A monoclonal antibody against Trop2 and its applications

Abstract

The present invention relates to the field of biomedicine. Specifically, the present invention relates to an isolated monoclonal antibody against Trop2 or an antigen-binding fragment thereof, and the use of the antibody or fragment in tumor treatment and diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof against Trop2, and the use of the antibody or fragment in tumor treatment and diagnosis. Background of the Invention

[0002] Human trophoblast cell surface antigen 2 (Trop2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a transmembrane glycoprotein encoded and expressed by the Tacstd2 gene. It is a 36kDa polypeptide composed of 323 amino acids. Trop2 protein was first discovered in human trophoblast cells and is a surface marker of trophoblast cells. It is highly expressed in embryonic progenitor stem cells, promotes the proliferation of embryonic progenitor stem cells, and plays an important role in tissue and organ formation and embryonic development. It is worth noting that Trop2 is also highly expressed in a variety of epithelial malignancies, such as oral squamous cell carcinoma, pancreatic cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, gastric cancer, colon cancer, and lung cancer. At present, the mechanism of Trop2-mediated signaling pathways is not fully understood. It mainly promotes tumor cell growth, proliferation, invasion, and metastasis by upregulating intracellular calcium concentration, regulating the expression of cell cycle proteins, and reducing the effect of fibronectin. Clinical studies have found that Trop2 antigen is highly expressed in tumor tissues and is closely related to the reduction of patient survival and poor prognosis. Immunohistochemistry results show that anti-Trop2 antibodies can be highly efficient and specific in affinity to tumor tissues. Therefore, Trop2 is a highly promising therapeutic target in tumor targeted therapy. In recent years, research on anti-tumor drugs targeting Trop2, such as antibodies, fusion proteins, chemical inhibitors, nanoformulations, and antibody-drug conjugates (ADCs), has gradually developed, especially ADC drug research has made rapid progress. The ADC drug IMMU-132, which is currently in the clinical phase II / III trial stage, is a humanized anti-Trop2 monoclonal antibody hRS7 coupled with a topoisomerase inhibitor camptothecin derivative SN-38. The drug has a good anti-tumor effect and is clinically used for recurrent and refractory triple-negative breast cancer that has failed to respond to multiple treatment options. The objective remission rate of patients after medication is 30%, the clinical efficacy is 46%, and the average time from medication to onset is only 1.9 months, which greatly improves patient compliance and prolongs the patient's progression-free survival by 6 months and the overall survival by 16.6 months. In the Phase II clinical trial of small cell lung cancer, the objective response rate of patients treated with IMMU-132 was 19%, the median response time was 6 months, the clinical efficacy rate was 43%, the median progression-free survival was 5.2 months, and the median overall survival was 9.5 months. Therefore, IMMU-132 has brought new hope to patients with triple-negative breast cancer and refractory small cell lung cancer.

[0003] The hybridoma fusion technology was first developed by Kohler and Milstein in 1975 and won the Nobel Prize in Physiology or Medicine in 1984. Since B cells cannot proliferate indefinitely and have a survival time of no more than 20 days in vitro, they cannot produce monoclonal antibodies on a large scale. Tumor cells, on the other hand, can proliferate and survive indefinitely. Therefore, under the action of a PEG (polyethylene glycol) fusion agent, B cells that can produce specific antibodies are fused with myeloma cells (or electrofused), and after multiple positive clone screenings, a monoclonal hybridoma cell line that can stably secrete antibodies against specific antigens is obtained. The antibodies produced are monoclonal antibodies against an antigenic determinant, which have the characteristics of high specificity, high purity, good homogeneity, high affinity, high titer, and low cost. In recent years, tumor-targeted therapy based on monoclonal antibodies has been considered one of the most promising and prominent tumor treatment strategies. According to statistics, as of 2018, the FDA has approved 24 monoclonal antibody drugs for the treatment of solid tumors, which target CD antigens (including CD19, CD20, CD30, CD33, CD38, and CD53), tumor cell surface molecules (HER2, EGFR, PD-L1, GD2, PMSA, and SLAMF7), inhibitory receptors PD-1 and CTLA-4 on the surface of immune cells, and inhibit tumor angiogenesis. However, at present, the targets available for monoclonal antibody drug research and development are very limited. Therefore, discovering new tumor-specific antigens, especially antigens with high expression levels and important functions in tumor tissues, and studying and preparing monoclonal antibodies targeting these antigens are of great significance for expanding the application of monoclonal antibodies in the field of tumor-targeted therapy. Brief description of the invention

[0004] In one aspect, the present invention provides a hybridoma cell line that was deposited with the China General Microbiological Culture Collection Center on June 25, 2019, under the accession number CGMCC No. 18167.

[0005] In one aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody is produced by a murine hybridoma cell line that was deposited with the China General Microbiological Culture Collection Center on June 25, 2019, under the accession number CGMCC No. 18167.

[0006] In one aspect, the present invention provides an isolated monoclonal antibody or an antigen-binding fragment thereof against Trop2, wherein the monoclonal antibody comprises the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of a monoclonal antibody produced by a murine hybridoma cell line that was deposited with the China General Microbiological Culture Collection Center on June 25, 2019, under the accession number CGMCC No. 18167.

[0007] In one aspect, the present invention provides an isolated monoclonal antibody against Trop2 or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region of a monoclonal antibody produced by a mouse hybridoma cell deposited with the China General Microbiological Culture Collection Center on June 25, 2019 under the accession number CGMCC No. 18167.

[0008] In one aspect, the present invention provides an isolated monoclonal antibody against Trop2 or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

[0009] The light chain variable region comprises:

[0010] VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 2,

[0011] VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 3, and

[0012] VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 4;

[0013] The heavy chain variable region comprises:

[0014] VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 10,

[0015] VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 11, and

[0016] VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 12.

[0017] In some embodiments, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

[0018] The light chain variable region comprises:

[0019] VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:2,

[0020] VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:3, and

[0021] VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:4;

[0022] The heavy chain variable region comprises:

[0023] VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO:10,

[0024] VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO:11, and

[0025] VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO:12.

[0026] In some embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:1.

[0027] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:9.

[0028] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 (variable region of the humanized heavy chain version).

[0029] In some embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 (variable region of the humanized light chain version).

[0030] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34.

[0031] In another aspect, the present invention provides a pharmaceutical composition comprising the monoclonal antibody of the present invention or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0032] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof is conjugated to a therapeutic moiety selected from a cytotoxin, a radioisotope or a bioactive protein.

[0033] In another aspect, the present invention provides a method for treating and / or preventing Trop2-related diseases in a patient, the method comprising administering to the patient an effective amount of the monoclonal antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention.

[0034] In some embodiments, the Trop2-related disease is a malignancy with high Trop2 expression. In some embodiments, the malignancy is an epithelial malignancy. In some embodiments, the malignancy is selected from male / female reproductive system tumors (such as endometrial cancer, uterine cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer), digestive system tumors (such as pancreatic cancer, colon cancer, gastric cancer, esophageal squamous cell carcinoma, esophageal cancer, cholangiocarcinoma, intestinal cancer), head and neck tumors (such as oral squamous cell carcinoma, pharyngeal cancer), nervous system tumors (such as glioma) and respiratory system tumors (such as lung cancer, such as small cell lung cancer).

[0035] In some embodiments, the method further comprises administering to the patient other anti-tumor treatment means, such as administering chemotherapeutic agents, antibodies targeting other tumor-specific antigens or radiotherapy.

[0036] In another aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating and / or preventing Trop2-related diseases.

[0037] In some embodiments, the Trop2-related disease is a tumor with high Trop2 expression. In some embodiments, the malignancy is an epithelial malignancy. In some embodiments, the malignancy is selected from male / female reproductive system tumors (such as endometrial cancer, uterine cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer), digestive system tumors (such as pancreatic cancer, colon cancer, gastric cancer, esophageal squamous cell carcinoma, esophageal cancer, cholangiocarcinoma, intestinal cancer), head and neck tumors (such as oral squamous cell carcinoma, pharyngeal cancer), nervous system tumors (such as glioma) and respiratory system tumors (such as lung cancer, such as small cell lung cancer).

[0038] In another aspect, the present invention further provides a method for detecting the presence or expression level of Trop2 in a biological sample, comprising contacting the biological sample and a control sample with the monoclonal antibody or antigen-binding fragment thereof against Trop2 of the present invention under conditions capable of forming a complex between the monoclonal antibody or antigen-binding fragment thereof against Trop2 of the present invention and Trop2. Then detecting the formation of the complex, wherein the difference in the formation of the complex between the biological sample and the control sample indicates the presence or expression level of Trop2 in the sample.

[0039] In another aspect, the present invention also provides a method for detecting the presence of a malignant tumor in a patient, comprising:

[0040] a) contacting a biological sample obtained from the patient with the monoclonal antibody or antigen-binding fragment thereof of the present invention;

[0041] b) detecting the binding of the monoclonal antibody or antigen-binding fragment thereof of the present invention to a target antigen in the biological sample, wherein a positive detection indicates the presence of a malignant tumor in the patient.

