Anti-Trop-2 antibodies and uses thereof

Abstract

The present invention belongs to the field of biology and relates to anti-Trop-2 antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and applications thereof. The anti-Trop-2 antibodies and antigen-binding fragments of the present invention can effectively bind to the human Trop-2 protein, exhibit excellent specificity and antigen affinity, and possess superior internalization activity, with promising applications.

Description

Technical Field

[0001] The invention belongs to the field of biology and relates to an anti-Trop-2 antibody. Background Art

[0002] Tumor-associated calcium signal transducer 2 (Trop-2) is a single-pass transmembrane cell surface glycoprotein encoded by the TACSTD2 gene. It is also known as tumor-associated calcium signal transducer 2 (TACSTD2) and belongs to the same family of tumor-associated calcium signal transducers as the epithelial adhesion molecule Ep-CAM (Trop1). Trop-2 consists of 323 amino acids, including a hydrophobic leader peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic tail. This tail contains a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequence that overlaps with a protein kinase C phosphorylation site, suggesting that PIP2 plays a key role in Trop-2 signal transduction. The N-terminus of the Trop-2 protein is an extracellular domain (Trop-2 EC), which is connected to a short intracellular tail (Trop-2 IC) by a unidirectional transmembrane helix (TM), anchoring it to the cell membrane. Phosphorylation of Trop-2 IC leads to significant conformational changes in functional regions, including the C-terminal sequence. These structural features may be important in regulating the activity of Trop-2.

[0003] Trop-2 is not only involved in the regulation of intracellular calcium ion concentration, cyclin D1, and phosphokinase C (PKC), but may also activate the ERK pathway, thereby regulating tumor cell growth, invasion, and metastasis. Trop-2 is highly expressed in 65% to 90% of tumors, such as pancreatic cancer, colon cancer, gastric cancer, lung cancer, prostate cancer, cervical cancer, ovarian cancer, and pancreatic cancer, but is absent or rarely expressed in normal human tissues. Clinical data indicate that high expression of Trop-2 in tumor cells can promote tumor proliferation, invasion, and metastatic spread, and is closely associated with poor prognosis and shortened survival in cancer patients, making it an important target for the development of targeted anti-tumor drug therapies. In recent years, antibodies and ADCs targeting Trop-2 have become a new hotspot in the treatment of Trop-2-positive tumors. Summary of the Invention

[0004] The present invention aims to provide a novel anti-Trop-2 antibody that can specifically bind to human Trop-2 and has great potential for tumor treatment. At the same time, the antibody has high internalization ability and is suitable for the development of ADC drugs.

[0005] In one aspect, the present invention provides an anti-Trop-2 antibody or antigen-binding fragment thereof that binds to Trop-2 or a fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three CDRs, namely, VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises three CDRs, namely, VL CDR1, VLCDR2, and VL CDR3; wherein,

[0006] The VH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106 or 114, the VH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107 or 115, the VH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108 or 116, and the VL CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 5 NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110 or 118, or consist of the amino acid sequence thereof, the VL CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111 or 119, and the VL CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112 or 120.

[0007] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0008] The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 2;

[0009] The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 3;

[0010] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:4;

[0011] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:6;

[0012] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:7;

[0013] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:8;

[0014] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0015] The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 10;

[0016] The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 11;

[0017] The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 12;

[0018] The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 14;

[0019] The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 15;

[0020] The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 16;

[0021] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0022] The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 18;

[0023] The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 19;

[0024] The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 20;

[0025] The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 22;

[0026] The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 23;

[0027] The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 24;

[0028] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0029] The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 26;

[0030] The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 27;

[0031] The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 28;

[0032] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:30;

[0033] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:31;

[0034] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:32;

[0035] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0036] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:34;

[0037] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:35;

[0038] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:36;

[0039] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:38;

[0040] The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 39;

[0041] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:40;

[0042] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0043] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:42;

[0044] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:43;

[0045] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:44;

[0046] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:46;

[0047] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:47;

[0048] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:48;

[0049] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0050] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:50;

[0051] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:51;

[0052] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:52;

[0053] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:54;

[0054] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:55;

[0055] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:56;

[0056] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0057] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:58;

[0058] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:59;

[0059] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:60;

[0060] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:62;

[0061] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:63;

[0062] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:64;

[0063] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0064] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:66;

[0065] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:67;

[0066] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:68;

[0067] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:70;

[0068] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:71;

[0069] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:72;

[0070] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0071] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:74;

[0072] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:75;

[0073] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:76;

[0074] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:78;

[0075] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:79;

[0076] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:80;

[0077] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0078] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:82;

[0079] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:83;

[0080] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:84;

[0081] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:86;

[0082] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:87;

[0083] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:88;

[0084] In a preferred embodiment, the antibody comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3; wherein,

[0085] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:90;

[0086] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:91;

[0087] The amino acid sequence of VH CDR3 is shown in SEQ ID NO:92;

[0088] The amino acid sequence of VL CDR1 is shown in SEQ ID NO:94;

[0089] The amino acid sequence of VL CDR2 is shown in SEQ ID NO:95;

[0090] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:96;

[0091] or,

[0092] The amino acid sequence of VH CDR1 is shown in SEQ ID NO:98;

[0093] The amino acid sequence of VH CDR2 is shown in SEQ ID NO:99;

[0094] The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 100;

[0095] The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 102;

[0096] The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 103;

[0097] The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 104;

[0098] or,

[0099] The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 106;

[0100] The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 107;

[0101] The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 108;

[0102] The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 110;

[0103] The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 111;

[0104] The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 112;

[0105] or,

[0106] The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 114;

[0107] The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 115;

[0108] The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 116;

[0109] The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 118;

[0110] The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 119;

[0111] The amino acid sequence of VL CDR3 is shown in SEQ ID NO:120.

[0112] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105 or 113, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the above sequence.

[0113] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109 or 117, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the above sequence.

[0114] In certain preferred embodiments, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the IgBlast system.

[0115] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention may further comprise one or more of a heavy chain constant region, a light chain constant region, and an Fc region. In further preferred embodiments, the light chain constant region is a lambda chain or a kappa chain constant region. In some preferred embodiments, the antibodies or antigen-binding fragments thereof are of IgG1, IgG2, IgG3, or IgG4 type.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody or antigen-binding fragment thereof.

[0117] In some embodiments, the heavy chain of the antibody comprises or consists of the amino acid sequence shown in SEQ ID NO: 121 or SEQ ID NO: 123, and the light chain of the antibody comprises or consists of the amino acid sequence shown in SEQ ID NO: 122 or SEQ ID NO: 124.

[0118] In one aspect, the present invention provides a biomaterial comprising:

[0119] (1) A nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention;

[0120] (2) A vector, host cell or microorganism comprising (1);

[0121] (3) The expression product, suspension or supernatant of (2) above.

[0122] Those skilled in the art can easily select and prepare vectors, host cells, or microorganisms containing the antibody coding sequence based on the antibody amino acid sequence, and can also know how to culture such host cells or microorganisms to obtain the corresponding expression products, suspensions, supernatants, etc., to obtain the corresponding antibodies. These are all routine techniques in the art.

