" in the present invention refers to an antibody with a Kd of 10 -8 M or higher.

[0120] An "affinity matured" antibody is one with one or more modifications in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such modifications.

[0121] The "Harbour HCAb mouse" (WO2002 / 085945A2), as used herein, is a transgenic mouse carrying a human immunoglobulin repertoire that produces novel, heavy-chain-only antibodies half the size of traditional IgG antibodies. The antibodies produced possess only the human heavy chain variable domain and the mouse Fc constant domain. By lacking light chains, these antibodies virtually eliminate the problems of light chain mispairing and heterodimerization, enabling the development of products that are difficult to achieve with traditional antibody platforms.

[0122] The term "chimeric antigen receptor" refers to a chimeric antigen receptor (CAR) T cell. The antigen-binding site of an antibody that recognizes a tumor antigen is coupled in vitro to the intracellular portion of the CD3-δ chain or FcεRIγ to form a chimeric protein. This protein is then transduced into the patient's T cells through gene transduction to express the CAR, effectively reprogramming the patient's T cells to generate large numbers of tumor-specific CAR-T cells. When these reprogrammed CAR T cells are introduced into the patient's body, they act like a GPS, specifically tracking, identifying, and guiding T cells to kill tumor cells. Most CARs consist of an extracellular antigen-binding domain (composed of light and heavy chains derived from monoclonal antibodies, connected by a flexible hinge region to form a single-chain antibody), a transmembrane region, and an intracellular signaling domain. The CAR structure is created by genetically recombining in vitro a scFv that recognizes a tumor-associated antigen with the intracellular signaling domain, the immunoreceptor tyrosine-based activation motif.

[0123] The term "immune cell" includes cells that have hematopoietic origin and play a role in the immune response, such as lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

[0124] The term "antibody-drug conjugate" refers to both the antigen-binding protein portion and the conjugated portion. The antigen-binding protein portion includes the "antigen-binding protein" described above, as well as antigen-binding fragments derived therefrom. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', Fv fragments, F(ab')2, scFv, di-scFv, and / or dAbs. The conjugated portion may contain at least one drug payload. The drug payload may include a pharmaceutically active ingredient and / or the aforementioned labeling molecule. The drug payload is a class of pharmaceutically active small molecule compounds or toxins or other drug molecules, and may be, but is not limited to, small molecule compounds, toxin molecules, oligonucleotides, protein degradation targeting chimeras (PROTACs), affinity ligands, fluorescent groups, radionuclide groups, and the like. Various drug payloads are known in the art. For example, small molecule compounds generally refer to substances with strong cytotoxicity. Exemplary small molecule compounds may exert such cytotoxic and cytostatic effects through mechanisms including, but not limited to, tubulin binding, DNA binding, inhibition of RNA polymerase, protein synthesis, or inhibition of topoisomerases. For example, the small molecule compound can be a microtubule inhibitor; for example, the microtubule inhibitor can be a maytansine (e.g., DM1 or DM4) and an auristatin (e.g., MMAE or MMAF), etc. For example, the small molecule compound can be a DNA damaging agent; for example, the DNA damaging agent can be calicheamicins, pyrrolobenzodiazepines (PBD, pyrrolobenzodiazepines), etc. For example, protein degradation targeting chimeras (PROTACs) are a class of compounds that can cause target protein degradation by inducing polyubiquitination of target proteins; for example, the PROTAC can be a BET protein degrader. The drug conjugate can also include at least one linker. For example, the linker can include a cleavable linker or a non-cleavable linker. The linker is used to connect one or more payload drugs to the antigen binding protein. In the present application, the cleavable linker can be a "cleavable" linker that facilitates the release of the drug. For example, the cleavable linker can include but is not limited to an acid-sensitive linker, a protease-sensitive linker, a light-sensitive linker, or a disulfide-containing linker. The linker may comprise one or more linker components, and a variety of linker components are known in the art, for example, maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit or vc), and paminobenzyloxycarbonyl (PAB).

[0125] The term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments thereof disclosed herein and other chemical components, such as physiologically / pharmaceutically acceptable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity. The "pharmaceutical composition" of the present invention may include a first active ingredient and a second active ingredient, a combination of two active ingredients present together in a unit dose or a single entity, wherein the first active ingredient and the second active ingredient are present in a mixture for simultaneous administration, such as in a formulation.

[0126] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0127] The "kit of parts" of the present invention are used in a manner known to those skilled in the art and are defined as a combination in which the first active ingredient (kit A) and the second active ingredient (kit B) are present in more than one unit. An example of a kit of parts is a combination in which the first active ingredient and the second active ingredient are present separately. The components of the kit of parts can be administered separately, sequentially, simultaneously, concurrently, or in a chronologically staggered manner. The antibodies or antigen-binding proteins thereof of the present invention can be administered as a single agent or in combination with one or more other agents, wherein the combination does not cause unacceptable adverse reactions.

