Anti-nectin-4 antibody and its application

Abstract

Provided is an antibody molecule or a fragment thereof that binds to human nectin-4. The antibody is obtained through hybridoma screening and humanization technology, and is used to prevent or treat cancer and can be used as a clinical lead drug molecule.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of priority to Chinese invention patent application number CN202010320420.3, filed on April 21, 2020, the entire contents of which are hereby incorporated by reference. Technical Field

[0003] The present invention belongs to the field of antibody drugs, and in particular, relates to antibodies against human nectin-4 and uses thereof in preparing drugs. Background Art

[0004] Nectin-4 (also known as PVRL4, poliovirus receptor-like molecule 4) is a type I transmembrane glycoprotein with a size of 66KD. It belongs to the IG superfamily protein molecule of the Nectin family. Its extracellular domain is composed of three Ig-like domains (VCC type) and participates in the formation and maintenance of adhesion junctions together with cadherins.

[0005] Nectin-4 is closely linked to the development and progression of various tumor cells. Nectin-4 has been found to be expressed in a variety of solid tumors, particularly bladder cancer. In breast, ovarian, and lung cancers, it acts as a tumor-associated antigen, with detection rates in 50% of breast, 49% of ovarian, and 86% of lung cancer tissues, respectively. Nectin-4 plays a key role in the development, invasion, and metastasis of these epithelial malignancies. Therefore, nectin-4 has become an important target for the diagnosis and treatment of many solid tumors.

[0006] Currently, the leading drug targeting nectin-4 is enfortumab vedotin, an antibody-drug conjugate (ADC) consisting of an anti-nectin-4 monoclonal antibody conjugated to the cell-killing drug monomethyl auristatin E (MMAE). It is primarily used to treat bladder cancer, particularly urothelial carcinoma, and received FDA Breakthrough Therapy Designation in March 2018. Furthermore, other studies have shown that the adhesion factor Nnectin-4 is not only an effective prognostic factor for breast cancer, but also a therapeutic target for triple-negative breast cancer (TNBC). In vitro and in vivo studies have confirmed that anti-nectin-4 antibody-drug conjugates (ADCs) have a promising therapeutic effect in both localized and metastatic TNBC. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to obtain a high-affinity antibody that specifically binds to nectin-4 through hybridoma screening and humanization technology, wherein a fully human antibody sequence is obtained through humanization transformation.

[0008] In response to the above technical problems, the present invention aims to provide an antibody molecule or fragment thereof that specifically binds to nectin-4, particularly human nectin-4, and to provide uses thereof. The antibody molecule fragments described herein encompass various functional fragments of antibodies, such as antigen-binding portions thereof, such as Fab, F(ab')2, or scFv fragments.

[0009] The technical solutions of the present invention are as follows:

[0010] In one aspect, the present invention provides an antibody molecule or fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region respectively comprise a combination of heavy chain CDRs and light chain CDRs selected from the following:

[0011] (1) CDR-H1 (GYTFTTY), CDR-H2 (YPGNVN), CDR-H3 (GLYYFDY) shown in SEQ ID NOs: 35, 39, and 43, respectively; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 47, and 49, respectively;

[0012] (2) CDR-H1 (GYTFTTYYIH), CDR-H2 (WIYPGNVNTK), CDR-H3 (GLYYFDY) shown in SEQ ID NOs: 36, 40, and 43, respectively; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 47, and 49, respectively;

[0013] (3) CDR-H1 (TYYIH), CDR-H2 (WIYPGNVNTKYNEKFKG), CDR-H3 (GLYYFDY) shown in SEQ ID NOs: 37, 41, and 43, respectively; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 47, and 49, respectively;

[0014] (4) CDR-H1 (TTYYIH), CDR-H2 (WIGWIYPGNVNTK), CDR-H3 (ARGLYYFD) shown in SEQ ID NOs: 38, 42, and 44, respectively; and CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRY), CDR-L3 (QQDYSSPY) shown in SEQ ID NOs: 46, 48, and 50, respectively;

[0015] (5) CDR-H1 (GFSLIDY), CDR-H2 (WGDGK), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 51, 55, and 59, respectively; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 61, 63, and 65, respectively;

[0016] (6) CDR-H1 (GFSLIDYGVS), CDR-H2 (VIWGDGKIY), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 52, 56, and 59, respectively; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 61, 63, and 65, respectively;

[0017] (7) CDR-H1 (DYGVS), CDR-H2 (VIWGDGKIYYNSVLKS), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 53, 57, and 59, respectively; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 61, 63, and 65, respectively;

[0018] (8) CDR-H1 (IDYGVS), CDR-H2 (WLGVIWGDGKIY), CDR-H3 (AKQGGLLFYAMD) shown in SEQ ID NOs: 54, 58, and 60, respectively; and CDR-L1 (LNSYSQKNYLAWY), CDR-L2 (LLIYFASTRE), CDR-L3 (QQHYNTPF) shown in SEQ ID NOs: 62, 64, and 66, respectively;

[0019] (9) CDR-H1 (GFSLIDY), CDR-H2 (WGDGK), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 51, 55, and 59, respectively; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 67, 63, and 65, respectively;

[0020] (10) CDR-H1 (GFSLIDY), CDR-H2 (WGDAK), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 51, 68, and 59, respectively; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 67, 63, and 65, respectively;

[0021] (11) CDR-H1 (GFSLIDY), CDR-H2 (WGGGK), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 51, 69, and 59, respectively; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 67, 63, and 65, respectively;

[0022] (12) CDR-H1 (GYTFTSY), CDR-H2 (YPGNAN), CDR-H3 (SVYYFDY) shown in SEQ ID NOs: 70, 74, and 78, respectively; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 80, and 49, respectively;

[0023] (13) CDR-H1 (GYTFTSYYIH), CDR-H2 (WIYPGNANNK), CDR-H3 (SVYYFDY) shown in SEQ ID NOs: 71, 75, and 78, respectively; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 80, and 49, respectively;

[0024] (14) CDR-H1 (SYYIH), CDR-H2 (WIYPGNANNKYNENFKG), CDR-H3 (SVYYFDY) shown in SEQ ID NOs: 72, 76, and 78, respectively; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 80, and 49, respectively;

[0025] (15) CDR-H1 (TSYYIH), CDR-H2 (WIGWIYPGNANNK), CDR-H3 (ARSVYYFD) shown in SEQ ID NOs: 73, 77, and 79, respectively; and CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRN), CDR-L3 (QQDYSSPY) shown in SEQ ID NOs: 46, 81, and 50, respectively;

[0026] (16) CDR-H1 (GYSFTDY), CDR-H2 (NPNNGN), CDR-H3 (EDRYAFAY) shown in SEQ ID NOs: 82, 86, and 90, respectively; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), CDR-L3 (QHTWEIPYT) shown in SEQ ID NOs: 92, 94, and 96, respectively;

[0027] (17) CDR-H1 (GYSFTDYYMH), CDR-H2 (RVNPNNGNTL), CDR-H3 (EDRYAFAY) shown in SEQ ID NOs: 83, 87, and 90, respectively; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), CDR-L3 (QHTWEIPYT) shown in SEQ ID NOs: 92, 94, and 96, respectively;

[0028] (18) CDR-H1 (DYYMH), CDR-H2 (RVNPNNGNTLYNQKFRG), CDR-H3 (EDRYAFAY) shown in SEQ ID NOs: 84, 88, and 90, respectively; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), CDR-L3 (QHTWEIPYT) shown in SEQ ID NOs: 92, 94, and 96, respectively;

[0029] (19) CDR-H1 (TDYYMH), CDR-H2 (WIGRVNPNNGNTL), CDR-H3 (AREDRYAFA) shown in SEQ ID NOs: 85, 89, and 91, respectively; and CDR-L1 (STSSYTYMHWY), CDR-L2 (LLIKYASNLE), CDR-L3 (QHTWEIPY) shown in SEQ ID NOs: 93, 95, and 97, respectively.