[0042] In some embodiments of the foregoing aspects, the biological sample includes a blood sample, a lymph sample or a component thereof. In some embodiments, the malignant tumor is selected from male / female reproductive system tumors (such as endometrial cancer, uterine cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer), digestive system tumors (such as pancreatic cancer, colon cancer, gastric cancer, esophageal squamous cell carcinoma, esophageal cancer, cholangiocarcinoma, intestinal cancer), head and neck tumors (such as oral squamous cell carcinoma, pharyngeal cancer), nervous system tumors (such as glioma) and respiratory system tumors (such as lung cancer, such as small cell lung cancer).

[0043] In another aspect, the present invention provides a diagnostic agent for detecting and / or diagnosing Trop2-related diseases such as malignant tumors, which comprises the monoclonal antibody or antigen-binding fragment thereof against Trop2 of the present invention, and optionally a physiologically acceptable carrier.

[0044] In another aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof against Trop2 of the present invention in the preparation of a diagnostic agent for detecting and / or diagnosing Trop2-related diseases such as malignant tumors.

[0045] In another aspect, the present invention provides a method for detecting and / or diagnosing Trop2-related diseases such as malignant tumors in an object, comprising administering to the object the monoclonal antibody or antigen-binding fragment thereof against Trop2 of the present invention or the diagnostic agent of the present invention.

[0046] In another aspect, the present invention further provides an isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0047] In some embodiments, the nucleic acid molecule encodes the CDRs of the light chain variable region and the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention. Specifically, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 18-20 or 26-28.

[0048] In some embodiments, the nucleic acid molecule encodes the light chain variable region or the heavy chain variable region of an antibody or an antigen-binding fragment thereof of the present invention. In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 17 or 25. In some embodiments, the nucleic acid molecule has the nucleotide sequences shown in SEQ ID NOs: 35-36.

[0049] In some embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

[0050] In another aspect, the present invention also provides an expression vector comprising the nucleic acid molecule of the present invention.

[0051] In another aspect, the present invention also provides a host cell transformed with the nucleic acid molecule of the present invention or the expression vector of the present invention.

[0052] In another aspect, the present invention also provides a method for producing a monoclonal antibody against Trop2 or an antigen-binding fragment thereof, comprising:

[0053] (i) culturing the host cell of the present invention under conditions suitable for the expression of the nucleic acid molecule or the expression vector of the present invention, and

[0054] (ii) isolating and purifying the antibody or an antigen-binding fragment thereof expressed by the nucleic acid molecule or the expression vector. Brief Description of the Drawings

[0055] Figure 1 It is a diagram for identifying the chromosome number of hybridoma cells. The average chromosome number is 104, which is in line with the theoretical value of hybridoma cell chromosomes, and there are characteristic chromosomes of hybridoma cells (marked with circles).

[0056] Figure 2 It is a diagram for detecting the titer of the antibody by ELISA method, and the titer is as high as 8,000,000.

[0057] Figure 3 It is a SDS-PAGE electrophoresis diagram of the antibody (Lane M: Marker, Lane 1: IMB1636, Lane 2: VH, VL).

[0058] Figure 4 It is a diagram for detecting the purity of the antibody by HPLC, and the purity is as high as 99%.

[0059] Figure 5 It is a diagram for identifying the antibody subtype. The result determines that the antibody subclass is IgG1 and the monoclonal antibody light chain is Kappa.

[0060] Figure 6 It is the antibody-antigen affinity analysis by Biacore method, and the ka (1 / Ms) = 1.057×10 6, kd(1 / s) = 7.297×10 -4 , KD = 4.842×10 -10 。

[0061] Figure 7 The antibody-antigen affinity reaction curve was obtained by ELISA method, and the affinity constant Ka = 3.9×10 10 。

[0062] Figure 8 It is a graph showing the analysis of the binding ability of the antibody to tumor cells by immunofluorescence method, demonstrating that the antibody has a high membrane binding activity with the tumor cell membrane BxPC-3 with high expression of Trop2.

[0063] Figure 9 It is the analysis of the affinity activity of the antibody to tumor cells based on the Confocal method, demonstrating that the antibody can be efficiently internalized by Trop2-expressing positive cells HCC-827.

[0064] Figure 10 It is a graph showing the analysis of the binding ability of the antibody to tumor cells by flow cytometry, demonstrating that the antibody has a high affinity activity with the tumor cell membrane with high expression of Trop2 antigen.

[0065] Figure 11 It is the in vivo imaging of mice to detect that the antibody can highly target tumor tissues in vivo and retain in tumor tissues for more than 11 days.

[0066] Figure 12 It is the detection of human tumor tissue microarray to show that the antibody can specifically bind to human lung squamous cell carcinoma tumor tissues.

[0067] Figure 13 It is the amino acid sequence information of murine anti-Trop2 antibody.

[0068] Figure 14 It is the nucleic acid sequence information of murine anti-Trop2 antibody.

[0069] Figure 15 It is the amino acid sequence information of humanized anti-Trop2 antibody.

[0070] Figure 16 It is the nucleic acid sequence information of humanized anti-Trop2 antibody.

[0071] Figure 17 Shows the in vivo antitumor effect of Anti-Trop2-LDM. Detailed description of the invention

[0072] I. Definitions

[0073] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used herein are terms and conventional procedures widely used in the relevant fields. Meanwhile, to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0074] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether produced naturally or in part or in whole synthetically (e.g., recombinantly), including any fragment that retains the ability to bind specifically with the full-length immunoglobulin and contains at least a portion of the variable region of the immunoglobulin molecule. Thus, an antibody includes any protein having a binding domain homologous or substantially homologous to the antigen-binding domain of an immunoglobulin (antibody-binding site). Antibodies include antibody fragments, such as anti-tumor cell antibody fragments. As used herein, therefore, the term antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as but not limited to Fab fragments, Fab' fragments, F(ab’)2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd’ fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the foregoing antibodies. The antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0075] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but that includes at least the portion of the variable region of the antibody that binds the antigen (e.g., one or more CDRs and / or one or more antibody-binding sites), and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies forCancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may include multiple chains linked together, such as by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids and typically at least or about 200 amino acids. An antigen-binding fragment includes any antibody fragment that, when inserted into an antibody framework (e.g., by replacement of the corresponding region), acquires the ability to bind an antigen immunospecifically (i.e., exhibits a Ka of at least or at least about 10 7 -10 8 M -1 ).

[0076] As used herein, a "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to the other antibody molecules. This property is in contrast to the properties of a polyclonal population of antibodies, which contains antibodies having a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, such as by fusing them with myeloma cells to produce a hybridoma cell line or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence encoding the antibody.

[0077] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (usually a myeloma or lymphoma cell) produced by fusing antibody-producing lymphocytes with non-antibody-producing cancer cells. As is known to those of ordinary skill in the art, hybridomas can proliferate and continuously supply specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, e.g., Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell", it also includes subclones and progeny cells of the hybridoma.

[0078] As used herein, "conventional antibody" refers to an antibody that comprises two heavy chains (which may be designated as H and H') and two light chains (which may be designated as L and L'), and two antigen-binding sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional domain thereof that retains antigen-binding ability (e.g., the heavy chain includes, but is not limited to, V H chain, V H -C H 1 chain, and V H -C H 1-C H 2-C H 3 chain), and each light chain may be a full-length light chain or any functional domain (e.g., the light chain includes, but is not limited to, V L chain and V L -C L chain). Each heavy chain (H and H') pairs with a light chain (L and L', respectively).

[0079] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., V H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4) and two full-length light chains (V L -C L ), and a hinge region, such as an antibody naturally produced by an antibody-secreting B cell and an antibody synthetically produced having the same domains.

[0080] As used herein, dsFv refers to an Fv having engineered intermolecular disulfide bonds that stabilize the V H -V L pair.

[0081] As used herein, a Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment having the same structure synthetically produced, for example, by recombinant methods. The Fab fragment comprises a light chain (comprising V Land C L ) and another chain, said another chain comprising the variable domain of the heavy chain (V H ) and one constant domain of the heavy chain (C H 1).

[0082] As used herein, an F(ab’)2 fragment is an antibody fragment resulting from pepsin digestion of an immunoglobulin at pH 4.0 - 4.5, or a fragment having the same structure produced, for example, by recombinant methods. The F(ab’)2 fragment essentially comprises two Fab fragments, wherein each heavy chain portion contains several additional amino acids, including cysteine that forms a disulfide bond linking the two fragments.

[0083] As used herein, a Fab’ fragment is a fragment comprising half of an F(ab’)2 fragment (one heavy chain and one light chain).

[0084] As used herein, an scFv fragment refers to an antibody fragment comprising a variable light chain (V L ) and a variable heavy chain (V H ) covalently linked in any order by a polypeptide linker. The linker length is such that the two variable domains bridge with substantially no interference. An exemplary linker is (Gly - Ser) n residues, interspersed with some Glu or Lys residues to increase solubility.

[0085] The term “chimeric antibody” refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a murine antibody and the constant region sequence is derived from a human antibody.