[0128] The term "cancer" refers to a disorder characterized by unregulated cell growth in mammals. Examples of cancer include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of cancer include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or cervical cancer, salivary gland cancer, kidney cancer or ureteral cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, bile duct cancer, central nervous system (CNS) tumors, spinal axis tumors, brain stem gliomas, glioblastoma multiforme, astrocytoma, glioma multiforme ... The present invention includes but is not limited to: melanoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (such as that associated with brain tumors) and Meigs syndrome, brain tumors and brain cancer, and head or neck cancer and related metastatic cancers.

[0129] "Treatment" refers to the administration of a therapeutic agent, whether internal or external, such as a composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein or a nucleic acid molecule encoding the same, to a patient suffering from one or more diseases or symptoms for which the therapeutic agent has a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate the one or more diseases or symptoms in the patient or population being treated, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable degree.

[0130] The term "preventing cancer" refers to delaying, inhibiting, or preventing the onset of cancer in a mammal in which the initiation of carcinogenesis or tumorigenesis has not been demonstrated, but in which the mammal has been identified as having a predisposition to cancer, as determined, for example, by genetic screening or other methods. The term also includes treating a mammal with a precancerous condition to halt the progression of the precancerous condition to a malignant tumor or to cause its regression.

[0131] The term "drug delivery device" primarily refers to packaging materials and injection systems. Packaging materials include, but are not limited to, vials, syringes, or test tubes; injection systems include, but are not limited to, syringes, infusion pumps, injection pens, needle-free devices, or subcutaneous patch delivery devices. The components of the injection device may be conventional in the art, including a container, a sealant, and an injection needle. The container includes, but is not limited to, a vial, syringe, or test tube. The container may be made of conventional materials in the art, such as glass or plastic. The sealant includes, but is not limited to, a sealing plug or a sealing ring. The sealant may be made of conventional materials in the art, such as rubber, plastic, or polymers. The injection needle includes, but is not limited to, a water-based injection, a single needle, or a microneedle assembly. The injection needle may be made of conventional materials in the art, such as metal, silicon, silicon dioxide, glass, nickel, titanium, or a biodegradable polymer. The water-based injection includes, but is not limited to, a vial-based injection, an ampoule-based injection, or a prefilled injection system. The ampoule-based injection may be a glass or plastic ampoule. The prefilled injection system can be conventional in the art, such as a prefilled syringe.

[0132] In the art, the CDRs of antibodies can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th edition, National Institutes of Health, Bethesda, Maryland (1991)) and the Chothia definition rules based on structural loop region positions (see, Al-Lazikani et al., J Mol Biol 273:927-48, 1997). The application can also use the Combined definition rules comprising the Kabat definition and the Chothia definition to determine the amino acid residues in the variable domain sequence and the full-length antibody sequence. In the present invention, each sequence is determined as shown in the table below according to the Combined definition rules.

[0133] Method for defining antibody CDRs in this application (see http: / / bioinf.org.uk / abs / )

[0134] Example 1 Preparation of antigens and stably transfected cell lines

[0135] a. Preparation of Antigens and Other Proteins

[0136] Human TROP2-ECD-Fc (abbreviated as hTROP2-ECD-Fc) protein was purchased from Acrobiosystems, Cat#: TR2-H5253;

[0137] Human TROP2-ECD-his protein (abbreviated as hTROP2-ECD-his) was purchased from Acrobiosystems, Cat#: TR2-H5223;

[0138] cyno TROP2-ECD-his, purchased from Acrobiosystems, Cat#: TR2-H52H3.

[0139] b. Preparation of Stable Cell Lines

[0140] HEK293T-hTROP2 (human TROP2) stable transfectant was prepared in the laboratory;

[0141] HEK293T cell suspension was added to a 6-well plate at a density of 5E5 / well and cultured overnight at 37°C. The plasmid encoding the human TROP2 gene was added to the tube and then diluted with Opti-MEM (Gibco, Cat. No. 31985070). The transfection reagent PEI was added to a new tube and diluted with Opti-MEM as well. The ratio of PEI to DNA was 3:1. The diluted PEI was added to the diluted plasmid, mixed well, and incubated at room temperature for 15 minutes. The above mixture was added to the cells, gently shaken to mix, and incubated at 37°C overnight. The supernatant was then discarded and replaced with fresh culture medium supplemented with 2μg / ml puromycin. The culture was incubated at 37°C, and fresh culture medium containing puromycin was replaced every three days. After the cells have grown fully, perform FACS verification. Dilute the verified cell pool to 5 cells / ml and seed five 96-well plates with 100 μl / well. Culture at 37°C for 14 days. Pick a single clone for FACS verification. After verification, continue to expand the culture and freeze.