[0030] According to the domain composition of the heavy chain variable region and the light chain variable region in antibodies known in the art, the heavy chain variable region or the light chain variable region in the antibody molecule of the present invention or its fragment comprises the above-mentioned domain components in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR is a framework region.

[0031] Preferably, in the antibody molecule or fragment thereof provided by the present invention, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, 9 or 10, or an amino acid sequence having at least 75% identity thereto; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, 11 or 12, or an amino acid sequence having at least 75% identity thereto; or,

[0032] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13, 15, 16, 18, 19, 20 or 21, or an amino acid sequence that is at least 75% identical to said amino acid sequence; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, 22, 23 or 25, or an amino acid sequence that is at least 75% identical to said amino acid sequence; or

[0033] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, 29 or 30, or an amino acid sequence that is at least 75% identical to said amino acid sequence; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, 31 or 32, or an amino acid sequence that is at least 75% identical to said amino acid sequence; or

[0034] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence that is at least 75% identical to the amino acid sequence; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence that is at least 75% identical to the amino acid sequence.

[0035] According to a specific embodiment of the present invention, the heavy chain variable region and the light chain variable region in the antibody molecule or fragment thereof are selected from a combination of the following amino acid sequences:

[0036] (1) the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 7; and, the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 8;

[0037] (2) the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 9; and, the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 11;

[0038] (3) the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 9; and, the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 12;

[0039] (4) the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 10; and, the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 11;

[0040] (5) the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 10; and, the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 12;

[0041] (6) the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 13; and, the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 14;

[0042] (7) the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 16; and, the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 22;

[0043] (8) the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 16; and, the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 23;

[0044] (9) the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 16; and, the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 25;

[0045] (10) the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 19; and, the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 25;

[0046] (11) the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 21; and, the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 25;

[0047] (12) the amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 27; and, the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 28;

[0048] (13) the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 29; and, the amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 32;

[0049] (14) the amino acid sequence set forth in SEQ ID NO: 30 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 30; and, the amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 32; or

[0050] (15) the amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 33; and, the amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 34.

[0051] Based on the specific amino acid sequences of the above-mentioned heavy chain variable regions or light chain variable regions provided by the present invention, those skilled in the art can routinely determine the amino acid sequences of the heavy chain CDRs and light chain CDRs contained therein. The heavy and light chain CDRs and their combinations determined by other methods known in the art are also included in the scope of the present invention.

[0052] The antibody molecule or fragment thereof provided by the present invention binds to poliovirus receptor-like molecule 4 (Nectin-4), preferably mammalian Nectin-4, more preferably primate Nectin-4, further preferably human or monkey Nectin-4, especially human Nectin-4.

[0053] Preferably, the antibody molecule is a murine antibody, a chimeric antibody or a fully or partially humanized antibody; the fragment is any fragment of the antibody molecule that can specifically bind to nectin-4, such as scFv, dsFv, (dsFv)2, Fab, Fab′, F(ab′)2 or Fv fragment.

[0054] Preferably, the antibody molecule is a monoclonal antibody or a single-chain antibody.

[0055] Preferably, the antibody molecule or fragment thereof further comprises a human or mouse constant region, preferably a mouse or human heavy chain constant region (CH) and / or a light chain constant region (CL); preferably, the antibody molecule or fragment thereof comprises a heavy chain and a light chain, for example, two heavy chains and two light chains. More preferably, the antibody molecule or fragment thereof comprises an IgG, IgA, IgM, IgD or IgE heavy chain constant region and / or a κ or λ type light chain constant region.

[0056] According to a specific embodiment of the present invention, the antibody molecule provided herein is a monoclonal antibody, preferably a humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 type, and the light chain constant region is of the κ type. For example, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence that is at least 75% identical to the amino acid sequence; and the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence that is at least 75% identical to the amino acid sequence.

[0057] In the context of the present invention, "at least 75% identity" is any percentage number between 75% and 100% identity, for example 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0058] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the light chain variable region, heavy chain variable region, heavy chain or light chain contained in the antibody molecule or fragment thereof described in the present invention.

[0059] The nucleic acid molecules of the present invention can be cloned into vectors and then transformed or transfected into host cells. Therefore, in another aspect, the present invention provides a vector comprising the nucleic acid molecules of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0060] The vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter a host cell in any way for purposes such as preservation or expression of antibodies. Therefore, in another aspect, the present invention provides a host cell comprising a nucleic acid molecule and / or vector of the present invention, or the host cell is transformed or transfected by a nucleic acid molecule and / or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

[0061] The antibody molecules provided by the present invention can be obtained by any method known in the art. For example, the heavy chain variable region and / or light chain variable region of the antibody can be obtained from the nucleic acid molecules provided by the present invention, or the heavy chain and / or light chain of the antibody molecule can be obtained, and then assembled into an antibody with any other domains of the antibody molecule; or, the host cell provided by the present invention is allowed to express the heavy chain variable region and / or light chain variable region of the antibody molecule or the heavy chain and / or light chain of the antibody molecule to assemble into the antibody, and the host cell is cultured. Optionally, the method further comprises the step of recovering the produced antibody molecule.

[0062] The antibody molecules or fragments thereof, nucleic acid molecules, vectors, host cells or fusion proteins provided by the present invention can be included in compositions, more particularly in pharmaceutical preparations, and thus used for various purposes according to actual needs. Therefore, in another aspect, the present invention also provides a composition comprising the antibody molecules or fragments thereof, nucleic acid molecules, vectors and / or host cells provided by the present invention. Preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

[0063] In another aspect, the present invention also provides use of the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell and / or composition in the preparation of a reagent for detecting or diagnosing a disease or disorder.

[0064] Accordingly, the present invention also provides a method for detecting or diagnosing a disease or disorder, comprising contacting an antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition of the present invention with a sample from a subject, wherein the subject is a mammal, preferably a primate, and more preferably a human.

[0065] In another aspect, the present invention also provides use of the antibody molecule or fragment thereof in the preparation of an antibody-drug conjugate.

[0066] Accordingly, the present invention provides an antibody drug conjugate, which is formed by coupling the antibody molecule or fragment thereof of the present invention with a cytotoxic moiety.

[0067] Preferably, the cytotoxic moiety is a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binder. Preferably, the tubulin inhibitor is selected from a maytansine derivative, monomethyl auristatin E (MMAE), monomethylauristatin F (MMAF), monomethyl dolastatin 10, a tubulysin derivative, a cryptophycin derivative, and a taltobulin. Preferably, the topoisomerase inhibitor is selected from a doxorubicin metabolite PNU-159682 derivative and an irinotecan (CPT-11) metabolite SN38 derivative. Preferably, the DNA binder is selected from a PBD derivative and a duocarmycin derivative.

[0068] In another aspect, the present invention also provides use of the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, composition and / or antibody-drug conjugate in the preparation of a medicament for preventing or treating a disease or disorder.

[0069] In another aspect, the present invention further provides a method for preventing or treating a disease or disorder, comprising administering the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, composition, and / or antibody-drug conjugate of the present invention to a subject in need thereof, wherein the subject is a mammal, more preferably a human.

[0070] Accordingly, in another aspect, the present invention provides a kit comprising an antibody molecule or fragment thereof, a nucleic acid molecule, a vector, a host cell, a composition and / or an antibody drug conjugate of the present invention. The kit can be used for therapeutic, detection or diagnostic purposes, such as treating, detecting or diagnosing a disease or disorder.

