An anti-Nectin-4 antibody and its application

Abstract

This invention provides an antibody molecule or fragment thereof that binds to human Nectin-4 and its application in the prevention or treatment of cancer. The antibody provided by this invention is obtained through hybridoma screening and humanization technology, and is a high-affinity antibody that specifically binds to Nectin-4, which can serve as an effective clinical lead drug molecule.

Description

Technical Field

[0001] This invention belongs to the field of antibody drugs. Specifically, this invention relates to antibodies against human Nectin-4 and their use in the preparation of drugs. Background Technology

[0002] Nectin-4 (also known as PVRL4, poliovirus receptor-like molecule 4) is a 66 kDa transmembrane glycoprotein belonging to the IG superfamily of the Nectin family. Its extracellular domain consists of three Ig-like domains (VCC type), and it participates in the formation and maintenance of adhesion junctions together with cadherin.

[0003] Nectin-4 is closely related to the occurrence and development of various tumor cells. It has been found to be expressed in various solid tumors, especially bladder cancer. In breast cancer, ovarian cancer, and lung cancer, as a tumor-associated antigen, it accounts for 50% of tissue expression detection rates in breast cancer, 49% in ovarian cancer, and 86% in lung cancer, respectively, and plays a crucial role in the occurrence, invasion, and metastasis of these epithelial malignancies. Therefore, Nectin-4 has become an important target for the diagnosis and treatment of many solid tumors.

[0004] Currently, the main drug targeting Nectin-4 is Enfortumab vedotin, an antibody-drug conjugate (ADC) composed of an anti-Nectin-4 monoclonal antibody and the cell-killing drug monomethyl auristatin E (MMAE). It is primarily used to treat bladder cancer, especially urothelial carcinoma, and received FDA Breakthrough Therapy Designation in March 2018. Furthermore, studies have shown that the adhesion factor Nectin-4 can not only serve as an effective prognostic factor for breast cancer but also as an effective 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 good efficacy against localized and metastatic TNBC. Summary of the Invention

[0005] 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 modification.

[0006] To address the aforementioned technical problems, the purpose of this invention is to provide an antibody molecule or fragment thereof that specifically binds to Nectin-4, particularly human Nectin-4, and to provide its uses. The fragments of the antibody molecule described in this invention encompass various functional fragments of antibodies, such as their antigen-binding portions, like Fab, F(ab')2, or scFv fragments.

[0007] The technical solution of the present invention is as follows:

[0008] On one hand, the present invention provides an antibody molecule or a fragment thereof, the antibody molecule or the 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:

[0009] (1) CDR-H1(GYTFTTY), CDR-H2(YPGNVN), and CDR-H3(GLYYFDY) shown in sequence in SEQ ID NO: 35, 39, and 43; and CDR-L1(KASQSVSNDVA), CDR-L2(YASNRYT), and CDR-L3(QQDYSSPYT) shown in sequence in SEQ ID NO: 45, 47, and 49;

[0010] (2) CDR-H1 (GYTFTTYYIH), CDR-H2 (WIYPGNVNTK), and CDR-H3 (GLYYFDY) shown in sequence in SEQ ID NO: 36, 40, and 43; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), and CDR-L3 (QQDYSSPYT) shown in sequence in SEQ ID NO: 45, 47, and 49;

[0011] (3) CDR-H1(TYYIH), CDR-H2(WIYPGNVNTKYNEKFKG), and CDR-H3(GLYYFDY) are shown in sequence in SEQ ID NO: 37, 41, and 43; and CDR-L1(KASQSVSNDVA), CDR-L2(YASNRYT), and CDR-L3(QQDYSSPYT) are shown in sequence in SEQ ID NO: 45, 47, and 49;

[0012] (4) CDR-H1 (TTYYIH), CDR-H2 (WIGWIYPGNVNTK), and CDR-H3 (ARGLYYFD) shown in SEQ ID NO: 38, 42, and 44 respectively; and CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRY), and CDR-L3 (QQDYSSPY) shown in SEQ ID NO: 46, 48, and 50 respectively;

[0013] (5) CDR-H1 (GFSLIDY), CDR-H2 (WGDGK), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, 55, and 59 respectively; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 61, 63, and 65 respectively;

[0014] (6) CDR-H1 (GFSLIDYGVS), CDR-H2 (VIWGDGKIY), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 52, 56, and 59 respectively; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 61, 63, and 65 respectively;

[0015] (7) CDR-H1(DYGVS), CDR-H2(VIWGDGKIYYNSVLKS), and CDR-H3(QGGLLFYAMDY) are shown in sequence in SEQ ID NO: 53, 57, and 59; and CDR-L1(KSSQSLLNSYSQKNYLA), CDR-L2(FASTRES), and CDR-L3(QQHYNTPFT) are shown in sequence in SEQ ID NO: 61, 63, and 65;

[0016] (8) CDR-H1 (IDYGVS), CDR-H2 (WLGVIWGDGKIY), and CDR-H3 (AKQGGLLFYAMD) shown in sequence in SEQ ID NO: 54, 58, and 60; and CDR-L1 (LNSYSQKNYLAWY), CDR-L2 (LLIYFASTRE), and CDR-L3 (QQHYNTPF) shown in sequence in SEQ ID NO: 62, 64, and 66;

[0017] (9) CDR-H1 (GFSLIDY), CDR-H2 (WGDGK), and CDR-H3 (QGGLLFYAMDY) shown in sequence in SEQ ID NO: 51, 55, and 59; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in sequence in SEQ ID NO: 67, 63, and 65;

[0018] (10) CDR-H1 (GFSLIDY), CDR-H2 (WGDAK), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, 68, and 59 respectively; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 67, 63, and 65 respectively;

[0019] (11) CDR-H1(GFSLIDY), CDR-H2(WGGGK), and CDR-H3(QGGLLFYAMDY) shown in sequence at SEQ ID NO: 51, 69, and 59; and CDR-L1(KSSQSLLNTYSQKNYLA), CDR-L2(FASTRES), and CDR-L3(QQHYNTPFT) shown in sequence at SEQ ID NO: 67, 63, and 65;

[0020] (12) CDR-H1(GYTFTSY), CDR-H2(YPGNAN), and CDR-H3(SVYYFDY) shown in SEQ ID NO: 70, 74, and 78 respectively; and CDR-L1(KASQSVSNDVA), CDR-L2(YASNRNT), and CDR-L3(QQDYSSPYT) shown in SEQ ID NO: 45, 80, and 49 respectively;

[0021] (13) CDR-H1(GYTFTSYYIH), CDR-H2(WIYPGNANNK), and CDR-H3(SVYYFDY) shown in SEQ ID NO: 71, 75, and 78 respectively; and CDR-L1(KASQSVSNDVA), CDR-L2(YASNRNT), and CDR-L3(QQDYSSPYT) shown in SEQ ID NO: 45, 80, and 49 respectively;