[0123] In another aspect, the present invention provides a conjugate, which is a conjugate of the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof is conjugated to a drug, wherein the drug is a cytotoxic agent, such as an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent, and an RNA polymerase II inhibitor, or any active pharmaceutical ingredient that interferes with a specific cellular pathway.

[0124] In another aspect, the present invention provides a composition comprising the antibody or antigen-binding fragment thereof or antibody or antigen-binding fragment conjugate of the present invention; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

[0125] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising: culturing the above-mentioned host cells or microorganisms to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the host cells.

[0126] In another aspect, provided is a use of the antibody or antigen-binding fragment thereof, the conjugate, the composition, or the biomaterial of the present invention in the preparation of a medicament for treating tumors, wherein the medicament is used to treat Trop-2-overexpressing cancers; preferably, the Trop-2-overexpressing cancers are gastric cancer, pancreatic cancer, prostate cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, bile duct cancer, urothelial carcinoma, breast cancer, or cervical cancer.

[0127] In another aspect, there is provided use of the antibody or antigen-binding fragment thereof of the present invention, the conjugate of the present invention, the composition of the present invention, or the biomaterial of the present invention in preparing a product for binding Trop-2.

[0128] In another aspect, the invention provides the use of an antibody or antigen-binding fragment thereof, a composition, a conjugate, or a biomaterial of the invention in combination with one or more other cancer therapeutic agents in the preparation of a medicament for treating tumors. Other cancer therapeutic agents include, but are not limited to, paclitaxel, mitomycin, vincristine, colchicine, doxorubicin, daunorubicin, dactinomycin D, emetine, puromycin, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan, docetaxel, pemetrexed, sorafenib, oxaliplatin, 5-FU, lapatinib, and analogs or homologs thereof. The cancer therapeutic agents also include radiotherapeutic agents and biomacromolecule drugs.

[0129] The antibodies of the present invention have good internalization ability, and chimeric and humanized antibodies can promote the internalization of Trop-2 in human cells. Therefore, the antibodies of the present invention have the potential to be used for the development of ADC drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1 The binding of the chimeric antibody to the Trop-2 protein is shown.

[0131] Figure 2 The chimeric antibody binds to Trop-2 on the cell membrane.

[0132] Figure 3 The figure shows the internalization amount of Trop-2 on the MDA-MB-468 cell membrane by the chimeric antibody.

[0133] Figure 4 The results of antigen-binding epitope analysis of the Trop-2 antibody are shown.

[0134] Figure 5 shows the binding of humanized antibodies to Trop-2 protein. (A) ELISA detected the binding of humanized antibodies HuA3-1, HuA3-3 and HuA3-4 to Trop-2 protein. (B) ELISA detected the binding of humanized antibody HuA3-2 to Trop-2 protein.

[0135] Figure 6 shows the binding of humanized antibodies to Trop-2 protein. (A) ELISA detected the binding of humanized antibodies HuA4-1, HuA4-3 and HuA4-6 to Trop-2 protein. (B) ELISA detected the binding of humanized antibodies HuA4-2, HuA4-4 and HuA4-5 to Trop-2 protein.

[0136] Figure 7 shows the binding of humanized monoclonal antibodies to Trop-2 on the cell membrane. (A) FACS detection of the binding of humanized antibodies HuA3-1 to HuA3-4 to Trop-2 on the cell membrane. (B) FACS detection of the binding of humanized antibodies HuA4-1 to HuA4-6 to Trop-2 on the cell membrane.

[0137] Figure 8 shows the amount of Trop-2 on the MDA-MB-468 cell membrane internalized by humanized antibodies. (A) FACS detection of the amount of Trop-2 on the MDA-MB-468 cell membrane internalized by humanized antibodies HuA3-1 to HuA3-4. (B) FACS detection of the amount of Trop-2 on the MDA-MB-468 cell membrane internalized by humanized antibodies HuA4-1 to HuA4-6.

[0138] Figure 9 shows the thermal stability analysis of humanized antibodies, (A) ELISA detection of the binding of humanized antibodies HuA3-1 to HuA3-4 to Trop-2 protein at different denaturation temperatures, (B) ELISA detection of the binding of humanized antibodies HuA4-1 to HuA4-6 to Trop-2 protein at different denaturation temperatures. DETAILED DESCRIPTION

[0139] The present invention will be described below with reference to specific examples. Unless otherwise specified, the reagents and instruments used in the following methods are commonly used in the art and can be obtained commercially. The methods used are all conventional methods in the art, and those skilled in the art can undoubtedly perform the methods and obtain the corresponding results based on the contents described in the examples.

[0140] definition:

[0141] As used herein, the term "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or completely synthetically (e.g., recombinantly), including any fragment that contains at least a portion of the variable region of an immunoglobulin molecule and retains the binding specificity of a full-length immunoglobulin. The term encompasses intact monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), single-chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site with the desired specificity. Antibodies include antibodies of any class or subclass (e.g., IgG1, IgG2, IgG3, and IgG4; IgM, IgD, IgE, and IgA).

[0142] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to a portion of an immunoglobulin molecule that binds to an antigen. Antigen-binding fragments can be synthetic, enzymatically obtainable, or genetically engineered polypeptides, including but not limited to Fab, Fab', F(ab'), 2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments and other fragments. The fragments may include multiple chains linked together, for example, 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. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding regions), results in an antibody that immunospecifically binds to an antigen.

[0143] As used herein, "conventional antibodies" refer to antibodies comprising two heavy chains (which may be designated H and H') and two light chains (which may be designated L and L') and two antigen-binding sites. The variable domain or variable region is the domain of the antibody heavy or light chain that recognizes and specifically binds to an antigen and comprises an amino acid sequence that varies between different antibodies. The heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, namely CH1, CH2, and CH3. The light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by relatively conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0144] As used herein, "hypervariable region," "complementarity determining region," and "CDR" are used interchangeably to refer to the amino acid sequences of the variable regions of the heavy and light chains of immunoglobulins that provide the primary contact residues for antibody binding to antigen or epitope.

[0145] As used herein, framework region (FR) refers to the variable domain residues excluding the hypervariable region residues. The FR of the variable domain is generally composed of four FR regions: FR1, FR2, FR3 and FR4. In terms of amino acid sequence, the FR region is relatively more conserved than the hypervariable region.

[0146] As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that comprises an amino acid sequence that is relatively more conserved than that of the variable region domain.

[0147] The term "murine antibody" herein refers to a mouse monoclonal antibody against human Trop-2 prepared according to the knowledge and skill in the art by injecting a test subject mouse with a Trop-2 antigen and then isolating a hybridoma expressing an antibody having the desired sequence or functional properties.

[0148] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, the gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and replaced with an equivalent portion of the gene encoding the human Fc constant region, which is then inserted into a human vector and expressed as a chimeric antibody molecule.

[0149] The term "humanized antibody" refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework. The constant region of the antibody molecule is derived from the constant region of a human antibody. Murine antibodies or antibodies from other species can be humanized using techniques well known in the art.