[0071] According to the various technical solutions provided by the present invention, the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, composition and / or antibody-drug conjugate can be used for the prevention, treatment, detection or diagnosis of diseases or disorders associated with high expression of Nectin-4. Preferably, the disease or disorder is a tumor or cancer with high expression of Nectin-4, in particular a solid tumor. For example, the disease or disorder is bladder cancer, pancreatic cancer, breast cancer (including triple-negative subtype and basal subtype), non-small cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer, etc.; in particular, bladder cancer, breast cancer, ovarian cancer or lung cancer.

[0072] Compared to existing technologies, this invention utilizes hybridoma screening and humanization techniques to obtain a high-affinity antibody that specifically binds to nectin-4. Humanization results in a fully human antibody sequence. Furthermore, studies of the physicochemical properties and cellular activity of this molecule confirm the identification of an effective clinical lead drug sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0074] Figure 1 The results of flow cytometry identification of antigen-expressing cell lines are shown. Figure 1 A: HT-1376 bladder cancer cells, small Figure 1 B: CHO-huNectin4S8 cells.

[0075] Figure 2 The results of FACS binding activity assay of hybridoma cell culture supernatants are shown. Figure 2 A: First round of screening, small Figure 2 B: Second round of screening.

[0076] Figure 3 Shown are the results of FACS binding experiments of antibodies to BT474-expressing cells.

[0077] Figure 4 The results of the BT474 cell endocytosis activity assay of the antibodies are shown.

[0078] Figure 5 The results of FACS binding experiments of antibodies to different cells expressing huNectin4, muNectin4, and cynoNectin4 are shown.

[0079] Figure 6 The results of the antibody binding experiment with Nectin protein family members are shown. Figure 6 A: Nectin-1, small Figure 6 B: Nectin-2, small Figure 6 C: Nectin-3, small Figure 6 D: Nectin-4.

[0080] Figure 7 The monkey serum stability test results of the antibody are shown. Figure 7 A: Enfortumab, a control antibody Figure 7 B:42D20 hz10.

[0081] Figure 8 The metabolism of antibodies in mice is shown. Figure 8A: Enfortumab, a control antibody Figure 8 B:42D20 hz10. DETAILED DESCRIPTION

[0082] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0083] The experimental methods in the following examples are all conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are all commercially available products unless otherwise specified.

[0084] The heavy chain amino acid sequence of the control antibody Enfortumab is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2.

[0085] The antibody sequences of the present invention are shown in Appendix I to Appendix IV.

[0086] The antigen NECTIN4 recombinant protein (sequence number: NP_002178.2, 32aa-349aa) is shown in SEQ ID NO: 3.

[0087] Example 1 Synthesis and expression of control antibodies

[0088] The fully synthetic Enfortumab antibody light chain variable region and heavy chain variable region genes were cloned into the eukaryotic expression vector pCDNA3.1, which carries the genes encoding the human kappa light chain constant region and the human IgG1 heavy chain constant region, respectively. Enfortumab light and heavy chain expression plasmids were generated and transformed into Escherichia coli for amplification. Large quantities of plasmids containing the Enfortumab antibody light chain (SEQ ID NO: 2) and heavy chain (SEQ ID NO: 1) were isolated and mixed with polyethyleneimine (PEI) before co-transfection into HEK293 cells. Five to six days after cell transfection, the culture supernatant was collected and purified using a Mabselect affinity chromatography column to obtain the Enfortumab antibody.

[0089] Example 2 Preparation of Nectin-4 Antigen-Expressing Cell Line

[0090] The nectin-4 reading frame was cloned from a nectin-4 cDNA vector (Beijing Sino Biological, Cat: HG19771-UT) by PCR and cloned into a stable expression vector containing a glutamine synthetase (GS) selection gene by enzyme digestion. The nectin-4 reading frame was electroporated (Nucleofector IIb, Lonza) into suspension-cultured CHO-K1 cells. The transfected cells were plated in CD CHOAGT™ medium (Gibco, Cat: 12490-025) containing 50 μM MSX (Sigma, Cat: M5379) and cultured in 96-well cell culture plates at 37°C in 5% CO2 for 2-3 weeks. Nine wells of cell growth were pre-screened under a microscope using MSX pressure selection and expanded to 24-well cell culture plates. Finally, the antigen-high-expressing clone S8 was selected by flow cytometry analysis (FACS) for expansion and cryopreservation.

[0091] The high-expression clone S8 was named CHO-huNectin4 S8, and the results of the comparative identification between this cell and HT-1376 bladder cancer cells endogenously expressing Nectin-4 are shown in Figure 1 .

[0092] Example 3 Screening and identification of hybridoma cells

[0093] 1. Mouse Immunization

[0094] Ten 8-week-old Balb / c mice were divided into two groups and immunized with conventional immunization and rapid immunization, respectively. The CHO-huNectin4 S8 engineered cell line was used as the immunizing agent for conventional immunization, and the recombinant protein human Nectin4 (purchased from Nearshore Protein, Cat: CJ19) was used as the immunizing agent for rapid immunization.

[0095] Blood samples were collected from mice before immunization as a negative control. The two immunizing agents were injected intraperitoneally, with the second and third immunizations performed 2 weeks apart. Blood titers were measured one week after the third immunization, and mice with high titers were selected for a boost immunization 3 days before fusion.

[0096] 2. Fusion Screening

[0097] Resuscitate and expand SP20 myeloma cells to a desired size. Change the cell culture medium the day before fusion to ensure optimal cell growth. On the day of fusion, harvest the myeloma cells, centrifuge, resuspend in basal medium, and count until ready for use.

[0098] The spleen and lymph nodes of mice were sterilely removed and ground to prepare a cell suspension. The cells were filtered through a cell strainer and then lysed. After lysis, the red blood cells were combined and counted. B cells and SP20 myeloma cells were mixed at a ratio of 1:2. After centrifugation, the mixed cells were washed twice with electrofusion solution and resuspended to adjust the density to 1-2×10 7 The cell suspension was added to an electroporation cuvette for fusion. After fusion, the cells were added to complete culture medium and placed in a 37°C, 8% CO2 incubator to recover for 30-240 minutes. Then, HAT medium was added and plated into 384-well plates for culture. The medium was replenished on the 5th day, and replaced with HT medium on the 7th day. Positive hybridoma screening was performed on the 8th-10th day as described below.

[0099] Hybridoma cell culture supernatants were analyzed by FACS to identify positive wells that bind to CHO-huNECTIN4S8 cells stably expressing human Nectin4 antigen on their cell surface, while not binding to blank CHOK1 cells. Positive wells were isolated by limiting dilution, and subcloning was terminated when two consecutive subclones showed 100% positivity. The resulting hybridoma cell line secreted only one antibody.

[0100] The results of FACS binding activity assay of hybridoma cell culture supernatant are shown in Figure 2 and Tables 1 to 3.

[0101] Table 1. Clones selected in the first round of screening

[0102] ID FACS <![CDATA[FACS(*10 5 )]]> 5M21 1731598.512 173.1599 7I18 900458.7143 90.04587 26G13 493254.2857 49.32543 29B12 685057.4296 68.50574 30J3 440992.4 44.09924

[0103] Table 2. Clones selected in the second round of screening

[0104] ID FACS <![CDATA[FACS(*10 5 )]]> 4A15 221093.1035 22.1 21N4 264347.4081 26.4 28J3 208131.2025 20.8 32A13 677419.4598 67.7 38D17 266536.8786 26.7 41024 1749182.695 174.9 42D20 533378.824 53.3 45F2 393898.7755 39.4 49C23 1510386.33 151.0 50I15 1299424.85 129.9

[0105] Table 3. Clones selected in the third round of screening

[0106]

[0107]

[0108] The obtained mouse monoclonal antibody is named after the hybridoma cell line ID.