[0142] CHO-K1-huTROP2 (human TROP2) stable transfectant was prepared in the laboratory;

[0143] CHO-K1 cells were harvested, centrifuged at 150 × g for 5 minutes, and resuspended in Opti-MEM (Gibco, Cat. No. 31985070) to a concentration of 1E7 cells / ml. 500 μl of the cell suspension was then added with 10 μg of a plasmid encoding the human TROP2 gene.

[0144] Place the cell suspension in an electroporation cuvette (Bio Rad, Cat. No. 1652081) and gently pipette to mix. Place the electroporation cuvette in a Gene Pulser Xcell electroporator (BioRad). After electroporation, transfer the cell suspension to a cell culture dish containing 10 ml of culture medium and culture overnight. Discard the supernatant and replace with fresh culture medium supplemented with 8 μg / ml puromycin. Culture at 37°C, replacing the fresh culture medium containing puromycin every three days. After the cells have grown confluently, perform FACS verification. Dilute the verified cell pool to 5 cells / ml and seed five 96-well plates at 100 μl / well. Culture at 37°C for 14 days. Pick a single clone for FACS verification. After verification, continue to expand the culture of the verified clone and freeze it.

[0145] CHO-K1-cynoTROP2 (cynomolgus TROP2) stable transfectants were prepared in the laboratory. The preparation method of the stable transfectants is as follows:

[0146] CHO-K1 cells were harvested, centrifuged at 150 × g for 5 minutes, and resuspended in Opti-MEM (Gibco, Catalog No. 31985070) to 1E7 cells / ml. 500 μl of the cell suspension was added to an electroporation cuvette (Bio-Rad, Catalog No. 1652081) containing 10 μg of a plasmid encoding the macaque TROP2 gene and gently pipetted to mix. The electroporation cuvette was placed in a Gene Pulser Xcell electroporator (Bio-Rad). After electroporation, the cell suspension was transferred to a cell culture dish containing 10 ml of culture medium and incubated overnight. The supernatant was then discarded and replaced with fresh culture medium supplemented with 8 μg / ml puromycin. The cells were cultured at 37°C, with fresh culture medium containing puromycin replaced every three days. After the cells have grown fully, perform FACS verification. Dilute the verified cell pool to 5 cells / ml and seed five 96-well plates with 100 μl / well. Culture at 37°C for 14 days. Pick a single clone for FACS verification. After verification, continue to expand the culture and freeze.

[0147] Example 2 Immunization of Harbour transgenic mice

[0148] The Harbour HCAb mouse (Harbour Antibodies BV, WO 2002 / 085945 A3) is a transgenic mouse carrying a human immunoglobulin repertoire, capable of producing novel "heavy chain"-only antibodies that are half the size of traditional IgG antibodies. The antibodies it produces possess only the human "heavy chain" variable domain and the mouse Fc constant domain. By lacking light chains, these antibodies virtually eliminate the problems of light chain mispairing and heterodimerization, enabling the development of products that are difficult to achieve with traditional antibody platforms.

[0149] Harbour HCAb Mouse Protein Immunization: Human TROP2-ECD-Fc protein was used to immunize 6-8 week old Harbour HCAb transgenic mice. All transgenic mice were housed in a SPF environment. For the primary immunization, 50 μg of Human TROP2-ECD-Fc protein was emulsified with Freund's complete adjuvant (Sigma, F5881) (the volume ratio of TROP2 protein to the total volume of PBS to CFA was 1:1; the injection volume per mouse was 250 μL, including 125 μL of CFA. The mixture was shaken at high speed to fully emulsify before use) and then injected intraperitoneally into the mouse. For subsequent booster immunizations, 25 μg of Human TROP2-ECD-Fc protein was mixed with RIBI adjuvant (Sigma, S6322) (first dissolve one bottle of RIBI with 1.25 ml of PBS, then add 25 μg of protein to 125 μL with PBS, and then mix evenly with 125 μL of dissolved RIBI at a 1:1 ratio) and injected intraperitoneally into mice. The interval between booster immunizations was 2 weeks. Boost immunizations were performed 3 to 4 times. Blood was collected 7 days after each booster immunization to detect serum antibody titers using ELISA, and mice with high serum titers were selected for subsequent single B cell screening experiments.

[0150] Harbour HCAb mouse cell immunization: HEK293T-hTROP2 cells were used to immunize 6-8 week old Harbour HCAb transgenic mice. All transgenic mice were housed in an SPF environment. In the initial immunization, 1×10 7 HEK293T-hTROP2 cells were resuspended in PBS and injected intraperitoneally into mice. For subsequent booster immunizations, 1×10 7 HEK293T-hTROP2 cells were resuspended in PBS and injected intraperitoneally into mice. The interval between the primary immunization and the first booster immunization was 2 weeks, and the interval between subsequent boosters was 3 weeks. Blood samples were collected 7 days after each booster immunization to measure serum antibody titers using ELISA. Mice with high serum titers were selected for subsequent single B cell screening experiments.