[0086] A “humanized” antibody is a form of a non - human (e.g., murine) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen - binding subsequences of an antibody) that contains minimal sequences derived from a non - human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the complementarity - determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non - human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0087] In addition, in humanization, amino acid residues within the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may also be mutated, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, by PCR-mediated mutagenesis, and the effects of such mutations on antibody binding or other functional properties can be evaluated using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs generally do not exceed one or two. Thus, the humanized antibodies described herein also encompass antibodies that contain one or two amino acid mutations within the CDRs.

[0088] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitope determinants usually consist of the chemical active surface features of a molecule, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0089] As used herein, the variable domain or variable region is a specific Ig domain of an antibody heavy or light chain that contains an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has a variable domain V L and V H . The variable domain provides antigen specificity and is thus responsible for antigen recognition. Each variable region contains complementarity-determining regions (CDRs) and framework regions (FRs), and the CDRs are part of the antigen-binding site domain.

[0090] As used herein, "antigen-binding domain" and "antigen-binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with a cognate antigen. A natural, conventional full-length antibody molecule has two conventional antigen-binding sites, each containing a portion of the heavy chain variable region and a portion of the light chain variable region. A conventional antigen-binding site contains loops that connect anti-parallel β-strands within the variable domain. The antigen-binding site can contain other portions of the variable domain. Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions are also known as complementarity-determining regions (CDRs).

[0091] As used herein, "hypervariable region", "HV", "complementary determining region", "CDR", and "antibody CDR" are used interchangeably to refer to one of multiple portions within each variable region that together form the antigen-binding site of an antibody. Each variable domain contains three CDRs, designated CDR1, CDR2, and CDR3. For example, the light chain variable domain contains three CDRs, designated VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable domain contains three CDRs, designated VH CDR1, VH CDR2, and VH CDR3. The three CDRs in a variable region are not contiguous along the linear amino acid sequence but are proximal in the folded polypeptide. The CDRs are located within loops connecting the parallel strands of the β-sheets of the variable domain. As described herein, those skilled in the art know and can identify CDRs based on Kabat or Chothia numbering (see, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

[0092] As used herein, a framework region (FR) is a domain within an antibody variable domain that lies within the β-sheets; in terms of amino acid sequence, the FR regions are relatively more conserved than the hypervariable regions.

[0093] As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that contains an amino acid sequence that is relatively more conserved than the amino acid sequence of the variable domain. In a conventional full-length antibody molecule, each light chain has a single light chain constant region (C L ) domain, while each heavy chain contains one or more heavy chain constant regions (C H ) domains, including C H 1, C H 2, C H 3, and C H 4. Full-length IgA, IgD, and IgG isotypes contain C H 1, C H 2, C H 3, and a hinge region, while IgE and IgM contain C H 1, C H 2, C H 3, and C H 4. C H 1 and C LThe Fab arms of the domain-extended antibody molecule thus contribute to antigen interaction and rotation of the antibody arms. The antibody constant regions can serve effector functions, such as but not limited to clearing antigens, pathogens, and toxins specifically bound by the antibody, for example by interacting with various cells, biomolecules, and tissues.

[0094] As used herein, a functional region of an antibody is an antibody portion that comprises at least the V H 、V L 、C H (such as C H 1, C H 2 or C H 3), C L or the hinge region domain or at least a functional region thereof.

[0095] As used herein, a functional region of a V H domain is at least a portion of the intact V H domain that retains at least partial binding specificity of the intact V H domain (such as by retaining one or more CDRs of the intact V H domain), such that the functional region of the V H domain binds antigen either alone or in combination with another antibody domain (such as a V L domain) or a region thereof. An exemplary functional region of a V H domain is a region that comprises CDR1, CDR2, and / or CDR3 of the V H domain.

[0096] As used herein, a functional region of a V L domain is at least a portion of the intact V L domain that retains at least partial binding specificity of the intact V L domain (such as by retaining one or more CDRs of the intact V L domain), such that the functional region of the V L domain binds antigen either alone or in combination with another antibody domain (such as a V H domain) or a region thereof. An exemplary functional region of a V L domain is a region that comprises CDR1, CDR2, and / or CDR3 of the V L domain.

[0097] As used herein, "specifically binds" or "immunologically specifically binds" with respect to an antibody or an antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody-binding site of the antibody and the antigen. The antigen can be an isolated antigen or present on a tumor cell. Generally, an antibody that immunologically specifically binds (or specifically binds) an antigen binds the antigen with an affinity constant Ka of about 1×10 7 M -1 or 1×10 8 M -1 or greater (or a dissociation constant (K d )) of 1×10 -7 M or 1×10 -8 M or lower). The affinity constant can be determined by standard kinetic methods of antibody reaction, e.g., immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Patent No. 7,229,619 which describes exemplary SPR and ITC methods for calculating the binding affinity of an antibody). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare LifeSciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0098] As used herein, the term "compete", with respect to antibodies, means that a first antibody or an antigen-binding fragment thereof binds to an epitope in a manner sufficiently similar to a second antibody or an antigen-binding fragment thereof such that the binding of the first antibody to its associated epitope is detectably reduced in the presence of the second antibody as compared to the absence of the second antibody. Alternatively, this need not be the case, provided that the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, in cases where each antibody detectably inhibits the binding of the other antibody to its associated epitope or ligand, whether to the same, greater or lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes. Competing and cross-competing antibodies are both encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or fragment thereof), one of ordinary skill in the art will recognize, based on the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed by the present invention and can be used in the methods disclosed herein.

[0099] As used herein, "polypeptide" refers to two or more amino acids covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

[0100] "Isolated protein", "isolated polypeptide", or "isolated antibody" means the protein, polypeptide, or antibody (1) is not associated with the natural components that accompany it in its natural state, (2) does not contain other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a polypeptide synthesized chemically or in a cell system different from the cell system of its natural source will be "isolated" from its natural associated components. Proteins can also be isolated to render them substantially free of natural associated components, i.e., using protein purification techniques well known in the art.

[0101] In peptides or proteins, suitable conservative amino acid substitutions are known to those of ordinary skill in the art and generally can be made without altering the biological activity of the resulting molecule. Typically, those of ordinary skill in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0102] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are typically linked together by phosphodiester bonds.

[0103] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that has been separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule such as a cDNA molecule can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

[0104] Sequence "identity" has its recognized meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (See, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0105] As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter may be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

[0106] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying the polypeptide in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0107] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate and amplify a vector. A host cell can also be used to express a polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. A host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

[0108] "Codon optimization" refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of a native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for a particular amino acid. Codon bias (differences in codon usage between organisms) is often related to the translational efficiency of messenger RNA (mRNA), which is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs within a cell generally reflects the codons most frequently used for peptide synthesis. Thus, a gene can be customized for optimal gene expression based on codon optimization in a given organism. Codon usage tables are readily available, for example, in the www.kazusa.orjp / codon / Codon Usage Database available at [website], and these tables can be adapted for use in different ways. See, Nakamura Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0109] As used herein, a "vector" is a replicable nucleic acid which, when transformed into a suitable host cell, can express one or more heterologous proteins therefrom. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction enzyme digestion and ligation. Vectors also include those which contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. Vectors generally remain free, but can be designed to integrate a gene or portion thereof into the chromosomes of the genome. Vectors which are artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those of skill in the art.

[0110] As used herein, a vector also includes a "viral vector" or "vector of a virus". A vector of a virus is an engineered virus which is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

[0111] As used herein, an "expression vector" includes a vector capable of expressing DNA which is operably linked to regulatory sequences capable of affecting the expression of such DNA fragments, such as a promoter region. Such additional fragments can include promoter and terminator sequences and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally are derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector which, when introduced into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those of skill in the art and include expression vectors replicable in eukaryotic cells and / or prokaryotic cells and expression vectors which remain free or integrate into the genome of the host cell.

[0112] As used herein, "treating" an individual having a disease or medical condition means that the symptoms of the individual are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, therapy and / or cure. Prevention refers to preventing a potential disease and / or preventing the worsening of symptoms or the progression of a disease. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof and any composition provided herein.

[0113] As used herein, "efficacy" means the effect resulting from the treatment of an individual which modifies, typically ameliorates or improves the symptoms of a disease or medical condition, or cures the disease or medical condition.

[0114] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or disorder.

[0115] As used herein, "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the occurrence or recurrence of a disease or symptom. A fully prophylactically effective dose need not occur by administration of a single dose and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0116] As used herein, the term "patient" refers to a mammal, such as a human.

[0117] II. Monoclonal Antibodies Against Trop2

[0118] The present invention is through the hybridoma fusion technology. Immunized spleen cells are prepared by immunizing BALB / C mice with Trop2 antigen, and fused with SP2 / 0 cells to establish a hybridoma cell line library secreting anti-Trop2 monoclonal antibodies. The hybridoma cell line Y1636-1 with stable and high antibody secretion is screened out from it. Monoclonal antibodies are produced in large quantities by the ascites induction method to prepare murine anti-Trop2 monoclonal antibodies with high titer, high affinity activity, and high specificity, and the amino acid sequence determination of the antibody and the humanization transformation of the murine monoclonal antibody are completed. The murine hybridoma cell line Y1636-1 that produces anti-Trop2 monoclonal antibodies was deposited on June 25, 2019, with the deposit number CGMCC No. 18167 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms.