[0022] (14) CDR-H1(SYYIH), CDR-H2(WIYPGNANNKYNENFKG), and CDR-H3(SVYYFDY) shown in SEQ ID NO: 72, 76, and 78 respectively; and CDR-L1(KASQSVSNDVA), CDR-L2(YASNRNT), and CDR-L3(QQDYSSPYT) shown in SEQ ID NO: 45, 80, and 49 respectively;

[0023] (15) CDR-H1(TSYYIH), CDR-H2(WIGWIYPGNANNK), and CDR-H3(ARSVYYFD) shown in sequence in SEQ ID NO: 73, 77, and 79; and CDR-L1(SNDVAWY), CDR-L2(LLIYYASNRN), and CDR-L3(QQDYSSPY) shown in sequence in SEQ ID NO: 46, 81, and 50;

[0024] (16) CDR-H1 (GYSFTDY), CDR-H2 (NPNNGN), and CDR-H3 (EDRYAFAY) shown in SEQ ID NO: 82, 86, and 90 respectively; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), and CDR-L3 (QHTWEIPYT) shown in SEQ ID NO: 92, 94, and 96 respectively;

[0025] (17) CDR-H1 (GYSFTDYYMH), CDR-H2 (RVNPNNGNTL), and CDR-H3 (EDRYAFAY) shown in SEQ ID NO: 83, 87, and 90 respectively; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), and CDR-L3 (QHTWEIPYT) shown in SEQ ID NO: 92, 94, and 96 respectively;

[0026] (18) CDR-H1 (DYYMH), CDR-H2 (RVNPNNGNTLYNQKFRG), and CDR-H3 (EDRYAFAY) are shown in sequence in SEQ ID NO: 84, 88, and 90; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), and CDR-L3 (QHTWEIPYT) are shown in sequence in SEQ ID NO: 92, 94, and 96;

[0027] (19) CDR-H1 (TDYYMH), CDR-H2 (WIGRVNPNNGNTL), and CDR-H3 (AREDRYAFA) are shown in sequence in SEQ ID NO: 85, 89, and 91; and CDR-L1 (STSSYTYMHWY), CDR-L2 (LLIKYASNLE), and CDR-L3 (QHTWEIPY) are shown in sequence in SEQ ID NO: 93, 95, and 97.

[0028] In accordance with the structural domain composition of heavy chain variable regions and light chain variable regions in antibodies known in the art, the heavy chain variable regions or light chain variable regions in the antibody molecules or fragments of the present invention contain the above-mentioned structural domain components in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR is a framework region.

[0029] Preferably, in the antibody molecule or fragment thereof provided by the present invention, the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO: 7, 9 or 10 or an amino acid sequence having at least 75% identity with said amino acid sequence; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO: 8, 11 or 12 or an amino acid sequence having at least 75% identity with said amino acid sequence; or,

[0030] The heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO: 13, 15, 16, 18, 19, 20 or 21 or an amino acid sequence having at least 75% identity with said amino acid sequence; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO: 14, 22, 23 or 25 or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0031] The heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO: 27, 29 or 30 or an amino acid sequence having at least 75% identity with said amino acid sequence; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO: 28, 31 or 32 or an amino acid sequence having at least 75% identity with said amino acid sequence; or,

[0032] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity with 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 having at least 75% identity with the amino acid sequence.

[0033] 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 its fragment are selected from combinations of the following amino acid sequences:

[0034] (1) An amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 7; and an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 8;

[0035] (2) An amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 9; and an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 11;

[0036] (3) An amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 9; and an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 12;

[0037] (4) An amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 10; and an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 11;

[0038] (5) An amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 10; and an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 12.

[0039] (6) An amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 13; and an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 14.

[0040] (7) An amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 16; and an amino acid sequence as shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 22;

[0041] (8) An amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 16; and an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 23;

[0042] (9) An amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 16; and an amino acid sequence as shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 25.

[0043] (10) An amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 19; and an amino acid sequence as shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 25;

[0044] (11) An amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 21; and an amino acid sequence as shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 25;

[0045] (12) An amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 27; and an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 28;

[0046] (13) An amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 29; and an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 32;

[0047] (14) An amino acid sequence as shown in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 30; and an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 32; or

[0048] (15) An amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 33; and an amino acid sequence as shown in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO: 34.

[0049] Based on the specific amino acid sequences of the heavy chain variable region or light chain variable region provided by the present invention, those skilled in the art can routinely determine the amino acid sequences of the heavy chain CDR and light chain CDR contained therein. Heavy and light chain CDRs and their combinations obtained by other known methods in the art are also covered within the scope of the present invention.

[0050] 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, and even more preferably human or monkey Nectin-4, especially human Nectin-4.

[0051] 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 capable of specifically binding to Nectin-4, such as scFv, dsFv, (dsFv)2, Fab, Fab′, F(ab′)2, or Fv fragment.

[0052] Preferably, the antibody molecule is a monoclonal antibody or a single-chain antibody.

[0053] Preferably, the antibody molecule or fragment thereof further comprises a human or mouse constant region, more preferably comprising 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 a light chain. More preferably, the antibody molecule or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a K or IR-type light chain constant region.

[0054] According to specific embodiments of the present invention, the antibody molecule provided by the present invention 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 contains the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 5.

[0055] In the context of this invention, "at least 75% identity" means any percentage of identity between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0056] On the other hand, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a light chain variable region, a heavy chain variable region, a heavy chain, or a light chain contained in the antibody molecule or a fragment thereof described in the present invention.

[0057] The nucleic acid molecules of this invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, in another aspect, this invention provides a vector containing the nucleic acid molecules of this invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector, etc.

[0058] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells or enter host cells in any way for purposes such as preserving or expressing antibodies. Therefore, in another aspect, the present invention provides a host cell containing the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant, or animal cells.

[0059] The antibody molecules provided by this invention can be obtained using 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 first from the nucleic acid molecules provided by this invention, or the heavy chain and / or light chain of the antibody molecule can be obtained, and then assembled with optional other structural domains of the antibody molecule to form an antibody; or, the host cells can be cultured while allowing the host cells provided by this invention 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 the antibody. Optionally, the method further includes the step of recovering the generated antibody molecules.

[0060] The antibody molecules or fragments thereof, nucleic acid molecules, vectors, host cells, or fusion proteins provided by this invention can be included in compositions, and more particularly in pharmaceutical formulations, for use in various purposes as needed. Therefore, in another aspect, this invention also provides a composition comprising the antibody molecules or fragments thereof, nucleic acid molecules, vectors, and / or host cells provided by this invention. Preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, excipient, or excipient.

[0061] Furthermore, the present invention also provides the use of the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition in the preparation of a medicament for the prevention or treatment of cancer. Preferably, the cancer is bladder cancer, breast cancer, ovarian cancer, or lung cancer.

