CN113227148B

CN113227148B - anti-GPC 3 antibody, antigen-binding fragment thereof, and medical use thereof

Info

Publication number: CN113227148B
Application number: CN202080006983.3A
Authority: CN
Inventors: 花海清; 余华星; 何娟梅; 包如迪
Original assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Current assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Priority date: 2019-12-05
Filing date: 2020-12-03
Publication date: 2022-11-01
Anticipated expiration: 2040-12-03
Also published as: TW202134286A; WO2021110095A1; CN113227148A

Abstract

Chimeric, humanized antibodies against the CDR regions of the GPC3 antibody are provided; also provided are pharmaceutical compositions comprising an anti-GPC 3 antibody or an antigen-binding fragment thereof; and its use as an anti-cancer agent.

Description

anti-GPC 3 antibody, antigen-binding fragment thereof, and medical use thereof

The present application claims priority from a chinese patent application (application number 201911236102.2) filed on year 2019, 12, 05 and a chinese patent application (application number 202010910640.1) filed on year 2020, 09, 02.

Technical Field

The present application relates to an anti-GPC 3 antibody specifically immunoreactive to human GPC3, and an antigen-binding fragment thereof, a chimeric antibody comprising CDR regions of the anti-GPC 3 antibody, a humanized antibody, and a pharmaceutical composition comprising the human anti-GPC 3 antibody and the antigen-binding fragment thereof, and uses thereof as a cell growth inhibitor and an anticancer drug, and for detecting or diagnosing tumors.

Background

Glypican3 (GPC 3) is an approximately 70kd membrane protein, belongs to the family of Glypicans, and plays a cell adhesion role as an extracellular matrix protein in organogenesis or a receptor of a cell growth factor. After GPC3 expression, it is cleaved by furin enzyme to generate an N-terminal approximately 40kDa soluble moiety and an approximately 30kDa moiety, which is anchored to the C-terminal of the cell membrane via a GPI molecule.

GPC3 is expressed in embryonic tissues (particularly the liver and kidney) and is an extracellular matrix protein associated with organ formation. In adult tissues, no expression of GPC3 was observed except for placenta, but expression was observed in various cancer tissues such as hepatocellular carcinoma, melanoma, clear ovarian cell carcinoma, and lung squamous cell carcinoma. It can be seen that GPC3 is a protein expressed in embryonic tissue, and is classified as an embryonic cancer antigen, like proteins such as alpha-fetoprotein (AFP) and Carcinoembryonic antigen (CEA). Specifically, GPC3 is characterized by being not expressed in normal tissue cells but specifically expressed in cancer cells, and therefore can be used as a target molecule for cancer therapy or a tumor marker.

In addition, genomics and functional studies show that GPC3 plays an important role in maintaining the activation of Wnt pathway and Hedgehogs pathway. For example, GPC 3-coupled heparan sulfate molecules can enhance the binding of Wnts to their receptors, and thus have an important role in maintaining the Wnt pathway. GPC3 is expressed in the brain, gut, bladder, gonads and skin, and is highly expressed on the surface of hepatocellular carcinomas; the Wnt pathway plays an important role in liver cancer development, such as 20% hepatocellular carcinoma beta-Catenin pathway mutation and Frizzled-7 receptor overexpression, so GPC3 may play a promoting role in the development of partial hepatocellular carcinoma.

The current targeted GPC3 macromolecular drugs that enter the clinical phase and disclose clinical outcome are Codrituzumab developed by roche (cotrutuzumab; alias GC 33), patent No.: US20100248359, the antibody drug targets the C-terminal 524-563 amino acids of human GPC3, belonging to the human IgG1, kappa subtype. GC33 induces antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and has a significant effect of inhibiting tumor growth in a HuH-7 tumor pharmacodynamic mouse model. GC33 is in clinical stage II (NCT 01507168), and indications include hepatocellular carcinoma (HCC), metastatic hepatocellular carcinoma, liver cancer, metastatic liver cancer and the like. However, because of poor clinical efficacy results, roche stopped the development of Codrituzumab.

The stability of an antibody is strongly related to the effect of the drug, its metabolism in the human body and its safety, for example, the better the stability of an antibody, the less immunogenic it will be.

Disclosure of Invention

The present application provides an anti-GPC 3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region.