[0109] Example 4 Sequence Identification of the Variable Region of Murine Monoclonal Antibodies

[0110] After the hybridoma cells secreting anti-human nectin-4 antibodies were expanded and cultured, total cellular RNA was extracted according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); the hybridoma cell total RNA was reverse transcribed into cDNA using 5× PrimeScript RT MasterMix (Takara); the antibody light chain variable region IgVL (κ) and heavy chain variable region VH sequences were amplified using degenerate primers (Anke Krebber.1997) and Extaq PCR reagent (Takara); the PCR amplification products were purified using a PCR clean-up Gel extraction kit (Macherey-Nagel); the amplified PCR products were ligated into T vectors according to the instructions of the pClone007 Simple Vector Kit (Qingke Biotechnology Co., Ltd.) and transformed into Escherichia coli competent cells. After strain amplification and plasmid extraction, DNA sequencing was performed to obtain the monoclonal antibody variable region sequences.

[0111] Example 5 Preparation of chimeric antibodies

[0112] The heavy chain variable region sequence of a murine anti-human nectin-4 monoclonal antibody was spliced with a published heavy chain constant region sequence of a human monoclonal antibody of the IgG1 subclass (SEQ ID NO: 4) and constructed into a mammalian cell expression vector. The light chain variable region sequence of a murine anti-human nectin-4 monoclonal antibody was spliced with a published light chain constant region sequence of a human monoclonal antibody of the κ subclass (SEQ ID NO: 5) and constructed into a mammalian cell expression vector. The constructed heavy chain and light chain vectors of the anti-human nectin-4 chimeric antibody were mixed and transfected into HEK293 cells using PEI. Approximately 7 days later, the cell supernatant was collected and purified using MabSelect to obtain the anti-human nectin-4 chimeric antibody protein.

[0113] The resulting chimeric antibody is referred to herein as "murine antibody designation xiIgG".

[0114] Example 6 Humanization of mouse antibodies and preparation of humanized antibodies

[0115] Comprehensive antibody encoding schemes are used to determine the amino acid sequence regions of the six antigen complementarity determining clusters (CDRs) of the heavy and light chains of the mouse antibody, as well as the framework region that supports the conserved three-dimensional conformation of the antibody. Subsequently, by analyzing and searching known human antibody sequences, the human antibody heavy chain variable region sequence that is most similar to the mouse antibody is selected, such as IGHV1|IGHJ4*01. This antibody framework region sequence is used as a template, and the mouse antibody heavy chain CDRs are combined with the human antibody framework region to ultimately generate the humanized antibody heavy chain variable region sequence. The same process is used to generate the humanized antibody light chain variable region sequence.

[0116] Antibodies with mouse antibody CDRs directly transplanted to human framework regions often experience a sharp drop in binding activity, so it is necessary to change individual amino acids in the framework region from human to mouse. To determine the back mutation site, one is to compare the designed humanized antibody sequence with the original mouse antibody sequence to check which amino acids are different; the second is to check whether these amino acids play an important role in supporting the antibody structure or in binding to the antigen. When humanizing the designed sequence, it is necessary to check whether there are some potential post-translational modification sites, such as N (asparagine) glycosylation sites, N deamidation sites, D (aspartic acid) isomerization sites, etc.

[0117] The humanized heavy chain variable region and light chain variable region were combined in pairs, and humanized antibodies were obtained by referring to the preparation of chimeric antibodies described in Example 5. The humanized antibodies were named "mouse antibody naming hzmn", where m and n are the numbers of the humanized (hz) modified sequences of the murine antibody VH and VL, respectively (VH_hz and VL_hz).

[0118] Example 7 Preparation of Antibody Drug Conjugates (ADCs)

[0119] Antibodies were reduced with 2.0-2.6 equivalents of TECP in PBS (pH 7.4) for 2 hours. A vcMMAE solution in DMA was then added to the reduced antibody solution (molar ratio of vcMMAE to antibody: 6:1). After stirring at 2-8°C for 1 hour, the solution was ultrafiltered to remove DMA and small molecule residues. The absorbance of the conjugate at 248-280 nm was measured by UV spectrophotometry, and the conjugate concentration was calculated. Samples were aliquoted into cryovials and stored at -80°C. The DAR value (4.0 ± 1) of the samples was determined by HPLC-HIC.

[0120] The ADC corresponding to the antibody is named "Antibody Name-E".

[0121] Example 8 In vitro cell binding assay

[0122] The anti-human nectin-4 control antibody Enfortumumab, the antibody of the present invention, or the ADC was diluted 2-fold starting from a starting concentration of 100 nM, for a total of 16 concentration points. 10 μl of the antibody at each concentration point was added to a 384-well plate.

[0123] BT474 cells (breast cancer cells) expressing nectin-4 on the cell surface were collected by centrifugation at 100 g for 5 minutes at room temperature, washed once with PBS containing 0.5% BSA, centrifuged at 100 g for 5 minutes at room temperature, and resuspended at a density of approximately 2×10 6 10 μl of culture medium was added to the wells of a 384-well plate containing the antibody. After incubation at 4°C for 1 hour, a fluorescently labeled goat anti-human IgG secondary antibody was added. Incubation was continued at 4°C for another 1 hour, and the mean fluorescence reading of the cell population was analyzed by flow cytometry.

[0124] The results of the FACS binding experiment between the mouse antibody of the present invention and BT474 cells are shown in Figure 3 in Figure 3 A and Tables 4 and 5.

[0125] Table 4. Binding of mouse monoclonal antibodies to BT474 cells

[0126]

[0127] Table 5. Binding of mouse monoclonal antibodies to BT474 cells

[0128]

[0129] The results of the FACS binding experiment between the humanized modified molecule of the present invention and BT474 cells are shown in Figure 3 in Figure 3 B. Figure 3 C and Tables 6 and 7.

[0130] Table 6. Binding of humanized antibodies to BT474 cells

[0131]

[0132] Table 7. Binding of humanized antibodies to BT474 cells

[0133]

[0134]

[0135] The results of the FACS binding experiment between the ADC of the present invention and BT474 cells are shown in Figure 3 in Figure 3 D and Table 8.

[0136] Table 8. Binding of Antibody Drug Conjugates to BT474 Cells

[0137]

[0138] Example 9 In vitro cell-based assays of antibodies

[0139] 9.1 BT474 cell endocytosis assay

[0140] 1. Collect BT474 cells, centrifuge at 1200 rpm for 8 minutes, and wash twice with DPBS (Gibco Cat.: 14190-136).

[0141] 2. Seed 1E5 cells per well and dilute the antibody starting at 10 μg / ml in a 1:2 gradient to make 7 spots, with the last spot being a blank well. Incubate the mixture on ice for 1 hour.

[0142] 3. Wash twice with ice-cold PBS, centrifuge at 1200 rpm for 8 minutes, resuspend the cells in RPMI 1640 medium supplemented with L-glutamine and HEPES, divide the cells into corresponding aliquots according to the time point, and incubate them at 37°C for different time periods. One aliquot should be kept on ice as the zero-point no-endocytosis control, and the group without antibody addition should be used as the NC control.

[0143] 4. Wash with pH 2.7 citric acid for 3.5 minutes, neutralize with 1M Tris-HCl solution (pH 9.5), wash twice with PBS, resuspend in an appropriate amount of 1% BSA-PBS, and detect on an IQplus instrument.

[0144] 5. Analyze and process the data using GraphPad Prism software.

[0145] The results of the BT474 cell endocytosis activity test of the mouse antibody of the present invention are shown in Figure 4 in Figure 4 A and Table 9.