[0151] Example 3 is based on Single B cell screening using the Optofluidic system

[0152] Optofluidic system uses optical-electric positioning (OEP TM) technology to move individual cells. The Beacon optoelectronic system is an automated bioinstrumentation device that allows for simultaneous execution of multiple operations, including biological function testing, experimental analysis, and positive clone selection, under cell culture conditions. The Beacon platform can perform these tasks in a massively parallel, automated manner on thousands of cells.

[0153] This application uses a plasma cell discovery workflow, which can screen up to 14,000 single plasma cells in each experiment to select antigen-specific plasma cells that secrete antibodies. These plasma cells that secrete specific antibodies are then exported to 96-well plates containing cell lysate for subsequent single B cell sequencing to identify the heavy chain sequence of the antibody produced by a single B cell (monoclonal).

[0154] Example 4 Single B cell sequencing and acquisition of fully human HCAb

[0155] RNA was extracted from B cells and reverse transcribed into cDNA (SuperScript IV First-Strand synthesis system, Invitrogen, 18091200). The human VH gene was then amplified using PCR using specific primers: 5'-GGTGTCCAGTGTSAGGTGCAGCTG-3' (SEQ ID NO: 92) and 5'-AATCCCTGGGC ACTGAAGAGACGGTGACC-3' (SEQ ID NO: 93). The amplified VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the Fc domain sequence of the human IgG1 antibody heavy chain. A fully human HCAb antibody was obtained.

[0156] After obtaining the variable domain sequence encoding the antibody molecule, conventional recombinant DNA technology can be used to fuse the heavy chain variable domain VH sequence with the corresponding human antibody light and heavy chain constant domain sequences to obtain a recombinant antibody molecule.

[0157] In this example, the antibody heavy chain variable domain sequence (VH) was synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode the full-length heavy chain of the IgG1 antibody. Specifically, HEK293 cells were cultured in FreeStyle TM Before transient transfection, adjust the cell density to (6-8) × 10 5 cells / mL, and cultured in a shaker at 37°C, 8% CO2 for 24 hours. The cell concentration was 1.2×106 Cells / mL. Prepare 30mL of cultured cells, dissolve the above-mentioned HCAb-encoding heavy chain plasmid in 1.5mL Opti-MEM reduced serum medium (Thermo, #31985088), and filter sterilize with a 0.22μm filter membrane. Take another 1.5mL Opti-MEM and dissolve 120μL of 1mg / mL PEI (Polysciences Inc, #23966-2) and let it stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drip the plasmid PEI mixed solution while shaking the culture bottle, and culture in a 37℃ 8% CO2 shaker for 5 days. Observe cell viability after 5 days. Collect the culture, centrifuge at 3300g for 10 minutes, and then take the supernatant; then centrifuge the supernatant at high speed to remove impurities. Use PBS (pH7.4) to balance the culture medium containing MabSelect TM A gravity column (Bio-Rad, #7311550) (GE Healthcare Life Science, #71-5020-91AE) was used for rinsing with 2-5 column volumes. The supernatant sample was passed through the column. The column was rinsed with 5-10 column volumes of PBS. The target protein was then eluted with 0.1 M glycine (pH 3.5), adjusted to neutrality with Tris-HCl (pH 8.0), and finally concentrated using an ultrafiltration tube (Millipore, #UFC901024) and exchanged with PBS buffer to obtain a purified antibody solution. The concentration was then determined using a NanoPhotometer NP80 (IMPLEN), and the solution was aliquoted and stored for future use.

[0158] The heavy chain determining region (CDR) sequences of the 17 human TROP2 antibodies screened and obtained in the present invention are shown in Table 1, the framework regions are shown in Table 2, and the VH sequences are shown in Table 3.

[0159] Table 1

[0160] Table 2

[0161] Table 3

[0162] Example 5 Production, purification and validation of recombinant antibodies

[0163] a. Recombinant Antibody Production and Purification

[0164] 293F cells were used for antibody expression. The cell density was adjusted to 3×10 6cells / mL. Add the antibody plasmid to the tube and then dilute it with Opti-MEM (Gibco, Cat. No. 31985070). Add the transfection reagent PEI to a new tube and dilute it with Opti-MEM as well. The ratio of PEI to DNA is 3:1. Add the diluted PEI to the diluted plasmid, mix well, and incubate at room temperature for 15 minutes. Add the above mixture to the cells, and culture the transfected cells in a 37°C, 5% CO2 incubator with shaking at 125 rpm. 24 hours after transfection, supplement with 10% volume of OPM-293 ProFeed (OPM, Cat. No. F081918). After 7 days of cell culture, collect the supernatant. Purify the transfection supernatant using a protein A column.