[0062] On the other hand, the present invention also provides a method for preventing or treating cancer, the method comprising administering an antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition of the present invention to a subject in need. Preferably, the cancer is bladder cancer, breast cancer, ovarian cancer, or lung cancer. The subject is a mammal, more preferably a human.

[0063] Furthermore, the present invention also provides the use of the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition in the preparation of reagents for diagnosing cancer. Preferably, the cancer is bladder cancer, breast cancer, ovarian cancer, or lung cancer.

[0064] Accordingly, the present invention also provides a method for diagnosing cancer, the method comprising contacting an antibody molecule or fragment thereof, a nucleic acid molecule, a carrier, a host cell, and / or a composition thereof with a sample from a subject. Preferably, the cancer is bladder cancer, breast cancer, ovarian cancer, or lung cancer. The subject is a mammal, more preferably a human.

[0065] 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, and / or a composition thereof. The kit can be used for therapeutic or diagnostic purposes.

[0066] In another aspect, the present invention also provides the use of the antibody molecule or fragment thereof in the preparation of antibody-drug conjugates.

[0067] Accordingly, the present invention provides an antibody-drug conjugate formed by conjugating an antibody molecule or fragment thereof of the present invention with a cytotoxic portion.

[0068] Preferably, the cytotoxic component is a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binding agent. Preferably, the tubulin inhibitor is selected from maytansine derivatives, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl dolastatin 10, tubulysin derivatives, cryptophycin derivatives, and taltobulin. Preferably, the topoisomerase inhibitor is selected from doxorubicin metabolite PNU-159682 derivatives and irinotecan (CPT-11) metabolite SN38 derivatives. Preferably, the DNA binding agent is selected from PBD derivatives and duocarmycin derivatives.

[0069] Compared to existing technologies, this invention obtains a high-affinity antibody that specifically binds to Nectin-4 through hybridoma screening and humanization techniques, wherein a fully human antibody sequence is obtained through humanization. Furthermore, physicochemical properties and cellular activity studies of this molecule have confirmed the acquisition of an effective clinical lead drug molecule sequence. Attached Figure Description

[0070] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0071] Figure 1 The flow cytometry results of the antigen-expressing cell lines are shown, among which small Figure 1 A: HT-1376 bladder cancer cells, small Figure 1 B: CHO-huNectin4 S8 cells.

[0072] Figure 2 The results of FACS binding activity assays in hybridoma cell culture supernatants are shown, in which small Figure 2 A: First round of screening, small Figure 2 B: Second round of screening, small Figure 2 C: Third round of screening.

[0073] Figure 3 The results of the antibody binding experiment with FACS on BT474-expressing cells are shown.

[0074] Figure 4 The results of the antibody's BT474 cell endocytosis activity assay are shown.

[0075] Figure 5 The results of FACS binding experiments of the antibody with different cells expressing huNectin4, muNectin4, and cynoNectin4 are shown.

[0076] Figure 6 The results of the antibody binding experiment to members of the Nectin protein family are shown, among which small... Figure 6 A: Nectin-1, small Figure 6 B: Nectin-2, small Figure 6 C: Nectin-3, small Figure 6 D: Nectin-4.

[0077] Figure 7 The results of the antibody stability assay in monkey serum were shown, in which small Figure 7 A: Control antibody Enfortumab, small Figure 7 B: 42D20 hz10.

[0078] Figure 8 The study showed the metabolism of the antibody in mice, where small Figure 8 A: Control antibody Enfortumab, small Figure 8 B: 42D20 hz10. Detailed Implementation

[0079] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the medicinal materials and reagents used in the following examples are commercially available products. Among them:

[0081] 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.

[0082] The antibody sequences of the present invention are shown in Appendix I to Appendix IV.

[0083] The recombinant NECTIN4 antigen (sequence number: NP_002178.2, 32aa-349aa) is shown in SEQ ID NO: 3.

[0084] Example 1 Synthesis and expression of control antibodies

[0085] The fully synthesized Enfortumab antibody light chain variable region and heavy chain variable region genes were cloned into the eukaryotic expression vector pCDNA3.1, which contains the coding genes for the human-kappa light chain constant region and the human IgG1 heavy chain constant region, respectively. Enfortumab light and heavy chain expression plasmids were obtained, transformed into *E. coli* for amplification, and a large number of plasmids containing the Enfortumab antibody light chain (SEQ ID NO: 2) and heavy chain (SEQ ID NO: 1) were isolated. These plasmids were then mixed with polyethyleneimine (PEI) and co-transfected into HEK293 cells. Five to six days after transfection, the culture supernatant was collected and purified using a Mabselect affinity chromatography column to obtain the Enfortumab antibody.

[0086] Example 2 Preparation of Nectin-4 antigen expression cell lines

[0087] The reading frame of the Nectin-4 gene was cloned from a vector containing Nectin-4 cDNA (Beijing Yiqiao Shenzhou, Cat: HG19771-UT) using PCR. The reading frame of the Nectin-4 gene was then cloned into a stable expression vector containing a glutamyl synthase (GS) selection gene using enzyme digestion. CHO-K1 cells in suspension culture were electroporated (Nucleofector IIb, Lonza). The transfected cells were then seeded in CD CHOAGT™ medium (Gibco, Cat: 12490-025) containing 50 μM MSX (Sigma, Cat: M5379) and incubated in 96-well cell culture plates at 37°C with 5% CO2 for 2-3 weeks. Nine cells were pre-screened under a microscope using MSX pressure screening and then scaled up to 24-well cell culture plates. Finally, the antigen-high expression clone S8 was selected by flow cytometry (FACS) and then scaled up for further culture and cryopreservation.

[0088] The highly expressed clone S8 was named CHO-huNectin4 S8. The comparative identification results of this cell with HT-1376 bladder cancer cells endogenously expressing Nectin-4 are shown below. Figure 1 .

[0089] Example 3 Screening and identification of hybridoma cells

[0090] 1. Mouse immunization

[0091] Ten 8-week-old Balb / c mice were divided into two groups and immunized using two methods: conventional immunization and rapid immunization. Conventional immunization used CHO-huNectin4 S8 engineered cell line as the immunizing agent, while rapid immunization used recombinant human Nectin4 protein (purchased from Nearshore Protein, Cat: CJ19) as the immunizing agent.

[0092] Blood samples were collected from mice before immunization as a negative control. Two immunizing agents were administered intraperitoneally, with a two-week interval between the second and third immunizations. Blood samples were collected one week after the third immunization to measure the titer. Mice with high titers were selected for a sprint immunization three days before fusion.

[0093] 2. Fusion Screening

[0094] SP20 myeloma cells were resuscitated and cultured to a certain scale. The medium was changed the day before fusion to ensure good cell growth at the time of fusion. On the day of fusion, myeloma cells were collected, centrifuged, resuspended in basal medium, and counted for later use.