[0119] Thus, in one aspect, the present invention provides a hybridoma cell that was deposited on June 25, 2019, with the deposit number CGMCC No. 18167 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms.

[0120] In one aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody is produced by murine hybridoma cells deposited on June 25, 2019, with the deposit number CGMCC No. 18167 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms.

[0121] In one aspect, the present invention provides an isolated monoclonal antibody against Trop2 or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of a monoclonal antibody produced by mouse hybridoma cells deposited with the China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC No. 18167 on June 25, 2019.

[0122] In one aspect, the present invention provides an isolated monoclonal antibody against Trop2 or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises the light chain variable region and the heavy chain variable region of a monoclonal antibody produced by mouse hybridoma cells deposited with the China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC No. 18167 on June 25, 2019.

[0123] Accordingly, the present invention provides an isolated monoclonal antibody against Trop2 or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region.

[0124] The light chain variable region comprises:

[0125] VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions, or additions relative to SEQ ID NO: 2.

[0126] VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions, or additions relative to SEQ ID NO: 3, and

[0127] VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions, or additions relative to SEQ ID NO: 4.

[0128] The heavy chain variable region comprises:

[0129] VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions, or additions relative to SEQ ID NO: 10.

[0130] VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions, or additions relative to SEQ ID NO: 11, and

[0131] VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:12.

[0132] In some embodiments, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region,

[0133] The light chain variable region comprises:

[0134] VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:2,

[0135] VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:3, and

[0136] VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:4;

[0137] The heavy chain variable region comprises:

[0138] VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO:10,

[0139] VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO:11, and

[0140] VH CDR3, which comprises the amino acid sequence shown in SEQ ID NO:12.

[0141] In some embodiments, the monoclonal antibody is a humanized antibody.

[0142] In some embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:1. In some embodiments, the light chain variable region comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO:1.

[0143] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:9. In some embodiments, the heavy chain variable region comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO:9.

[0144] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33 (variable region of the humanized heavy chain version).

[0145] In some embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34 (variable region of the humanized light chain version).

[0146] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34.

[0147] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention is derived from a monoclonal antibody produced by a mouse hybridoma cell deposited with the China General Microbiological Culture Collection Center under the accession number CGMCC No. 18167 on June 25, 2019. In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention binds to the same epitope on Trop2 as the monoclonal antibody produced by the mouse hybridoma cell deposited with the China General Microbiological Culture Collection Center under the accession number CGMCC No. 18167 on June 25, 2019. In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention competes with the monoclonal antibody produced by the mouse hybridoma cell deposited with the China General Microbiological Culture Collection Center under the accession number CGMCC No. 18167 on June 25, 2019 for binding to Trop2.

[0148] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention specifically targets tumor cells. The tumor cells specifically targeted by the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention include, but are not limited to, male / female reproductive system tumor cells (such as endometrial cancer cells, uterine cancer cells, cervical cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells), digestive system tumor cells (such as pancreatic cancer cells, colon cancer cells, gastric cancer cells, esophageal squamous cell carcinoma cells, esophageal cancer cells, cholangiocarcinoma cells, intestinal cancer cells), head and neck tumor cells (such as oral squamous cell carcinoma cells, pharyngeal cancer cells), nervous system tumor cells (such as glioma cells), and respiratory system tumor cells (such as lung cancer cells, such as small cell lung cancer).

[0149] III. Nucleic Acids, Vectors, and Antibody Production Methods

[0150] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention described above. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized for the host cell to be expressed. In some embodiments, the nucleic acid molecule of the present invention is operably linked to an expression regulatory sequence.

[0151] In some embodiments, the nucleic acid molecule encodes the CDRs of the light chain variable region and the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention. Specifically, the nucleic acid molecule encodes VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 or VH CDR3. Specifically, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 18-20 or 26-28.

[0152] In some embodiments, the nucleic acid molecule encodes the light chain variable region or the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 17 or a nucleotide sequence having at least 85%, at least 90%, at least 95% or higher sequence identity relative to SEQ ID NO: 17. In some embodiments, the nucleic acid molecule has a nucleotide sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 17.

[0153] In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 25 or a nucleotide sequence having at least 85%, at least 90%, at least 95% or higher sequence identity relative to SEQ ID NO: 25. In some embodiments, the nucleic acid molecule has a nucleotide sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity relative to SEQ ID NO: 25.

[0154] In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 35.

[0155] In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 36.

[0156] The present invention also provides an expression vector comprising at least one nucleic acid molecule of the present invention described above.

[0157] The present invention also provides a host cell transformed with at least one nucleic acid molecule or expression vector of the present invention described above.

[0158] In another aspect, the present invention provides a method for producing the antibody or antigen-binding fragment thereof of the present invention, comprising:

[0159] (i) culturing the host cell of the present invention under conditions suitable for the expression of the nucleic acid molecule or expression vector, and

[0160] (ii) isolating and purifying the antibody or antigen-binding fragment thereof expressed by the host cell.

[0161] The present invention also relates to an isolated antibody or antigen-binding fragment thereof obtained by the method of the present invention as described above, which is capable of specifically binding to Trop2.

[0162] In some embodiments, the nucleic acid molecule of the present invention comprises the nucleotide sequences shown in SEQ ID NO: 17-20, SEQ ID NO: 25-28, and SEQ ID NO: 35-36.

[0163] IV. Antibody Conjugates

[0164] The present invention also provides an antibody conjugate, which comprises the antibody or antigen-binding fragment thereof of the present invention and a therapeutic moiety conjugated to the antibody or antigen-binding fragment thereof. In some embodiments, the therapeutic moiety includes, for example, cytotoxins, radioisotopes, or bioactive proteins, etc.

[0165] Cytotoxins include any agent that is harmful to cells (e.g., cell killing). Examples include: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide glucuronide, etoposide thioglucoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, didehydroxy anthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs.

[0166] Therapeutic moieties that can be used for conjugation also include, for example: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., nitrogen mustard, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly called daunomycin) and doxorubicin), antibiotics (e.g., actinomycin D (formerly called actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0167] Other preferred examples of therapeutic cytotoxins that can be conjugated to the Trop2 - specific antibodies or fragments thereof of the present invention include esperamicin, calicheamicin, maytansine, auristatin, and their derivatives.

[0168] The cytotoxins can be conjugated to the antibodies of the present invention using linker technologies employed in the art. Examples of linker types that have been used to conjugate cytotoxins to Trop2 - specific antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide - containing linkers. Optionally, for example, linkers that are cleavable at low pH in the lysosomal compartment or are cleavable by proteases, such as proteases that are preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsin B, C, D), can be selected.

[0169] The antibodies of the present invention can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radio - antibody conjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine - 131, indium - 111, yttrium - 90, and lutetium - 177. Methods for preparing radio - antibody conjugates are well - established in the art.

[0170] The antibodies of the present invention can also be conjugated to proteins having the desired biological activity, which can be used to modify specific biological responses. Such bioactive proteins include, for example: toxins with enzymatic activity or their active fragments, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins, such as tumor necrosis factor or interferon - γ; or biological response modifiers, such as lymphokines, interleukin - 1 (“IL - 1”), interleukin - 2 (“IL - 2”), interleukin - 6 (“IL - 6”), interleukin - 10 (“IL - 10”), granulocyte - macrophage colony - stimulating factor (“GM - CSF”), granulocyte colony - stimulating factor (“G - CSF”), or other immune factors such as IFN, etc.

[0171] In some specific embodiments, the therapeutic moiety is lidamycin. For example, lidamycin comprises the apoprotein LDP shown in SEQ ID NO:37, and the chromophore AE shown in Formula I that binds to LDP. In some embodiments, the LDP is linked to the antibody of the present invention, for example, to the N - terminus of the light chain of the antibody of the present invention, through a linker such as the linker shown in SEQ ID NO:38.

[0172]

[0173] V. Disease Treatment and / or Prevention

[0174] The present invention provides a method for treating and / or preventing Trop2-related diseases such as malignancies in a patient, the method comprising administering to the patient an effective amount of an antibody against Trop2 or an antigen-binding fragment thereof of the present invention or an antibody conjugate of the present invention.

[0175] Trop2-related diseases that can be treated and / or prevented by the method of the present invention are, for example, tumors with high Trop2 expression, including but not limited to male / female reproductive system tumors, such as endometrial cancer, uterine cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer; digestive system tumors, such as pancreatic cancer, colon cancer, gastric cancer, esophageal squamous cell carcinoma, esophageal cancer, cholangiocarcinoma, intestinal cancer; head and neck tumors, such as oral squamous cell carcinoma, throat cancer; nervous system tumors, such as glioma; and respiratory system tumors, such as lung cancer, preferably small cell lung cancer.

[0176] In some embodiments, the method further comprises administering to the patient other anti-tumor treatment means, such as administering chemotherapeutic agents, antibodies targeting other tumor-specific antigens, or radiotherapy.