[0150] The term "epitope" refers to any protein determinant capable of specifically binding to an immunoglobulin or otherwise interacting with a molecule. Epitope determinants typically consist of chemically active surface groupings of molecules, such as amino acids or carbohydrates or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be "linear" or "conformational." If two antibodies exhibit competitive binding to an antigen, they are likely to bind to the same epitope on the antigen.

[0151] As used herein, "expression" refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. These terms encompass the transcription of a gene into RNA. These terms also encompass the translation of RNA into one or more polypeptides, and also encompass all naturally occurring post-transcriptional and post-translational modifications. The expression or production of an antibody or antigen-binding fragment thereof can be within the cytoplasm of a cell, or in an extracellular environment such as a growth medium for a cell culture.

[0152] As used herein, "host cell" includes individual cells or cell cultures that can or have received a vector for the introduction of a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. Host cells can be eukaryotic or prokaryotic. Suitable host cells include, but are not limited to, CHO cells, HeLa cells, HEK cells, such as HEK 293 cells.

[0153] As used herein, an "expression vector" is a replicon, a vector capable of expressing DNA, in which a segment can be operably inserted into another nucleic acid segment to cause replication or expression of that segment. An expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, cosmid, virus, or other vector, which, when introduced into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, as well as expression vectors that remain episomal or that integrate into the host cell genome.

[0154] As used herein, "treatment" refers to any success or indication of success in alleviating or ameliorating an injury, condition, or disorder, including any objective or subjective parameter, such as symptom relief, remission, weakening, or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, reducing the degree of degenerative endpoint failure, improving the physical or mental health of the subject, or prolonging survival. Treatment can be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluation.

[0155] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to the dose or concentration of a drug that is effective in treating the disease or condition of interest, i.e., the amount necessary to prevent, cure, ameliorate, retard, or partially retard the symptoms of a disease or condition. Similarly, as used herein, a "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, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0156] As used herein, the term "subject" refers to a mammal, such as a human.

[0157] Antibodies of the present invention

[0158] The present invention provides an antibody against Trop-2, namely an anti-Trop-2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically recognizes and binds to Trop-2.

[0159] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can specifically bind to Trop-2, have excellent internalization activity in target tumor cells expressing hTrop-2, and have the potential to be used for the development of ADC drugs.

[0160] In some embodiments, the tumors targeted include but are not limited to those described below with respect to neoplastic diseases. In other embodiments, the antibodies or antigen-binding fragments thereof of the present invention can inhibit tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, and more preferably at least about 50%.

[0161] The anti-Trop-2 antibodies or antigen-binding fragments thereof of the present invention comprise substitutions, insertions, or deletions. The anti-Trop-2 antibodies of the present invention include modifications to the light chain variable region, heavy chain variable region, light chain, or heavy chain, resulting in an amino acid sequence that differs from the amino acid sequence from which the antibody was derived. For example, an amino acid sequence derived from the same designated protein can be similar to the starting sequence, e.g., have a certain percentage identity, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the starting sequence.

[0162] In certain embodiments, amino acid modifications, which may be one or more, may be introduced into the Fc region of an antibody provided herein to generate an Fc variant. The Fc variant may comprise a human Fc region sequence comprising an amino acid modification at one or more amino acid positions.

[0163] The "antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, or de-immunized antibodies, or Fab fragments, Fab' fragments, F(ab') 2 Fragments, single-chain antibodies, nanobodies, and epitope-binding fragments of any of the foregoing.

[0164] In some embodiments, the antibodies of the invention can be monospecific, bispecific, or multispecific.An anti-Trop-2 antibody can be linked to another antibody or antibody fragment to generate a bispecific or multispecific antibody with a second or more binding specificities.

[0165] In certain embodiments, antibodies can be further modified to add functional components. Suitable moieties for antibody derivatization include, but are not limited to, the following examples: PEG, dextran, proteins, lipids, therapeutic agents, or toxins. Antibodies can be modified by phosphorylation, acetylation, glycosylation, PEGylation, amidation, or conjugation to other proteins.

[0166] Neoplastic diseases

[0167] The antibodies or antigen-binding fragments thereof of the present invention can be used to treat neoplastic diseases. The antibodies or antigen-binding fragments thereof of the present invention can be used to prevent and / or treat various tumors that express hTROP-2. Non-limiting examples of treatable cancers include, but are not limited to, one or more of various tumors such as gastric cancer, pancreatic cancer, prostate cancer, intestinal cancer, breast cancer (such as triple-negative breast cancer), ovarian cancer, lung cancer (such as squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, etc.), bile duct cancer, urothelial carcinoma, and cervical cancer.

[0168] Nucleic acids, vectors, and antibody production methods

[0169] The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention. Those skilled in the art can easily determine the nucleotide sequence of the nucleic acid based on the amino acid sequence of the antibody or antigen-binding fragment thereof and select appropriate codons to express the antibody or antigen-binding fragment thereof according to actual needs.

[0170] The present invention also provides an expression vector, which can be easily selected and prepared based on the nucleic acid sequence of the antibody of the present invention.

[0171] One aspect of the present invention further provides a host cell or microorganism, which is transformed by at least one of the aforementioned expression vectors.

[0172] Furthermore, the present invention also provides an expression product, suspension or supernatant of the host cell or microorganism. As is known in the art, such an expression product, suspension or supernatant comprises the antibody or antigen-binding fragment thereof of the present invention.

[0173] Yet another aspect of the present invention relates to a method for producing an anti-Trop-2 antibody or an antigen-binding fragment thereof, the method comprising: (i) culturing the host cell or microorganism of the present invention under conditions suitable for expression by an expression vector, and (ii) isolating and purifying the antibody or antigen-binding fragment thereof expressed by the expression vector.

[0174] Pharmaceutical composition

[0175] The present invention also provides a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment or antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier.

[0176] As used herein, an “antibody drug conjugate” (abbreviated as “conjugate”) relates to an antibody or antigen-binding fragment, an antibody-based binding protein, or an antibody fragment thereof as described herein coupled to a therapeutically active substance or active pharmaceutical ingredient. The therapeutically active substance or active pharmaceutical ingredient includes, but is not limited to, an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent, and an RNA polymerase II inhibitor, or any active pharmaceutical ingredient that interferes with a specific cellular pathway that is essential for cell survival and / or for a specific cellular pathway. The therapeutically active substance or active pharmaceutical ingredient may be conjugated to the antibody or antibody fragment non-site-specifically via a lysine or cysteine ​​side chain; or it may be conjugated site-specifically via chemical, chemo-enzymatic, or enzymatic conjugation known in the art.

[0177] As used herein, "pharmaceutically acceptable carrier" can be any suitable and pharmaceutically acceptable carrier. It can be any compatible solid or liquid filler, diluent, other excipient, or coating material administered to the user. Examples of suitable carriers, diluents, and / or excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and any combination thereof. In many cases, it is preferred that the composition include an isotonic agent, such as a sugar, a polyol, or sodium chloride. The pharmaceutical composition of the present invention may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants.

[0178] The compositions of the present invention may be in a variety of forms including liquid, semi-solid and solid dosage forms, but the preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions.

[0179] The pharmaceutical composition of the present invention may be administered via any of enteral, topical, and parenteral routes.

