Monoclonal antibody molecule and use thereof

Abstract

The present application relates to an antibody or antigen-binding fragment targeting human 5T4, and a use thereof in the preparation of a detection kit or detection reagent for diagnosing diseases related to 5T4 positive tumors, and a use thereof in the preparation of a drug for treating diseases related to 5T4 positive tumors.

Description

Monoclonal antibody molecules and their uses Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antibody or antigen-binding fragment targeting human 5T4, and its use in preparing a detection kit or preparing a drug. Background Art

[0002] Tumor is a disease caused by abnormal cell proliferation under the influence of various internal and external carcinogenic factors. It is divided into two major categories: benign and malignant according to its invasive ability and the presence of distant metastasis. Metastasis is a characteristic of malignant tumors and the main cause of death in cancer patients. Once metastasis occurs, it has a huge impact on the patient's quality of life and prognosis. In the process of continuous exploration of the mechanisms of tumor occurrence and progression, studies have found that normal placental trophoblasts and malignant tumor cells share some common physiological processes, including invasion of local tissues and avoidance of immune surveillance. 5T4 is a glycoprotein found in embryonic trophoblast cells, also known as Wnt activation inhibitory factor 1 (WAIF1) or trophoblast cell glycoprotein (TPBG). It was selected as a solid tumor target because of its restricted expression in normal adult tissues but high expression in many solid tumor malignancies.

[0003] Due to its differential expression in normal and cancerous tissues, 5T4 has emerged as a potential therapeutic target for tumors. However, the mechanism of action of this molecule in tumors remains poorly understood. However, existing studies suggest that 5T4 may act through the Wnt signaling pathway, epithelial-mesenchymal transition (EMT), and CXCR4 / CXCL12 pathway. The Wnt signaling pathway is a key pathway in embryonic development and is crucial for normal development, cellular homeostasis, and regeneration. Wnt has two pathways: the canonical Wnt / β-catenin pathway and the non-canonical Wnt pathway. The Wnt signaling pathway is inactive in normal mature cells but is enhanced in tumor cells. Studies have demonstrated that 5T4 interferes with Wnt / β-catenin signaling by inhibiting the endocytosis of LRP6 and Wnt receptors, thereby inhibiting cell adhesion and cytoskeletal formation, and promoting tumor migration and spread. There is also evidence that 5T4 participates in the non-canonical Wnt pathway in breast and gastric cancer cells, similarly promoting cancer cell migration and invasion. Other research results have confirmed that 5T4 is involved in the epithelial-mesenchymal transition phenomenon that contributes to the occurrence, development, invasion, metastasis and spread of malignant tumors, as well as the expression of CXCL12 / CXCR4 on the surface of embryonic and tumor cells.

[0004] However, not only is the mechanism of 5T4's tumor action still unclear, but there are currently no drugs targeting 5T4 on the market globally. According to clinical trial data, there are currently approximately nine 5T4-targeted drugs in development, with only a little over ten in the clinical pipeline, and these drugs are highly diverse. In summary, the development of multifunctional antibodies or drugs targeting 5T4 has the potential to provide significant clinical benefits in the field of anti-cancer treatment. Summary of the Invention

[0005] In one aspect, the present application provides an antibody or antigen-binding fragment targeting human 5T4, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a complementarity determining region (HCDR) as shown in any one of the following tables:

[0006] In some embodiments, the VL of the antibody or antigen-binding fragment comprises a complementarity determining region (LCDR) as shown in any one of the following tables:

[0007] In some embodiments, the antibody or antigen-binding fragment comprises any one of the following complementarity determining regions (CDRs):

[0008] 1) the VH comprises HCDR1 as shown in SEQ ID NO: 88, HCDR2 as shown in SEQ ID NO: 101, and HCDR3 as shown in SEQ ID NO: 113, and the VL comprises LCDR1 as shown in SEQ ID NO: 129, LCDR2 as shown in SEQ ID NO: 137, and LCDR3 as shown in SEQ ID NO: 139; or

[0009] 2) the VH comprises HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 108, and the VL comprises LCDR1 as shown in SEQ ID NO: 126, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 141; or

[0010] 3) the VH comprises HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 95, and HCDR3 as shown in SEQ ID NO: 114, and the VL comprises LCDR1 as shown in SEQ ID NO: 125, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 142; or

[0011] 4) the VH comprises HCDR1 as shown in SEQ ID NO:82, HCDR2 as shown in SEQ ID NO:91, and HCDR3 as shown in SEQ ID NO:112, and the VL comprises LCDR1 as shown in SEQ ID NO:129, LCDR2 as shown in SEQ ID NO:137, and LCDR3 as shown in SEQ ID NO:139; or

[0012] 5) the VH comprises HCDR1 as shown in SEQ ID NO: 87, HCDR2 as shown in SEQ ID NO: 100, and HCDR3 as shown in SEQ ID NO: 111, and the VL comprises LCDR1 as shown in SEQ ID NO: 120, LCDR2 as shown in SEQ ID NO: 136, and LCDR3 as shown in SEQ ID NO: 148; or

[0013] 6) the VH comprises HCDR1 as shown in SEQ ID NO:80, HCDR2 as shown in SEQ ID NO:93, and HCDR3 as shown in SEQ ID NO:110, and the VL comprises LCDR1 as shown in SEQ ID NO:120, LCDR2 as shown in SEQ ID NO:136, and LCDR3 as shown in SEQ ID NO:147; or

[0014] 7) the VH comprises HCDR1 as shown in SEQ ID NO: 81, HCDR2 as shown in SEQ ID NO: 104, and HCDR3 as shown in SEQ ID NO: 109, and the VL comprises LCDR1 as shown in SEQ ID NO: 127, LCDR2 as shown in SEQ ID NO: 133, and LCDR3 as shown in SEQ ID NO: 139; or

[0015] 8) the VH comprises HCDR1 as shown in SEQ ID NO: 86, HCDR2 as shown in SEQ ID NO: 97, and HCDR3 as shown in SEQ ID NO: 116, and the VL comprises LCDR1 as shown in SEQ ID NO: 129, LCDR2 as shown in SEQ ID NO: 130, and LCDR3 as shown in SEQ ID NO: 139; or

[0016] 9) the VH comprises HCDR1 as shown in SEQ ID NO: 89, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 117, and the VL comprises LCDR1 as shown in SEQ ID NO: 121, LCDR2 as shown in SEQ ID NO: 134, and LCDR3 as shown in SEQ ID NO: 149; or

[0017] 10) the VH comprises HCDR1 as shown in SEQ ID NO: 84, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 107, and the VL comprises LCDR1 as shown in SEQ ID NO: 123, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 140; or

[0018] 11) the VH comprises HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 94, and HCDR3 as shown in SEQ ID NO: 115, and the VL comprises LCDR1 as shown in SEQ ID NO: 125, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 141; or

[0019] 12) the VH comprises HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 114, and the VL comprises LCDR1 as shown in SEQ ID NO: 124, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 143; or

[0020] 13) the VH comprises HCDR1 as shown in SEQ ID NO: 90, HCDR2 as shown in SEQ ID NO: 102, and HCDR3 as shown in SEQ ID NO: 106, and the VL comprises LCDR1 as shown in SEQ ID NO: 128, LCDR2 as shown in SEQ ID NO: 131, and LCDR3 as shown in SEQ ID NO: 146; or

[0021] 14) the VH comprises HCDR1 as shown in SEQ ID NO: 83, HCDR2 as shown in SEQ ID NO: 92, and HCDR3 as shown in SEQ ID NO: 105, and the VL comprises LCDR1 as shown in SEQ ID NO: 128, LCDR2 as shown in SEQ ID NO: 131, and LCDR3 as shown in SEQ ID NO: 145; or

[0022] 15) the VH comprises HCDR1 as shown in SEQ ID NO: 87, HCDR2 as shown in SEQ ID NO: 99, and HCDR3 as shown in SEQ ID NO: 111, and the VL comprises LCDR1 as shown in SEQ ID NO: 119, LCDR2 as shown in SEQ ID NO: 135, and LCDR3 as shown in SEQ ID NO: 150; or

[0023] 16) the VH comprises HCDR1 as shown in SEQ ID NO: 79, HCDR2 as shown in SEQ ID NO: 103, and HCDR3 as shown in SEQ ID NO: 118, and the VL comprises LCDR1 as shown in SEQ ID NO: 122, LCDR2 as shown in SEQ ID NO: 132, and LCDR3 as shown in SEQ ID NO: 144; or

[0024] 17) The VH comprises HCDR1 as shown in SEQ ID NO: 151, HCDR2 as shown in SEQ ID NO: 152, and HCDR3 as shown in SEQ ID NO: 153, and the VL comprises LCDR1 as shown in SEQ ID NO: 154, LCDR2 as shown in SEQ ID NO: 155, and LCDR3 as shown in SEQ ID NO: 156.

[0025] In some embodiments, the VH comprises an amino acid sequence of any one of SEQ ID NOs: 1-16, 63-67 and 157 (see table below), or comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence homology or identical to the amino acid sequence of any one of SEQ ID NOs: 1-16, 63-67 and 157, and the differences in the amino acid sequences are all in the non-CDR regions.

[0026] In some embodiments, the VL comprises an amino acid sequence of any one of SEQ ID NOs: 17-31, 68-70 and 158 (see table below), or comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence homology or identical to the amino acid sequence of any one of SEQ ID NOs: 17-31, 68-70 and 158, and the differences in the amino acid sequences are all in the non-CDR regions.