[0146] Table 9. Endocytic activity of mouse monoclonal antibodies in BT474 cells

[0147] Endocytosis efficiency (%) Enfortumab 63 mIgG 5M21 69 mIgG 26G13 37 mIgG 29B12 57 mIgG 30J3 56 mIgG 7I18 66 Isotype control 16

[0148] The results of the BT474 cell endocytosis activity test of the humanized modified molecule of the present invention are shown in Figure 4 in Figure 4 B, 4C and Tables 10 and 11.

[0149] Table 10. Endocytic activity of humanized antibodies in BT474 cells

[0150] Endocytosis efficiency (%) Enfortumab 62.4 5M21 xiIgG 56.9 5M21 hz00 54.8 5M21 hz01 62.2 5M21 hz10 64.8 5M21 hz11 64.4

[0151] Table 11. Endocytic activity of humanized antibodies in BT474 cells

[0152]

[0153]

[0154] The experimental results of BT474 cell endocytosis activity of ADC of the present invention are shown in Figure 4 in Figure 4 D and Table 12.

[0155] Table 12. BT474 cell endocytic activity of antibody drug conjugates

[0156] Endocytosis efficiency (%) Enfortumab-E 58.9 42D20 hz10-E 74 42D20 hz43-E 68.9 42D20 hz44-E 71.1 42D20 hz63-E 67.9 42D20 hz64-E 66.6 20M12 xiIgG-E 67 20M12 hz01-E 56.6 20M12 hz11-E 58.4 Isotype control --

[0157] 9.2 BT474 Cell Proliferation Inhibition Assay

[0158] Nectin4-expressing breast cancer BT474 cells were cultured, harvested by trypsinization, centrifuged at 400 g for 5 minutes, and the supernatant discarded. Plates were plated at a density of 4,000 cells per well and incubated at 37°C, 5% CO2 for 24 hours. The test antibody was dissolved in medium containing 1% BSA at a starting concentration of 200 μg / ml. Three-fold dilutions were made into nine dilutions, including zero, and 100 μl of each antibody was added to a 96-well plate. 100 μl of the diluted antibody sample was mixed with 100 μl of cells and transferred to a 96-well plate. The plate was incubated at 37°C, 5% CO2 for 120 hours. CCK-8 was prepared at a concentration of 5 μM in medium containing 1% BSA. 20 μl of CCK-8 was added to the 96-well plate and incubated at 37°C, 5% CO2 for 4 hours. Finally, the plate was removed from the plate and allowed to stand at room temperature for 15 minutes. The plate was then thoroughly vortexed to mix. The plate was read at a wavelength of 450 nm. The working concentrations of naked antibody and ADC samples (ng / ml) were used as the X-axis, and the measured absorbance value was used as the Y-axis. SoftMaxPro was used for four-parameter fitting to obtain the EC50 values of naked antibody samples and ADC samples.

[0159] The results are shown in Tables 13, 14 and 15.

[0160] Table 13. BT474 cell proliferation inhibitory activity of mouse antibodies

[0161] EC50 (ng / ml) Percentage of cell killing effect at the highest concentration Enfortumab 3.367 43.3％ mIgG 5M21 42.34 33.5％ mIgG 29B12 387.0 33.2％ mIgG 30J3 16.88 52.8％ Isotype control N / A N / A

[0162] Table 14. BT474 cell proliferation inhibitory activity of ADC

[0163]

[0164] Table 15. BT474 cell proliferation inhibitory activity of ADC

[0165]

[0166]

[0167] Example 10 Antibody cross-antigen activity analysis-FACS

[0168] cell:

[0169] CHO-huNectin4 S8;

[0170] HEK293 cells expressing murine Nectin4 (NP_082169): HEK-muNectin4; and

[0171] HEK293 cells expressing Cyno Nectin4 (SEQ ID NO: 6): HEK-cynoNectin4.

[0172] The experiment was carried out with reference to Example 8, and the results are shown in FIG. Figure 5 of Figure 5 A, 5B and 5C, and Tables 16, 17 and 18.

[0173] Table 16. Binding of humanized antibodies to CHO-huNectin4 S8 cells

[0174]

[0175] Table 17. Binding of humanized antibodies to HEK-muNectin4 cells

[0176]

[0177]

[0178] Table 18. Binding of humanized antibodies to HEK-cynoNectin4 cells

[0179]

[0180] Example 11 In vitro binding affinity and kinetics experiments of antibodies

[0181] The antibody-antigen interaction was measured using a GE BIAcore S200 instrument. Following the GE BiotinCAPture Kit operating instructions, a His-tagged human NECTIN4 antigen was first coupled to both the CAP analysis channel and the control sample channel of the sensor chip. A gradient dilution of the antibody sample (starting at 20 nM, diluted 1:3 over eight concentrations, with a repeat at 0.741 nm) was then passed through both the analysis and control sample channels. The resulting light response was measured. The binding constant, Kon, and dissociation constant, Koff, as well as the affinity constant, KD, were determined using instrument software fitting analysis (1:1 binding mode).

[0182] The results are shown in Table 19.

[0183] Table 19. Binding affinity and kinetic results of antibodies

[0184] ka(1 / Ms) kd(1 / s) KD(M) Enfortumab 1.12E+06 5.52E-03 4.94E-09 42D20 xiIgG 7.90E+05 7.98E-04 1.01E-09 42D20 hz10 8.31E+05 9.46E-04 1.14E-09 42D20 hz11 1.19E+06 1.15E-03 9.60E-10 42D20 hz13 1.19E+06 1.08E-03 9.04E-10 42D20 hz63 8.73E+05 1.57E-03 1.80E-09

[0185] Example 12 Verification of cross-binding activity between antibodies and antigenic proteins of the same family

[0186] antigen:

[0187] human Nectin-1(C-6His)Novoprotein Cat#C492

[0188] human Nectin-2(C-6His)Novoprotein Cat#C440

[0189] human Nectin-3(C-6His)Novoprotein Cat#C630

[0190] human Nectin-4(C-6His)Novoprotein Cat#CJ19

[0191] Antibody (primary antibody): an antibody of the present invention;

[0192] control antibody;

[0193] CD111 / Nectin-1 / PVRL1 Antibody, Rabbit Fab, 80244-RP01-100, Sinobiologics.

[0194] Anti-Nectin 2 antibody(ab233085), Rabbit antibody, Abcam.

[0195] Anti-Nectin 3 antibody (ab137961), Rabbit antibody, Abcam.

[0196] Secondary Antibody:

[0197] Goat anti-Rabbit IgG-Fc Secondary antibody(HRP)Cat#SSA003, JacksonImmuno.

[0198] The plate was coated with 1ug / ml antigen and incubated overnight at 4°C. Then, serially diluted antibodies were added, and finally, HRP-labeled secondary antibodies were added to detect the absorbance of A450. Figure 6 of Figure 6 A, 6B, 6C and 6D.

[0199] The results show that the antibodies of the present invention and the control antibodies exhibit consistent properties, both can specifically recognize the nectin-4 antigen, have a dose-dependent binding effect, and do not have cross-binding activity with other proteins in the nectin-4 family.

[0200] Example 13 Study on the stability of antibodies in monkey serum in vitro

[0201] Experimental materials: test antibody, FBS, test antibody-bound antigen, anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-035-098).

[0202] Experimental instruments: 37°C incubator, microplate reader.

[0203] Experimental steps:

[0204] Sample preparation:

[0205] 1) Adjust the concentration of the test antibody to 20 μg / ml, filter sterilize, and aliquot into 250 μl tubes for later use;

[0206] 2) Add an equal volume of monkey serum to the aliquoted antibody to be tested, i.e., the final concentration is 50% serum concentration and 10 μg / ml antibody concentration;

[0207] 3) Prepare a total of 7 samples, seal with parafilm, and store at 37°C. Maintain sterility throughout the process.

[0208] 4) Samples were collected on days 0, 3, 7, 10, 14, and 21 and stored at 4°C for testing. The sample collected on day 21 should be stored at 4°C for at least one day.