[0165] b. Validation of recombinant antibodies

[0166] (1) Verification of antibody binding properties

[0167] For characterization using FACS, the recombinant antibody was serially diluted from 100 nM to eight different concentrations, incubated with CHO-K1-huTROP2, CHO-K1, CHO-K1-cynoTROP2, and HCC1954 and BxPC-3 cell lines (endogenously expressing TROP2; HCC1954: ATCC, Catalog No. CRL-2338; BxPC-3: ATCC, Catalog No. CRL-1687) for 1 hour, followed by incubation with a secondary antibody (Alexa Fluor 488-conjugated AffiniPure Anti-human IgG Fc Fragment Speci?c, Jackson, Cat.: 109-545-098) for 1 hour. The results are shown in Figures 3-7 and Table 4 below.

[0168] Table 4 Results of antibody binding ability test with highly expressed hu-TROP2 at the cellular level

[0169] Bench marker 1 was sacituzumab govitecan (heavy chain amino acid sequence shown in SEQ ID NO: 67, light chain amino acid sequence shown in SEQ ID NO: 74) prepared in-house from Immunomedics (now Gilead), with an hIgG1 isotype, purchased from Crownbio, Cat#: C0001-4.

[0170] The results showed that the recombinant antibodies all had very good binding to CHO-K1-huTROP2, and the binding effects of most antibodies were comparable to those of the reference prior art antibodies. Among them, the binding effects of PR304362, PR304375, PR304377, and PR304474 were better than those of the prior art antibodies. In addition, the aforementioned recombinant antibodies all had strong binding to CHO-K1-cynoTROP2 cells, indicating that these recombinant antibodies all had cross-reactivity with cynoTROP2 and were better than the reference prior art antibodies. All antibodies did not bind to CHO-K1 empty cells (Figure 4). The binding experimental results with HCC1954 and BxPC-3 showed that all antibodies had varying degrees of binding, especially the antibodies numbered PR304362, PR304375, and PR304377, which had better binding effects than the reference prior art antibodies.

[0171] The binding ability of the 14 antibodies prepared in Examples 1-5 of the present invention to the hTROP2-ECD-his protein and cynoTROP2-ECD-his protein was tested based on the ELISA assay. The binding ability of different antibodies to the hTROP2-ECD-his protein was determined by comparing the binding curves of the different antibodies. The specific experimental process is as follows: human hTROP2-ECD-his was first diluted to a concentration of 1 μg / mL and added to a 96-well plate, 100 μL per well, and incubated at 4°C overnight. After washing the 96-well plate three times with PBST solution, a 2% BSA solution in PBS was added and incubated at 37°C for 1 hour. The antibody to be tested was diluted in a concentration gradient (100nM, 10-fold dilution), added to the 96-well plate, and incubated at 37°C for 1 hour. After washing three times with PBST solution, anti-human IgG H+L-HRP secondary antibody (5000× dilution) was added and incubated at 37°C for 30-60 minutes. After washing three times with PBST solution, TMB color development solution was added for 5-15 minutes, and then stop solution was added to stop color development.

[0172] Table 5 Results of antibody binding ability test with hTROP2-ECD-his and cynoTROP2-ECD-his at the protein level

[0173] The test results are shown in Table 5, Figures 1 and 2. All 14 antibodies have the ability to bind to huTROP2-ECD-his and cyno-TROP2-ECD-his proteins. Among them, the binding abilities of antibodies PR304462, PR304473, PR304474, PR304505, and PR304519 are comparable to Bench marker 1.

[0174] (2)K D Value detection

[0175] The K of the above 14 antibodies was detected using conventional procedures in the art. D The specific steps are as follows: human TROP2 protein was diluted to a final concentration of 1.5 μg / mL and directly immobilized on the HIS1K biosensor with an immobilization height of 0.3 nm. For kinetic measurements, the candidate antibody was diluted to 100 nM with PBST, bound for 300 s, dissociated for 600 s, and regenerated with 10 mM glycine HCl (pH 1.5) for 15 s. A simple one-to-one Languir binding model (Octet Red96 data analysis software) was used to calculate the association rate (kon) and dissociation rate (kdis). The equilibrium dissociation constant (K D ) was calculated as kdis / kon. The results are shown in Table 6 below.

[0176] Table 6 Antibody affinity determination

[0177] Table 6 shows the K values ​​of 14 antibodies. D These results show that antibodies such as PR304375, PR304473, PR304474, and PR304505 have comparable affinity to the reference prior art antibodies.

[0178] In summary, the present invention immunized and screened 14 fully human antibodies with good binding ability and affinity to hTROP2 and cynoTROP2, reaching levels comparable to or better than the prior art Sacituzumab govitecan antibody.