[0095] Spleens and lymph nodes of mice were aseptically harvested, ground, and used to prepare cell suspensions. The suspensions were filtered through a cell filter, followed by erythrocyte lysis. The lysed cells were then pooled and counted. B cells were mixed with SP20 myeloma cells at a ratio of 1:2. After centrifugation, the mixed cells were washed twice with electrofusion buffer, resuspended, and the density adjusted to 1-2 × 10⁻⁶ cells / mL. 7 Approximately / ml. The cell suspension was added to an electroporation cuvette for fusion, and then added to intact culture medium. The cells were incubated at 37°C with 8% CO2 for 30-240 minutes. HAT medium was then added and the cells were seeded into 384-well plates for culture. Medium was added on day 5, and HT medium was used on day 7. Positive hybridoma screening was performed on days 8-10.

[0096] Hybridoma cell culture supernatant was analyzed using FACS to screen for positive wells that could bind to CHO-huNECTIN4 S8 cells that stably expressed human Nectin4 antigen on their cell surface, while not binding to blank CHOK1 cells. The selected positive wells were then single-celled using limiting dilution. Subcloning was stopped when two consecutive subclones showed 100% positivity. The resulting hybridoma cell lines secreted only one antibody.

[0097] The results of the FACS binding activity assay of hybridoma cell culture supernatant are shown in the figure. Figure 2 See Tables 1 to 3.

[0098] Table 1. Clones selected in the first round of screening

[0099] 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

[0100] Table 2. Clones selected in the second round of screening

[0101] 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 41O24 1749182.695 174.9 42D20 533378.824 53.3 45F2 393898.7755 39.4 49C23 1510386.33 151.0 50I15 1299424.85 129.9

[0102] Table 3. Clones selected in the third round of screening

[0103] ID FACS <![CDATA[FACS(*10 5 ) <!-- 8 -->]]> 4E9 180199.602 180.199 9M13 448161.147 44.816 12C7 639255.386 63.926 16E10 515026.703 51.503 20M12 783128.784 78.313 30L7 505457.216 50.546 30L18 284554.941 28.455

[0104] The obtained mouse monoclonal antibody was named after the hybridoma cell line ID.

[0105] Example 4 Identification of the variable region sequence of murine monoclonal antibodies

[0106] After expanding the culture of hybridoma cells secreting anti-human Nectin-4 antibody, total RNA was extracted from the cells according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.). The total RNA from the hybridoma cells 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 a T vector and transformed into E. coli competent cells according to the instructions of the pClone007 Simple Vector Kit (Qingke Biotechnology Co., Ltd.). After amplification and plasmid extraction, DNA sequencing was performed to obtain the monoclonal antibody variable region sequence.

[0107] Example 5 Preparation of chimeric antibodies

[0108] The heavy chain variable region sequence of a murine anti-human Nectin-4 monoclonal antibody and the published heavy chain constant region sequence of a human monoclonal antibody IgG1 subclass (SEQ ID NO: 4) were spliced ​​together and constructed into a mammalian cell expression vector. The light chain variable region sequence of the murine anti-human Nectin-4 monoclonal antibody and the published light chain constant region sequence of a human monoclonal antibody K subclass (SEQ ID NO: 5) were spliced ​​together 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 paired and mixed, and HEK293 cells were transfected with PEI. After approximately 7 days, the cell supernatant was collected and purified using Mabselect to obtain the anti-human NECTIN4 chimeric antibody protein.

[0109] The resulting chimeric antibody is referred to as “mouse antibody xiIgG” in this paper.

[0110] Example 6Humanization of mouse antibodies and preparation of humanized antibodies

[0111] Based on the comprehensive antibody encoding scheme, the amino acid sequence regions of the six antigen complementarity determinants (CDRs) of the heavy and light chains of the murine antibody, as well as the framework region supporting the conserved three-dimensional conformation of the antibody, were determined. Subsequently, by analyzing and searching known human antibody sequences, the variable region sequence of the heavy chain of the human antibody most similar to that of the murine antibody, such as IGHV1|IGHJ4*01, was selected. Its antibody framework region sequence was used as a template to bind the murine antibody heavy chain CDRs to the human antibody framework region, ultimately generating the humanized antibody heavy chain variable region sequence. The same process was used to generate the humanized antibody light chain variable region sequence.

[0112] Antibodies whose CDRs are directly transplanted into the human framework region often exhibit a sharp decrease in binding activity. Therefore, it is necessary to revert certain amino acids in the framework region from human to mouse origin. Identifying the reversion mutation sites involves two steps: first, comparing the designed humanized antibody sequence with the original mouse antibody sequence to examine which amino acids differ; second, examining whether these amino acids play a crucial role in supporting the antibody structure or in binding to the antigen. Simultaneously, the humanized sequence needs to be examined for potential post-translational modification sites, such as N-(asparagine) glycosylation sites, N-deamidation sites, and D-(aspartic acid) isomerization sites.

[0113] By combining the humanized heavy chain variable region and light chain variable region in pairs, and referring to the preparation of chimeric antibodies described in Example 5, humanized antibodies were obtained. The humanized antibody was named "mouse antibody name hzmn", where m and n are the humanized (hz) modified sequences (VH_hz and VL_hz) of the mouse antibodies VH and VL, respectively.

[0114] Example 7 Preparation of antibody-drug conjugates (ADCs)

[0115] After reducing the antibody in PBS at pH 7.4 with 2.0-2.6 equivalents of TECP for 2 hours, a DMA solution of vcMMAE was added to the reduced antibody solution (molar ratio of vcMMAE to antibody was 6:1). The mixture was stirred at 2-8°C for 1 hour, followed by ultrafiltration to remove DMA and small molecule residues. The absorbance of the conjugate was measured at 248 nm–280 nm using a UV spectrophotometer, and the concentration of the conjugate was calculated. The samples were aliquoted into cryovials and stored at -80°C. The DAR value (4.0 ± 1) of the samples was determined by HPLC-HIC.

[0116] The ADC corresponding to the antibody is named using the format "antibody naming-E".

[0117] Example 8 In vitro cell binding experiment

[0118] The anti-human Nectin-4 control antibody Enfortumumab, the antibody of the present invention, or the ADC were serially diluted 2-fold starting from an initial concentration of 100 nM, resulting in a total of 16 concentration points. 10 μL of the antibody at each concentration point was added to a 384-well plate.

[0119] BT474 cells (breast cancer cells) expressing Nectin-4 were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended to a cell density of approximately 2 x 10⁻⁶ cells / mL. 6 Cells / ml, 10 μL was added to the wells of a 384-well plate containing antibody. After incubation at 4°C for 1 hour, fluorescently labeled goat anti-human IgG secondary antibody was added. After further incubation at 4°C for 1 hour, the mean fluorescence reading of the cell population was analyzed by flow cytometry.