[0209] Detection method:

[0210] 1) PBS was used to coat the antigen and anti-IgG Fab monoclonal antibody in a 96-well enzyme-linked microtiter plate at 0.2 μg / ml and 100 μl / well at 4°C overnight.

[0211] 2) Prepare the required reagents:

[0212] Blocking solution 5% BSA + PBS

[0213] Antibody diluent 5% BSA + PBS + 50% FBS

[0214] ELISA plate washing solution 0.1% Tween + PBS

[0215] 3) Wash the coated ELISA plate three times with PBS, 300 μl / well, to remove free uncoated antigen;

[0216] 4) Add blocking solution, 200 μl / well, and block at 37°C for 1 hour;

[0217] 5) Dilute the test antibody to 2 μg / ml using antibody diluent and dilute it 3-fold in a total of 8 steps;

[0218] 6) Discard the blocking solution and add 100 μl of diluted antibody to each well of the two coating methods, incubating at 37°C for 1 hour.

[0219] 7) Wash the plate 3 times with PBST;

[0220] 8) Dilute the secondary antibody at 1:5000 and add 100 μl / well to the washed ELISA plate. Incubate at 37°C for 40 min.

[0221] 9) Wash the plate 3 times with PBST;

[0222] 10) TMB color development, 100 μl / well, protected from light for 10 min;

[0223] 11) Add 50 μl of 2M HCL to terminate the reaction and read the result at 450 nm.

[0224] Result processing:

[0225] The binding curve was developed based on the ELISA color value, and the changes in the binding curve at different storage times were observed to evaluate the stability of the antibody binding activity after storage. Figure 7 of Figure 7 A. 7B.

[0226] The results show that after incubation at 37°C for 21 days, the effective antibody content of the antibody of the present invention does not change at all, that is, it can exist stably at 37°C for more than 21 days.

[0227] Example 14Drug metabolism analysis in antibody mice

[0228] Experimental materials: the antibody to be tested, serum collected from mice at different time points, the binding antigen of the antibody to be tested, human nectin-4, anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), and HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-035-098).

[0229] Experimental methods:

[0230] Serum collection:

[0231] 1) Female Balb / C mice, 3 mice / group, 200 μg / mouse via tail vein or intraperitoneal administration;

[0232] 2) Blood was collected from the tail vein at the time points of the experimental design. The blood samples were placed at room temperature for at least 30 minutes. The serum was collected at 4000 rpm for 15 minutes and stored at -20°C. To prevent serum evaporation, the final serum collection volume should be greater than 20 μl.

[0233] 3) The final serum collection should be frozen at -20°C for at least 24 hours.

[0234] Detection method:

[0235] 1) PBS was used to coat the antigen and anti-IgG Fab monoclonal antibody in a 96-well enzyme-linked microtiter plate at 0.2 μg / ml and 100 μl / well at 4°C overnight.

[0236] 2) Prepare the required reagents:

[0237] Blocking solution 5% BSA + PBS

[0238] Antibody diluent 5% BSA + PBS + 20% blank mouse serum

[0239] ELISA plate washing solution 0.1% Tween + PBS

[0240] 3) Wash the coated ELISA plate three times with PBS, 300 μl / well;

[0241] 4) Add blocking solution, 200 μl / well, and block at 37°C for 1 hour;

[0242] 5) Dilute the starting serum to an appropriate concentration using blocking buffer and then dilute to an appropriate concentration range using antibody diluent. The specific dilution factor should be adjusted based on preliminary experiments. In principle, the final color value of the tested serum should be within the color value range of the standard.

[0243] 6) Dilute the antibody standard with antibody diluent. The dilution of the standard should still be adjusted based on the preliminary experiment so that the standard fits a linear curve (if suitable software is available, an S-shaped curve can also be fitted).

[0244] 7) Discard the blocking solution from the ELISA plate. Add the diluted antibody standard and test serum to the ELISA plate for both coating methods, 100 μl / well, and incubate at 37°C for 1 hour.

[0245] 8) Wash the plate 3 times with PBST;

[0246] 9) Dilute the secondary antibody at 1:5000 and add 100 μl / well to the washed ELISA plate. Incubate at 37°C for 40 min.

[0247] 10) Wash the plate three times with PBST;

[0248] 11) TMB color development, 100 μl / well, protected from light for 10 min;

[0249] 12) Add 50 μl of 2M HCL to terminate the reaction and read the result at 450 nm.

[0250] See the results Figure 8 of Figure 8 A, 8B and Table 20.

[0251] Table 20. Metabolism results of antibodies in mice

[0252]

[0253] Concentration: μg / ml

[0254] NA: below the detection limit, which is 9.77 ng / ml.