[0177] VI. Pharmaceutical Compositions

[0178] The present invention also provides a pharmaceutical composition comprising an antibody against Trop2 or an antigen-binding fragment thereof of the present invention or an antibody conjugate of the present invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition is used for treating and / or preventing Trop2-related diseases such as malignancies in a patient.

[0179] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody molecule, immunoconjugate, can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0180] The pharmaceutical composition of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0181] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents.

[0182] Microbial presence can be ensured against by a sterilization procedure or by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. In many cases, an isosmotic agent is preferably included in the composition, for example, sugars, polyols such as mannitol, sorbitol or sodium chloride. Prolonged absorption of injectable drugs can be achieved by adding a delayed absorbent, such as monostearate and gelatin, to the composition.

[0183] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except to the extent that any are incompatible with the active compound, may be in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

[0184] Therapeutic compositions generally must be sterile and stable under the conditions of preparation and storage. The compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using a coating, such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants, appropriate fluidity can be maintained.

[0185] A sterile injectable solution can be prepared by incorporating the active compound in the required amount into a suitable solvent and, if necessary, adding one or a combination of the ingredients listed above, followed by sterile microfiltration. Generally, a dispersion is prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and the other required ingredients listed above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), to obtain a powder of the active ingredient plus any additional required ingredients from its pre-sterile filtered solution.

[0186] The amount of active ingredient that can be combined with the carrier material to produce a single dosage form varies depending on the subject to be treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Usually, on a 100% basis, this amount ranges from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, in combination with a pharmaceutically acceptable carrier.

[0187] The dosage regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be proportionally decreased or increased as required by the exigencies of the treatment situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form which are readily administrable and provide for a uniform dosage. The unit dosage form used herein refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined quantity of the active compound which, in association with the required pharmaceutical carrier, is calculated to produce the desired therapeutic effect. The specification of the particular unit dosage forms of the invention is limited to and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of the individual sensitivity.

[0188] For the administration of antibody molecules, the dosage range is from about 0.0001 to 100 mg / kg, more usually 0.01 to 20 mg / kg recipient body weight. For example, the dose can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight or 20 mg / kg body weight, or in the range of 1 - 20 mg / kg. Exemplary treatment regimens call for administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3 - 6 months, or an initial dosing interval slightly shorter (such as once a week to once every three weeks) with a lengthened dosing interval later (such as once a month to once every 3 - 6 months).

[0189] Alternatively, the antibody molecule can also be administered as a sustained release formulation, in which case less frequent dosing is required. The dose and frequency vary according to the half-life of the antibody molecule in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The administered dose and frequency vary according to whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over a long period of time. Some patients are treated continuously for the rest of their lives. In therapeutic applications, sometimes higher doses are required to be administered at shorter intervals until the progression of the disease is alleviated or stopped, preferably until the patient shows partial or complete improvement of the disease symptoms. Thereafter, the patient can be administered in a prophylactic regimen.

[0190] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may vary to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, and that is non-toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention being applied, the route of administration, the time of administration, the excretion rate of the specific compound being applied, the duration of treatment, other drugs, compounds, and / or materials being co-administered with the specific composition being applied, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0191] An "effective amount" of an antibody or antigen-binding fragment thereof of the present invention or an antibody conjugate of the present invention preferably results in a decrease in the severity of the symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or the prevention of damage or disability caused by the affliction of the disease. For example, for the treatment of tumors, an "effective amount" of an antibody or antigen-binding fragment thereof of the present invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80% relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy against human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which inhibition can be measured in vitro by assays well known to those skilled in the art. An effective amount of an antibody or antigen-binding fragment thereof of the present invention is capable of reducing tumor size or otherwise alleviating the symptoms of the subject, such as preventing and / or treating metastasis or recurrence. Those skilled in the art can determine such amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0192] The antibody or antigen-binding fragment thereof of the present invention, the antibody conjugate of the present invention, or the pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more methods well known in the art. Those skilled in the art will understand that the route of administration and / or mode varies depending on the desired result. Preferred routes of administration of the antibody of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0193] Alternatively, the antibody or antigen-binding fragment thereof, antibody conjugate, or pharmaceutical composition of the present invention may also be administered by non-parenteral routes, such as local, epidermal, or mucosal routes, for example, intranasal, oral, vaginal, rectal, sublingual, or topical.

[0194] The active compounds can be prepared with carriers that protect the compounds from rapid release, for example, controlled-release formulations, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are protected by patents or are generally known to those skilled in the art. See, for example, Sustained and controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0195] In certain embodiments, the antibodies of the present invention can be formulated to ensure proper distribution in the body. For example, the blood-brain barrier (BBB) blocks many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention can cross the BBB (if needed), they can be formulated in, for example, liposomes.

[0196] VII. Combination Therapies

[0197] The Trop2-targeting antibody or antigen-binding fragment thereof, antibody conjugate, or pharmaceutical composition of the present invention can be administered in combination with a chemotherapeutic agent or an antibody targeting other tumor antigens.

[0198] There are no particular limitations on the chemotherapeutic agents or antibodies targeting other tumor antigens that can be used in combination with the antibodies of the present invention or the pharmaceutical compositions of the present invention. Examples of such chemotherapeutic agents and antibodies targeting other tumor antigens include, but are not limited to: ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine, tegafur, tegafur-uracil, TS-1, doxifluridine, nelarabine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrexate, irinotecan, etoposide, eribulin, sobuzoxane, docetaxel, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine, actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, nesiritide, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, mitoxantrone, oxaliplatin, carboplatin, cisplatin, nedaplatin, anastrozole, exemestane, ethinylestradiol, chlormadinone acetate, goserelin, tamoxifen, dexamethasone, bicalutamide, toremifene, flutamide, prednisolone, diethylstilbestrol, mitotane, methyltestosterone, leuprorelin, letrozole, medroxyprogesterone acetate, ibritumomab tiuxetan, imatinib, everolimus, erlotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, tretinoin, pembrolizumab, bevacizumab, bortezomib, and lapatinib. In a specific embodiment, the chemotherapeutic agent is a platinum-containing chemotherapeutic agent, such as cisplatin.

[0199] The antibodies of the present invention or the antibody conjugates of the present invention and the chemotherapeutic agents or antibodies targeting other tumor antigens can all be administered at one time or separately. When administered separately (in the case of using mutually different administration regimens), they can be administered continuously without interruption or at a predetermined interval.

[0200] There are no particular limitations on the combined doses of the antibodies of the present invention or the antibody conjugates of the present invention and the chemotherapeutic agents or antibodies targeting other tumor antigens in the pharmaceutical compositions of the present invention. As described above, the dose of the antibodies of the present invention can be determined by referring to the dose when the antibodies are used alone. The chemotherapeutic agents and antibodies targeting other tumor antigens can be used according to the doses indicated on their respective drugs or can be reduced (taking into account the combined effect with the antibodies of the present invention).

[0201] The antibodies of the present invention or the antibody conjugates of the present invention or the pharmaceutical compositions of the present invention can also be combined with radiotherapy, for example, including administering ionizing radiation to a patient, which is earlier than, during, and / or later than the administration process of the antibodies or pharmaceutical compositions of the present invention.

[0202] VIII. Detection and Diagnosis

[0203] In another aspect, the present invention also provides a method for detecting the presence of Trop2 or the expression level of Trop2 in a biological sample, comprising contacting the biological sample and a control sample with the monoclonal antibody against Trop2 of the present invention or its antigen-binding fragment under conditions where a complex can be formed between the monoclonal antibody against Trop2 of the present invention or its antigen-binding fragment and Trop2. Then, the formation of the complex is detected, wherein the difference in the formation of the complex between the biological sample and the control sample indicates the presence of Trop2 or the expression level of Trop2 in the sample.

[0204] In some embodiments, the biological sample is an ex vivo sample.

[0205] It has been found that Trop2 is highly expressed in many tumors, especially epithelial malignancies. Therefore, the monoclonal antibody against Trop2 of the present invention or its antigen-binding fragment can be used for diagnosing Trop2-related malignancies.

[0206] Accordingly, the present invention also provides a method for detecting the presence of malignancy in a patient, comprising detecting the expression level of Trop2 in a biological sample from the patient by the method of the present invention, wherein a higher expression level of Trop2 in the biological sample of the patient than that in a control biological sample indicates the presence of malignancy in the patient.

[0207] In some embodiments, the control biological sample is a biological sample from a healthy individual. In some embodiments, the control biological sample is a biological sample from an individual known not to have malignancy.

[0208] The biological sample includes but is not limited to tissue samples, blood samples, lymph samples, etc.

[0209] In another aspect, the present invention provides a diagnostic agent for detecting and / or diagnosing Trop2-related diseases such as malignancies, which comprises the monoclonal antibody against Trop2 of the present invention or its antigen-binding fragment, and optionally a physiologically acceptable carrier. In some embodiments, the diagnostic agent is a contrast agent.

[0210] In another aspect, the present invention provides the use of the monoclonal antibody against Trop2 of the present invention or its antigen-binding fragment in the preparation of a diagnostic agent for detecting and / or diagnosing Trop2-related diseases such as malignancies. In some embodiments, the diagnostic agent is a contrast agent.