[0120] The results of the FACS binding experiment of the mouse antimolecule of the present invention with BT474 cells are shown in the figure. Figure 3 In Figure 3 A and Tables 4 and 5.

[0121] Table 4. Binding of murine monoclonal antibodies to BT474 cells

[0122]

[0123]

[0124] Table 5. Binding of murine monoclonal antibodies to BT474 cells

[0125]

[0126] The results of the FACS binding experiment between the humanized modified molecule of this invention and BT474 cells are shown in the figure. Figure 3 In Figure 3 B. Figure 3 C and Tables 6 and 7.

[0127] Table 6. Binding of humanized antibodies to BT474 cells

[0128]

[0129] Table 7. Binding of humanized antibodies to BT474 cells

[0130]

[0131]

[0132] The experimental results of the binding of the ADC of this invention to FACS in BT474 cells are shown below. Figure 3 In Figure 3 D and Table 8.

[0133] Table 8. Binding of antibody-drug conjugates to BT474 cells

[0134]

[0135] Example 9 In vitro cell experiments of antibodies

[0136] 9.1 BT474 Cell Endocytosis Assay

[0137] 1. Collect BT474 cells, centrifuge at 1200 rpm for 8 minutes, and wash twice with DPBS (Gibco Cat.: 14190-136).

[0138] 2.1E5 cells were seeded in each well. Antibody was diluted at a concentration of 10ug / ml and applied to 7 wells in a 1:2 gradient. The last well was a blank well. The mixture was incubated on ice for 1 hour.

[0139] 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 equal portions according to the time points, and incubate at 37°C for different time periods. One portion is kept on ice as a control without endocytosis at 0 point, and the group without antibody is set up as the NC control.

[0140] 4. Wash with citric acid at pH 2.7 for 3.5 minutes, neutralize with 1M Tris-HCl solution at pH 9.5, wash twice with PBS, resuspend in an appropriate amount of 1% BSA-PBS, and detect on an IQplus instrument.

[0141] 5. Use GraphPad Prism software to analyze and process the data.

[0142] The experimental results of the BT474 cell endocytosis activity of the mouse antimolecule of the present invention are shown in the figure. Figure 4 In Figure 4 A and Table 9.

[0143] Table 9. BT474 cell endocytosis activity of murine monoclonal antibodies

[0144] Internalization efficiency (%) Enfortumab 63 mIgG 5M21 69 mIgG 26G13 37 mIgG 29B12 57 mIgG 30J3 56 mIgG 7I18 66 isotype control 16

[0145] The experimental results of the BT474 cell endocytosis activity of the humanized modified molecule of the present invention are shown in the figure. Figure 4 In Figure 4 B, 4C and Tables 10 and 11.

[0146] Table 10. BT474 cell endocytic activity of humanized antibodies

[0147] Internalization efficiency (%) Enfortumab 62.4 5M21 xiIgG 56.9 5M21 hz00 54.8 5M21 hz01 62.2 5M21 hz10 64.8 5M21 hz11 64.4

[0148] Table 11. BT474 cell endocytic activity of humanized antibodies

[0149]

[0150]

[0151] The experimental results of the BT474 cell endocytosis activity of the ADC of the present invention are shown in the figure. Figure 4 In Figure 4 D and Table 12.

[0152] Table 12. BT474 cell endocytosis activity of antibody-drug conjugates

[0153] Internalization 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 hzo1-E 56.6 20M12 hz11-E 58.4 isotype control --

[0154] 9.2 BT474 Cell Proliferation Inhibition Assay

[0155] BT474 breast cancer cells expressing Nectin4 were cultured, and cells were collected by trypsin digestion. The cells were centrifuged at 400g for 5 minutes, and the supernatant was discarded. Cells were seeded into plates at a density of 4000 cells per well and cultured at 37°C with 5% CO2 for 24 hours. The antibody to be tested was dissolved in medium containing 1% BSA at an initial concentration of 200 μg / ml, and then diluted 3-fold (9 dilutions, including 0 concentration). 100 μL of antibody at each concentration was added to a 96-well plate. 100 μL of the diluted antibody sample was mixed with 100 μL of cells and incubated at 37°C with 5% CO2 for 120 hours. 5 μM CCK-8 was prepared using medium containing 1% BSA, and 20 μL of CCK-8 was added to a 96-well plate. The plate was incubated at 37°C with 5% CO2 for 4 hours. Finally, the cell plate was removed and allowed to stand at room temperature for 15 minutes before being thoroughly shaken to mix. The plate was read at a detection wavelength of 450 nm. Using the working concentration (ng / ml) of the bare antibody and ADC samples as the X-axis and the absorbance value as the Y-axis, the EC50 values ​​of the bare antibody and ADC samples were obtained by performing four-parameter fitting using SoftMaxPro.

[0156] The results are shown in Tables 13, 14 and 15.

[0157] Table 13. Inhibitory activity of mouse anti-BT474 cell proliferation.

[0158] 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

[0159] Table 14. Inhibitory activity of ADC on BT474 cell proliferation

[0160]

[0161] Table 15. Inhibitory activity of ADC on BT474 cell proliferation

[0162]

[0163]

[0164] Example 10 Antibody cross-antigen activity analysis - Facs

[0165] cell:

[0166] CHO-huNectin4 S8;

[0167] HEK293 cells expressing mouse Nectin4 (NP_082169): HEK-muNectin4; and

[0168] HEK293 cells expressing Cyno Nectin4 (SEQ ID NO: 6): HEK-cynoNectin4.

[0169] The experiment was conducted as described in Example 8, and the results are shown below. Figure 5 of Figure 5 A, 5B and 5C, as well as Tables 16, 17 and 18.

[0170] Table 16. Binding of humanized antibodies to CHO-huNectin4 S8 cells

[0171]

[0172] Table 17. Binding of humanized antibodies to HEK-muNectin4 cells

[0173]

[0174] Table 18. Binding of humanized antibodies to HEK-cynoNectin4 cells

[0175]

[0176] Example 11 In vitro binding affinity and kinetics of antibodies

[0177] Antibody-antigen interaction was determined using a GE BIAcore S200 instrument. Following the GE BiotinCAPture Kit operating instructions, the His-tagged human NECTIN4 antigen was first coupled to both the analytical and control sample channels of the sensor chip. Then, serially diluted antibody samples (starting concentration 20 nM, 1:3 dilution at 8 concentration points, with repetition at the 0.741 nm concentration point) were flowed together through both the analytical and control sample channels. The photoreaction values ​​following antibody-antigen binding were measured. After fitting and analysis using the instrument software (1:1 binding mode), the binding constant Kon, dissociation constant Koff, and affinity constant KD of the antibody were finally obtained.

[0178] The results are shown in Table 19.