In some embodiments, the anti-GPC 3 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region and/or a light chain variable region selected from:

the heavy chain variable region comprises at least 1 HCDR1 selected from the group consisting of SEQ ID NOs: SEQ ID NO:7, SEQ ID NO:8,

and the variable region of the antibody heavy chain comprises at least 1 HCDR2 selected from the group consisting of SEQ ID NOs: the amino acid sequence of SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11,

and the variable region of the antibody heavy chain comprises at least 1 HCDR3 selected from the group consisting of SEQ ID NOs: the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14; and

the variable region of the antibody light chain comprises at least 1 LCDR1 selected from the following sequences: SEQ ID NO:15, SEQ ID NO:16,

and the antibody light chain variable region comprises at least 1 LCDR2 selected from the group consisting of seq id no: SEQ ID NO:17, SEQ ID NO:18,

and the antibody light chain variable region comprises at least 1 LCDR3 selected from the group consisting of seq id no: SEQ ID NO:19, SEQ ID NO:20.

in some embodiments of the present application, the CDR sequences of the antibodies or antigen-binding fragments thereof of the present application may have 1-3 amino acid mutations, wherein a 1-3 amino acid mutation refers to any 1-3 amino acid insertion, deletion, or substitution; specifically, the mutation of 1 to 3 amino acids is an insertion, deletion or substitution of 1 to 3 amino acids capable of optimizing the activity of an antibody, the stability of an antibody or reducing the immunogenicity.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises:

are respectively shown as SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:12 HCDR1, HCDR2 and HCDR3;

respectively shown in SEQ ID NO: 7. SEQ ID NO:10 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3.

Or as shown in SEQ ID NO: 8. SEQ ID NO:11 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 as shown in fig. 14.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody light chain variable region comprises:

respectively shown in SEQ ID NO: 15. SEQ ID NO:17 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in 19;

or as shown in SEQ ID NO: 16. SEQ ID NO:18 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 20.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises: are respectively shown as SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:12 HCDR1, HCDR2 and HCDR3; the light chain variable region comprises: respectively shown in SEQ ID NO:15SEQ ID NO:17 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in 19.

The present application also relates to a specific embodiment, an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, comprising a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises:

SEQ ID NO:7 or a variant of SEQ ID NO:7 compared with HCDR1 with 1-3 amino acid mutations,

The amino acid sequence of SEQ ID NO:9 or a sequence identical to SEQ ID NO:9 HCDR2 and having 1-3 amino acid mutations compared to

The amino acid sequence of SEQ ID NO:12 or a variant of SEQ ID NO:12 HCDR3 with 1-3 amino acid mutations compared to;

the light chain variable region comprises:

SEQ ID NO:15 or a variant of SEQ ID NO:15 to LCDR1 with 1-3 amino acid mutations,

SEQ ID NO:17 or a variant of SEQ ID NO:17 LCDR2 with 1-3 amino acid mutations compared to

SEQ ID NO:19 or a variant of SEQ ID NO:19 compared to LCDR3 with 1-3 amino acid mutations.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, comprising a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises: are respectively shown as SEQ ID NO: 7. SEQ ID NO:10 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3;

the light chain variable region comprises: respectively shown in SEQ ID NO: 15. SEQ ID NO:17 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in 19.

the heavy chain variable region comprises:

SEQ ID NO:10 or a sequence identical to SEQ ID NO:10 compared to HCDR2 and having 1-3 amino acid mutations

SEQ ID NO:13 or a sequence identical to SEQ ID NO:13 HCDR3 with 1-3 amino acid mutations compared;

the antibody light chain variable region comprises:

SEQ ID NO:17 or a variant of SEQ ID NO:17 LCDR2 and having 1-3 amino acid mutations compared

The amino acid sequence of SEQ ID NO:19 or a variant of SEQ ID NO:19 compared to LCDR3 with 1-3 amino acid mutations.

the heavy chain variable region comprises:

respectively shown in SEQ ID NO: 8. SEQ ID NO:11 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 as shown in 14;

the light chain variable region comprises: respectively shown in SEQ ID NO: 16. SEQ ID NO:18 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 20.