[0179] Example 6 Characterization of the endocytosis of recombinant antibodies in A431 tumor cells

[0180] Trop2 antibodies can mediate the internalization of Trop2 protein expressed on the cell surface by binding to the extracellular surface of Trop2. In this example, the internalization of Trop2 antibodies was tested in a short time (6 hours).

[0181] Following the method described in Example 5, FACS was used to characterize the binding of recombinant antibodies PR304681, PR304462, PR304505, and PR304375 to breast cancer cells A431 (CRL-1555, ATCC), which overexpress Trop2. The results are shown in Figure 8 . The saturation values ​​for binding of PR304681, PR304505, and PR304375 to A431 were high and comparable, while the saturation value for binding of PR304462 to A431 was lower.

[0182] Antibodies used for endocytosis detection were labeled with pHAb reactive dye (Promega, #G9841). After labeling, the dye-to-antibody ratio (DAR) for each labeled antibody was measured. A431 cells were cultured and expanded in T-75 flasks using F12K medium (Gibco, #21127-022) supplemented with 10% serum (BI, fetal bovine serum, #04-002-1A). After reaching 90% confluency, the medium was aspirated and the cells were washed twice with PBS. The cells were treated with trypsin (Invitrogen, #15050065) for approximately 1 minute and then neutralized with culture medium. The cells were transferred to a sterile 15 ml centrifuge tube and centrifuged at 1000 rpm at room temperature for 5 minutes to pellet the cells. The medium was aspirated and the cells were resuspended in their respective culture medium. The cells were gently pipetted to obtain a single-cell suspension. The A431 cells were counted using a cell counting plate and then resuspended in ice-cold FACS buffer (PBS + 2% FBS) to a volume of 2 × 10 6 Cells / ml, 100ul of cell suspension per well was transferred to a 96-well V-plate (Corning, #3894), and centrifuged at 400g for 5 minutes. After discarding the supernatant, the following procedure was followed for the determination of no endocytosis: 100ul of FACS buffer containing PR304681 or PR304462 or PR304505 or PR304375 or hIgG1 at a final concentration of 100nM was added, and incubated at 4°C for 6 hours. Unbound antibodies were removed by washing twice with ice-cold FACS buffer. For the determination of endocytosis, the cells after adding antibodies were placed at 37°C for 6 hours, and then the cells were transferred to 4°C and pre-cooled PBS was added to prevent the endocytosis of antibodies. The fluorescence intensity of the cells was analyzed using an Incyte iQue Plus flow cytometer. Endocytosis = (MFI 37℃ -MFI 4℃ ) / DAR.

[0183] The results are shown in Figure 9. At saturated antibody binding concentrations, the three antibodies PR304681, PR304505, and PR304375, which have high saturation values ​​for binding to A431, were internalized more strongly than PR304462 in Trop2-positive tumor cells A431. Among them, PR304375 was internalized most strongly.

[0184] Example 7: Killing of target cells A431 by constructing ADC with recombinant antibody coupled to Dxd

[0185] The Trop2 HCAb antibody was dialyzed overnight at 4°C to displace the coupling buffer to the desired pH, resulting in a concentration of 3 mg / ml. The solution was then added with an equivalent amount of TCEP (SIGMA-ALDRICH, 75259) and incubated at room temperature with shaking for 3 hours. Subsequently, an equivalent amount of MC-GGFG-DXD (MedChemExpress, HY-13631D) was added, and the reaction was incubated at room temperature with shaking for 2 hours. The reaction was terminated by the addition of a predetermined amount of cysteine. The reaction solution was then dialyzed against PBS at 4°C overnight to remove small molecules, resulting in a clean product.

[0186] 8,000 A431 cells were seeded per well in a 96-well plate. After overnight culture, serially diluted PR304681-Dxd, PR304462-Dxd, PR304505-Dxd, PR304375-Dxd, IgG1-Dxd, or Dxd, or the corresponding antibody for each ADC, were added. Six days later, cell viability was assessed using CellTiter Glo (Promega, #G7572). As shown in Figures 10A-10D, ADCs constructed with the three antibodies PR304681, PR304505, and PR304375, which were well internalized, exhibited significant killing effects.

[0187] Example 8 PK experiment of Trop2 heavy chain antibody in rats

[0188] The purpose of this study was to determine the pharmacokinetic parameters of an antibody following intravenous administration in female Sprague Dawley rats. The specific experimental procedure was as follows: Blood samples were obtained from Sprague Dawley rats prior to tail vein injection of the antibody. Following tail vein injection of 5 mg / kg of the antibody, blood samples were collected prior to dosing, and at 10 minutes, 2 hours, 8 hours, 24 hours, and on days 3, 7, 10, 14, 21, and 28 for serum antibody concentration determination. Three rats were assigned to each group for PR300516 and PR301168. The PK experimental design is shown in Table 8.