[0211] In another aspect, the present invention provides a method for detecting and / or diagnosing Trop2-related diseases such as malignancies in a subject, comprising administering to the subject a monoclonal antibody against Trop2 of the present invention or an antigen-binding fragment thereof or a diagnostic agent of the present invention.

[0212] In some embodiments of the above aspect of the present invention, the monoclonal antibody or an antigen-binding fragment thereof of the present invention is further conjugated with a fluorescent dye, chemical substance, polypeptide, enzyme, isotope, tag, etc. that can be used for detection or detected by other reagents.

[0213] The malignancies include but are not limited to male / female reproductive system tumors, such as endometrial cancer, uterine cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer; digestive system tumors, such as pancreatic cancer, colon cancer, gastric cancer, esophageal squamous cell carcinoma, esophageal cancer, cholangiocarcinoma, intestinal cancer; head and neck tumors, such as oral squamous cell carcinoma, pharyngeal cancer; nervous system tumors, such as glioma; and respiratory system tumors, such as lung cancer, preferably small cell lung cancer.

[0214] IX. Kits

[0215] Also within the scope of the present invention is a kit for the method of the present invention, which kit comprises a monoclonal antibody or an antigen-binding fragment thereof of the present invention or an antibody conjugate of the present invention or a pharmaceutical composition of the present invention or a diagnostic agent of the present invention, and instructions for use. The kit may further comprise at least one additional detection reagent for detecting the presence of the monoclonal antibody of the present invention. The kit generally comprises a label indicating the intended use and / or method of use of the contents of the kit. The term label includes any written or recorded material provided on, with, or otherwise with the kit. Examples

[0216] Example 1. Establishment of hybridoma cell line

[0217] 1. Animal immunization

[0218] Primary immunization: Trop2 antigen was mixed with complete Freund's adjuvant in equal volume and emulsified completely under ice bath conditions by ultrasonic treatment, and 300 μl per mouse was injected intraperitoneally.

[0219] Secondary immunization: After a two-week interval, the antigen solution was mixed with incomplete Freund's adjuvant in equal volume and emulsified completely by ultrasonic treatment under ice bath conditions, and 300 μl per mouse was injected intraperitoneally.

[0220] Tertiary immunization: The operation was the same as that of the secondary immunization step. One week later, the antibody titer of the mouse serum was detected by ELISA, and the serum titer reached one million.

[0221] Quaternary immunization: 300 μl of 50 μg antigen solution was injected intraperitoneally into each mouse, and cell fusion was carried out three days later.

[0222] 2. Preparation of peritoneal macrophages

[0223] Take healthy 5-week-old female BALB / c mice, sacrifice them by cervical dislocation, immerse them in 75% alcohol for 5 minutes for disinfection, aseptically open the abdominal skin in a dissection tray in a laminar flow hood, expose the peritoneal muscle layer, lift the abdominal muscles with sterile forceps, inject 5 ml of incomplete RPMI 1640 culture medium into the abdominal cavity, gently massage the abdomen for 1 - 2 minutes, then aspirate the peritoneal fluid and transfer it to a 50 ml plastic centrifuge tube. Repeat steps 2 - 3 times. Centrifuge at 1000 rpm for 5 minutes to remove the supernatant. Resuspend with complete 1640 medium supplemented with HAT, plate it on a 96-well culture plate, and incubate it in an incubator at 37°C and 5% CO2 for one day. Check for contamination under a microscope and reserve it for cell fusion.

[0224] 3. Treatment of myeloma cells

[0225] Inoculate SP2 / 0 cells on the back of BALB / c mice. When the tumor mass grows to about 500 mm 3 or so, dissect the tumor mass, grind it into a cell suspension, perform a viable cell count using 0.4% trypan blue staining solution (take 0.1 ml of the cell suspension and add it to 0.9 ml of trypan blue staining solution for counting. Cells not stained blue are viable cells). Myeloma cells with a survival rate greater than 90% can be used for fusion and are placed at 37°C for standby.

[0226] 4. Preparation of splenic lymphocytes

[0227] Take BALB / c mice with serum antibody titers reaching the fusion index, sacrifice them and soak them in 75% alcohol for 5 minutes for disinfection. Take out the spleen in a laminar flow hood, gently wash it, remove the surrounding connective tissue, cut the spleen into small pieces with sterile scissors, crush the spleen by extruding it with the inner core of a 2.5 ml syringe, and filter it through a 200-mesh copper mesh to obtain a splenocyte suspension. Centrifuge at 1000 rpm for 5 minutes, wash it twice with serum-free medium, centrifuge as above, and perform a viable cell count on the above suspension using 0.4% trypan blue staining solution and reserve it for use.

[0228] 5. Cell fusion

[0229] Take 1×10 8 spleen cells and 2×10 7-5 ×10 7Myeloma cells SP2 / 0-Ag14 (usually at a ratio of 10:1 - 10:5) are mixed in a 50 ml centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant is aspirated completely, the bottom of the centrifuge tube is gently tapped to make the precipitated cells loose and uniform, and it is preheated in a 40 °C water bath for standby. When in use, 1 ml of 50% PEG-1450 solution (pH 8.0) preheated to 40 °C is added evenly and slowly within 45 s. While adding, the centrifuge tube is gently rotated to make the PEG solution fully and evenly contact the loose cells. 1 ml of serum-free medium preheated to 40 °C is slowly and evenly added to the bottom of the centrifuge tube within 60 s while rotating the centrifuge tube; 5 ml of serum-free medium preheated to 40 °C is slowly and evenly added to the bottom of the centrifuge tube within 60 s while rotating the centrifuge tube; 10 ml of serum-free medium preheated to 40 °C is slowly and evenly added to the bottom of the centrifuge tube within 90 s while rotating the centrifuge tube; repeat once; centrifuge at 1000 rpm for 5 min and discard the supernatant. Add 1640 complete medium supplemented with HAT culture medium, gently pipette to form a cell suspension, inoculate into 7 - 8 well-checked 96-well plates containing macrophages, and then culture in an incubator at 37 °C and 5% CO2. After 5 days, 1 / 2 of the medium is replaced with HAT medium. After 7 - 10 days, the HAT medium is replaced with HT medium. After the 14th day, ordinary complete medium can be used. Observe the growth of hybridoma cells. When they grow to more than 1 / 10 of the bottom area of the well, aspirate the supernatant for antibody detection, and perform the second antibody detection at the same interval. Compare the results of the two positive values, pick out the wells with increased or unchanged positive values, initially determine them as positive wells, and further screen and clone the positive wells.

[0230] 6. Screening and subcloning of positive hybridomas

[0231] The dilution culture method is used for re-screening and subcloning of hybridoma cells. Prepare mouse peritoneal cells as above. Pick up the hybridoma cell suspension from the positive wells, dilute the hybridoma cells with HT medium containing 20% serum to different dilution cell suspensions containing 2.5, 15, and 50 cells per milliliter, plate them on 96-well plates, 0.2 ml / well, and the number of hybridoma cells per well is 0.5, 3, and 10 respectively. Culture at 37 °C and 5% CO2 for 7 - 10 days. When visible clones appear, observe under the microscope, pick out the wells with only single clone growth (the clone is round and the growth trajectory arc of the edge cells is smooth, which is a monoclonal), and perform antibody detection. Take the cells from the wells with positive antibody detection and perform 2 - 3 times of the above dilution culture subcloning screening, then expand the culture and freeze them.

[0232] 7. Identification of hybridoma cell stability

[0233] The hybridoma cell line was continuously cultured before and after cryopreservation, and the antibody titer in the cell supernatant was detected by the indirect ELISA method. The results showed that there was no significant change in the antibody titer during passage and after re-thawing, demonstrating that the hybridoma cells could stably secrete anti-Trop2 monoclonal antibody( Figure 2 ).

[0234] The hybridoma cell was deposited with the China General Microbiological Culture Collection Center (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) on June 25, 2019 under the accession number CGMCC No. 18167.

[0235] Example 2 Preparation of Anti-Trop2 Monoclonal Antibody

[0236] I. Antibody Preparation

[0237] Female BALB / c mice at 7-8 weeks old were pretreated by intraperitoneal injection of 500 μl Freund's incomplete adjuvant. After 7-10 days, hybridoma cells in good growth state were collected, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The hybridoma cells were resuspended in PBS to prepare a cell suspension with a concentration of 1×10 7 cells / ml. 200 μl of the cell suspension was injected into the peritoneal cavity of each mouse. The abdominal swelling of the mice was observed every day after 3 days of injection. Usually, ascites was collected after 7-10 days. The collected ascites was centrifuged at 10000 rpm for 30 min, the supernatant was collected and filtered through a 0.45 μm filter membrane to remove large lipid masses, and the ascites was preliminarily extracted by the ammonium sulfate precipitation method and purified by a Protein G (1 ml) affinity chromatography column to prepare anti-Trop2 monoclonal antibody.