[0255] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264] Sequence Listing <110> Maiwei (Shanghai) Biotechnology Co., Ltd. <120> Anti-nectin-4 antibody and its application <130> LC21210004P-CN <140> CN2021800302777 <141> 2021-04-21 <150> CN202010320420.3 <151> 2020-04-21 <160> 97 <170> PatentIn version 3.3 <210> 1 <211> 447 <212> PRT <213> artificial sequence <220> <223> Enfortumab, HC <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr 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> 2 <211> 214 <212> PRT <213> artificial sequence <220> <223> Enfortumab, LC <400> 2 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 Gln Gly Ile Ser Gly Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln 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 Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val 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 <210> 3 <211> 318 <212> PRT <213> Homo sapiens <400> 3 Gly Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp 1 5 10 15 Ala Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly 20 25 30 Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu 35 40 45 Ala Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu 50 55 60 Gly Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser 65 70 75 80 Val Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys 85 90 95 Arg Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu 100 105 110 Arg Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu 115 120 125 Glu Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly 130 135 140 Ser Pro Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr 145 150 155 160 Ser Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu 165 170 175 Phe His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys 180 185 190 Val Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile 195 200 205 Leu His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp 210 215 220 Gln Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys Leu 225 230 235 240 Ser Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu Asp Gly 245 250 255 Pro Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe Pro 260 265 270 Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser Asn 275 280 285 Glu Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp Pro 290 295 300 Gln Glu Asp Ser Gly Lys Gln Val Asp Leu Val Ser Ala Ser 305 310 315 <210> 4 <211> 330 <212> PRT <213> Homo sapiens <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Leu Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Leu Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Leu Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 5 <211> 107 <212> PRT <213> Homo sapiens <400> 5 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 6 <211> 510 <212> PRT <213> Cyno <400> 6 Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly 20 25 30 Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala 35 40 45 Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln 50 55 60 Val Ala Trp Ala Arg Ala Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala 65 70 75 80 Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly 85 90 95 Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val 100 105 110 Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg 115 120 125 Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg 130 135 140 Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu 145 150 155 160 Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser 165 170 175 Pro Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser 180 185 190 Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe 195 200 205 His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val 210 215 220 Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile Leu 225 230 235 240 His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp Gln 245 250 255 Asn Leu Trp His Val Gly Arg Glu Gly Ala Met Leu Lys Cys Leu Ser 260 265 270 Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu Asp Gly Pro 275 280 285 Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe Pro Pro 290 295 300 Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser Asn Glu 305 310 315 320 Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp Pro Gln 325 330 335 Glu Asp Ser Gly Lys Gln Val Asp Leu Val Ser Ala Ser Val Val Val 340 345 350 Val Gly Val Ile Ala Ala Leu Leu Phe Cys Leu Leu Val Val Val Val 355 360 365 Val Leu Met Ser Arg Tyr His Arg Arg Lys Ala Gln Gln Met Thr Gln 370 375 380 Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg 385 390 395 400 Leu His Ser His His Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val 405 410 415 Gly Leu Arg Ala Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser 420 425 430 Cys Ser Val Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu 435 440 445 Thr Thr Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly 450 455 460 Ser Gly Arg Thr Glu Glu Glu Glu Asp Gln Asp Glu Gly Ile Lys Gln 465 470 475 480 Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys Pro 485 490 495 Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg Gly His Leu Val 500 505 510 <210> 7 <211> 116 <212> PRT <213> artificial sequence <220> <223> VH <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Thr Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys 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 Gly Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> artificial sequence <220> <223> VL <400> 8 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Leu Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 116 <212> PRT <213> artificial sequence <220> <223> VH <400> 9 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 Thr Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly 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 Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 10 <211> 116 <212> PRT <213> artificial sequence <220> <223> VH <400> 10 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 Thr Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys 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 Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val × 100 105 110 Thr Val Ser Ser 115 <210> 11[[ID=?53]] <211> 107 <212> PRT <213> Artificial sequence <220> <223> VL <400> 11 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 Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr 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 Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 107 <212> PRT <213> Artificial sequence <220> <223> VL <400> 12 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 Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr 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 Leu Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 13 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Ser Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Ser Val Thr Ser Val 115 <210> 14 <211> 113 <212> PRT <213> artificial sequence <220> <223> VL <400> 14 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly 1 5 10 15 Gln Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 His Tyr Asn Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 15 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 15 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr[[ID=四十一]] [[ID=四十二]]20 25 30[[ID=四十三]] [[ID=四十四]]Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile[[ID=四十五]] [[ID=四十六]]35 40 45[[ID=四十七]] Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 16 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 16 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 17 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Glu Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 18 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 19 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 19 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Asp Ala Lys Ile Tyr Tyr Asn Ser Val Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 20 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 20 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Gly Asp Lys Ile Tyr Tyr Asn Ser Val Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 21 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Gly Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 22 <211> 114 <212> PRT <213> Artificial sequence <220> <223> VL <400> 22 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 Lys Ser Ser Gln Ser Leu Leu Asn Ser[[ID=]17] 20 25 30 Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Arg <210> 23 <211> 114 <212> PRT <213> Artificial sequence <220> <223> VL <400> 23 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 Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln 85 90 95 His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Arg <210> 24 <211> 114 <212> PRT <213> artificial sequence <220> <223> VL <400> 24 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 Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln 85 90 95 His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Arg <210> 25 <211> 114 <212> PRT <213> artificial sequence <220> <223> VL <400> 25 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 Lys Ser Ser Gln Ser Leu Leu Asn Thr 20 25 30 Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln 85 90 95 His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Arg <210> 26 <211> 114 <212> PRT <213> artificial sequence <220> <223> VL <400> 26 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 Lys Ser Ser Gln Ser Leu Leu Asn Lys 20 25 30 Tyr Ser Gln Lys Asm Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Arg <210> 27 <211> 116 <212> PRT <213> artificial sequence[[ID=2S]] <220> <223> VH <400> 27 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys 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 Ser Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 28 <211> 107 <212> PRT <213> artificial sequence <220> <223> VL <400> 28 Ser Val Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Asn Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 29 <211> 116 <212> PRT <213> artificial sequence <220> <223> VH <400> 29 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 Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys Tyr Asn Glu Asn Phe 50 55 60 Lys Gly 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 Ser Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 30 <211> 116 <212> PRT <213> artificial sequence <220> <223> VH <400> 30 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 Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys 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 Ser Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 31 <211> 107 <212> PRT <213> Artificial sequence <220> <223> VL <400> 31 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Asn Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 32 <211> 107 <212> PRT <213> Artificial sequence <220> <223> VL <400> 32 Glu Val Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Asn Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 33 <211> 117 <212> PRT <213> artificial sequence <220> <223> VH <400> 33 Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Ser Arg Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Val Asn Pro Asn Asn Gly Asn Thr Leu Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Arg Tyr Ala Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 34 <211> 111 <212> PRT <213> artificial sequence <220> <223> VL <400> 34 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 Gln Ser Val Ser Thr Ser 20 25 30 Ser Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Thr Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR-H1 <400> 35 Gly Tyr Thr Phe Thr Thr Tyr 1 5 <210> 36 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR-H1 <400> 36 Gly Tyr Thr Phe Thr Thr Tyr Tyr Ile His 1 5 10 <210> 37 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR-H1 <400> 37 Thr Tyr Tyr Ile His 1 5 <210> 38 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 38 Thr Thr Tyr Tyr Ile His 1 5 <210> 39 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 39 Tyr Pro Gly Asn Val Asn 1 5 <210> 40 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 40 Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys 1 5 10 <210> 41 <211> 17 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 41 Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 42 <211> 13 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 42 Trp Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys 1 5 10 <210> 43 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-H3 <400> 43 Gly Leu Tyr Tyr Phe Asp Tyr 1 5 <210> 44 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-H3 <400> 44 Ala Arg Gly Leu Tyr Tyr Phe Asp 1 5 <210> 45 <211> 11 <212> PRT <213> artificial sequence <220> <223> CDR-L1 <400> 45 Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala 1 5 10 <210> 46 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-L1 <400> 46 Ser Asn Asp Val Ala Trp Tyr 1 5 <210> 47 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 47 Tyr Ala Ser Asn Arg Tyr Thr 1 5 <210> 48 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 48 Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr 1 5 10 <210> 49 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-L3 <400> 49 Gln Gln Asp Tyr Ser Ser Pro Tyr Thr 1 5 <210> 50 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-L3 <400> 50 Gln Gln Asp Tyr Ser Ser Pro Tyr 1 5 <210> 51 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 51 Gly Phe Ser Leu Ile Asp Tyr 1 5 <210> 52 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 52 Gly Phe Ser Leu Ile Asp Tyr Gly Val Ser 1 5 10 <210> 53 <211> 5 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 53 Asp Tyr Gly Val Ser 1 5 <210> 54 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 54 Ile Asp Tyr Gly Val Ser 1 5 <210> 55 <211> 5 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 55 Trp Gly Asp Gly Lys 1 5 <210> 56 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 56 Val Ile Trp Gly Asp Gly Lys Ile Tyr 1 5 <210> 57 <211> 16 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 57 Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys Ser 1 5 10 15 <210> 58 <211> 12 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 58 Trp Leu Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr 1 5 10 <210> 59 <211> 11 <212> PRT <213> artificial sequence [[ID=1)7]]<220> <223> CDR-H3 <400> 59 Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr 1 5 10 <210> 60 <211> 12 <212> PRT <213> artificial sequence <220> <223> CDR-H3 <400> 60 Ala Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp 1 5 10 <210> 61 <211> 17 <212> PRT <213> artificial sequence <220> <223> CDR-L1 <400> 61 Lys Ser Ser Gln Ser Leu Leu Asn Ser Tyr Ser Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 62 <211> 13 <212> PRT <213> artificial sequence <220> <223> CDR-L1 <400> 62 Leu Asn Ser Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr 1 5 10 <210> 63 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 63 Phe Ala Ser Thr Arg Glu Ser 1 5 <210> 64 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 64 Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu 1 5 10 <210> 65 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-L3 <400> 65 Gln Gln His Tyr Asn Thr Pro Phe Thr 1 5 <210> 66 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-L3 <400> 66 Gln Gln His Tyr Asn Thr Pro Phe 1 5 <210> 67 <211> 17 <212> PRT <213> artificial sequence <220> <223> CDR-L1 <400> 67 Lys Ser Ser Gln Ser Leu Leu Asn Thr Tyr Ser Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 68 <211> 5 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 68 Trp Gly Asp Ala Lys 1 5 <210> 69 <211> 5 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 69 Trp Gly Gly Gly Lys 1 5 <210> 70 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 70 Gly Tyr Thr Phe Thr Ser Tyr 1 5 <210> 71 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 71 Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His 1 5 10 <210> 72 <211> 5 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 72 Ser Tyr Tyr Ile His 1 5 <210> 73 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 73 Thr Ser Tyr Tyr Ile His 1 5 <210> 74 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 74 Tyr Pro Gly Asn Ala Asn 1 5 <210> 75 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 75 Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys 1 5 10 <210> 76 <211> 17 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 76 Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys Tyr Asn Glu Asn Phe Lys 1 5 10 15 Gly <210> 77 <211> 13 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 77 Trp Ile Gly Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys 1 5 10 <210> 78 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-H3 <400> 78 Ser Val Tyr Tyr Phe Asp Tyr 1 5 <210> 79 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-H3 <400> 79 Ala Arg Ser Val Tyr Tyr Phe Asp 1 5 <210> 80 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 80 Tyr Ala Ser Asn Arg Asn Thr 1 5 <210> 81 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 81 Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Asn 1 5 10 <210> 82 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 82 Gly Tyr Ser Phe Thr Asp Tyr 1 5 <210> 83 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 83 Gly Tyr Ser Phe Thr Asp Tyr Tyr Met His 1 5 10 <210> 84 <211> 5 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 84 Asp Tyr Tyr Met His 1 5 <210> 85 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-H1 <400> 85 Thr Asp Tyr Tyr Met His 1 5 <210> 86 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 86 Asn Pro Asn Asn Gly Asn 1 5 <210> 87 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 87 Arg Val Asn Pro Asn Asn Gly Asn Thr Leu 1 5 10 <210> 88 <211> 17 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 88 Arg Val Asn Pro Asn Asn Gly Asn Thr Leu Tyr Asn Gln Lys Phe Arg 1 5 10 15 Gly <210> 89 <211> 13 <212> PRT <213> artificial sequence <220> <223> CDR-H2 <400> 89 Trp Ile Gly Arg Val Asn Pro Asn Asn Gly Asn Thr Leu 1 5 10 <210> 90 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-H3 <400> 90 Glu Asp Arg Tyr Ala Phe Ala Tyr 1 5 <210> 91 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-H3 <400> 91 Ala Arg Glu Asp Arg Tyr Ala Phe Ala 1 5 <210> 92 <211> 15 <212> PRT <213> artificial sequence <220> <223> CDR-L1 <400> 92 Arg Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr Thr Tyr Met His 1 5 10 15 <210> 93 <211> 11 <212> PRT <213> artificial sequence <220> <223> CDR-L1 <400> 93 Ser Thr Ser Ser Tyr Thr Tyr Met His Trp Tyr 1 5 10 <210> 94 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 94 Tyr Ala Ser Asn Leu Glu Ser 1 5 <210> 95 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR-L2 <400> 95 Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu 1 5 10 <210> 96 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-L3 <400> 96 Gln His Thr Trp Glu Ile Pro Tyr Thr 1 5 <210> 97 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-L3 <400> 97 Gln His Thr Trp Glu Ile Pro Tyr 1 5