the heavy chain variable region comprises: the amino acid sequence of SEQ ID NO:8 or a variant of SEQ ID NO:8 compared to HCDR1, SEQ ID NO:11 or a variant of SEQ ID NO:11 HCDR2 having 1-3 amino acid mutations compared to SEQ ID NO:14 or a sequence identical to SEQ ID NO:14 HCDR3 with 1-3 amino acid mutations compared;

the light chain variable region comprises: SEQ ID NO:16 or a sequence identical to SEQ ID NO:16 LCDR1 having 1-3 amino acid mutations compared to SEQ ID NO:18 or a sequence identical to SEQ ID NO:18 LCDR2 having 1-3 amino acid mutations compared to SEQ ID NO:20 or a sequence identical to SEQ ID NO:20 compared to LCDR3 with 1-3 amino acid mutations.

the heavy chain variable region comprises: respectively shown in SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:12 HCDR1, HCDR2 and HCDR3; the light chain variable region comprises: respectively shown in SEQ ID NO:16SEQ ID NO:18 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 20.

the heavy chain variable region comprises: SEQ ID NO:7 or a sequence identical to SEQ ID NO:7 compared to HCDR1, SEQ ID NO:9 or a sequence identical to SEQ ID NO:9 HCDR2 having 1-3 amino acid mutations compared to SEQ ID NO:12 or a variant of SEQ ID NO:12 HCDR3 with 1-3 amino acid mutations compared to; the light chain variable region comprises: the amino acid sequence of SEQ ID NO:16 or a sequence identical to SEQ ID NO:16 compared to LCDR1, SEQ ID NO:18 or a sequence identical to SEQ ID NO:18 LCDR2 having 1-3 amino acid mutations compared to SEQ ID NO:20 or a variant of SEQ ID NO:20 compared to LCDR3 with 1-3 amino acid mutations.

the heavy chain variable region comprises: respectively shown in SEQ ID NO: 7. SEQ ID NO:10 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3; the light chain variable region comprises: respectively shown in SEQ ID NO: 16. SEQ ID NO:18 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 20.

the heavy chain variable region comprises: SEQ ID NO:7 or a sequence identical to SEQ ID NO:7 compared to HCDR1, SEQ ID NO:10 or a sequence identical to SEQ ID NO:10 compared to HCDR2 with 1-3 amino acid mutations and SEQ ID NO:13 or a sequence identical to SEQ ID NO:13 HCDR3 with 1-3 amino acid mutations compared; the light chain variable region comprises: SEQ ID NO:16 or a sequence identical to SEQ ID NO:16 compared to LCDR1, SEQ ID NO:18 or a sequence identical to SEQ ID NO:18 LCDR2 having 1-3 amino acid mutations compared to SEQ ID NO:20 or a sequence identical to SEQ ID NO:20 compared to LCDR3 with 1-3 amino acid mutations.

the heavy chain variable region comprises: respectively shown in SEQ ID NO: 8. SEQ ID NO:11 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 14; the light chain variable region comprises: are respectively shown as SEQ ID NO: 15. SEQ ID NO:17 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in 19.

the heavy chain variable region comprises: SEQ ID NO:8 or a variant of SEQ ID NO:8 compared to HCDR1, SEQ ID NO:11 or a variant of SEQ ID NO:11 compared to HCDR2 with 1-3 amino acid mutations and SEQ ID NO:14 or a sequence identical to SEQ ID NO:14 HCDR3 with 1-3 amino acid mutations compared; the light chain variable region comprises: the amino acid sequence of SEQ ID NO:15 or a variant of SEQ ID NO:15 compared to LCDR1 with 1-3 amino acid mutations, SEQ ID NO:17 or a sequence identical to SEQ ID NO:17 compared to LCDR2 with 1-3 amino acid mutations and SEQ ID NO:19 or a variant of SEQ ID NO:19 compared to LCDR3 with 1-3 amino acid mutations.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody is a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, a human antibody or fragment thereof, and a humanized antibody or fragment thereof.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or an antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, igG3, or IgG4, or a variant thereof.

In particular embodiments, the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, or IgG4.

In specific embodiments, the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG 1.