[0027] Based on the embodiments described above, the antibodies or antigen-binding fragments provided herein may comprise any one of the following groups of VH and VL sequence combinations: 1) a VH as described in SEQ ID NO: 1, and a VL as described in SEQ ID NO: 17, or 2) a VH as described in SEQ ID NO: 2, and a VL as described in SEQ ID NO: 18, or 3) a VH as described in SEQ ID NO: 3, and a VL as described in SEQ ID NO: 19, or 4) a VH as described in SEQ ID NO: 4, and a VL as described in SEQ ID NO: 17, or 5) a VH as described in SEQ ID NO: 5, and a VL as described in SEQ ID NO: 20, or 6) a VH as described in SEQ ID NO: 6, and a VL as described in SEQ ID NO: 21, or 7) a VH as described in SEQ ID NO: 7, and a VL as described in SEQ ID NO: 22, or 8) a VH as described in SEQ ID NO: 8, and a VL as described in SEQ ID NO: 23, or 9) a VH as described in SEQ ID NO: 9, and a VL as described in SEQ ID NO: 10. NO:24, or 10) the VH according to SEQ ID NO:10, and the VL according to SEQ ID NO:25, or 11) the VH according to SEQ ID NO:11, and the VL according to SEQ ID NO:26, or 12) the VH according to SEQ ID NO:12, and the VL according to SEQ ID NO:27, or 13) the VH according to SEQ ID NO:13, and the VL according to SEQ ID NO:28, or 14) the VH according to SEQ ID NO:14, and the VL according to SEQ ID NO:29, or 15) the VH according to SEQ ID NO:15, and the VL according to SEQ ID NO:30, or 16) the VH according to SEQ ID NO:16, and the VL according to SEQ ID NO:31, or 17) the VH according to SEQ ID NO:157, and the VL according to SEQ ID NO:158.

[0028] More preferably, the antibody or antigen-binding fragment provided herein may comprise any one of the following groups of VH and VL sequence combinations: 1) the VH of SEQ ID NO: 63, and the VL of SEQ ID NO: 68, or 2) the VH of SEQ ID NO: 63, and the VL of SEQ ID NO: 69, or 3) the VH of SEQ ID NO: 63, and the VL of SEQ ID NO: 70, or 4) the VH of SEQ ID NO: 64, and the VL of SEQ ID NO: 68, or 5) the VH of SEQ ID NO: 64, and the VL of SEQ ID NO: 69, or 6) the VH of SEQ ID NO: 64, and the VL of SEQ ID NO: 70, or 7) the VH of SEQ ID NO: 65, and the VL of SEQ ID NO: 68, or 8) the VH of SEQ ID NO: 65, and the VL of SEQ ID NO: 69, or 9) the VH of SEQ ID NO: 66. NO:65, and the VL as described in SEQ ID NO:70, or 10) the VH as described in SEQ ID NO:66, and the VL as described in SEQ ID NO:68, or 11) the VH as described in SEQ ID NO:66, and the VL as described in SEQ ID NO:69, or 12) the VH as described in SEQ ID NO:66, and the VL as described in SEQ ID NO:70, or 13) the VH as described in SEQ ID NO:67, and the VL as described in SEQ ID NO:68, or 14) the VH as described in SEQ ID NO:67, and the VL as described in SEQ ID NO:69, or 15) the VH as described in SEQ ID NO:67, and the VL as described in SEQ ID NO:70.

[0029] In some embodiments, the antibody or antigen-binding fragment may be a chimeric or humanized antibody or antigen-binding fragment, For example, the chimeric or humanized antibody or antigen-binding fragment comprises residues derived from a non-human species, wherein the residues from the non-human species may be from a complementarity determining region (CDR), a mutated and / or truncated complementarity determining region, or any other residue involved in antigen (e.g., 5T4) binding. For example, at least about 98%, at least about 98.1%, at least about 98.2%, at least about 98.3%, at least about 98.4%, at least about 98.5%, at least about 98.6%, at least about 98.7%, at least about 98.8%, at least about 98.9%, at least about 99%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 99.99% of the partial sequence of the chimeric or humanized antibody or antigen-binding fragment is derived from human antibody sequences, and the residues of the partial sequence derived from human antibody sequences can be derived from residues of the framework regions, and / or constant regions of the humanized antibody. The non-human species mentioned herein generally refers to a species used for immunization with an antigen, for example, a species selected from mice, rats, rabbits, non-human primates or non-human mammals.

[0030] In some embodiments, the antigen-binding fragment is selected from one or more of scFv, Fv, Fab and F(ab)2, preferably Fab.

[0031] In some embodiments, the antibody or antigen-binding fragment may comprise any of the following groups of heavy chain variable regions and heavy chain constant region 1 (Fd fragment) and light chain sequence combinations: 1) the Fd fragment of SEQ ID NO: 159, and the light chain of SEQ ID NO: 176, or 2) the Fd fragment of SEQ ID NO: 160, and the light chain of SEQ ID NO: 177, or 3) the Fd fragment of SEQ ID NO: 161, and the light chain of SEQ ID NO: 178, or 4) the Fd fragment of SEQ ID NO: 162, and the light chain of SEQ ID NO: 192, or 5) the Fd fragment of SEQ ID NO: 163, and the light chain of SEQ ID NO: 179, or 6) the Fd fragment of SEQ ID NO: 164, and the light chain of SEQ ID NO: 180, or 7) the Fd fragment of SEQ ID NO: 165, and the light chain of SEQ ID NO: 181, or 8) the Fd fragment of SEQ ID NO: 166. NO: 182, or 9) the Fd fragment of SEQ ID NO: 167, and the light chain of SEQ ID NO: 183, or 10) the Fd fragment of SEQ ID NO: 168, and the light chain of SEQ ID NO: 184, or 11) the Fd fragment of SEQ ID NO: 169, and the light chain of SEQ ID NO: 185, or 12) the Fd fragment of SEQ ID NO: 170, and the light chain of SEQ ID NO: 186, or 13) the Fd fragment of SEQ ID NO: 171, and the light chain of SEQ ID NO: 187, or 14) the Fd fragment of SEQ ID NO: 172, and the light chain of SEQ ID NO: 188, or 15) the Fd fragment of SEQ ID NO: 173, and the light chain of SEQ ID NO: 189, or 16) the Fd fragment of SEQ ID NO: 174 17) the Fd fragment of SEQ ID NO: 174, and the light chain of SEQ ID NO: 190, or 17) the Fd fragment of SEQ ID NO: 175, and the light chain of SEQ ID NO: 191.

[0032] In some embodiments, the antibody or antigen-binding fragment may comprise any of the following groups of heavy chain variable regions and heavy chain constant region 1 (Fd fragment) and light chain sequence combinations: 1) the Fd fragment of SEQ ID NO: 226, and the light chain of SEQ ID NO: 231, or 2) the Fd fragment of SEQ ID NO: 226, and the light chain of SEQ ID NO: 232, or 3) the Fd fragment of SEQ ID NO: 226, and the light chain of SEQ ID NO: 233, or 4) the Fd fragment of SEQ ID NO: 227, and the light chain of SEQ ID NO: 231, or 5) the Fd fragment of SEQ ID NO: 227, and the light chain of SEQ ID NO: 232, or 6) the Fd fragment of SEQ ID NO: 227, and the light chain of SEQ ID NO: 233, or 7) the Fd fragment of SEQ ID NO: 228, and the light chain of SEQ ID NO: 231, or 8) the Fd fragment of SEQ ID NO: 229. NO: 228, and the light chain as described in SEQ ID NO: 232, or 9) the Fd fragment as described in SEQ ID NO: 228, and the light chain as described in SEQ ID NO: 233, or 10) the Fd fragment as described in SEQ ID NO: 229, and the light chain as described in SEQ ID NO: 231, or 11) the Fd fragment as described in SEQ ID NO: 229, and the light chain as described in SEQ ID NO: 232, or 12) the Fd fragment as described in SEQ ID NO: 229, and the light chain as described in SEQ ID NO: 233, or 13) the Fd fragment as described in SEQ ID NO: 230, and the light chain as described in SEQ ID NO: 231, or 14) the Fd fragment as described in SEQ ID NO: 230, and the light chain as described in SEQ ID NO: 232, or 15) the Fd fragment as described in SEQ ID NO: 230, and the light chain as described in SEQ ID NO: 233.

[0033] In some embodiments, the antibody or antigen-binding fragment has a specific affinity for human 5T4 of about 1×10 -9 M is about 1×10 -8 For example, the antibodies or antigen-binding fragments provided herein have a binding affinity of at least about 1×10 -9 M, at least about 2×10 -9 M, at least about 3×10 -9 M, at least about 4×10 -9 M, at least about 5×10 -9M, at least about 6×10 -9 M, at least about 7×10 -9 M, at least about 8×10 -9 M, at least about 9×10 -9 M, or at least about 1×10 -8 M, and can specifically bind to the human 5T4.

[0034] In another aspect, the present application provides a fusion protein comprising the antibody or antigen-binding fragment described herein and at least one heterologous polypeptide fragment. For example, the heterologous polypeptide fragment may refer to: a polypeptide derived from a different entity than the antibody or antigen-binding fragment; a polypeptide not naturally produced by mammalian cells or a given host cell; or a polypeptide that is not endogenous to a given host cell. For example, the heterologous polypeptide fragment provided herein that can be fused to the antibody or antigen-binding fragment may be another antibody or antigen-binding fragment, an enzyme, a cytokine, a growth factor, a marker, or a combination thereof. For example, the heterologous polypeptide fragment is an immunoglobulin Fc polypeptide or a human serum albumin polypeptide.

[0035] In some embodiments, the fusion protein is a multispecific antibody comprising two or more antigen binding sites targeting human 5T4 and other antigenic epitopes. For example, the fusion protein is a bispecific antibody or bispecific antigen-binding fragment comprising a target for human 5T4 and an antigenic epitope or antigenic determinant other than human 5T4. For example, the fusion protein is a trispecific antibody or trispecific antigen-binding fragment comprising a target for human 5T4 and two antigenic epitopes or antigenic determinants other than human 5T4. The binding fragment of the bispecific antibody, bispecific antigen-binding fragment, trispecific antibody, or trispecific antigen-binding fragment capable of binding to 5T4 comprises an antigen-binding fragment provided by the present invention. In some embodiments, the antigenic epitope or antigenic determinant other than human 5T4 is derived from an immune cell surface molecule, a cell surface receptor molecule, and / or a cancer cell.

[0036] On the other hand, the present application provides a chimeric antigen receptor (CAR), which comprises an antibody or antigen binding fragment described herein of an extracellular region and an intracellular signaling domain. In some embodiments, the chimeric antigen receptor may further comprise a hinge region and / or a transmembrane region of the extracellular region.

[0037] In another aspect, the present application provides a conjugate comprising the antibody or antigen-binding fragment described herein and at least one effector. The conjugate further comprises a linker molecule capable of directly or indirectly conjugating the antibody or antigen-binding fragment to the effector. For example, the linker molecule is adapted to cause the conjugate to cleave upon entry into a cell, or to cause separation of the antibody or antigen-binding fragment from the effector.