[0238] II. Antibody Identification

[0239] 1. Antibody Purity Identification

[0240] The purity of the anti-Trop2 monoclonal antibody was detected by HPLC. The detection conditions of HPLC were as follows: gel column: SEC S3000; flow rate: 1 ml / min; sample loading volume: 20 μl; mobile phase: the acetonitrile ratio increased from 5-95% in a gradient from 0-16 min, with a total duration of 20 min; detector: 280 nm; the purity of the prepared monoclonal antibody was judged by the integral peak area of the monoclonal antibody characteristic absorption at 280 nm( Figure 4 ).

[0241] Detection of antibody purity by SDS-PAGE electrophoresis: Take 10 μl of the anti-Trop2 monoclonal antibody sample, add 1 / 4 volume of 5× loading buffer, and boil it in boiling water for 5 min for denaturation and use. Prepare a separating gel with a concentration of 12% and a stacking gel with a concentration of 5%. Connect the power supply and perform electrophoresis at 80 v for 30 min. After the sample enters the separating gel, adjust the voltage to 120 v and perform electrophoresis for 1.5 h. After electrophoresis, take out the gel, put it into Coomassie Brilliant Blue staining solution for staining for 3 h, then decolorize it overnight at room temperature with the decolorizing solution, and take a picture with a gel imaging system( Figure 3 ).

[0242] 2. Identification of antibody class and subclass

[0243] Use the SBA Clonotyping System-HRP mouse monoclonal antibody typing kit to identify the subclass of the anti-Trop2 monoclonal antibody. Coat the enzyme-linked immunosorbent assay (ELISA) plate with the capture antibody at a concentration of 5-10 μg / ml, 100 μl / well, and incubate overnight at 4 °C; wash 3 times with PBST, 5 min each time. Block with 200 μl / well of PBST containing 2% BSA at 37 °C for 2 h, and wash as above; add the diluted anti-Trop2 monoclonal antibody solution diluted by an appropriate multiple, incubate at 37 °C for 2 h, and wash as above; add different types of secondary antibodies at appropriate dilution factors, including: Goat Anti-Mouse Ig, Human ads-HRP (positive control); Goat Anti-Mouse IgA-HRP; Goat Anti-Mouse IgG1, Human ads-HRP; Goat Anti-Mouse IgG2a, Human ads-HRP; Goat Anti-Mouse IgG2b, Human ads-HRP; Goat Anti-Mouse IgG3, Human ads-HRP; Goat Anti-Mouse IgM, Human ads-HRP; Goat Anti-Mouse Kappa-HRP; Goat Anti-Mouse Lambda-HRP; incubate at 37 °C for 1.5 h, and wash as above; add 100 μl / well of TMB substrate for color development, place it in the dark at room temperature for 15 min, then add 100 μl / well of 2M H2SO2 to terminate the reaction, and measure the absorbance value at A450. The positive value well is the corresponding antibody subclass type( Figure 5 ).

[0244] 3. Determination of antibody amino acid sequence

[0245] Extract the RNA of hybridoma cells, reverse transcribe to obtain a cDNA library, and amplify the monoclonal antibody gene sequence by PCR with special primers, so as to obtain the amino acid sequences of the heavy and light chains of the monoclonal antibody.

[0246] 4. Humanization of Murine Antibodies

[0247] Design and Synthesis of Humanized Antibodies: Based on the murine antibody sequence, humanization design was carried out. After synthesizing the humanized antibody sequence, a humanized antibody expression vector was constructed; at the same time, a control antibody expression vector of a human-mouse chimeric antibody was constructed as a control. The above-prepared expression vectors were subjected to large-scale plasmid extraction to prepare transfection-grade plasmids.

[0248] Expression and Purification of Humanized Antibodies: The above-prepared humanized antibody expression vector was transiently transfected into mammalian cells, and the recombinant antibody was purified using Protein A. After concentration, it was quantified using the BCA method; using the target protein provided by Party A as an antigen, the antigen was coated on a 96-well plate, and the binding of the humanized antibody to the target protein was detected by ELISA.

[0249] Determination of the Affinity of Humanized Antibodies: The BiaCore T200 was used to detect the affinity of the above-prepared humanized antibody to the target protein.

[0250] Example 3. Determination of the Affinity Activity of Anti-Trop2 Monoclonal Antibodies

[0251] 1. Determination of the Antigen-Antibody Affinity Constant by ELISA

[0252] The Trop2 antigen was diluted to three dilutions (0.1 μg / ml, 0.2 μg / ml, 0.4 μg / ml) and coated on an enzyme-linked immunosorbent assay (ELISA) plate. The anti-Trop2 monoclonal antibody was diluted at concentrations of (1, 0.2, 0.04, 0.008, 0.0016, 0.00032, 0.000064, 0.0000128 μg / ml). The antigen-antibody reaction curve was determined by indirect ELISA. When the curve approached the plateau, it indicated that all the antigen was bound. The antibody concentration (mol / L) corresponding to 50% of the maximum binding to the antigen was found on the curve and substituted into the following formula to calculate Ka. Ka = (n - 1) / 2(n[ab] - [ab]t). The unit of Ka is M -1 . Among them, [Ab] is the antibody concentration corresponding to A = 1 / 2Amax when the antigen concentration is [Ag]; [Ab]t represents the antibody concentration corresponding to A = 1 / 2Amax when the antigen concentration is [Ag]t; [Ag], [Ag]t: represent the antigen concentrations; n is the dilution factor between the antigen [Ag] and [Ag]t.( Figure 7 )

[0253] 2. Detection of the Antigen-Antibody Affinity by Biacore

[0254] The anti-Trop2 antibody was amino-coupled to the CM5 chip at pH 5.0, with an antibody concentration of 10 μg / ml, an injection time of 60 s, a flow rate of 10 μl / min, and a coupling amount of 378.6. Different concentration gradients of Trop2 antigen (0, 0.814, 1.628, 3.256, 6.512, 13.025, 26.05, 52.1 μg / ml) were passed over the chip, with an injection time of 60 s, a flow rate of 30 μl / min, a dissociation time of 600 s, and a regeneration condition of glycine-HCl, pH 1.5 (100 s, 30 μl / min) to obtain the Biacore affinity diagram and the affinity constant.( Figure 6 )

[0255] 3. Detection of the binding ability of the antibody to tumor cells by immunofluorescence

[0256] NIH3T3, MDA-MB-231, and BXPC-3 cells were cultured separately in cell slides at 1×10 4 cells / well and cultured at 37 °C for 24 h, then fixed with 4% paraformaldehyde for 30 min, washed 3 times with pre-cooled PBS at 4 °C, blocked overnight at 4 °C with a PBST solution containing 1% BSA, added the purified anti-Trop2 antibody at a concentration of 10 μg / ml and incubated overnight at 4 °C. The SP2 / 0 ascites was used as a negative control, washed 3 times with pre-cooled PBST at 4 °C, added the rhodamine-labeled goat anti-mouse IgG secondary antibody diluted 1:200, incubated at room temperature in the dark for 1.5 h, washed 3 times with PBST, added 1 drop of DAPI (1 mg / ml) to each well and incubated for 10 min, and observed and photographed under a fluorescence microscope.( Figure 8 )

[0257] 4. Confocal immunofluorescence detection of anti-Trop2 antibody

[0258] The HCC-827 cells in the logarithmic growth phase were made into a single-cell suspension. After cell counting, they were inoculated at 1×10 4 cells / well / 200 μl in cell slides and cultured at 37 °C for 24 h, then added the anti-Trop2 antibody at 10 μg / mL, 200 μL / well; for the binding of the antibody to the cell surface, after adding the antibody IMB1636, incubated at 4 °C for 30 min, collected the cells, washed 3 times with PBS, fixed with 4% paraformaldehyde (200 μL / well) for 15 min, washed 3 times with PBST, permeabilized with 0.2% Triton-X100 (200 μl + 100 ml PBS) at 200 μL / well for 10 min, washed 3 times with PBST, blocked with 5% BSA at 37 °C for 30 min. Added the secondary antibody (goat anti-mouse AF488) at 3 μL + 1.5 mL PBST, 200 μL / well, incubated at 37 °C for 30 min, washed 5 times with PBST, stained with DAPI for 15 min, washed 3 times with PBST, added the anti-fluorescence quencher, and observed under a confocal microscope.