[0180] Combination therapy

[0181] The present invention also provides a combination therapy comprising the anti-Trop-2 antibody or pharmaceutical composition of the present invention and at least one other cancer therapeutic agent.

[0182] The other other cancer therapeutic agent includes, but is not limited to, paclitaxel, mitomycin, vincristine, colchicine, doxorubicin, daunorubicin, dactinomycin D, emetine, puromycin, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan, docetaxel, pemetrexed, sorafenib, oxaliplatin, 5-FU, lapatinib, and analogs or homologs thereof. The cancer therapeutic agent also includes a radiotherapeutic agent and a biomacromolecule drug.

[0183] The antibody of the present invention and the cancer therapeutic agent may be administered all at once or separately. When administered separately (in the case of using different administration schedules), they may be administered continuously without interruption or at predetermined intervals.

[0184] Anti-Trop-2 antibody sequences according to the present invention

[0185] Table 1: Amino acid sequence numbers of VH, VH-CDR1, VH-CDR2, VH-CDR3, VL, VL-CDR1, VL-CDR2, VL-CDR3 of the antibodies of the present invention

[0186]

[0187]

[0188] Table 2: Sequence information of the present invention

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] Table 3 Amino acid sequences of heavy and light chains of cA3 antibody

[0195]

[0196]

[0197] Table 4 Amino acid sequences of heavy and light chains of cA4 antibody

[0198]

[0199] Example 1: Preparation of anti-Trop-2 antibody mouse monoclonal antibody

[0200] This example describes a method for producing mouse anti-human Trop-2 monoclonal antibodies using hybridoma technology. First, the extracellular domain of the Trop-2 protein (Uniprot: P09758) was expressed as an immunogen. A 6xFc tag was added to the C-terminus of the extracellular domain of the Trop-2 protein amino acid sequence (His27-Thr274) and then cloned into the V152 vector (provided by Huaan Monoclonal Antibodies) to generate V152-Trop-2 ECD-Fc. This vector was transiently transfected into 293 cells (source: ATCC). After 5 days, the cell culture fluid was collected and the supernatant was purified using a nickel column (manufacturer: Solarbio, catalog number: I8090) to obtain the Trop-2 protein used in this experiment. To produce anti-human Trop-2 mouse monoclonal antibodies, 4-week-old BAL B / c mice (provided by Huaan Monoclonal Antibodies) were first immunized with 100 μg of Trop-2 protein. On days 14 and 28 after the initial immunization, mice were re-immunized with 50 μg of Trop-2 protein. Serum titers from immunized mice were assessed by ELISA. Trop-2 protein was diluted to 1 μg / ml in PBS (Corning, Catalog No. 21-040-CV) and coated onto microplates overnight at 4°C. Plates were then blocked with 1% BSA-PBST blocking buffer at 37°C for 1 hour. After washing with PBST, diluted serum from immunized mice was added to the plates and incubated at 37°C for 1 hour. After washing, HRP-conjugated goat anti-mouse IgG (Abcam, Catalog No. Ab205719, 1:10,000 dilution) was added and incubated at 37°C for 0.5 hour. After washing, TMB solution (Huzhou Yingchuang, Catalog No. TMB-S-001) was added and incubated at room temperature in the dark for 5 minutes. The reaction was then terminated with 2N H2SO4. The absorbance was measured at 450 nm on a microplate reader (Molecular Devices, M5). On day 42 after immunization, mice with sufficient anti-Trop-2 antibody titers were boosted with 50 μg of Trop-2 protein. Three to five days later, mice were sacrificed, and splenocytes were harvested and washed two to three times with IMDM (Shanghai Yuanpei, Catalog No. L610KJ) basal medium by centrifugation. The cells were then mixed with mouse myeloma SP2 / 0 cells (provided by Huaan Monoclonal Antibody) at a 1:1 ratio. PEG (Roche, Catalog No. 25771700) was added to the mixed cells, gently stirred, and incubated at 37°C for 30 seconds. After fusion, cells were diluted into IMEM selective medium supplemented with 15% fetal bovine serum (Zhejiang Tianhang Biological, Catalog No. 11011-8611) and 1× HUAT (Sigma, Catalog No. H0262-1VL). 200 μl were added to each well of a 96-well cell culture plate and placed in a 5% CO2, 37°C incubator. After 10-14 days, anti-Trop-2 antibodies were detected in hybridoma supernatants by ELISA.Five different hybridoma clones were identified, including 17F1-G2, A2-H4, 24H2-D10, 31C-1, and D7-1.

[0201] Flow cytometry (FACS) was used to examine the binding of hybridoma supernatants to Trop-2 endogenously expressed on the membranes of the human breast cancer cell line MDA-MB-468 (source: Shanghai Cell Bank). MDA-MB-468 cells were incubated with various dilutions of hybridoma supernatants at 4°C for 30 minutes. After washing the cells twice, FITC-conjugated goat anti-mouse IgG (Abcam, Catalog No. ab6785, 1:1000 dilution) was added and incubated at 4°C in the dark for 30 minutes. After washing the cells twice, the cells were analyzed using a BD C6 flow cytometer, confirming that all five hybridoma supernatants bound to Trop-2 on the membranes of MDA-MB-468 cells (data not shown).

[0202] Example 2: Mouse Antibody-Mediated Trop-2 Internalization

[0203] Hybridoma supernatant-mediated Trop-2 protein internalization in MDA-MB-468 cells was detected by flow cytometry (FACS). MDA-MB-468 cells were reacted with hybridoma supernatant at 4°C for 30 minutes. After washing twice, the cells were split and incubated at 4°C and 37°C for 4 hours, respectively. After washing twice, FITC-conjugated goat anti-mouse IgG (Abcam, Catalog No. ab6785, 1:1000 dilution) was added and incubated at 4°C in the dark for 30 minutes. After washing twice, the cells were analyzed by flow cytometry, and the endocytosis rate was calculated based on the decrease in cell surface fluorescence at 37°C compared to 4°C. The endocytosis rate was calculated as follows: endocytosis rate (%) = (4°C fluorescence intensity MFI - 37°C fluorescence intensity MFI) / 4°C fluorescence intensity MFI * 100%. The above method was used to detect the internalization rate of the hybridoma supernatants, confirming that the supernatants of the five hybridomas can mediate the internalization of Trop-2 (data not shown).

[0204] Example 3: Sequencing of light and heavy chain variable regions and expression of human-mouse chimeric antibodies

[0205] RNA was extracted from hybridoma cells expressing 17F1-G2, A2-H4, 24H2-D10, 31C-1, and D7-1 and reverse transcribed into cDNA according to conventional methods. The antibody gene sequences were then sequenced to obtain the heavy and light chain variable region sequences of 17F1-G2, A2-H4, 24H2-D10, 31C-1, and D7-1. Furthermore, amino acid sequences were analyzed, and CDR sequences were confirmed using the IgBlast database.