[0179] Table 19. Results of antibody binding affinity and kinetics

[0180] 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

[0181] Example 12 Verification of cross-binding activity between antibody and its family of antigen proteins

[0182] antigen:

[0183] human Nectin-1(C-6His)Novoprotein Cat#C492

[0184] human Nectin-2(C-6His)Novoprotein Cat#C440

[0185] human Nectin-3(C-6His)Novoprotein Cat#C630

[0186] human Nectin-4(C-6His)Novoprotein Cat#CJ19

[0187] Antibody (primary antibody): The antibody of this invention;

[0188] Control antibody;

[0189] CD111 / Nectin-1 / PVRL1 Antibody, Rabbit Fab, 80244-RP01-100, Sinobiologics.

[0190] Anti-Nectin 2antibody(ab233085), Rabbit antibody, Abcam.

[0191] Anti-Nectin 3antibody(ab137961), Rabbit antibody, Abcam.

[0192] Secondary antibody:

[0193] Goat anti-Rabbit IgG-Fc Secondary antibody(HRP)Cat#SSA003, JacksonImmuno.

[0194] The A450 absorbance was measured by coating the plate with 1 μg / ml antigen, incubating overnight at 4°C, then adding serially diluted antibodies, and finally adding HRP-labeled secondary antibody. Results are shown below. Figure 6 of Figure 6 A, 6B, 6C, and 6D.

[0195] The results show that the antibody of the present invention and the control antibody exhibit the same properties, both of which 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.

[0196] Example 13 In vitro stability study of antibody in monkey serum

[0197] Experimental materials: test antibody, FBS, test antibody binding antigen, anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-035-098).

[0198] Experimental instruments: 37℃ incubator, ELISA reader.

[0199] Experimental steps:

[0200] Sample preparation:

[0201] 1) Adjust the concentration of the antibody to be tested to 20ug / ml, filter and sterilize, and aliquot into 250ul tubes for later use;

[0202] 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 10ug / ml antibody concentration;

[0203] 3) A total of 7 samples were prepared, sealed with sealing film, and placed at 37°C, maintaining sterility throughout the process;

[0204] 4) Samples were taken on days 0, 3, 7, 10, 14 and 21 and stored at 4°C for testing. The sample taken on day 21 should be stored at 4°C for at least one day.

[0205] Detection method:

[0206] 1) Coat the bound antigen and anti-IgG Fab monoclonal antibody separately with PBS in 96-well ELISA plates, 0.2ug / ml, 100ul / well, and incubate overnight at 4°C;

[0207] 2) Prepare the required reagents:

[0208] Blocking solution 5% BSA + PBS

[0209] Antibody dilution solution: 5% BSA + PBS + 50% FBS

[0210] ELISA plate washing buffer: 0.1% Tween + PBS

[0211] 3) Wash the coated ELISA plate three times with PBS, 300ul / well, to remove free uncoated antigen;

[0212] 4) Add blocking solution, 200 μL / well, and seal at 37°C for 1 hour;

[0213] 5) The antibody to be tested was diluted to 2ug / ml using antibody dilution buffer, and then diluted 3-fold for a total of 8 gradients;

[0214] 6) Discard the blocking solution, add diluted antibody to each of the two coating methods (100 μL / well), and incubate at 37°C for 1 hour.

[0215] 7) Wash the plate three times with PBST;

[0216] 8) Dilute the secondary antibody 1:5000, add it to the washed ELISA plate, 100 μL / well, and incubate at 37°C for 40 min;

[0217] 9) Wash the plate three times with PBST;

[0218] 10) TMB color development, 100ul / well, protected from light for 10 minutes;

[0219] 11) Add 50ul of 2M HCl to terminate, and take a reading at 450nm.

[0220] Result processing:

[0221] Binding curves were constructed based on ELISA colorimetric values. Changes in the binding curves were observed at different storage times to assess the stability of antibody binding activity after storage. Results are shown below. Figure 7 of Figure 7 A, 7B.

[0222] The results showed that the effective antibody content of the present invention did not change after incubation at 37°C for 21 days, meaning that it could remain stable at 37°C for more than 21 days.

[0223] Example 14Antibody drug metabolism analysis in mice

[0224] Experimental materials: test antibody, mouse serum collected at different time points, test antibody binding antigen human Nectin-4, anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-035-098).

[0225] Experimental methods:

[0226] Serum collection:

[0227] 1) Female Balb / C mice, 3 mice / group, were administered the drug via tail vein or intraperitoneal vein, 200ug / mouse;

[0228] 2) Collect blood from the tail vein at the time points specified in the experimental design, keep the blood samples at room temperature for more than 30 minutes, collect serum at 4000 rpm for 15 minutes, and store at -20℃. To prevent serum evaporation, the final serum collection volume should be greater than 20 μL.

[0229] 3) The last serum collection should be frozen at -20°C for at least 24 hours.

[0230] Detection method:

[0231] 1) Coat the bound antigen and anti-IgG Fab monoclonal antibody separately with PBS in 96-well ELISA plates, 0.2ug / ml, 100ul / well, and incubate overnight at 4°C;

[0232] 2) Prepare the required reagents:

[0233] Blocking solution 5% BSA + PBS

[0234] Antibody dilution solution: 5% BSA + PBS + 20% blank mouse serum

[0235] ELISA plate washing buffer: 0.1% Tween + PBS

[0236] 3) Wash the coated ELISA plate three times with PBS, 300ul / well;

[0237] 4) Add blocking solution, 200 μL / well, and seal at 37°C for 1 hour;

[0238] 5) The initial serum was diluted to an appropriate concentration using blocking buffer and then diluted to an appropriate concentration range using antibody diluent. The specific dilution factor needs to be adjusted based on the preliminary experiment. In principle, the final colorimetric value of the serum to be tested should be within the range of the colorimetric value of the standard.

[0239] 6) Dilute the antibody standard with antibody diluent. The dilution of the standard should still be adjusted according to the preliminary experiment to fit the standard to a linear curve (if suitable software is available, an S-shaped curve can also be fitted).

[0240] 7) Discard the blocking solution in the ELISA plate. Add diluted antibody standard and serum to be tested to the ELISA plates of the two coating methods, 100 μL / well, and incubate at 37°C for 1 h.

[0241] 8) Wash the plate three times with PBST;

[0242] 9) Dilute the secondary antibody 1:5000, add it to the washed ELISA plate, 100 μL / well, and incubate at 37°C for 40 min;

[0243] 10) Wash the plate three times with PBST;

[0244] 11) TMB color development, 100ul / well, protected from light for 10 minutes;

[0245] 12) Add 50ul of 2M HCl to terminate, and take a reading at 450nm.

[0246] See results Figure 8 of Figure 8 A, 8B and Table 20.

[0247] Table 20. Results of antibody metabolism in mice

[0248]

[0249] Concentration: ug / ml

[0250] NA: Below the detection limit, which is 9.77 ng / ml.