[0259] For the case of cell endocytosis of antibodies, antibody IMB1636 was added and incubated at 37°C for 2h. In addition, to prove that the endocytosed antibodies were engulfed and degraded by lysosomes, 200μL / well of LAMP-1 antibody (1:200) targeting lysosomal surface protein was incubated at 37°C for 2h after the cells were blocked, and LAMP-1 was labeled with donkey anti-rabbit AF555 secondary antibody so that lysosomes were labeled with red fluorescence. The other steps were the same as above to observe the co-localization of antibody IMB1636 and lysosomes. Figure 9 )

[0260] 5. Flow cytometry to detect the ability of antibodies to bind to tumor cells

[0261] Take 5×10 cells of BXPC-3, MDA-MB-231, and NIH 3T3 6 Place the cells in EP tubes, wash with 100 μl 4°C precooled PBS containing 2% FBS, resuspend with 100 μl PBS containing 2% FBS, add 100 μl / tube of anti-Trop2 antibody at concentrations of 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 μg / ml, respectively, use SP2 / 0 ascites as negative control, incubate at 4°C for 1.5h, wash with PBS containing 2% FBS, add 200 μl FITC-labeled goat anti-mouse IgG secondary antibody (1:200 dilution), incubate at 4°C in the dark for 1h, wash with PBS three times, and detect by flow cytometry. Figure 10 )

[0262] 6. Determination of antibody binding ability to human tumor tissue microarray

[0263] The tumor tissue chips used in this experiment were purchased from Shanghai Xinchao Biotechnology Co., Ltd. The main experimental steps included: baking, dewaxing, antigen repair, endogenous peroxidase blocking, primary antibody incubation, secondary antibody incubation, DAB color development, hematoxylin counterstaining, and sealing. Figure 12 )

[0264] 7. In vivo imaging of mice to monitor the ability of antibodies to target tumors

[0265] BXPC-3, MDA-MB-231, and NIH 3T3 cells were inoculated into the axilla of mice, and 6×10 4 / , tumor volume to reach 200-300cm 2, the anti-Trop2 antibody was labeled with the Dylight 680 antibody kit and injected into the tail vein at 20 mg / kg. In vivo imaging was performed at 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 24 h, 30 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, 120 h, 132 h, 144 h, 156 h, 168 h, 192 h, 216 h, 240 h, and 264 h for photography.( Figure 11 )

[0266] 8. Killing Activity of anti-Trop2-LDM and LDM against Tumor Cells Cultured in Vitro

[0267] The conjugate anit-Trop2-LDM of the anti-Trop2 antibody and the antitumor antibiotic lidamycin (LDM) was prepared (for the preparation method, see the reference: Wang R, Li L, Duan A, Li Y, Liu X, Miao Q, Gong J, Zhen Y. Crizotinibe enhances anti-CD30-LDM induced antitumor efficacy in NPM-ALK positive anaplastic large cell lymphoma. Cancer Lett. 2019 Apr 28; 448: 84-93.). The apoprotein of lidamycin is LDP, and the amino acid sequence of LDP is shown in SEQ ID NO: 37. LDP was fused to the N-terminus of the light chain of the humanized anti-Trop2 antibody shown in SEQ ID NO: 34 through the linker shown in SEQ ID NO: 38, and then conjugated with the heavy chain to form anit-Trop2-LDp.

[0268] The highly active pure product of lidamycin (LDM) was taken and the active chromophore AE shown in Figure I was separated by a C4 column.

[0269]

[0270] The mobile phase was water: acetonitrile: trifluoroacetic acid = 78%: 22%: 0.1%. The absorbance at 350 nm was detected, and the chromophore AE was collected. The anti-Trop2-LDP protein and the chromophore AE were mixed at a ratio of 1:4, placed on a shaker and slowly shaken, and reacted overnight at 4 °C in the dark. Then, the mixture of the two was ultrafiltered and centrifuged at 4 °C and 3500 rpm for 4 - 6 times to remove the unassembled free chromophore. Ultrafiltration was stopped when no chromophore was detected in the ultrafiltered solution. At this time, the mixture was the antibody-drug conjugate Anti-Trop2-LDM solution. After ultrafiltration and concentration, the antibody-drug conjugate Anti-Trop2-LDM was obtained, and the absorbance of Anti-Trop2-LDM at 350 nm was detected by reverse-phase HPLC (C4, 300A).

[0271] Select tumor cells that have grown to the logarithmic growth phase after passage, prepare them into a single-cell suspension and count, and adjust the cell concentration to 3×10 3 cells / well, 80 μL per well, and inoculate into a 96-well plate. After culturing for 24 h, add 20 μL of anti-Trop2-IgG, LDM, and Anti-Trop2-IgG-LDM diluted to different concentrations, and set three parallel wells for each concentration. After culturing for 48 h, add 10 μL of CCK-8 reagent to each well in the dark, shake for 3 min, and place in an incubator for 1 h. Take out the 96-well plate and shake for 2 min, and detect the OD value at 450 nm with an enzyme-linked immunosorbent assay reader. In addition to the above drug administration groups with different concentrations, three parallel wells of a drug-free control group and a cell-free blank group were also set. The results are shown in the following table:

[0272]

[0273] 9. Antitumor effects of Anti-Trop2-LDM and LDM in vivo

[0274] Using the human lung cancer tumor cell line HCC827 with high expression of TROP2, a subcutaneous xenograft tumor model was established to evaluate the in vivo anti-tumor effect of Anti-Trop2-LDM. Female, 6-week-old BALB / c nude mice were selected, and HCC827 tumor cells were inoculated subcutaneously in the right axilla of the mice, with 3×106 cells inoculated per mouse. Subsequently, the tumor growth was observed. When the tumor volume reached 100 mm3 - 150 mm3 8 days after tumor inoculation, the mice were randomly divided into 6 groups of 6 mice each, and different drugs were given for treatment as follows: Different concentrations of Anti-Trop2-LDM (0.4 mg / kg, 0.6 mg / kg, 0.8 mg / kg), LDM (0.05 mg / kg), and TROP2-mAb (0.8 mg / kg) were administered via the tail vein once at 8 days, 16 days, and 24 days, respectively, for a total of 3 administrations. The control group was given an equal volume of normal saline. Starting from the 8th day, the length and width of the tumor (a: length, b: width) were measured every 4 days until the 40th day. The tumor volume was calculated using the formula V = ab2 / 2, and the tumor growth curve was plotted. At the same time, the body weight of the mice was weighed to plot the body weight change curve. The results are as Figure 17 shown.

[0275]

[0276]

[0277]

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof against Trop2, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, The light chain variable region comprises: VL CDR1, which has the amino acid sequence shown in SEQ ID NO:2, VL CDR2, which has the amino acid sequence shown in SEQ ID NO:3, and VL CDR3, which has the amino acid sequence shown in SEQ ID NO:4; The heavy chain variable region comprises: VH CDR1, which has the amino acid sequence shown in SEQ ID NO:10, VH CDR2, which has the amino acid sequence shown in SEQ ID NO:11, and VH CDR3, which has the amino acid sequence shown in SEQ ID NO:

12.

2. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

1.

3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

9.

4. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

33.

5. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

34.

6. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

34.

7. A monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody is produced by a mouse hybridoma cell deposited with the China General Microbiological Culture Collection Center under the accession number CGMCC No. 18167 on June 25, 2019.

8. A hybridoma cell deposited with the China General Microbiological Culture Collection Center under the accession number CGMCC No. 18167 on June 25, 2019.

9. An antibody conjugate comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-7, and a therapeutic moiety selected from a cytotoxin, a radioisotope or a bioactive protein conjugated to the monoclonal antibody or antigen-binding fragment thereof.

10. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-7 or the antibody conjugate according to claim 9, and a pharmaceutically acceptable carrier.

11. Use of a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-7, an antibody conjugate according to claim 9, or a pharmaceutical composition according to claim 10 in the preparation of a medicament for treating Trop2-related diseases, wherein the Trop2-related diseases are selected from uterine cancer, breast cancer, ovarian cancer, pancreatic cancer, esophageal cancer, or lung cancer.

12. The use according to claim 11, wherein the uterine cancer is endometrial cancer.

13. The use according to claim 11, wherein the esophageal cancer is esophageal squamous cell carcinoma.

14. The use according to claim 11, wherein the lung cancer is small cell lung cancer.

15. Use of a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-7, an antibody conjugate according to claim 9, or a pharmaceutical composition according to claim 10 in the preparation of a medicament for treating Trop2-related diseases, wherein the Trop2-related diseases are selected from epithelial malignancies.

16. Use of a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-7 in the preparation of a diagnostic agent for detecting the presence of a malignancy in a patient, wherein the malignancy is selected from uterine cancer, breast cancer, ovarian cancer, pancreatic cancer, esophageal cancer, or lung cancer.

17. The use according to claim 16, wherein the uterine cancer is endometrial cancer.

18. The use according to claim 16, wherein the esophageal cancer is esophageal squamous cell carcinoma.

19. The use according to claim 16, wherein the lung cancer is small cell lung cancer.

20. Use of a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-7 in the preparation of a diagnostic agent for detecting the presence of a malignancy in a patient, wherein the malignancy is selected from epithelial malignancies.

21. An isolated nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-7.

22. The isolated nucleic acid molecule of claim 21, wherein the nucleic acid molecule is operably linked to an expression control sequence.

23. The isolated nucleic acid molecule of claim 21, wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 17 and 25, or SEQ ID NO: 18-20 and 26-28, or SEQ ID NO: 35-36.

24. An expression vector comprising the nucleic acid molecule according to any one of claims 21-23.

25. A host cell transformed with the nucleic acid molecule according to any one of claims 21-23 or the expression vector according to claim 24.

26. A method for producing a monoclonal antibody against Trop2 or an antigen-binding fragment thereof, comprising: (i) culturing the host cell of claim 25 under conditions suitable for the expression of the nucleic acid molecule or the expression vector, and (ii) isolating and purifying the antibody or an antigen-binding fragment thereof expressed by the nucleic acid molecule or the expression vector.

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