[0038] In some embodiments, the effector is a detectable label. In some embodiments, the detectable label is a fluorescent marker, a radionuclide, avidin, biotin, and / or an enzyme. In other embodiments, the effector is a therapeutic drug. For example, the effector is a cytotoxic molecule; it can also be one or more of a radioisotope, a chemical drug, an immunomodulatory agent, an anti-angiogenic agent, an antiproliferative agent, a pro-apoptotic agent, a cytostatic agent, a lytic enzyme, and / or a therapeutic nucleic acid molecule. For example, the cytotoxin is an antibiotic.

[0039] In another aspect, the present application provides a nucleic acid encoding the antibody or antigen-binding fragment described herein. For example, the nucleic acid comprises DNA and / or mRNA. For example, the nucleic acid is a therapeutic nucleic acid.

[0040] In some embodiments, the nucleic acid is a DNA comprising a nucleotide sequence encoding a heavy chain variable region, wherein the nucleotide sequence encoding the heavy chain variable region (VH) comprises the nucleotide sequence shown in any one of SEQ ID NOs: 32-47, 71-75, and 242. In some embodiments, the nucleic acid is a DNA comprising a nucleotide sequence encoding a light chain variable region (VL), wherein the nucleotide sequence encoding the light chain variable region comprises the nucleotide sequence shown in any one of SEQ ID NOs: 48-62, 76-78, and 243.

[0041] Specifically, the nucleic acid is a DNA comprising: 1) a nucleotide sequence encoding VH as shown in SEQ ID NO:32 and a nucleotide sequence encoding VL as shown in SEQ ID NO:48, or 2) a nucleotide sequence encoding VH as shown in SEQ ID NO:33 and a nucleotide sequence encoding VL as shown in SEQ ID NO:49, or 3) a nucleotide sequence encoding VH as shown in SEQ ID NO:34 and a nucleotide sequence encoding VL as shown in SEQ ID NO:50, or 4) a nucleotide sequence encoding VH as shown in SEQ ID NO:35 and a nucleotide sequence encoding VL as shown in SEQ ID NO:48, or 5) a nucleotide sequence encoding VH as shown in SEQ ID NO:36 and a nucleotide sequence encoding VL as shown in SEQ ID NO:51, or 6) a nucleotide sequence encoding VH as shown in SEQ ID NO:37 and a nucleotide sequence encoding VL as shown in SEQ ID NO:52, or 7) a nucleotide sequence encoding VH as shown in SEQ ID NO:38 and a nucleotide sequence encoding VL as shown in SEQ ID NO:53, or 8) a nucleotide sequence encoding VH as shown in SEQ ID NO:39 and a nucleotide sequence encoding VL as shown in SEQ ID NO:44. 9) the nucleotide sequence encoding VH as set forth in SEQ ID NO:39 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:54, or 9) the nucleotide sequence encoding VH as set forth in SEQ ID NO:40 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:55, or 10) the nucleotide sequence encoding VH as set forth in SEQ ID NO:41 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:56, or 11) the nucleotide sequence encoding VH as set forth in SEQ ID NO:42 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:57, or 12) the nucleotide sequence encoding VH as set forth in SEQ ID NO:43 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:58, or 13) the nucleotide sequence encoding VH as set forth in SEQ ID NO:44 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:59, or 14) the nucleotide sequence encoding VH as set forth in SEQ ID NO:45 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:60, or 15) the nucleotide sequence encoding VH as set forth in SEQ ID NO:46 16) the nucleotide sequence encoding VH as shown in SEQ ID NO:46 and the nucleotide sequence encoding VL as shown in SEQ ID NO:61, or 17) the nucleotide sequence encoding VH as shown in SEQ ID NO:47 and the nucleotide sequence encoding VL as shown in SEQ ID NO:62,or 17) a nucleotide sequence encoding VH as shown in SEQ ID NO: 242 and a nucleotide sequence encoding VL as shown in SEQ ID NO: 243.

[0042] More preferably, the nucleic acid is a DNA comprising: 1) a nucleotide sequence encoding VH as shown in SEQ ID NO: 71 and a nucleotide sequence encoding VL as shown in SEQ ID NO: 76, or 2) a nucleotide sequence encoding VH as shown in SEQ ID NO: 71 and a nucleotide sequence encoding VL as shown in SEQ ID NO: 77, or 3) a nucleotide sequence encoding VH as shown in SEQ ID NO: 71 and a nucleotide sequence encoding VL as shown in SEQ ID NO: 78, or 4) a nucleotide sequence encoding VH as shown in SEQ ID NO: 72 and a nucleotide sequence encoding VL as shown in SEQ ID NO: 76, or 5) a nucleotide sequence encoding VH as shown in SEQ ID NO: 72 and a nucleotide sequence encoding VL as shown in SEQ ID NO: 77, or 6) a nucleotide sequence encoding VH as shown in SEQ ID NO: 72 and a nucleotide sequence encoding VL as shown in SEQ ID NO: 78, or 7) a nucleotide sequence encoding VH as shown in SEQ ID NO: 73 and a nucleotide sequence encoding VL as shown in SEQ ID NO: NO:76, or 8) the nucleotide sequence encoding VH as set forth in SEQ ID NO:73 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:77, or 9) the nucleotide sequence encoding VH as set forth in SEQ ID NO:73 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:78, or 10) the nucleotide sequence encoding VH as set forth in SEQ ID NO:74 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:76, or 11) the nucleotide sequence encoding VH as set forth in SEQ ID NO:74 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:77, or 12) the nucleotide sequence encoding VH as set forth in SEQ ID NO:74 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:78, or 13) the nucleotide sequence encoding VH as set forth in SEQ ID NO:75 and the nucleotide sequence encoding VL as set forth in SEQ ID NO:76, or 14) the nucleotide sequence encoding VH as set forth in SEQ ID NO:75 and the nucleotide sequence encoding VL as set forth in SEQ ID NO: 15) the nucleotide sequence encoding VH as shown in SEQ ID NO:75 and the nucleotide sequence encoding VL as shown in SEQ ID NO:78.

[0043] In some embodiments, the nucleic acid is a DNA comprising a nucleotide sequence encoding a heavy chain variable region and a heavy chain constant region 1 (Fd fragment), wherein the nucleotide sequence encoding the Fd fragment comprises the nucleotide sequence shown in any one of SEQ ID NOs: 193-209 and 234-238. In some embodiments, the nucleic acid is a DNA comprising a nucleotide sequence encoding a light chain, wherein the nucleotide sequence encoding the light chain comprises the nucleotide sequence shown in any one of SEQ ID NOs: 210-225, 239-241 and 244.

[0044] Specifically, the nucleic acid is a DNA comprising: 1) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 193 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 210, or 2) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 194 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 211, or 3) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 195 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 212, or 4) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 196 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 244, or 5) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 197 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 213, or 6) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 198 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 214, or 7) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 199 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 210. NO:199, and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:215; or 8) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:200 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:216; or 9) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:201 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:217; or 10) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:202 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:218; or 11) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:203 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:219; or 12) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:204 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:220; or 13) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:205 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO: 14) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 206 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO: 222, or 15) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 207 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO: 223,or 16) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 208 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO: 224, or 17) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 209 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO: 225.

[0045] More preferably, the nucleic acid is a DNA comprising: 1) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 234 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 239, or 2) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 234 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 240, or 3) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 234 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 241, or 4) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 235 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 239, or 5) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 235 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 240, or 6) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 235 and a nucleotide sequence encoding the light chain as shown in SEQ ID NO: 241, or 7) a nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO: 236. NO:236 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:239, or 8) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:236 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:240, or 9) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:236 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:241, or 10) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:237 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:239, or 11) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:237 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:240, or 12) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:237 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:241, or 13) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:238 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO: NO:239, or 14) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:238 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:240, or 15) the nucleotide sequence encoding the Fd fragment as shown in SEQ ID NO:238 and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:241.

[0046] In some embodiments, the nucleic acid is mRNA; one or more modification techniques can be used to produce more stable mRNA. Known mRNA modification techniques can be roughly divided into three categories: using artificially synthesized non-natural RNA to replace natural RNA to synthesize mRNA; adding 5'caps, 3'poly (A) "tails" and UTR (untranslated region) sequences; and using special new formulation technologies to effectively protect mRNA. Among them, the preferred mRNA modification technology is to synthesize mRNA by replacing natural RNA with artificially synthesized non-natural RNA. Chemical modifications on eukaryotic mRNA can be roughly divided into three categories: methylation, pseudouridine (Ψ), and hypoxanthine. For example, the chemical modification can be selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0047] On the other hand, the present application provides a recombinant vector comprising a nucleic acid as described herein. Specifically, the recombinant vector may refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. The recombinant vector may include single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, without free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA or both; and other types of polynucleotides known in the art. For example, a viral vector can be used. The viral vector may comprise a virally derived DNA or RNA sequence for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus AAV). Viruses and viral vectors can be used for in vitro, ex vivo and / or in vivo delivery. For example, the recombinant vector may be contained in a delivery vector. In certain embodiments, the delivery vector comprises an antibody or antigen-binding fragment as described herein, a nucleic acid as described herein, and / or a recombinant vector as described herein, and optionally comprises liposomes and / or lipid nanoparticles (LNPs). For example, the delivery vector can be introduced into a cell by a physical delivery method. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. In some embodiments, LNPs can be wrapped in cationic lipid particles (liposomes) for nucleic acid described herein, and can be delivered to cells relatively easily. In some instances, lipid nanoparticles do not contain any viral components, and this helps to reduce safety and immunogenicity problems to greatest extent. Lipid granules can be used for external, ex vivo and in vivo delivery. For example, the composition of LNPs can comprise cationic lipids, ionizable lipids, pegylated lipids and / or support lipids, and optional cholesterol components.

[0048] On the other hand, the present application provides a host cell comprising the recombinant vector described in the present application and / or the delivery vector described in the present application.

[0049] On the other hand, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment described herein, the multispecific antibody or fusion protein described herein, the conjugate described herein, the chimeric antigen receptor described herein, the nucleic acid described herein, the recombinant vector described herein, or the host cell described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0050] On the other hand, the present application provides the use of the antibody or antigen-binding fragment described herein, the multispecific antibody or fusion protein described herein, the conjugate described herein, the chimeric antigen receptor described herein, the nucleic acid described herein, the recombinant vector described herein, the host cell described herein, or the pharmaceutical composition described herein in the preparation of a medicament for treating 5T4-positive tumor-related diseases.