[0251] The above description of 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, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260] sequence list <110> Shanghai Puming Biotechnology Co., Ltd. <120> An anti-Nectin-4 antibody and its application <130> LC19110065 <160> 97 <170> SIPOSequenceListing 1.0 <210> 1 <211> 447 <212> PRT <213> Artificial sequence <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 <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> artificial sequence <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> artificial sequence <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> artificial sequence <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> artificial sequence <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 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 <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 <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 <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 <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 <211> 107 <212> PRT <213> artificial sequence <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 <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 <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 Val Ser Ser 115 <210> 14 <211> 113 <212> PRT <213> artificial sequence <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 <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 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 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 <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 <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 <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 <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 <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 <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 <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 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 <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 <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 <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 <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 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> 27 <211> 116 <212> PRT <213> artificial sequence <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 <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 <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 <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 <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 <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 <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 <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 <400> 35 Gly Tyr Thr Phe Thr Thr Tyr 1 5 <210> 36 <211> 10 <212> PRT <213> Artificial sequence <400> 36 Gly Tyr Thr Phe Thr Thr Tyr Tyr Ile His 1 5 10 <210> 37 <211> 5 <212> PRT <213> Artificial sequence <400> 37 Thr Tyr Tyr Ile His 1 5 <210> 38 <211> 6 <212> PRT <213> Artificial sequence <400> 38 Thr Thr Tyr Tyr Ile His 1 5 <210> 39 <211> 6 <212> PRT <213> Artificial sequence <400> 39 Tyr Pro Gly Asn Val Asn 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial sequence <400> 40 Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys 1 5 10 <210> 41 <211> 17 <212> PRT <213> Artificial sequence <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 <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 <400> 43 Gly Leu Tyr Tyr Phe Asp Tyr 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial sequence <400> 44 Ala Arg Gly Leu Tyr Tyr Phe Asp 1 5 <210> 45 <211> 11 <212> PRT <213> Artificial sequence <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 <400> 46 Ser Asn Asp Val Ala Trp Tyr 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial sequence <400> 47 Tyr Ala Ser Asn Arg Tyr Thr 1 5 <210> 48 <211> 10 <212> PRT <213> Artificial sequence <400> 48 Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr 1 5 10 <210> 49 <211> 9 <212> PRT <213> Artificial sequence <400> 49 Gln Gln Asp Tyr Ser Ser Pro Tyr Thr 1 5 <210> 50 <211> 8 <212> PRT <213> Artificial sequence <400> 50 Gln Gln Asp Tyr Ser Ser Pro Tyr 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial sequence <400> 51 Gly Phe Ser Leu Ile Asp Tyr 1 5 <210> 52 <211> 10 <212> PRT <213> Artificial sequence <400> 52 Gly Phe Ser Leu Ile Asp Tyr Gly Val Ser 1 5 10 <210> 53 <211> 5 <212> PRT <213> Artificial sequence <400> 53 Asp Tyr Gly Val Ser 1 5 <210> 54 <211> 6 <212> PRT <213> Artificial sequence <400> 54 Ile Asp Tyr Gly Val Ser 1 5 <210> 55 <211> 5 <212> PRT <213> Artificial sequence <400> 55 Trp Gly Asp Gly Lys 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial sequence <400> 56 Val Ile Trp Gly Asp Gly Lys Ile Tyr 1 5 <210> 57 <211> 16 <212> PRT <213> Artificial sequence <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 <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 <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 <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 <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 <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 <400> 63 Phe Ala Ser Thr Arg Glu Ser 1 5 <210> 64 <211> 10 <212> PRT <213> Artificial sequence <400> 64 Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu 1 5 10 <210> 65 <211> 9 <212> PRT <213> Artificial sequence <400> 65 Gln Gln His Tyr Asn Thr Pro Phe Thr 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial sequence <400> 66 Gln Gln His Tyr Asn Thr Pro Phe 1 5 <210> 67 <211> 17 <212> PRT <213> Artificial sequence <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 <400> 68 Trp Gly Asp Ala Lys 1 5 <210> 69 <211> 5 <212> PRT <213> Artificial sequence <400> 69 Trp Gly Gly Gly Lys 1 5 <210> 70 <211> 7 <212> PRT <213> Artificial sequence <400> 70 Gly Tyr Thr Phe Thr Ser Tyr 1 5 <210> 71 <211> 10 <212> PRT <213> Artificial sequence <400> 71 Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His 1 5 10 <210> 72 <211> 5 <212> PRT <213> Artificial sequence <400> 72 Ser Tyr Tyr Ile His 1 5 <210> 73 <211> 6 <212> PRT <213> Artificial sequence <400> 73 Thr Ser Tyr Tyr Ile His 1 5 <210> 74 <211> 6 <212> PRT <213> Artificial sequence <400> 74 Tyr Pro Gly Asn Ala Asn 1 5 <210> 75 <211> 10 <212> PRT <213> Artificial sequence <400> 75 Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys 1 5 10 <210> 76 <211> 17 <212> PRT <213> Artificial sequence <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 <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 <400> 78 Ser Val Tyr Tyr Phe Asp Tyr 1 5 <210> 79 <211> 8 <212> PRT <213> Artificial sequence <400> 79 Ala Arg Ser Val Tyr Tyr Phe Asp 1 5 <210> 80 <211> 7 <212> PRT <213> Artificial sequence <400> 80 Tyr Ala Ser Asn Arg Asn Thr 1 5 <210> 81 <211> 10 <212> PRT <213> Artificial sequence <400> 81 Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Asn 1 5 10 <210> 82 <211> 7 <212> PRT <213> Artificial sequence <400> 82 Gly Tyr Ser Phe Thr Asp Tyr 1 5 <210> 83 <211> 10 <212> PRT <213> Artificial sequence <400> 83 Gly Tyr Ser Phe Thr Asp Tyr Tyr Met His 1 5 10 <210> 84 <211> 5 <212> PRT <213> Artificial sequence <400> 84 Asp Tyr Tyr Met His 1 5 <210> 85 <211> 6 <212> PRT <213> Artificial sequence <400> 85 Thr Asp Tyr Tyr Met His 1 5 <210> 86 <211> 6 <212> PRT <213> Artificial sequence <400> 86 Asn Pro Asn Asn Gly Asn 1 5 <210> 87 <211> 10 <212> PRT <213> Artificial sequence <400> 87 Arg Val Asn Pro Asn Asn Gly Asn Thr Leu 1 5 10 <210> 88 <211> 17 <212> PRT <213> Artificial sequence <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 <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 <400> 90 Glu Asp Arg Tyr Ala Phe Ala Tyr 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial sequence <400> 91 Ala Arg Glu Asp Arg Tyr Ala Phe Ala 1 5 <210> 92 <211> 15 <212> PRT <213> Artificial sequence <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 <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 <400> 94 Tyr Ala Ser Asn Leu Glu Ser 1 5 <210> 95 <211> 10 <212> PRT <213> Artificial sequence <400> 95 Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu 1 5 10 <210> 96 <211> 9 <212> PRT <213> Artificial sequence <400> 96 Gln His Thr Trp Glu Ile Pro Tyr Thr 1 5 <210> 97 <211> 8 <212> PRT <213> Artificial sequence <400> 97 Gln His Thr Trp Glu Ile Pro Tyr 1 5