Claims

1. An anti-nectin-4 antibody molecule or fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region respectively comprise a combination of heavy chain CDRs and light chain CDRs selected from the following: CDR-H1, CDR-H2, CDR-H3 are shown in SEQ ID NOs: 51, 69, 59, respectively; and CDR-L1, CDR-L2, CDR-L3 are shown in SEQ ID NOs: 67, 63, 65, respectively.

2. The antibody molecule or fragment thereof according to claim 1, characterized in that The heavy chain variable region and light chain variable region of the antibody molecule or fragment thereof respectively comprise: the amino acid sequence shown in SEQ ID NO: 21; and, the amino acid sequence shown in SEQ ID NO:

25.

3. The antibody molecule or fragment thereof according to claim 1 or 2, characterized in that The antibody molecule is a murine antibody or a fully or partially humanized antibody.

4. The antibody molecule or fragment thereof according to claim 1 or 2, characterized in that The antibody molecule is a chimeric antibody.

5. The antibody molecule or fragment thereof according to claim 1 or 2, characterized in that The fragment is a scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv fragment of the antibody molecule.

6. The antibody molecule or fragment thereof according to claim 1 or 2, characterized in that The antibody molecule is a monoclonal antibody or a single-chain antibody.

7. The antibody molecule or fragment thereof according to claim 1 or 2, characterized in that The antibody molecule or fragment thereof further comprises a constant region.

8. The antibody molecule or fragment thereof according to claim 1 or 2, characterized in that The antibody molecule or fragment thereof further comprises a murine or human heavy chain constant region (CH) and / or a light chain constant region (CL).

9. The antibody molecule or fragment thereof according to claim 1 or 2, characterized in that The antibody molecule or fragment thereof comprises a heavy chain and a light chain.

10. The antibody molecule or fragment thereof according to claim 1 or 2, characterized in that The antibody molecule or fragment thereof comprises an IgG, IgA, IgM, IgD or IgE heavy chain constant region and / or a kappa or lambda type light chain constant region.

11. The antibody molecule or fragment thereof according to claim 10, characterized in that The antibody molecule is a monoclonal antibody.

12. The antibody molecule or fragment thereof according to claim 11, characterized in that The antibody molecule is a humanized monoclonal antibody.

13. The antibody molecule or fragment thereof according to claim 11 or 12, characterized in that The heavy chain constant region of the monoclonal antibody is of IgG1 type, and the light chain constant region is of κ type.

14. A nucleic acid molecule encoding the antibody molecule or fragment thereof according to any one of claims 1 to 13. A vector comprising the nucleic acid molecule according to claim 14 . 16 . A host cell comprising the nucleic acid molecule of claim 14 and / or the vector of claim 15 , or the host cell is transformed or transfected with the nucleic acid molecule of claim 14 and / or the vector of claim 15 . 17 . A composition comprising the antibody molecule or fragment thereof according to claim 1 , the nucleic acid molecule according to claim 14 , the vector according to claim 15 , or the host cell according to claim 16 .

18. The composition according to claim 17, characterized in that The composition is a pharmaceutical composition.

19. The composition according to claim 17 or 18, characterized in that The composition further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

20. Use of the antibody molecule or fragment thereof according to any one of claims 1 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, or the composition according to any one of claims 17 to 19 in the preparation of a reagent for detecting or diagnosing a disease or disorder, wherein the disease or disorder is bladder cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, gastric cancer, or esophageal cancer that expresses nectin-4.

21. The use according to claim 20, characterized in that The breast cancer includes triple-negative subtype and basal subtype.

22. The use according to claim 20, characterized in that The lung cancer is non-small cell lung cancer.

23. Use of the antibody molecule or fragment thereof according to any one of claims 1 to 13 in the preparation of an antibody-drug conjugate, wherein the antibody-drug conjugate is formed by coupling the antibody molecule or fragment thereof according to any one of claims 1 to 13 to a cytotoxic moiety, wherein the cytotoxic moiety is Monomethyl auristatin E (MMAE).

24. An antibody-drug conjugate, which is formed by coupling the antibody molecule or fragment thereof according to any one of claims 1 to 13 with a cytotoxic moiety, wherein the cytotoxic moiety is Monomethyl auristatin E (MMAE).

25. Use of the antibody molecule or fragment thereof according to any one of claims 1 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, the composition according to any one of claims 17 to 19, or the antibody-drug conjugate according to claim 24 in the preparation of a medicament for preventing or treating a disease or disorder, wherein the disease or disorder is bladder cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, gastric cancer, or esophageal cancer that expresses nectin-4.

26. The use according to claim 25, characterized in that The breast cancer includes triple-negative subtype and basal subtype.

27. The use according to claim 25, characterized in that The lung cancer is non-small cell lung cancer.

28. A kit comprising the antibody molecule or fragment thereof according to any one of claims 1 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, the composition according to any one of claims 17 to 19, or the antibody drug conjugate according to claim 24.

29. The kit according to claim 28, characterized in that The kit is used to treat, detect or diagnose a disease or disorder.

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