[0051] On the other hand, the present application provides use of the antibody or antigen-binding fragment described herein or the conjugate described herein in the preparation of a detection kit or a detection reagent, wherein the detection kit or the detection reagent is used to diagnose diseases associated with 5T4-positive tumors.

[0052] On the other hand, the present application provides a method for treating 5T4-positive tumor-related diseases, comprising administering to a subject in need thereof an effective therapeutic dose of the antibody or antigen-binding fragment described herein, the multispecific antibody or fusion protein described herein, the conjugate described herein, the chimeric antigen receptor described herein, the nucleic acid described herein, the recombinant vector described herein, the host cell described herein, or the pharmaceutical composition described herein.

[0053] On the other hand, the present application provides a method for diagnosing a 5T4-positive tumor-related disease, the method comprising providing a detection kit or detection reagent, the detection kit or detection reagent comprising the antibody or antigen-binding protein described herein and / or the conjugate described herein. In some embodiments, the method further comprises providing a system or instrument for using the detection kit or detection reagent. For example, the system or instrument can be a system or instrument suitable for detecting a detectable marker by spectroscopic means, photochemical means, biochemical means, immunochemical means, electrical means, optical means, or chemical means.

[0054] In some embodiments, the 5T4-positive tumor is a solid tumor. For example, the disease associated with a 5T4-positive tumor is cancer. For example, the 5T4-positive tumor and its associated diseases include: lung cancer, breast cancer, gastric cancer, endometrial cancer, colon cancer, ovarian cancer, oral cancer, prostate cancer, and / or kidney cancer.

[0055] The antibodies or antigen-binding fragments provided in this application have at least the following advantages: 1) they exhibit excellent specific binding activity against tumor cells that highly express 5T4; 2) the humanized antibodies have a degree of humanization exceeding 98% and possess good thermal stability and binding activity to human 5T4.

[0056] The antibodies or antigen-binding fragments thereof provided herein can have different uses.

[0057] In some embodiments, the antibody or its antigen-binding fragment has low endocytic activity, and the antibody or its antigen-binding fragment can be part of a T cell engager (TCE), a blocking antibody, a diagnostic reagent, or an adoptive cell (e.g., CAR-T, CAR-NK). In other embodiments, the antibody or its antigen-binding fragment has high endocytic activity, and the antibody or its antigen-binding fragment can be part of an antibody drug conjugate (ADC) or a receptor degrader. The endocytic activity specifically refers to the degree to which the antibody or its antigen-binding fragment is endocytosed by target cells. The endocytic activity can be characterized by incubating the antibody or its antigen-binding fragment with target cells and detecting the content of the free antibody or its antigen-binding fragment in the detection system.

[0058] More preferably, the endocytic activity of the antibody or antigen-binding fragment thereof can be compared with the endocytic effect using a control antibody to determine whether the endocytic activity is high or low. The control antibody can be an antibody that is widely used in the art and a product containing an antibody (e.g., ADC, TCE, CAR-T). Different control antibodies can be selected in different comparison scenarios; for example, when it is necessary to compare whether the endocytic activity of the antibody or antigen-binding fragment thereof is high, an ADC or receptor degrader that is widely used in the art is selected as a control antibody. If the endocytic activity of the antibody or antigen-binding fragment thereof is higher than that of the control antibody, it is determined that the endocytic activity is high; for another example, when it is necessary to compare whether the endocytic activity of the antibody or antigen-binding fragment thereof is low, a TCE, blocking antibody, diagnostic reagent or adoptive cell that is widely used in the art is selected as a control antibody. If the endocytic activity of the antibody or antigen-binding fragment thereof is lower than that of the control antibody, it is determined that the endocytic activity is low.

[0059] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0061] Figures 1A-1B show the binding activity curves of the antibody molecules of the present application on PANC-1 cells that highly express human 5T4.

[0062] Figures 2A-2B show the binding activity curves of the antibody molecules of the present application on T47D cells that highly express human 5T4.

[0063] Figures 3A-3B show the binding activity curves of the antibody molecules of the present application on A375 cells that highly express human 5T4.

[0064] Figures 4A-4B show the binding activity curves of the antibody molecules of the present application on PC-3 cells that highly express human 5T4.

[0065] Figures 5A-5B show the binding ability of the humanized antibody molecules of the present application to HEK293 cells overexpressing human 5T4. DETAILED DESCRIPTION

[0066] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0067] Definition of terms

[0068] In this application, the term "antibody" refers in particular to a protein comprising at least two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain constant region comprises three or (in the case of IgM- or IgE-type antibodies) four heavy chain constant domains (CH1, CH2, CH3 and CH4), wherein the first constant domain CH1 is adjacent to the variable region and can be connected to the second constant domain CH2 by a hinge region. The light chain constant region consists of only one constant domain. The variable region can be further subdivided into hypervariable regions (called complementary determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)), wherein each variable region comprises three CDRs and four FRs. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The heavy chain constant region can be of any type, such as a γ-, δ-, α-, μ- or ε-type heavy chain. Preferably, the heavy chain of the antibody is a γ chain. In addition, the light chain constant region can also be of any type, such as a κ- or λ-type light chain. Preferably, the light chain of the antibody is a κ chain. The terms "γ- (δ-, α-, μ- or ε-) type heavy chain" and "κ- (λ-) type light chain" refer to an antibody heavy chain or antibody light chain having an amino acid sequence of a heavy chain or light chain constant region derived from a naturally occurring, especially a human heavy chain or light chain constant region amino acid sequence. In particular, the amino acid sequence of the constant domain of a γ-type (especially γ1-type) heavy chain is at least 95%, in particular at least 98%, identical to the amino acid sequence of the constant domain of a human γ (especially one of the allotypes of human γ1) antibody heavy chain. In addition, the amino acid sequence of the κ-type light chain constant domain is in particular at least 95%, in particular at least 98% identical to the amino acid sequence of the constant domain of one of the allotypes of a human κ antibody light chain. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system (e.g., C1q, the first component of the classical activation pathway of the complement system). The antibodies can be, for example, humanized, human, or chimeric antibodies.

[0069] The antigen-binding portion of an antibody (also referred to herein as an "antigen-binding fragment") generally refers to the full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody binding fragments include a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; a F(ab)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region, each Fab fragment binding to the same antigen; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and a dAb fragment consisting of the VH domain.

[0070] In this application, the term "scFv" generally refers to a single chain variable region fragment (single chain fragment variable), sometimes also referred to as a single chain antibody, which is a fusion protein composed of the heavy chain variable region (VH) and light chain variable region (VL) of an antibody. In scFv, the connection order of VH and VL can be swapped, and VH can be placed at the N-terminus or VL can be placed at the N-terminus. VH and VL can be connected by a "linker" sequence. Single chain variable region fragments can also include heavy chain variable region fragments (Single variable domain on a heavy chain (VHH)), or nano antibodies for short. Due to the lack of light chains, nano antibodies only have 3 antigen recognition regions (CDR regions) belonging to the heavy chain. The molecular weight is about one-tenth of that of traditional antibodies and about one-half of that of antigen binding fragments (scFv, VH-VL).

[0071] In this application, the term "homology" or "identity" is generally used to describe the degree of similarity between two nucleotide sequences or two amino acid sequences, and has the same meaning as "percent identity". Preferably, it is determined according to the present invention over the entire length of a reference sequence or over the entire length of the corresponding portion of the reference sequence (which corresponds to the sequence defining homology or identity). Antibodies derived from parent antibodies (which are defined by one or more amino acid sequences (e.g., specific CDR sequences or specific variable region sequences)) are particularly antibodies having amino acid sequences, such as CDR sequences or variable region sequences, that are at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous or identical, especially identical, to each amino acid sequence of the parent antibody. In certain embodiments, the antibody derived from the parent antibody (i.e., its derivative) comprises the same CDR sequences as the parent antibody, but differs in the remaining sequences of the variable region. The percent homology of two sequences can be calculated as follows: after aligning the two sequences, the number of positions where the residues are identical is divided by the total length of the aligned sequences and multiplied by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, for example, the BLAST suite available on the NCBI website (Altschul, SF et al. (1990) J. Mol. Biol. 215:403-410).

[0072] In this application, the term "humanized antibody" generally refers to an antibody from a non-human species, which has one or more complementary determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. Conventionally, the major part of a humanized antibody is a human immunoglobulin (acceptor antibody), and contains a small amount of sequences derived from a non-human immunoglobulin, wherein the hypervariable region or complementary determining region (CDR) residues of the acceptor (antibody) are replaced with non-human species, such as hypervariable region residues or CDR residues with desired specificity, affinity and binding ability in the antibody (donor antibody) of mouse, rat, rabbit or non-human primate. In some cases, one or more framework region (FR) residues of a human immunoglobulin can be replaced with corresponding non-human residues or other residues according to a structural model, for example, to improve the affinity of the humanized antibody. In addition, a humanized antibody may contain residues that are not present in the acceptor antibody or the donor antibody. Carrying out these modifications can further optimize antibody performance. Humanized antibodies typically contain substantially all (residues) of at least one, typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are human immunoglobulin sequences. Humanized antibodies also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. For further details, see Neuberger, MS et al., 1985, Nature, 314: 268-270; Jones et al., 1986, Nature, 321: 522-525; Reichmann et al., 1988, Nature, 332: 323-329; Presta, 1992, Curr. Op. Struct. Biol., 2: 593-596; Queen et al., U.S. Pat. No. 5,585,089.

[0073] In this application, the term "chimeric antibody" generally refers to a monoclonal antibody whose variable regions (i.e., binding regions) are of the same origin or species, while at least a portion of its constant regions are of different origin or species. Such chimeric monoclonal antibodies are typically prepared using recombinant DNA technology. Chimeric antibodies composed of a murine variable region and a human constant region can be used in the present application to treat human diseases because such antibodies are easy to prepare and have less immunogenicity than pure murine monoclonal antibodies. Such murine / human chimeric antibodies are the expression products of immunoglobulin genes composed of a DNA segment encoding a murine immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Other forms of chimeric antibodies encompassed by the present invention are antibodies whose class or subclass has been modified or altered to be different from the original. Such "chimeric" antibodies are also referred to as "class-sandwich antibodies." Methods for preparing chimeric antibodies include conventional DNA recombination and gene transfection techniques currently well known in the art. See, eg, Morrison, SL et al., 1984, Proc. Nat'l Acad Sci., 81:6851.