Claims

1. An antibody molecule or antigen-binding fragment thereof against poliovirus receptor-like molecule 4 (Nectin-4), said antibody molecule or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) The heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 51, 55, and 59, and the amino acid sequence of the heavy chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 13; and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 61, 63, and 65, and the amino acid sequence of the light chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; (2) The heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 51, 55, and 59, and the amino acid sequence of the heavy chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 61, 63, and 65, and the amino acid sequence of the light chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 22; (3) The heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 51, 55, and 59, and the amino acid sequence of the heavy chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 61, 63, and 65, and the amino acid sequence of the light chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23; (4) The heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3, sequentially shown in SEQ ID NO: 51, 68, and 59, and the amino acid sequence of the heavy chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 19; and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3, sequentially shown in SEQ ID NO: 67, 63, and 65, and the amino acid sequence of the light chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 25; or (5) The heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 51, 69, and 59, and the amino acid sequence of the heavy chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 21; and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 67, 63, and 65, and the amino acid sequence of the light chain variable region has at least 75% identity with the amino acid sequence shown in SEQ ID NO: 25; Furthermore, the CDR is defined according to CHOTHIA.

2. The antibody molecule or its antigen-binding fragment according to claim 1, characterized in that, The aforementioned at least 75% identity refers to 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

3. The antibody molecule or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region and light chain variable region of the antibody molecule or its antigen-binding fragment are combinations of the following amino acid sequences: (1) The amino acid sequence shown in SEQ ID NO: 13; and the amino acid sequence shown in SEQ ID NO: 14; (2) The amino acid sequence shown in SEQ ID NO: 16; and the amino acid sequence shown in SEQ ID NO: 22; (3) The amino acid sequence shown in SEQ ID NO: 16; and the amino acid sequence shown in SEQ ID NO: 23; (4) The amino acid sequence shown in SEQ ID NO: 19; and the amino acid sequence shown in SEQ ID NO: 25; or (5) The amino acid sequence shown in SEQ ID NO: 21; and the amino acid sequence shown in SEQ ID NO:

25.

4. The antibody molecule or its antigen-binding fragment according to claim 1, characterized in that, The antibody molecule or its antigen-binding fragment binds to mammalian Nectin-4.

5. The antibody molecule or its antigen-binding fragment according to claim 1, characterized in that, The antibody molecule or its antigen-binding fragment binds to primate Nectin-4.

6. The antibody molecule or its antigen-binding fragment according to claim 1, characterized in that, The antibody molecule or its antigen-binding fragment binds to human or monkey Nectin-4.

7. The antibody molecule or its antigen-binding fragment according to claim 1, characterized in that, The antibody molecule is a murine antibody, a chimeric antibody, or a fully or partially humanized antibody.

8. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 7, characterized in that, The antigen-binding fragment is a fragment of the antibody molecule such as scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

9. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 7, characterized in that, The antibody molecule is a monoclonal antibody or a single-chain antibody.

10. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 7, characterized in that, The antibody molecule or its antigen-binding fragment also includes a constant region.

11. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 6, characterized in that, The antibody molecule or its antigen-binding fragment also includes a mouse or human heavy chain constant region (CH) and / or a light chain constant region (CL).

12. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 7, characterized in that, The antibody molecule or its antigen-binding fragment comprises a heavy chain and a light chain.

13. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 7, characterized in that, The antibody molecule or its antigen-binding fragment contains a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

14. The antibody molecule or its antigen-binding fragment according to claim 9, characterized in that, The antibody molecule is a humanized monoclonal antibody.

15. The antibody molecule or its antigen-binding fragment according to claim 14, characterized in that, The monoclonal antibody has a heavy chain constant region of type IgG1 and a light chain constant region of type κ.

16. A nucleic acid molecule comprising encoding an antibody molecule or an antigen-binding fragment thereof as described in any one of claims 1 to 15.

17. A vector comprising the nucleic acid molecule of claim 16.

18. A host cell comprising the nucleic acid molecule of claim 16 and / or the vector of claim 17, or said host cell being transformed or transfected by the nucleic acid molecule of claim 16 and / or the vector of claim 17; wherein said host cell is not a plant cell.

19. A composition comprising an antibody molecule or an antigen-binding fragment thereof as described in any one of claims 1 to 15, a nucleic acid molecule as described in claim 16, a vector as described in claim 17, or a host cell as described in claim 18.

20. The composition according to claim 19, characterized in that, The composition is a pharmaceutical composition.

21. The composition according to claim 20, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier, excipient, or excipient.

22. Use of the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 15, the nucleic acid molecule of claim 16, the vector of claim 17, the host cell of claim 18, or the composition of any one of claims 19 to 21 in the preparation of a medicament for the prevention or treatment of cancer, wherein the cancer is bladder cancer, breast cancer, ovarian cancer, or lung cancer.

23. A kit comprising an antibody molecule or antigen-binding fragment thereof as described in any one of claims 1 to 15, a nucleic acid molecule as described in claim 16, a vector as described in claim 17, a host cell as described in claim 18, or a composition as described in any one of claims 19 to 21.

24. Use of the antibody molecule or antigen-binding fragment thereof as described in any one of claims 1 to 15 in the preparation of antibody-drug conjugates.

25. An antibody-drug conjugate formed by conjugating an antibody molecule or its antigen-binding fragment according to any one of claims 1 to 15 with a cytotoxic portion.

26. The antibody-drug conjugate according to claim 25, characterized in that, The cytotoxic component is a microtubule inhibitor, a topoisomerase inhibitor, or a DNA binder.

27. The antibody-drug conjugate according to claim 26, characterized in that, The microtubule inhibitors are selected from maytansin derivatives, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl dolastatin 10, tubulysin derivatives, cryptophycin derivatives, and taltobulin.

28. The antibody-drug conjugate according to claim 26, characterized in that, The topoisomerase inhibitors are selected from the doxorubicin metabolite PNU-159682 derivative and the irinotecan (CPT-11) metabolite SN38 derivative.

29. The antibody-drug conjugate according to claim 26, characterized in that, The DNA binding agent is selected from PBD derivatives and Duocarmycin derivatives.

30. Use of the antibody-drug conjugate of any one of claims 25 to 29 in the preparation of a medicament for treating Nectin4-expressing cancers, characterized in that, The cancers expressed by Nectin4 are bladder cancer, breast cancer, ovarian cancer, or lung cancer.

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