[0206] To express chimeric antibodies, the heavy and light chain variable regions of 17F1-G2, A2-H4, 24H2-D10, 31C-1, and D7-1 were linked to the human antibody heavy chain IgG1 constant region and the human antibody light chain kappa constant region, respectively, and cloned into the vector V152 (provided by Huaan Monoclonal Antibody). The vectors were then transfected into 293 cells (source: ATCC) using the transfection reagent PEI (source: Polysciences). After culturing for about 5 days, the cell supernatant was centrifuged at 3000 rpm for 10 minutes and protein purified. The supernatant was purified from hybridoma A (manufacturer: Solarbio, catalog number: I8090) to a purity of >95%. The chimeric antibodies expressed in the supernatants of the 15 hybridomas 17F1-G2, A2-H4, 24H2-D10, 31C-1, and D7-1 were cA1, cA2, cA3, cA4, and cA5, respectively. The expression levels of the five chimeric antibodies were 24.63, 41.79, 135.77, 238.91, and 452.31 μg / ml, respectively.

[0207] Example 4: Binding of chimeric antibodies to Trop-2

[0208] The ELISA method was used to quantitatively detect the binding ability of the chimeric antibody to the human Trop-2 protein. Trop-2-His (manufacturer: Acrobiosystems, catalog number: TR2-H5223) was diluted in PBS at a concentration of 1 μg / ml and coated on a microplate at 4°C overnight. Then, the plate was blocked with 1% BSA-PBST blocking solution at 37°C for 1 hour; after washing the plate with PBST, cA1, cA2, cA3, cA4, and cA5 were diluted to different concentrations (starting concentration 68.5 nM, 3-fold gradient dilution, a total of 12 concentrations) and added to the plate and incubated at 37°C for 1 hour. After washing the plate, HRP-labeled goat anti-human IgG (manufacturer: Sigma, catalog number A0170, 1:10000 dilution) was added and reacted at 37°C for 1 hour. After washing the plate, TMB solution was added and reacted at room temperature in the dark for 10 minutes, and then 2N H2SO4 was added to terminate the reaction. The absorbance was detected at a wavelength of 450 nm on an enzyme reader. See the results. Figure 1 The EC50 values ​​of chimeric antibodies cA1, cA2, cA3, cA4, and cA5 binding to Trop-2 were 0.136 nM, 0.075 nM, 0.165 nM, 0.141 nM, and 0.114 nM, respectively, indicating that these chimeric molecules all have high binding activity to Trop-2.

[0209] The chimeric antibody was tested for binding to Trop-2 on the membrane of a human breast cancer cell line (MDA-MB-468) endogenously expressing Trop-2 using the FACS method described in Example 1. The antibody was diluted 3-fold to a total of 7 concentration points, and hRS7 was used as a positive control (hRS7 was generated according to the sequence disclosed in US20070212350). The results are shown in Table 1. Figure 2 The EC50 values ​​of chimeric antibodies cA1, cA2, cA3, cA4, and cA5 binding to Trop-2 on the MDA-MB-468 cell membrane were 2.234nM, 1.274nM, 1.211nM, 2.470nM, and 2.274nM, respectively. The EC50 of the control antibody hRS7 binding to Trop-2 on the membrane was 3.848nM, indicating that these chimeric molecules can bind to Trop-2 on the cell membrane and are stronger than the control antibody.

[0210] Example 5: Chimeric Antibody-Mediated Trop-2 Internalization

[0211] The FACS method described in Example 2 was used to detect the internalization of Trop-2 on the MDA-MB-468 cell membrane by chimeric antibodies. MDA-MB-468 cells were reacted with different concentrations of chimeric antibodies (initial concentration 2.5 nM, 3-fold gradient dilution, a total of 4 concentration points, namely 2.5, 0.8, 0.3, and 0.1 nM) at 4°C for 30 minutes. After washing the cells twice, the cells were divided into two parts and incubated at 4°C and 37°C for 4 hours respectively. After washing the cells twice, FITC-labeled goat anti-human IgG was added and reacted at 4°C in the dark for 30 minutes. The cells were washed and analyzed by flow cytometry. The amount of internalization = 4°C fluorescence intensity MFI - 37°C fluorescence intensity MFI. See the results. Figure 3 , chimeric antibodies cA1~cA5 were internalized to varying degrees at multiple concentrations.

[0212] Example 6: Analysis of Trop-2 Antibody Antigen Binding Epitopes

[0213] To determine the epitope of the chimeric antibody, human Trop-2-His protein (Acrobiosystems, Catalog No. TR2-H5223) was diluted in PBS at a concentration of 1 μg / ml and coated onto microplates overnight at 4°C. After washing, the plates were blocked with 1% BSA-PBST blocking buffer at 37°C for 1 hour. After washing with PBST, various concentrations of the antibody (starting at 137 nM, diluted three-fold for a total of 12 concentrations) and 0.9 nM biotinylated Trop-2 antibody hRS7 (ThermoFisher Antibody Labeling Kit, Catalog No. A39257) were added to the plates and incubated at 37°C for 1 hour. After washing, HRP-conjugated streptavidin (Abcam, Catalog No. ab7403) was added and incubated at 37°C for 30 minutes. After washing, TMB solution was added and incubated at room temperature in the dark for 30 minutes. The reaction was terminated with 2-NH2SO4 and the absorbance was measured at 450 nm using a microplate reader. The results are as follows Figure 4 As shown, the chimeric antibodies cA3 and cA5, similar to hRS7, can competitively bind to the Trop-2 protein bound to the biotin-labeled hRS7 antibody, indicating that cA3, cA5 and hRS7 share an epitope, while cA1, cA2, and cA4 cannot competitively bind to the Trop-2 protein bound to the biotin-labeled hRS7 antibody, indicating that cA1, cA2, cA4 and hRS7 recognize different epitopes.

[0214] Example 7: Humanization of chimeric antibodies

[0215] According to the results of Example 6, cA3 and cA5 recognize the same epitope, and cA1, cA2, and cA4 recognize the same epitope. Based on the results of Examples 4 and 5, from the cA3 and cA5 groups that recognize the same epitope, cA3, which has higher binding and endocytosis activity, was selected for humanization. From the cA1, cA2, and cA4 groups, cA4, which has the highest expression level (238.91 μg / ml), was selected for humanization. The heavy and light chain variable regions of the cA3 and cA4 antibodies were compared with an available human IgG gene sequence database to identify the best matching human germline IgG gene sequence. The CDR regions of the heavy and light chains of the chimeric antibodies were then transplanted onto the matching framework sequences of the heavy and light chain variable region genes, respectively. Specifically, the human germline IgG heavy and light chains selected by cA3 are (1) IGHV5-51*01, IGKV1-5*01; (2) IGHV5-51*01, IGKV1-12*01; (3) IGHV1-46*01, IGKV1-5*01; (4) IGHV1-46*01, IGKV3-20*01; the corresponding new humanized molecules are HuA3-1, HuA3-2, HuA3-3, and HuA3-4, respectively. The human germline IgG heavy and light chains selected by cA4 are (1) IGHV1-3*01, IGKV3-20*01; (2) IGHV1-3*01, IGKV1-27*01; (3) IGHV5-51*01, IGKV3-20*01; (4) IGHV5-51*01, IGKV4-1*01; (5) IGHV5-51*01, IGKV1-27*01; (6) IGHV1-18*01, IGKV1-33*01; the corresponding new humanized molecules are HuA4-1, HuA4-2, HuA4-3, HuA4-4, HuA4-5, and HuA4-6. After construction, the sequences of the heavy chain variable region, light chain variable region, and CDR region of each humanized antibody are shown in Table 2. The heavy and light chain sequences of the cA3 and cA4 antibodies are shown in Tables 3 and 4. The heavy and light chain constant regions of the humanized antibodies are identical to those of the cA3 and cA4 antibodies. Based on the information disclosed above, those skilled in the art can readily determine the complete amino acid sequences of the heavy and light chains of each humanized molecule.