[0074] In this application, the term "binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). In most cases, "specific binding" of an antibody to an antigen means that the antibody binds more strongly to its target, e.g., an epitope, for which it is specific, than to another target. The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K d ) is used to represent the dissociation constant (K d) is lower than the dissociation constant of the second target Y2, then its binding to the first target Y1 is stronger than the second target Y2. Affinity can be measured by common methods known in the art, including but not limited to surface plasmon resonance-based assays (such as the BIAcore assay described in PCT Application Publication No. WO2005 / 012359); enzyme-linked immunosorbent assay (ELISA); and competition assays (such as RIA). Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, all of which can be used for the purposes of the present invention. Preferably, the dissociation constant of the target to which the antibody specifically binds is more than 100 times, 200 times, 500 times or more than 1000 times lower than the dissociation constant of the target to which the antibody does not specifically bind; alternatively, in certain embodiments, the antibody or antigen-binding fragment that specifically binds to the target has the following dissociation constant (K d ): ≦1μM, ≦100nM, ≦10nM, ≦1nM or ≦0.1nM. In addition, the dissociation constant K d The reciprocal affinity constant K a It can also reflect binding affinity, which specifically refers to the degree of firmness with which an antibody and an antigen bind. Thus, "specific binding" of an antibody to an antigen can also refer to a binding affinity between the binding partners of at least 10 6 M -1 , preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 The affinity constant K a Antibodies that are specific for an antigen are specifically antibodies that can 6 M - 1 , preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 K a An antibody that binds to the antigen with an affinity.

[0075] In this application, the term "epitope" generally refers to any polypeptide determinant that is capable of specifically binding to a target antigen. In certain embodiments, epitope determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope can be an antigen or a region of an antigen to which a binding protein binds. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0076] In this application, the term "fusion protein" generally refers to a protein that contains polypeptide components derived from more than one parent protein or polypeptide. These proteins are composed of two or more polypeptides, which, although not usually bound in their native state, are bound together by peptide bonds at their respective amino and carboxyl termini to form a continuous polypeptide. It should be understood that the two or more polypeptide components can be directly bound or indirectly bound via a peptide linker / spacer. Fusion proteins can also generally be expressed by a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is attached in frame to a nucleotide sequence encoding a polypeptide sequence from a different protein, and optionally separated from it by a linker. The fusion gene can then be expressed as a single protein by a recombinant host cell.

[0077] In this application, the term "multispecific antibody" generally refers to an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL units have polyepitopic specificity (i.e., they can bind to two different epitopes on one biological molecule or each epitope on different biological molecules). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, antibody fragments such as Fab, Fv, dsFv, scFv, bivalent antibodies (diabodies), bispecific diabodies and trivalent antibodies (triabodies), and antibody fragments that have been covalently or non-covalently linked. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. "Monospecific" refers to the ability to bind to only one epitope.

[0078] In this application, the term "chimeric antigen receptor (CAR)" generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane domain, and an intracellular T cell receptor-activation signal domain that specifically binds to an antigen or target. The extracellular domain of CAR binds to the target antigen on the surface of the target cell, resulting in CAR clustering and transmitting the activation stimulus to the CAR-containing cell. CAR has the specificity of redirecting immune effector cells and triggers proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of cells expressing target antigens in a manner independent of the major histocompatibility complex (MHC).

[0079] In this application, the term "detectable label" generally refers to a portion that is attached to a polypeptide (such as an antibody), glycoprotein, or analyte so that a reaction thereof can be detected, and the polypeptide, glycoprotein, or analyte so labeled is referred to as "detectably labeled". Various methods of labeling polypeptides and glycoproteins are known in the art and can be applied to the schemes of the present application. Examples of labels for polypeptides include (but are not limited to) the following: radioisotopes or radionuclides (e.g., indium ( 111 In), iodine ( 131 I or 125 I), yttrium ( 90 Y), Lutetium ( 177 Lu), actinium ( 225 Ac), bismuth ( 212 Bior 213 Bi), sulfur ( 35 S), carbon ( 14 C) tritium ( 3 H), rhodium ( 188 Rh), technetium ( 99 mTc), praseodymium or phosphorus ( 32 P) or positron-emitting radionuclides, such as carbon-11 ( 11 C), potassium-40 ( 40 K), nitrogen-13 ( 13 N), oxygen-15 ( 15 O), fluorine-18 ( 18 F) and iodine-121( 121 I)), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups (which can be detected by labeled avidin (e.g., a molecule containing a streptavidin portion) and a fluorescent label or an enzymatic activity detectable optically or calorimetrically), and a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag).

[0080] As used herein, the term "cytotoxin" generally refers to any agent that is detrimental to cell growth and proliferation and that acts to reduce, inhibit or destroy cells or malignancies.

[0081] In this application, the term "nucleic acid" may include single-stranded and double-stranded nucleic acids and ribonucleic acids and deoxyribonucleic acids. It may include naturally occurring and synthetic nucleotides, and may be naturally or synthetically modified, for example, by methylation, 5'- and / or 3'-capping. In this application, the term "nucleic acid encoding ..." or "(nucleotide) sequence encoding ..." generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may also include start and stop signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized.

[0082] In this application, the term "recombinant vector" generally refers to a genetically modified oligonucleotide or polynucleotide construct. When the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and when the vector is contacted with a host cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the host cell, the genetically modified oligonucleotide or polynucleotide construct allows the host cell to express the mRNA, protein, polypeptide, or peptide, and is therefore also referred to as a "recombinant expression vector." The recombinant vectors of the present application are not entirely naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present application may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, may be synthetic or partially obtained from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may comprise naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector. Recombinant expression vectors are well known and readily available. Examples of recombinant expression vectors include plasmids, phage, viral vectors, and other nucleic acid molecules or vehicles containing nucleic acid molecules used to transform host cells and promote the expression of coding sequences.

[0083] In the present application, the terms "cell" and "host cell" are used interchangeably, and generally refer to cells containing exogenous recombinant DNA such as expression vectors, and also refer to cells containing polypeptides or proteins obtained by recombinant DNA expression. Such host cells can be used to produce antibodies for the present application. In certain embodiments, as a host cell, there are no particular restrictions as long as it is a host cell that can be used for the expression of polypeptide sequences such as antibodies for the present application. Recombinant host cells or host cells refer not only to specific subject cells, but also to the offspring of such cells. Because some modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from the parental cells, but such cells are still included in the scope of the terms "cell" or "host cell" used in the present application.

[0084] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier for administering therapeutic agents, such as antibodies or polypeptides, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and that can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may also be present in these carriers.

[0085] In this application, the terms "effective therapeutic dose" and "effective amount" are used interchangeably, and generally refer to an amount or dosage sufficient to produce a desired therapeutic outcome when a pharmaceutical composition comprising one or more antibodies or antigen-binding proteins is administered. More specifically, a therapeutically effective amount is sufficient to treat a specified condition, illness or disease over a period of time, and specifically may refer to an amount of a drug (e.g., an antibody or antigen-binding fragment provided herein) or a pharmaceutical composition thereof that improves, alleviates, mitigates and / or delays one or more of its symptoms. In some examples for cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce tumor growth rate (e.g., inhibit tumor growth) or prevent or delay other cells that do not wish to proliferate. In some embodiments, an effective amount is an amount sufficient to delay the development of a tumor or cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay the recurrence of a tumor or cancer. An effective amount can be administered in one or more administrations. In some embodiments, an effective amount of a drug or its pharmaceutical composition can: (1) reduce the number of cancer cells; (2) reduce the size of a tumor; (3) inhibit, delay, slow down, and preferably prevent, to some extent, the infiltration of cancer cells into surrounding organs; (4) inhibit (i.e., slow down or stop to some extent) tumor metastasis; (5) inhibit tumor growth; (6) prevent or delay the occurrence and / or recurrence of tumors; (7) alleviate to some extent one or more symptoms associated with cancer. The effective amount can vary depending on the specific antibody used and also depends on a variety of factors related to the patient being treated and the severity of the condition. For example, if an antibody drug is to be administered in vivo, factors such as the patient's age, weight, and health, as well as dose-response curves and toxicity data obtained in preclinical animal work, would be among the factors considered. Determining the effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of those skilled in the art.

[0086] In this application, the term "diagnosis" generally refers to the detection of a disease or condition, or the determination of the state or degree of a disease or condition. The term "diagnosis" also includes detecting the cause of a disease or condition, determining the therapeutic effect of a drug treatment, or predicting a response pattern to a drug treatment or a xenobiotic. The diagnostic methods described herein can be used independently or in combination with other diagnostic and / or staging methods for a specific disease or condition known in the medical field. As used herein, antibodies for diagnosing a disease can be bound to a target that is specifically associated with a specific disease or shows increased expression in the disease; can be used in, for example, an assay system (e.g., ELISA); can also be used, for example, to detect a target in a biological sample from a patient or for diagnostic imaging of a disease site (e.g., a tumor) in a patient. A "biologically functional fragment" of a diagnostic antibody will exhibit at least one (if not some or all) biological function possessed by a complete antibody, comprising at least specific binding to a target antigen.

[0087] In this application, the term "treatment" generally refers to therapeutic treatment and prophylactic or preventive measures. Patients in need of treatment include patients with a condition as well as patients susceptible to the condition or patients for whom the condition is to be prevented. When the term "treatment" means that the antibodies or antigen-binding fragments provided herein are used to treat tumors or cancer, the achievable or desired effects may include, but are not limited to: depletion of target cells, binding to and neutralizing the normal functions of target cells, blocking the activity of proteins required for target cell survival and causing the death of the target cells, binding to proteins on target cells and triggering their apoptotic signals, mediating ADCC (antibody-dependent cell-mediated cytotoxicity) or mediating CDC (complement-dependent cytotoxicity); the target cells may be tumor cells, cancer cells or virus-infected cells.

[0088] In this application, the term "tumor" generally refers to a group of cells or tissues formed by misregulated cell proliferation. Tumors may show partial or complete lack of structural organization and functional coordination with normal tissues and usually form a distinct tissue mass, which can be benign or malignant.