[0189] Table 8 PK experimental design

[0190] Table 9 Statistics of PK parameters in rats

[0191] As shown in Table 9 and Figures 11A and 11B , the half-lives of PR304681 and PR304462 in rats are 112 hours and 119 hours, respectively.

Claims

1. A TROP2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH), wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are respectively identical to the HCDR1, HCDR2 and HCDR3 of the VH having the amino acid sequence shown in SEQ ID NO:40-56.

2. The TROP2 antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region comprises a HCDR1 having a sequence as shown in any one of SEQ ID NOs: 1-10, a HCDR2 having a sequence as shown in any one of SEQ ID NOs: 11-25, and a HCDR3 having a sequence as shown in any one of SEQ ID NOs: 26-39.

3. The TROP2 antibody or antigen-binding fragment thereof according to claim 2, wherein: The heavy chain variable region comprises: HCDR1 with the sequence shown in SEQ ID NO: 1, HCDR2 with the sequence shown in SEQ ID NO: 11, and HCDR3 with the sequence shown in SEQ ID NO: 26; HCDR1 with the sequence shown in SEQ ID NO: 2, HCDR2 with the sequence shown in SEQ ID NO: 12, and HCDR3 with the sequence shown in SEQ ID NO: 27; HCDR1 with the sequence shown in SEQ ID NO: 3, HCDR2 with the sequence shown in SEQ ID NO: 13, and HCDR3 with the sequence shown in SEQ ID NO: 28; HCDR1 with the sequence shown in SEQ ID NO: 4, HCDR2 with the sequence shown in SEQ ID NO: 14, and HCDR3 with the sequence shown in SEQ ID NO: 28; HCDR1 with the sequence shown in SEQ ID NO: 1, HCDR2 with the sequence shown in SEQ ID NO: 15, and HCDR3 with the sequence shown in SEQ ID NO: 29; HCDR1 with the sequence shown in SEQ ID NO: 5, HCDR2 with the sequence shown in SEQ ID NO: 16, and HCDR3 with the sequence shown in SEQ ID NO: 30; HCDR1 with the sequence shown in SEQ ID NO: 1, HCDR2 with the sequence shown in SEQ ID NO: 17, and HCDR3 with the sequence shown in SEQ ID NO: 31; HCDR1 with the sequence shown in SEQ ID NO: 6, HCDR2 with the sequence shown in SEQ ID NO: 18, and HCDR3 with the sequence shown in SEQ ID NO: 32; HCDR1 with the sequence shown in SEQ ID NO:7, HCDR2 with the sequence shown in SEQ ID NO:19, and HCDR3 with the sequence shown in SEQ ID NO:33; HCDR1 with the sequence shown in SEQ ID NO: 8, HCDR2 with the sequence shown in SEQ ID NO: 20, and HCDR3 with the sequence shown in SEQ ID NO: 34; HCDR1 with the sequence shown in SEQ ID NO:9, HCDR2 with the sequence shown in SEQ ID NO:21, and HCDR3 with the sequence shown in SEQ ID NO:35; HCDR1 with the sequence shown in SEQ ID NO: 10, HCDR2 with the sequence shown in SEQ ID NO: 22, and HCDR3 with the sequence shown in SEQ ID NO: 36; HCDR1 with the sequence shown in SEQ ID NO: 8, HCDR2 with the sequence shown in SEQ ID NO: 23, and HCDR3 with the sequence shown in SEQ ID NO: 37; HCDR1 with the sequence shown in SEQ ID NO: 8, HCDR2 with the sequence shown in SEQ ID NO: 24, and HCDR3 with the sequence shown in SEQ ID NO: 38; Or, the HCDR1 sequence shown in SEQ ID NO: 1, the HCDR2 sequence shown in SEQ ID NO: 25, and the HCDR3 sequence shown in SEQ ID NO:

39.

4. The TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 40-56.

5. The TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein: The antigen-binding fragments include Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv, VHH, heavy chain antibody (HCAb) and / or dAb.

6. The TROP2 antibody or antigen-binding fragment thereof according to claim 5, wherein The heavy chain antibody includes a constant region comprising the amino acid sequence shown in SEQ ID NO:

57.

7. An isolated nucleic acid encoding the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid according to claim 7; preferably, the backbone of the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector, and the viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

9. A transformant, characterized in that The transformant comprises the recombinant expression vector according to claim 8; preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell, more preferably, the eukaryotic cell is a yeast cell or a mammalian cell; wherein the mammalian cell is, for example, a 293 cell or a CHO cell. 10 . A method for preparing a TROP2 antibody or an antigen-binding fragment thereof, comprising culturing the transformant according to claim 9 and obtaining the TROP2 antibody or the antigen-binding fragment thereof from the culture.