[0216] The antibodies of the present invention were expressed and purified in 293 cells (source: ATCC) as in Example 3, with an antibody purity of >95%. The expression levels for HuA3-1 to HuA3-4 were 38.63, 57.78, 86.58, and 112.57 μg / ml, respectively; and the expression levels for HuA4-1 to HuA4-6 were 349.45, 316.93, 325.37, 338.42, 333.89, and 279.83 μg / ml, respectively.

[0217] Example 8: Binding of humanized antibodies to Trop-2

[0218] The binding ability of human antibodies to human Trop-2 protein was quantitatively detected by ELISA method as described in Example 4. The initial concentration of the antibody was 68.5 nM, and a 3-fold gradient dilution was performed with a total of 12 concentration points. The results are shown in Figures 5 and 6. Figure 5A The results showed that the EC50 values ​​of antibodies HuA3-1, HuA3-3, and HuA3-4 binding to Trop-2 were 0.175nM, 0.229nM, and 0.223nM, respectively, and their binding activity was stronger than that of the control antibody hRS7 (EC50 was 0.440nM); Figure 5B The results showed that antibody HuA3-2 had comparable binding activity to hRS7, with EC50 values ​​of 0.132 nM and 0.215 nM, respectively; Figure 6A The results showed that the EC50 values ​​of antibodies HuA4-1, HuA4-3, HuA4-6, and hRS7 binding to Trop-2 were 0.197nM, 0.252nM, 0.183nM, and 0.312nM, respectively. Figure 6B The EC50 values ​​of antibodies HuA4-2, HuA4-4, HuA4-5, and hRS7 binding to Trop-2 were 0.314 nM, 0.268 nM, 0.231 nM, and 0.355 nM, respectively, indicating that the humanized molecules HuA4-1 to HuA4-6 are similar to hRS7 and all have high binding activity to Trop-2.

[0219] The binding of humanized antibodies to Trop-2 on the membrane of human breast cancer cell line (MDA-MB-468) endogenously expressing Trop-2 was detected by FACS method as described in Example 1. The initial concentration of the antibody was 7.6 nM, and the antibody was diluted 3 times for a total of 6 concentration points. The results are shown in Table 1. Figure 7A and 7B The EC50 values ​​for HuA3-1 to HuA3-4 binding to Trop-2 on the MDA-MB-468 cell membrane were 1.05 nM, 0.941 nM, 2.086 nM, and 1.646 nM, respectively. The EC50 values ​​for HuA4-1 to HuA4-6 binding to Trop-2 on the MDA-MB-468 cell membrane were 2.413 nM, 1.285 nM, 1.679 nM, 1.259 nM, 1.144 nM, and 1.530 nM, respectively. These results indicate that these humanized molecules exhibited superior binding activity to Trop-2 on the cell membrane compared to the control antibody hRS7 (EC50 of 5.239 nM). Furthermore, compared to cA3 and cA4, the humanized molecules showed no decrease in affinity for Trop-2 compared to the chimeric molecules.

[0220] Example 9: Humanized antibody-mediated Trop-2 endocytosis

[0221] The internalization of Trop-2 on the MDA-MB-468 cell membrane by human antibodies was detected by the FACS method described in Example 2. MDA-MB-468 cells were reacted with different concentrations of antibodies (starting concentration 2.5nM, 3-fold gradient dilution, a total of 4 concentration points) at 4°C for 30 minutes. After washing the cells twice, the cells were divided into two parts and incubated at 4°C and 37°C for 4 hours respectively. After washing the cells twice, FITC-labeled goat anti-human IgG (from Abcam, product number: ab97224) was added and reacted at 4°C in the dark for 30 minutes. The cells were washed and analyzed by flow cytometry. The results are shown in Figure 8A and 8B The Trop-2 internalization mediated by each humanized antibody was better than that of the control antibody hRS7.