[0089] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0090] In this application, the term "selected from" generally refers to the selected objects and all combinations thereof. For example, "selected from A, B and C" means all combinations of A, B and C, for example, A, B, C, A+B, A+C, B+C, or A+B+C.

[0091] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0092] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antibodies or antigen-binding fragments, preparation methods, and uses of the present invention, and are not intended to limit the scope of the present invention.

[0093] Example

[0094] Example 1

[0095] Screening of 5T4 antibodies from mouse immune antibody library

[0096] In this example, a mouse immune library was constructed by immunizing mice with target antigens to obtain mouse spleen tissue, and the constructed mouse immune library was screened using an immune tube and a magnetic bead screener in a solid-phase and liquid-phase cross-screening manner.

[0097] 1. Construction of Mouse Immune Library

[0098] Specifically, the immunization antigens used were human or monkey 5T4 antigens with different tags, including hu5T4-Fc, hu5T4-His, Cyno5T4-His, and Cyno5T4-Fc proteins, and mice were immunized in a single or mixed manner. The first immunization antigen was emulsified with Freund's complete adjuvant, with an antigen amount of 100 μg / mouse. The subsequent immunization adjuvant was Freund's incomplete adjuvant, with an antigen amount of 50 μg / mouse. The injection method was intraperitoneal and subcutaneous multiple-point injection. The serum of the immunized mice was taken, and the serum titer was detected by ELISA. The RNA of the mouse spleen tissue was extracted, and the cDNA was obtained by reverse transcription according to the method provided by the TaKaRa reverse transcription kit, and primers were designed to amplify the antibody light and heavy chain variable region genes. The light and heavy chain variable region gene amplification products were randomly identified by agarose gel electrophoresis and recovered by gel cutting. The obtained antibody light and heavy chain variable region gene amplification products were used as templates for adapter amplification, light and heavy chain ligation amplification, and antibody light and heavy chain ligation product (Fab full-length sequence). The Fab full-length sequence was recovered for enzyme digestion reaction identification. Referring to the method provided by the ligase kit, combined with the target library capacity, the Fab full-length sequence and the phage display vector PMID-1121B were digested, recovered, and ligated. The ligation product was recovered and finally transformed into competent Escherichia coli SS320 by electroporation. The transformed E. coli SS320 bacterial solution was inoculated and cultured to the logarithmic growth phase. VSCM13 was added to assist phage culture. The phage was preserved and used for subsequent phage screening.

[0099] 2. Mouse immune library screening

[0100] The obtained phage library was screened for antibodies using the Kingfisher method and the immunotube method respectively:

[0101] 1) Mouse immune library screening (Kingfisher method)

[0102] The prepared mouse immune library phage suspension was diluted and blocked with a final concentration of 2.5% BSA, co-incubated with Dynabeads, and the phages after negative screening were collected; the magnetic beads were bound and washed according to the Kingfisher magnetic bead screening system method, and 2.5% BSA was incubated with the magnetic beads; the phage suspension collected after negative screening was incubated with Dynabeads coated with biotin-labeled antigen and blocked, and then bound and washed; the phages were eluted with Trypsin; the eluted phage solution was thoroughly mixed with logarithmic phase SS320 cells and incubated at 37°C for 30 minutes, spread on 2YT-Car+-Tet+ plates, and cultured in a 37°C incubator overnight.

[0103] Calculate the phage input, output, etc., scrape the input phage and perform three rounds of screening. Each round of screening uses three gradients of antigen concentration to screen for antibody clones with higher affinity.

[0104] 2) Mouse immune library screening (immunotube method)

[0105] The prepared mouse immune library phage suspension was blocked with 5% PBSM; the diluted antigen was added to the immunotube, coated overnight at 4°C, then washed according to the immunotube screening system method and blocked with 5% PBSM; the phage suspension was incubated with the protein-coated and blocked immunotube, and combined and washed according to the immunotube screening system method; the phage was eluted with Trypsin; the eluted phage solution was fully mixed with logarithmic phase SS320 cells and incubated at 37°C for 30 minutes, spread on 2YT-Car+-Tet+ plates, and cultured in a 37°C incubator overnight.

[0106] The phage input and output were calculated, the input phage was prepared by scraping and three rounds of screening were performed. Each round of screening used a 3-fold gradient of decreasing antigen concentration to obtain antibody clones with higher affinity.

[0107] The expression supernatant from the selected pool from three rounds of screening was diluted fivefold using PBSM and affinity tested by ELISA to identify a well-enriched output pool. Phage monoclonal supernatant was centrifuged and subjected to a primary screening ELISA. A total of 672 clones were selected, and 444 positive clones with human-monkey cross-reactivity were obtained, of which 176 were sequence-unique molecules. Based on sequence diversity analysis and initial affinity screening, 113 molecules were selected for full-length construction.

[0108] Clones that bind well to HEK293 cells overexpressing human 5T4 antigen (Hu5T4-FL-HEK293-A6) and HEK293 cells overexpressing monkey 5T4 antigen (cyno5T4-FL-HEK293-A7) were screened by FACS, and 16 molecules were selected for subsequent testing.

[0109] Example 2

[0110] In vitro evaluation of the binding activity of candidate molecules from the mouse library on tumor cells

[0111] In this example, the binding activity of the preferred anti-5T4 antibody on PANC-1, T47D, A375, and PC-3 cells that highly express the human 5T4 antigen was detected by FACS. HEK293 cells overexpressing the human 5T4 antigen (Hu5T4-FL-HEK293-A6) were used as positive controls, and Raji and Jurkat cells that do not express human 5T4 were used as negative controls.

[0112] The results are shown in Figures 1A-1B, 2A-2B, 3A-3B and 4A-4B. FACS analysis showed that A103, A159, A239, A258, A264 and A265 among the anti-5T4 antibody molecules had better binding to PANC-1 than the positive control ASN004 (P64631); A056, A103, A264 and A265 had better binding to T47D cells than the positive control ASN004 (P64631); A103, A159, A239, A258, A264 and A265 had better binding to T47D cells than the positive control ASN004 (P64631); 4. The binding of A159, A234, A239 and A265 on PC-3 cells was better than that of the positive control ASN004 (P64631); the binding of A134 and A265 on A375 cells was better than that of the positive control ASN004 (P64631); the binding specificity of A001, A024, A027, A028, A031, A234, A258, A264 and A265 on Raji cells was higher than that of the positive control ASN004 (P64631). (Positive control ASN004 reference: Smith, Roger A et al. "ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models." Molecular cancer therapeutics vol.20,8(2021):1327-1337.doi:10.1158 / 1535-7163.MCT-20-0565)

[0113] Example 3

[0114] Humanized mouse antibody design

[0115] In this example, the murine antibody A265 was humanized using CDR grafting. First, the parental sequence was modeled using homology modeling. The parental antibody V region sequence was then compared against a database query to identify the human germline with the highest similarity. After defining the parental antibody CDR and framework regions, sequences with varying degrees of humanization were designed based on the differences in the framework regions. The degree of humanization was calculated using the formula = (number of amino acids in the full-length sequence - number of amino acids in the backmutation) / number of amino acids in the full-length sequence × 100%.

[0116] The results are summarized in Table 1. A total of 15 humanized molecules were designed based on the parental A265-mVH-A265-mVL sequence. Through gene synthesis, plasmid construction, protein expression, purification, and characterization, humanized antibody proteins were obtained, demonstrating binding activity comparable to that of the parental antibody. ELISA binding activity results are shown in Table 2, and affinity kinetics results are shown in Table 3. Binding activity to HEK293 cells overexpressing the human 5T4 antigen (Hu5T4-FL-HEK293-A6) is shown in Figures 5A-5B.

[0117] The thermal stability of humanized antibodies was determined by differential scanning fluorimetry (DSF). m Specifically, SYPRO Orange dye is used. SYPRO Orange is an environmentally sensitive hydrophobic dye. When the temperature rises, the protein unfolds and the hydrophobic part is exposed. The dye specifically binds to the hydrophobic part of the protein, and the fluorescence is enhanced. m The higher the value, the more stable the protein. m The test results of the value are shown in Table 4. Among them, T m 1 is the first melting temperature caused by the melting of the CH2 domain of the monoclonal antibody, T m 2 is the second melting temperature caused by the unfolding of the CH3 domain and Fab (see the unfolding temperature of antibody molecules detected by micro differential scanning fluorimetry (nanoDSF) in the literature “High-Throughput Melting-Temperature Analysis of a Monoclonal Antibody by Differential Scanning Fluorimetry in the Presence of Surfactants”).

[0118] Table 1. Summary of humanized antibody design

[0119] Table 2. EC values ​​of humanized antibodies binding to human 5T4 antigen detected by ELISA 50 Results Summary Table

[0120] Table 3. Affinity kinetics test results of humanized antibody molecules

[0121] Table 4. Humanized antibody molecules T m Value Summary Table

[0122] The nucleotide and amino acid sequences of Fab involved in this application are as follows (the bold underlined ones are the constant region sequences), wherein the heavy chain is represented as the Fd fragment. If there are any differences between the sequences involved in this section and the full text of the application, this section shall prevail:

[0123] Nucleotide sequence

[0124] Light chain:

[0125] Heavy chain:

[0126] Light chain:

[0127] Heavy chain:

[0128] Light chain:

[0129] Heavy chain:

[0130] Light chain:

[0131] Heavy chain:

[0132] Amino acid sequence (double underline marks CDR divisions)

[0133] Light chain:

[0134] Heavy chain:

[0135] Light chain:

[0136] Heavy chain:

[0137] Light chain:

[0138] Heavy chain:

[0139] Light chain:

[0140] Heavy chain:

Claims

1. An antibody or antigen-binding fragment targeting human 5T4, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the following complementarity determining regions (CDRs): 1) HCDR1 as shown in SEQ ID NO: 88, HCDR2 as shown in SEQ ID NO: 101 and HCDR3 as shown in SEQ ID NO: 113, or 2) HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 108, or 3) HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 95 and HCDR3 as shown in SEQ ID NO: 114, or 4) HCDR1 as shown in SEQ ID NO: 82, HCDR2 as shown in SEQ ID NO: 91 and HCDR3 as shown in SEQ ID NO: 112, or 5) HCDR1 as shown in SEQ ID NO: 87, HCDR2 as shown in SEQ ID NO: 100, and HCDR3 as shown in SEQ ID NO: 111, or 6) HCDR1 as shown in SEQ ID NO: 80, HCDR2 as shown in SEQ ID NO: 93, and HCDR3 as shown in SEQ ID NO: 110, or 7) HCDR1 as shown in SEQ ID NO: 81, HCDR2 as shown in SEQ ID NO: 104, and HCDR3 as shown in SEQ ID NO: 109, or 8) HCDR1 as shown in SEQ ID NO: 86, HCDR2 as shown in SEQ ID NO: 97, and HCDR3 as shown in SEQ ID NO: 116, or 9) HCDR1 as shown in SEQ ID NO: 89, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 117, or 10) HCDR1 as shown in SEQ ID NO: 84, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 107, or 11) HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 94, and HCDR3 as shown in SEQ ID NO: 115, or 12) HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 114, or 13) HCDR1 as shown in SEQ ID NO:90, HCDR2 as shown in SEQ ID NO:102, and HCDR3 as shown in SEQ ID NO:106, or 14) HCDR1 as shown in SEQ ID NO: 83, HCDR2 as shown in SEQ ID NO: 92, and HCDR3 as shown in SEQ ID NO: 105, or 15) HCDR1 as shown in SEQ ID NO: 87, HCDR2 as shown in SEQ ID NO: 99, and HCDR3 as shown in SEQ ID NO: 111, or 16) HCDR1 as shown in SEQ ID NO: 79, HCDR2 as shown in SEQ ID NO: 103, and HCDR3 as shown in SEQ ID NO: 118, or 17) HCDR1 as shown in SEQ ID NO: 151, HCDR2 as shown in SEQ ID NO: 152 and HCDR3 as shown in SEQ ID NO:

153.

2. The antibody or antigen-binding fragment of claim 1, wherein the VL comprises the following complementarity determining regions (CDRs): 1) LCDR1 as shown in SEQ ID NO: 129, LCDR2 as shown in SEQ ID NO: 137, and LCDR3 as shown in SEQ ID NO: 139, or 2) LCDR1 as shown in SEQ ID NO: 126, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 141, or 3) LCDR1 as shown in SEQ ID NO: 125, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 142, or 4) LCDR1 as shown in SEQ ID NO: 120, LCDR2 as shown in SEQ ID NO: 136, and LCDR3 as shown in SEQ ID NO: 148, or 5) LCDR1 as shown in SEQ ID NO: 120, LCDR2 as shown in SEQ ID NO: 136, and LCDR3 as shown in SEQ ID NO: 147, or 6) LCDR1 as shown in SEQ ID NO: 127, LCDR2 as shown in SEQ ID NO: 133, and LCDR3 as shown in SEQ ID NO: 139, or 7) LCDR1 as shown in SEQ ID NO: 129, LCDR2 as shown in SEQ ID NO: 130, and LCDR3 as shown in SEQ ID NO: 139, or 8) LCDR1 as shown in SEQ ID NO: 121, LCDR2 as shown in SEQ ID NO: 134, and LCDR3 as shown in SEQ ID NO: 149, or 9) LCDR1 as shown in SEQ ID NO: 123, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 140, or 10) LCDR1 as shown in SEQ ID NO: 125, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 141, or 11) LCDR1 as shown in SEQ ID NO: 124, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 143, or 12) LCDR1 as shown in SEQ ID NO: 128, LCDR2 as shown in SEQ ID NO: 131, and LCDR3 as shown in SEQ ID NO: 146, or 13) LCDR1 as shown in SEQ ID NO: 128, LCDR2 as shown in SEQ ID NO: 131, and LCDR3 as shown in SEQ ID NO: 145, or 14) LCDR1 as shown in SEQ ID NO: 119, LCDR2 as shown in SEQ ID NO: 135, and LCDR3 as shown in SEQ ID NO: 150, or 15) LCDR1 as shown in SEQ ID NO: 122, LCDR2 as shown in SEQ ID NO: 132, and LCDR3 as shown in SEQ ID NO: 144, or 16) LCDR1 as shown in SEQ ID NO:154, LCDR2 as shown in SEQ ID NO:155 and LCDR3 as shown in SEQ ID NO:

156.

3. The antibody or antigen-binding fragment according to claim 1 or 2, comprising any one of the following complementarity determining regions (CDRs): 1) the VH comprises HCDR1 as shown in SEQ ID NO: 88, HCDR2 as shown in SEQ ID NO: 101, and HCDR3 as shown in SEQ ID NO: 113, and the VL comprises LCDR1 as shown in SEQ ID NO: 129, LCDR2 as shown in SEQ ID NO: 137, and LCDR3 as shown in SEQ ID NO: 139; or 2) the VH comprises HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 108, and the VL comprises LCDR1 as shown in SEQ ID NO: 126, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 141; or 3) the VH comprises HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 95, and HCDR3 as shown in SEQ ID NO: 114, and the VL comprises LCDR1 as shown in SEQ ID NO: 125, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 142; or 4) the VH comprises HCDR1 as shown in SEQ ID NO:82, HCDR2 as shown in SEQ ID NO:91, and HCDR3 as shown in SEQ ID NO:112, and the VL comprises LCDR1 as shown in SEQ ID NO:129, LCDR2 as shown in SEQ ID NO:137, and LCDR3 as shown in SEQ ID NO:139; or 5) the VH comprises HCDR1 as shown in SEQ ID NO: 87, HCDR2 as shown in SEQ ID NO: 100, and HCDR3 as shown in SEQ ID NO: 111, and the VL comprises LCDR1 as shown in SEQ ID NO: 120, LCDR2 as shown in SEQ ID NO: 136, and LCDR3 as shown in SEQ ID NO: 148; or 6) the VH comprises HCDR1 as shown in SEQ ID NO:80, HCDR2 as shown in SEQ ID NO:93, and HCDR3 as shown in SEQ ID NO:110, and the VL comprises LCDR1 as shown in SEQ ID NO:120, LCDR2 as shown in SEQ ID NO:136, and LCDR3 as shown in SEQ ID NO:147; or 7) the VH comprises HCDR1 as shown in SEQ ID NO: 81, HCDR2 as shown in SEQ ID NO: 104, and HCDR3 as shown in SEQ ID NO: 109, and the VL comprises LCDR1 as shown in SEQ ID NO: 127, LCDR2 as shown in SEQ ID NO: 133, and LCDR3 as shown in SEQ ID NO: 139; or 8) the VH comprises HCDR1 as shown in SEQ ID NO: 86, HCDR2 as shown in SEQ ID NO: 97, and HCDR3 as shown in SEQ ID NO: 116, and the VL comprises LCDR1 as shown in SEQ ID NO: 129, LCDR2 as shown in SEQ ID NO: 130, and LCDR3 as shown in SEQ ID NO: 139; or 9) the VH comprises HCDR1 as shown in SEQ ID NO: 89, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 117, and the VL comprises LCDR1 as shown in SEQ ID NO: 121, LCDR2 as shown in SEQ ID NO: 134, and LCDR3 as shown in SEQ ID NO: 149; or 10) the VH comprises HCDR1 as shown in SEQ ID NO: 84, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 107, and the VL comprises LCDR1 as shown in SEQ ID NO: 123, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 140; or 11) the VH comprises HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 94, and HCDR3 as shown in SEQ ID NO: 115, and the VL comprises LCDR1 as shown in SEQ ID NO: 125, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 141; or 12) the VH comprises HCDR1 as shown in SEQ ID NO: 85, HCDR2 as shown in SEQ ID NO: 96, and HCDR3 as shown in SEQ ID NO: 114, and the VL comprises LCDR1 as shown in SEQ ID NO: 124, LCDR2 as shown in SEQ ID NO: 138, and LCDR3 as shown in SEQ ID NO: 143; or 13) the VH comprises HCDR1 as shown in SEQ ID NO: 90, HCDR2 as shown in SEQ ID NO: 102, and HCDR3 as shown in SEQ ID NO: 106, and the VL comprises LCDR1 as shown in SEQ ID NO: 128, LCDR2 as shown in SEQ ID NO: 131, and LCDR3 as shown in SEQ ID NO: 146; or 14) the VH comprises HCDR1 as shown in SEQ ID NO: 83, HCDR2 as shown in SEQ ID NO: 92, and HCDR3 as shown in SEQ ID NO: 105, and the VL comprises LCDR1 as shown in SEQ ID NO: 128, LCDR2 as shown in SEQ ID NO: 131, and LCDR3 as shown in SEQ ID NO: 145; or 15) the VH comprises HCDR1 as shown in SEQ ID NO: 87, HCDR2 as shown in SEQ ID NO: 99, and HCDR3 as shown in SEQ ID NO: 111, and the VL comprises LCDR1 as shown in SEQ ID NO: 119, LCDR2 as shown in SEQ ID NO: 135, and LCDR3 as shown in SEQ ID NO: 150; or 16) the VH comprises HCDR1 as shown in SEQ ID NO: 79, HCDR2 as shown in SEQ ID NO: 103, and HCDR3 as shown in SEQ ID NO: 118, and the VL comprises LCDR1 as shown in SEQ ID NO: 122, LCDR2 as shown in SEQ ID NO: 132, and LCDR3 as shown in SEQ ID NO: 144; or 17) The VH comprises HCDR1 as shown in SEQ ID NO: 151, HCDR2 as shown in SEQ ID NO: 152, and HCDR3 as shown in SEQ ID NO: 153, and the VL comprises LCDR1 as shown in SEQ ID NO: 154, LCDR2 as shown in SEQ ID NO: 155, and LCDR3 as shown in SEQ ID NO:

156.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the VH comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 16, 63 to 67 and 157, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence of any one of SEQ ID NOs: 1 to 16, 63 to 67 and 157.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the VL comprises an amino acid sequence of any one of SEQ ID NOs: 17 to 31, 68 to 70, and 158, or an amino acid sequence having at least 90% sequence homology to an amino acid sequence of any one of SEQ ID NOs: 17 to 31, 68 to 70, and 158.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 5, comprising any one of the following VH and VL sequence combinations: 1) the VH of SEQ ID NO: 1, and the VL of SEQ ID NO: 17, or 2) the VH of SEQ ID NO: 2, and the VL of SEQ ID NO: 18, or 3) the VH described in SEQ ID NO: 3, and the VL described in SEQ ID NO: 19, or 4) the VH of SEQ ID NO: 4, and the VL of SEQ ID NO: 17, or 5) the VH of SEQ ID NO: 5, and the VL of SEQ ID NO: 20, or 6) the VH of SEQ ID NO: 6, and the VL of SEQ ID NO: 21, or 7) the VH of SEQ ID NO: 7, and the VL of SEQ ID NO: 22, or 8) the VH of SEQ ID NO: 8, and the VL of SEQ ID NO: 23, or 9) the VH of SEQ ID NO: 9, and the VL of SEQ ID NO: 24, or 10) the VH of SEQ ID NO: 10, and the VL of SEQ ID NO: 25, or 11) VH according to SEQ ID NO: 11, and VL according to SEQ ID NO: 26, or 12) the VH of SEQ ID NO: 12, and the VL of SEQ ID NO: 27, or 13) the VH of SEQ ID NO: 13, and the VL of SEQ ID NO: 28, or 14) the VH of SEQ ID NO: 14, and the VL of SEQ ID NO: 29, or 15) the VH of SEQ ID NO: 15, and the VL of SEQ ID NO: 30, or 16) the VH of SEQ ID NO: 16, and the VL of SEQ ID NO: 31, or 17) The VH described in SEQ ID NO: 157, and the VL described in SEQ ID NO:

158.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 5, comprising any one of the following VH and VL sequence combinations: 1) the VH of SEQ ID NO: 63, and the VL of SEQ ID NO: 68, or 2) the VH of SEQ ID NO: 63, and the VL of SEQ ID NO: 69, or 3) the VH of SEQ ID NO: 63, and the VL of SEQ ID NO: 70, or 4) the VH of SEQ ID NO: 64, and the VL of SEQ ID NO: 68, or 5) the VH of SEQ ID NO: 64, and the VL of SEQ ID NO: 69, or 6) the VH of SEQ ID NO: 64, and the VL of SEQ ID NO: 70, or 7) the VH of SEQ ID NO: 65, and the VL of SEQ ID NO: 68, or 8) the VH of SEQ ID NO: 65, and the VL of SEQ ID NO: 69, or 9) the VH of SEQ ID NO: 65, and the VL of SEQ ID NO: 70, or 10) the VH of SEQ ID NO: 66, and the VL of SEQ ID NO: 68, or 11) the VH of SEQ ID NO: 66, and the VL of SEQ ID NO: 69, or 12) the VH of SEQ ID NO: 66, and the VL of SEQ ID NO: 70, or 13) the VH of SEQ ID NO: 67, and the VL of SEQ ID NO: 68, or 14) the VH of SEQ ID NO: 67, and the VL of SEQ ID NO: 69, or 15) The VH of SEQ ID NO: 67, and the VL of SEQ ID NO:

70.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 7, which is a chimeric or humanized antibody or antigen-binding fragment.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antigen-binding fragment is selected from one or more of scFv, Fv, Fab and F(ab)2, preferably Fab.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, comprising any one of the following combinations of heavy chain variable region, heavy chain constant region 1 (Fd fragment) and light chain sequence: 1) the Fd fragment of SEQ ID NO: 159, and the light chain of SEQ ID NO: 176, or 2) the Fd fragment of SEQ ID NO: 160, and the light chain of SEQ ID NO: 177, or 3) the Fd fragment of SEQ ID NO: 161, and the light chain of SEQ ID NO: 178, or 4) the Fd fragment of SEQ ID NO: 162, and the light chain of SEQ ID NO: 192, or 5) the Fd fragment of SEQ ID NO: 163, and the light chain of SEQ ID NO: 179, or 6) the Fd fragment of SEQ ID NO: 164, and the light chain of SEQ ID NO: 180, or 7) the Fd fragment of SEQ ID NO: 165, and the light chain of SEQ ID NO: 181, or 8) the Fd fragment of SEQ ID NO: 166, and the light chain of SEQ ID NO: 182, or 9) the Fd fragment of SEQ ID NO: 167, and the light chain of SEQ ID NO: 183, or 10) the Fd fragment of SEQ ID NO: 168, and the light chain of SEQ ID NO: 184, or 11) the Fd fragment of SEQ ID NO: 169, and the light chain of SEQ ID NO: 185, or 12) the Fd fragment of SEQ ID NO: 170, and the light chain of SEQ ID NO: 186, or 13) the Fd fragment of SEQ ID NO: 171, and the light chain of SEQ ID NO: 187, or 14) the Fd fragment of SEQ ID NO: 172, and the light chain of SEQ ID NO: 188, or 15) the Fd fragment of SEQ ID NO: 173, and the light chain of SEQ ID NO: 189, or 16) the Fd fragment of SEQ ID NO: 174, and the light chain of SEQ ID NO: 190, or 17) The Fd fragment described in SEQ ID NO: 175, and the light chain described in SEQ ID NO:

191.

11. The antibody or antigen-binding fragment according to any one of claims 1 to 9, comprising any one of the following combinations of heavy chain variable region, heavy chain constant region 1 (Fd fragment), and light chain sequence: 1) the Fd fragment of SEQ ID NO: 226, and the light chain of SEQ ID NO: 231, or 2) the Fd fragment of SEQ ID NO: 226, and the light chain of SEQ ID NO: 232, or 3) the Fd fragment of SEQ ID NO: 226, and the light chain of SEQ ID NO: 233, or 4) the Fd fragment of SEQ ID NO: 227, and the light chain of SEQ ID NO: 231, or 5) the Fd fragment of SEQ ID NO: 227, and the light chain of SEQ ID NO: 232, or 6) the Fd fragment of SEQ ID NO: 227, and the light chain of SEQ ID NO: 233, or 7) the Fd fragment of SEQ ID NO: 228, and the light chain of SEQ ID NO: 231, or 8) the Fd fragment of SEQ ID NO: 228, and the light chain of SEQ ID NO: 232, or 9) the Fd fragment of SEQ ID NO: 228, and the light chain of SEQ ID NO: 233, or 10) the Fd fragment of SEQ ID NO: 229, and the light chain of SEQ ID NO: 231, or 11) the Fd fragment of SEQ ID NO: 229, and the light chain of SEQ ID NO: 232, or 12) the Fd fragment of SEQ ID NO: 229, and the light chain of SEQ ID NO: 233, or 13) the Fd fragment of SEQ ID NO: 230, and the light chain of SEQ ID NO: 231, or 14) the Fd fragment of SEQ ID NO: 230, and the light chain of SEQ ID NO: 232, or 15) The Fd fragment described in SEQ ID NO: 230, and the light chain described in SEQ ID NO:

233.

12. The antibody or antigen-binding fragment according to any one of claims 1 to 11, which has a specific affinity for human 5T4 of about 1×10 -9 M is about 1×10 - 8 The binding affinity of M.

13. A fusion protein comprising the antibody or antigen-binding fragment of any one of claims 1 to 12, and at least one heterologous polypeptide fragment. The fusion protein according to claim 13 , which is a multispecific antibody comprising two or more antigen binding sites targeting human 5T4 and other antigenic epitopes.

15. A chimeric antigen receptor (CAR) comprising the extracellular region of the antibody or antigen-binding fragment of any one of claims 1-12 and an intracellular signaling domain.

16. A conjugate comprising the antibody or antigen-binding fragment of any one of claims 1 to 12 and at least one effector. The conjugate according to claim 16 , wherein the effector is a detectable label. The conjugate according to claim 17 , wherein the detectable label is a fluorescent label, a radionuclide, avidin, biotin and / or an enzyme. The conjugate according to claim 16 , wherein the effector is a cytotoxic molecule.

20. A nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-12.

21. The nucleic acid according to claim 20, comprising a nucleotide sequence encoding a heavy chain variable region, wherein the nucleotide sequence encoding the heavy chain variable region comprises the nucleotide sequence shown in any one of SEQ ID NOs: 32-47, 71-75 and 242.

22. The nucleic acid according to claim 20 or 21, comprising a nucleotide sequence encoding a light chain variable region, wherein the nucleotide sequence encoding the light chain variable region comprises the nucleotide sequence shown in any one of SEQ ID NOs: 48-62, 76-78 and 243.

23. The nucleic acid according to any one of claims 20 to 22, comprising a nucleotide sequence encoding a heavy chain variable region and a heavy chain constant region 1 (Fd fragment), wherein the nucleotide sequence encoding the Fd fragment comprises the nucleotide sequence shown in any one of SEQ ID NOs: 193 to 209 and 234 to 238.

24. The nucleic acid according to any one of claims 20 to 23, comprising a nucleotide sequence encoding a light chain, wherein the nucleotide sequence encoding the light chain comprises the nucleotide sequence shown in any one of SEQ ID NOs: 210 to 225, 239 to 241 and 244. A recombinant vector comprising the nucleic acid according to any one of claims 20 to 24. A host cell comprising the recombinant vector according to claim 25 .

27. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-12, the fusion protein of claim 13 or 14, the conjugate of any one of claims 16-19, the chimeric antigen receptor of claim 15, the nucleic acid of any one of claims 20-24, the recombinant vector of claim 25, or the host cell of claim 26, and a pharmaceutically acceptable carrier, diluent or excipient.

28. Use of the antibody or antigen-binding fragment of any one of claims 1-12, the fusion protein of claim 13 or 14, the conjugate of any one of claims 16-19, the chimeric antigen receptor of claim 15, the nucleic acid of any one of claims 20-24, the recombinant vector of claim 25, the host cell of claim 26, or the pharmaceutical composition of claim 27 in preparing a medicament for treating 5T4-positive tumor-related diseases.

29. Use of the antibody or antigen-binding fragment of any one of claims 1-12 or the conjugate of any one of claims 16-19 in the preparation of a detection kit or a detection reagent for diagnosing a disease associated with 5T4-positive tumors.

30. A method for treating a 5T4-positive tumor-related disease, the method comprising administering to a subject in need thereof an effective therapeutic dose of the antibody or antigen-binding fragment of any one of claims 1-12, the fusion protein of claim 13 or 14, the conjugate of any one of claims 16-19, the chimeric antigen receptor of claim 15, the nucleic acid of any one of claims 20-24, the recombinant vector of claim 25, the host cell of claim 26, or the pharmaceutical composition of claim 27.

31. A method for diagnosing a 5T4-positive tumor-related disease, the method comprising providing a detection kit or a detection reagent, wherein the detection kit or the detection reagent comprises the antibody or antigen-binding protein according to any one of claims 1-12 and / or the conjugate according to any one of claims 16-19.

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