11. A multispecific antibody, characterized in that The multispecific antibody comprises the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and other anti-tumor antibodies or fragments thereof or analogs thereof; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

12. An antigen-binding protein derivative, characterized in that: The antigen-binding protein derivative comprises the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and a detectable labeling molecule. Preferably, the detectable labeling molecule is an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

13. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate comprises a cytotoxic agent or a label, and the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; preferably, the cytotoxic agent is MMAF, MMAE or Dxd, and the label is a fluorescent agent.

14. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

15. A genetically modified cell, characterized in that The genetically modified cells comprise the TROP2 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6; the genetically modified cells are preferably eukaryotic cells, more preferably isolated human cells; further preferably immune cells such as T cells or NK cells.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to claim 11, the antigen-binding protein derivative according to claim 12, the antibody-drug conjugate according to claim 13, the chimeric antigen receptor according to claim 14, or the genetically modified cell according to claim 15, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further contains one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

17. A kit comprising the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to claim 11, the antigen-binding protein derivative according to claim 12, the antibody-drug conjugate according to claim 13, the chimeric antigen receptor according to claim 14, the genetically modified cell according to claim 15, or the pharmaceutical composition according to claim 16; Preferably, the kit further comprises (i) a device for administering the antibody or antigen-binding fragment thereof, antibody drug conjugate, chimeric antigen receptor, genetically modified cell or pharmaceutical composition; and / or (ii) instructions for use.

18. A medicine kit, characterized in that: The kit includes kit A and kit B: The drug kit A contains the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to claim 11, the antigen-binding protein derivative according to claim 12, the antibody-drug conjugate according to claim 13, the chimeric antigen receptor according to claim 14, the genetically modified cell according to claim 15, or the pharmaceutical composition according to claim 16; The drug kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

19. Use of the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to claim 11, the antigen-binding protein derivative according to claim 12, the antibody-drug conjugate according to claim 13, the chimeric antigen receptor according to claim 14, the genetically modified cell according to claim 15, the pharmaceutical composition according to claim 16, the kit according to claim 17, or the kit according to claim 18 in the preparation of a product for diagnosing, treating and / or preventing tumors; preferably, the tumor is a TROP2-positive tumor; more preferably, a hematological cancer or a solid cancer; The blood cancer is, for example, B-cell leukemia, lymphoma, acute myeloid leukemia, Burkitt's lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, or marginal zone lymphoma; The solid cancers include breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, or adrenal cancer.

20. A method for diagnosing, treating and / or preventing a TROP2-mediated disease or condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of a TROP2 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, a multispecific antibody as described in claim 11, an antigen-binding protein derivative as described in claim 12, an antibody-drug conjugate as described in claim 13, a chimeric antigen receptor as described in claim 14, a genetically modified cell as described in claim 15, or a pharmaceutical composition as described in claim 16 to treat the patient in need.

21. The method according to claim 20, wherein The disease or condition is a tumor, preferably a blood cancer or a solid cancer; The blood cancer is preferably as defined in claim 19; The solid cancer is preferably as defined in claim 19.

22. A method for immunodetection or determination of TROP2, characterized in that: The method comprises using the TROP2 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, the multispecific antibody as described in claim 11, the antigen-binding protein derivative as described in claim 12, the antibody-drug conjugate as described in claim 13, the chimeric antigen receptor as described in claim 14, the genetically modified cell as described in claim 15, the pharmaceutical composition as described in claim 16 or the kit as described in claim 17; preferably, the detection is for non-diagnostic, preventive and / or therapeutic purposes.

23. A combination therapy comprising administering to a patient in need thereof the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to claim 11, the antigen-binding protein derivative according to claim 12, the antibody-drug conjugate according to claim 13, the chimeric antigen receptor according to claim 14, the genetically modified cell according to claim 15, and / or the pharmaceutical composition according to claim 16; and a second therapeutic agent; the second therapeutic agent preferably comprises other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

24. A drug delivery device, characterized in that: The drug delivery device comprises the TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to claim 11, the antigen-binding protein derivative according to claim 12, the antibody-drug conjugate according to claim 13, the chimeric antigen receptor according to claim 14, the genetically modified cell according to claim 15, the pharmaceutical composition according to claim 16, the kit according to claim 17, and / or the kit according to claim 18; Preferably, the drug delivery device further comprises a component for containing or administering the TROP2 antibody or its antigen-binding fragment, the multispecific antibody, the antigen-binding protein derivative, the antibody-drug conjugate, the chimeric antigen receptor, the genetically modified cell, the pharmaceutical composition, the kit, or the set of kits to a subject, such as a syringe, an implantable drug delivery device, or an infusion device.

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