[0222] Example 10: Thermal stability analysis of antibodies

[0223] The thermal stability of humanized antibodies was detected by ELISA. Trop-2-His protein was diluted into PBS at a concentration of 1 μg / ml and coated on a microplate at 4°C overnight. Then it was blocked with 1% BSA-PBS blocking solution at 37°C for 1 hour; the antibody was diluted to 205.5nM with 1% BSA-PBS solution, and 50μl per well was placed in the PCR plate. Different temperatures were set on the PCR instrument: 58°C, 61.5°C, 64°C, 67.4°C, 71.8°C, 76°C, 78.6°C, and 80°C. The antibodies were placed at the above temperatures respectively. After reacting for 30 minutes, centrifuged at 1500rpm for 5 minutes to remove inactivated aggregated precipitated proteins. The treated antibody was diluted to 2.5nM and subjected to ELISA analysis according to the method of Example 4. Figure 9A and 9B As shown in the figure, the thermal stability temperatures of the humanized antibodies are all above 60°C, which have good thermal stability. Among them, HuA4-6 has the highest thermal stability temperature, reaching 73°C. Sequence Listing <110> Zhejiang Borui Biopharmaceutical Co., Ltd. Hangzhou Bozhirui Biopharmaceutical Co., Ltd. <120> Anti-Trop-2 antibodies and uses thereof <130> DSP1F212817ZX <140> 202111456440.4 <141> 2021-12-02 <160> 124 <170> SIPOSequenceListing 1.0 <210> 1 <211> 115 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Trp Ile Gly Trp Val Lys Lys Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Phe Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 Val Ser Ser 115 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <400> 2 Asn Tyr Trp Ile Gly 1 5 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <400> 3 Phe Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly 1 5 10 15 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <400> 4 Glu Ser Ala Met Asp Tyr 1 5 <210> 5 <211> 107 <212> PRT <213> Artificial Sequence <400> 5 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Val Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly<000055​​​ 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <400> 6 Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <400> 7 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <400> 8 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 9 <211> 115 <212> PRT <213> Artificial Sequence <400> 9 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Ser Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <400> 10 Ser Tyr Trp Leu Gly 1 5 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <400> 11 Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly 1 5 10 15 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <400> 12 Glu Ser Val Phe Asp Tyr 1 5 <210> 13 <211> 107 <212> PRT <213> Artificial Sequence <400> 13 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Val Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <400> 14 Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <400> 15 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 17 <211> 117 <212> PRT <213> Artificial Sequence <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ser Gln Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ser Arg Tyr Gly Ser Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 18 Asp Tyr Sees His 1 5 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 19 Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe Lys Gly 1 5 10 15 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <400> 20 Tyr Gly Ser Tyr Thr Met Asp Tyr 1 5 <210> 21 <211> 111 <212> PRT <213> Artificial Sequence <400> 21 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Val Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 22 <211> 15 <212> PRT <213> Artificial Sequence <400> twenty two Arg Ala Ser Glu Ser Val Asp Val Tyr Gly Asn Ser Phe Met His 1 5 10 15 <210> twenty three <211> 7 <212> PRT <213> Artificial Sequence <400> twenty three Arg Ala Ser Asn Leu Glu Ser 1 5 <210> twenty four <211> 9 <212> PRT <213> Artificial Sequence <400> twenty four Gln Gln Ser Asn Glu Asp Pro Trp Thr 1 5 <210> 25 <211> 115 <212> PRT <213> Artificial Sequence <400> 25 Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 Val Ser Ser 115 <210> 26 <211> 5 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 26 Ser Tyr Trp Met His 1 5 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 27 Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe Lys Ser 1 5 10 15 <210> 28 <211> 6 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 28 Gly Ser Ala Met Asp Tyr 1 5 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <400> 29 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Val Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <400> 30 Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <400> 31 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <400> 32 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 33 <211> 120 <212> PRT <213> Artificial Sequence <400> 33 Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Ser Asp Gly Ile Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Gly Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 34 Thr Tyr Ser Met Ser 1 5 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 35 Ile Asn Ser Asp Gly Ile Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly 1 5 10 15 <210> 36 <211> 11 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 36 Glu Gly Tyr Gly Asn Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 37 <211> 110 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 37 Asp Ile Val Leu Thr Gln Ser Pro Val Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln His Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <400> 38 Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His 1 5 10 15 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <400> 39 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <400> 40 Gln Gln Asn Asn Glu Asp Leu Thr 1 5 <210> 41 <211> 117 <212> PRT <213> Artificial Sequence <400> 41 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Ser Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 42 <211> 5 <212> PRT <213> Artificial Sequence <400> 42 Asp Tyr Ser Met His 1 5 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <400> 43 Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe Lys Gly 1 5 10 15 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <400> 44 Tyr Gly Ser Tyr Thr Met Asp Tyr 1 5 <210> 45 <211> 111 <212> PRT <213> Artificial Sequence <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Val Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 46 <211> 15 <212> PRT <213> Artificial Sequence <400> 46 Arg Ala Ser Glu Ser Val Asp Val Tyr Gly Asn Ser Phe Met His 1 5 10 15 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <400> 47 Arg Ala Ser Asn Leu Glu Ser 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <400> 48 Gln Gln Ser Asn Glu Asp Pro Trp Thr 1 5 <210> 49 <211> 117 <212> PRT <213> Artificial Sequence <400> 49 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Ser Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 50 <211> 5 <212> PRT <213> Artificial Sequence <400> 50 Asp Tyr Ser Met His 1 5 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <400> 51 Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe Lys Gly 1 5 10 15 <210> 52 <211> 8 <212> PRT <213> Artificial Sequence <400> 52 Tyr Gly Ser Tyr Thr Met Asp Tyr 1 5 <210> 53 <211> 111 <212> PRT <213> Artificial Sequence <400> 53 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Val Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 54 <211> 15 <212> PRT <213> Artificial Sequence <400> 54 Arg Ala Ser Glu Ser Val Asp Val Tyr Gly Asn Ser Phe Met His 1 5 10 15 <210> 55 <211> 7 <212> PRT <213> Artificial Sequence <400> 55 Arg Ala Ser Asn Leu Glu Ser 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <400> 56 Gln Gln Ser Asn Glu Asp Pro Trp Thr 1 5 <210> 57 <211> 117 <212> PRT <213> Artificial Sequence <400> 57 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met[[ID=2D]] ; 35 40 45 Gly Ile Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe 50 55 60 ; Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Ser Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 58 <211> 5 <212> PRT <213> Artificial Sequence <400> 58 Asp Tyr Ser Met His 1 5 <210> 59 <211> 16 <212> PRT <213> Artificial Sequence <400> 59 Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe Lys Gly 1 5 10 15 <210> 60 <211> 8 <212> PRT <213> Artificial Sequence <400> 60 Tyr Gly Ser Tyr Thr Met Asp Tyr 1 5 <210> 61 <211> 111 <212> PRT <213> Artificial Sequence <400> 61 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Val Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 62 <211> 15 <212> PRT <213> Artificial Sequence <400> 62 Arg Ala Ser Glu Ser Val Asp Val Tyr Gly Asn Ser Phe Met His 1 5 10 15 <210> 63 <211> 7 <212> PRT <213> Artificial Sequence <400> 63 Arg Ala Ser Asn Leu Glu Ser 1 5 <210> 64 <211> 9 <212> PRT <213> Artificial Sequence <400> 64 Gln Gln Ser Asn Glu Asp Pro Trp Thr 1 5 <210> 65 <211> 117 <212> PRT <213> Artificial Sequence <400> 65 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Ser Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 66 <211> 5 <212> PRT <213> Artificial Sequence <400> 66 Asp Tyr Ser Met His 1 5 <210> 67 <211> 16 <212> PRT <213> Artificial Sequence <400> 67 Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe Lys Gly 1 5 10 15 <210> 68 <211> 8 <212> PRT <213> Artificial Sequence <400> 68 Tyr Gly Ser Tyr Thr Met Asp Tyr 1 5 <210> 69 <211> 111 <212> PRT <213> Artificial Sequence <400> 69 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Val Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 70 <211> 15 <212> PRT <213> Artificial Sequence <400> 70 Arg Ala Ser Glu Ser Val Asp Val Tyr Gly Asn Ser Phe Met His 1 5 10 15 <210> 71 <211> 7 <212> PRT <213> Artificial Sequence <400> 71 Arg Ala Ser Asn Leu Glu Ser 1 5 <210> 72 <211> 9 <212> PRT <213> Artificial Sequence <400> 72 Gln Gln Ser Asn Glu Asp Pro Trp Thr 1 5 <210> 73 <211> 115 <212> PRT <213> Artificial Sequence <400> 73 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 74 <211> 5 <212> PRT <213> Artificial Sequence <400> 74 Ser Tyr Trp Met His 1 5 <210> 75 <211> 16 <212> PRT <213> Artificial Sequence <400> 75 Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe Lys Ser 1 5 10 15 <210> 76 <211> 6 <212> PRT <213> Artificial Sequence <400> 76 Gly Ser Ala Met Asp Tyr 1 5 <210> 77 <211> 107 <212> PRT <213> Artificial Sequence <400> 77 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Val Ser Glu Ser Ile Ser Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 78 <211> 11 <212> PRT <213> Artificial Sequence <400> 78 Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 79 <211> 7 <212> PRT <213> Artificial Sequence <400> 79 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial Sequence <400> 80 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 81 <211> 115 <212> PRT <213> Artificial Sequence <400> 81 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 82 <211> 5 <212> PRT <213> Artificial Sequence <400> 82 Ser Tyr Trp Met His 1 5 <210> 83 <211> 16 <212> PRT <213> Artificial Sequence <400> 83 Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe Lys Ser 1 5 10 15 <210> 84 <211> 6 <212> PRT <213> Artificial Sequence <400> 84 Gly Ser Ala Met Asp Tyr 1 5 <210> 85 <211> 107 <212> PRT <213> Artificial Sequence <400> 85 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Val Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 86 <211> 11 <212> PRT <213> Artificial Sequence <400> 86 Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 87 <211> 7 <212> PRT <213> Artificial Sequence <400> 87 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 88 <211> 9 <212> PRT <213> Artificial Sequence <400> 88 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 89 <211> 115 <212> PRT <213> Artificial Sequence <400> 89 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 90 <211> 5 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 90 Ser Tyr Trp Met His 1 5 <210> 91 <211> 16 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 91 Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe Lys Ser 1 5 10 15 <210> 92 <211> 6 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 92 Gly Ser Ala Met Asp Tyr 1 5 <210> 93 <211> 107 <212> PRT <213> Artificial Sequence <400> 93 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Val Ser Glu Ser Ile Ser Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 94 <211> 11 <212> PRT <213> Artificial Sequence <400> 94 Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 95 <211> 7 <212> PRT <213> Artificial Sequence <400> 95 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial Sequence <400> 96 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 97 <211> 115 <212> PRT <213> Artificial Sequence <400> 97 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 98 <211> 5 <212> PRT <213> Artificial Sequence <400> 98 Ser Tyr Trp Met His 1 5 <210> 99 <211> 16 <212> PRT <213> Artificial Sequence <400> 99 Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe Lys Ser 1 5 10 15 <210> 100 <211> 6 <212> PRT <213> Artificial Sequence <400> 100 Gly Ser Ala Met Asp Tyr 1 5 <210> 101 <211> 107 <212> PRT <213> Artificial Sequence <400> 101 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Val Ser Glu Ser Ile Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 102 <211> 11 <212> PRT <213> Artificial Sequence <400> 102 Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 103 <211> 7 <212> PRT <213> Artificial Sequence <400> 103 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 104 <211> 9 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 104 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 105 <211> 115 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 105 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 106 <211> 5 <212> PRT <213> Artificial Sequence <400> 106 Ser Tyr Trp Met His 1 5 <210> 107 <211> 16 <212> PRT <213> Artificial Sequence <400> 107 Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe Lys Ser 1 5 10 15 <210> 108 <211> 6 <212> PRT <213> Artificial Sequence <400> 108 Gly Ser Ala Met Asp Tyr 1 5 <210> 109 <211> 107 <212> PRT <213> Artificial Sequence <400> 109 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Val Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe [[ID=]18]85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 110 <211> 11 <212> PRT <213> Artificial Sequence <400> 110 ] Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 111 <211> 7 <212> PRT <213> Artificial Sequence <400> 111 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 112 <211> 9 <212> PRT <213> Artificial Sequence <400> 112 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 113 <211> 115 <212> PRT <213> Artificial Sequence <400> 113 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 114 <211> 5 <212> PRT <213> Artificial Sequence <400> 114 Ser Tyr Trp Met His 1 5 <210> 115 <211> 16 <212> PRT <213> Artificial Sequence <400> 115 Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe Lys Ser 1 5 10 15 <210> 116 <211> 6 <212> PRT <213> Artificial Sequence <400> 116 Gly Ser Ala Met Asp Tyr 1 5 <210> 117 <211> 107 <212> PRT <213> Artificial Sequence <400> 117 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ala Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Val Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 118 <211> 11 <212> PRT <213> Artificial Sequence <400> 118 Arg Ala Ser Gln Ser Ile Gly Ala Asn Ile His 1 5 10 <210> 119 <211> 7 <212> PRT <213> Artificial Sequence <400> 119 Tyr Val Ser Glu Ser Ile Ser 1 5 <210> 120 <211> 9 <212> PRT <213> Artificial Sequence <400> 120 Leu Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 121 <211> 447 <212> PRT <213> Artificial Sequence <400> 121 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ser Gln Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asp Ala Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ser Arg Tyr Gly Ser Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 122 <211> 218 <212> PRT <213> Artificial Sequence <400> 122 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Val Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 123 <211> 445 <212> PRT <213> Artificial Sequence <400> 123 Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Gly Ser Gly Asn Ile Lys Tyr Asp Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 124 <211> 214 <212> PRT <213> Artificial Sequence​​​​​​​​​​​​​​​​​​​​​65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Leu Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An anti-Trop-2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDRs, namely, VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises three CDRs, namely, VL CDR1, VL CDR2, and VL CDR3; wherein: The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 114; The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 115; The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 116; The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 118; The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 119; The amino acid sequence of VL CDR3 is shown in SEQ ID NO:

120.

2. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 113; the amino acid sequence of the light chain variable region has at least 90% sequence identity with SEQ ID NO:

117.

3. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 113; the amino acid sequence of the light chain variable region has at least 95% sequence identity with SEQ ID NO:

117.

4. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region has at least 96% sequence identity with SEQ ID NO: 113; the amino acid sequence of the light chain variable region has at least 96% sequence identity with SEQ ID NO:

117.

5. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region has at least 97% sequence identity with SEQ ID NO: 113; the amino acid sequence of the light chain variable region has at least 97% sequence identity with SEQ ID NO:

117.

6. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region has at least 98% sequence identity with SEQ ID NO: 113; the amino acid sequence of the light chain variable region has at least 98% sequence identity with SEQ ID NO:

117.

7. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 113; the amino acid sequence of the light chain variable region has at least 99% sequence identity with SEQ ID NO:

117.

8. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 113; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

117. 9 . The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 1 , further comprising one or more of a heavy chain constant region, a light chain constant region, and an Fc region. 10 . The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 9 , wherein the light chain constant region is a λ chain or a κ chain constant region.

11. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 9, wherein the antibody or antigen-binding fragment thereof is of IgG1, IgG2, IgG3 or IgG4 type.

12. The anti-Trop-2 antibody or antigen-binding fragment thereof according to claim 9, wherein The antibody heavy chain consists of the sequence shown in SEQ ID NO: 123, and the antibody light chain consists of the sequence shown in SEQ ID NO:

124.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier.

14. A biomaterial comprising: (1) A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12; (2) A vector, host cell or microorganism comprising (1); (3) The expression product of (2) above; the expression product is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the method comprising culturing the host cell or microorganism according to claim 14 to express the antibody or antigen-binding fragment thereof and isolating the antibody or antigen-binding fragment thereof.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the pharmaceutical composition according to claim 13, or the biomaterial according to claim 14 in the preparation of a medicament for treating a tumor, wherein the tumor is gastric cancer, pancreatic cancer, prostate cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, bile duct cancer, urothelial carcinoma, breast cancer, or cervical cancer.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13, or the biomaterial according to claim 14, in combination with one or more other cancer therapeutic agents, in the preparation of a medicament for treating tumors, wherein the tumor is gastric cancer, pancreatic cancer, prostate cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, bile duct cancer, urothelial carcinoma, breast cancer, or cervical cancer.

18. The use according to claim 17, wherein the other cancer therapeutic agent is paclitaxel, mitomycin, vincristine, colchicine, doxorubicin, daunorubicin, dactinomycin D, emetine, puromycin, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan, docetaxel, pemetrexed, sorafenib, oxaliplatin, 5-FU or lapatinib.

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