In specific embodiments, the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises an amino acid sequence as set forth in SEQ ID NO:41, or a heavy chain constant region.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a mutated IgG1 heavy chain constant region having enhanced ADCC toxicity compared to the parent amino acid. In specific embodiments, the mutated IgG1 heavy chain constant region is as set forth in SEQ ID NO: shown at 40.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or the antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, a lambda chain, or a mutant thereof;

preferably, the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain;

more preferably, the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises an amino acid sequence as set forth in SEQ ID NO:42, or a light chain constant region.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of seq id nos: the amino acid sequence of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:26.

the present application also relates to a preferred embodiment, an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that is at least 70%,75%,80%,85%,90%,95%, or 99% identical to the following sequence: the amino acid sequence of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:26.

in the context of the present application, reference to "at least 70% identity" means, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a range between any two numerical values.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or an antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of seq id nos: the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:25.

in some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the light chain variable region of the anti-GPC 3 antibody or an antigen-binding fragment thereof comprises a light chain variable region having at least 70%,75%,80%,85%,90%,95% or 99% identity compared to the following sequence: SEQ ID NO:22 or SEQ ID NO:25.

in some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, comprising:

SEQ ID NO:21, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto, and SEQ ID NO:22, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

SEQ ID NO:21, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto, and SEQ ID NO:25, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

SEQ ID NO:23, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto, and SEQ ID NO:22, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

SEQ ID NO:23, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto, and SEQ ID NO:25, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

SEQ ID NO:24, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto, and SEQ ID NO:22, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

SEQ ID NO:24, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto, and SEQ ID NO:25, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

the amino acid sequence of SEQ ID NO:26, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto, and SEQ ID NO:22, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

SEQ ID NO:26, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto, and SEQ ID NO:25, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity thereto.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or an antigen-binding fragment thereof comprises:

the heavy chain variable region is SEQ ID NO:21 and the light chain variable region is SEQ ID NO:22;

the heavy chain variable region is SEQ ID NO:21 and the light chain variable region is SEQ ID NO:25;

the heavy chain variable region is SEQ ID NO:23 and the light chain variable region is SEQ ID NO:22;

the heavy chain variable region is SEQ ID NO:23 and the light chain variable region is SEQ ID NO:25;

the heavy chain variable region is SEQ ID NO:24 and the light chain variable region is SEQ ID NO:22;

the heavy chain variable region is SEQ ID NO:24 and the light chain variable region is SEQ ID NO:25;

the heavy chain variable region is SEQ ID NO:26 and the light chain variable region is SEQ ID NO:22; or

The heavy chain variable region is SEQ ID NO:26 and the light chain variable region is SEQ ID NO:25.

in some embodiments of the present application, there is provided an anti-GPC 3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 80%,85%,90%,95%, 99%, or 100% identical to the following sequence: SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39.

in some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof comprises a light chain that is at least 80%,85%,90%,95%, 99%, or 100% identical to the following sequence: SEQ ID NO:28 or SEQ ID NO:31.

in other embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, comprising:

the amino acid sequence of SEQ ID NO:27, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and the heavy chain of SEQ ID NO:28, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

SEQ ID NO:27, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and the heavy chain of SEQ ID NO:31, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

the amino acid sequence of SEQ ID NO:29, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and SEQ ID NO:28, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

SEQ ID NO:29, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and SEQ ID NO:31, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

the amino acid sequence of SEQ ID NO:30, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and SEQ ID NO:28, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

SEQ ID NO:30, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and SEQ ID NO:31, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

the amino acid sequence of SEQ ID NO:32, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and a heavy chain of SEQ ID NO:28, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

SEQ ID NO:32, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and a heavy chain of SEQ ID NO:31, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto.

In some embodiments of the present application, there is provided an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody comprises:

the heavy chain is SEQ ID NO:27 and the light chain of SEQ ID NO:28;

the heavy chain is SEQ ID NO:27 and the light chain of SEQ ID NO:31;

the heavy chain is SEQ ID NO:29 and the light chain of SEQ ID NO:28;

the heavy chain is SEQ ID NO:29 and the light chain of SEQ ID NO:31;

the heavy chain is SEQ ID NO:30 and the light chain of SEQ ID NO:28;

the heavy chain is SEQ ID NO:30 and the light chain of SEQ ID NO:31;

the heavy chain is SEQ ID NO:32 and the light chain of SEQ ID NO:28; or

The heavy chain is SEQ ID NO:32 and the light chain of SEQ ID NO:31.

SEQ ID NO:36, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and a heavy chain of SEQ ID NO:28, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

SEQ ID NO:36, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and SEQ ID NO:31, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

the amino acid sequence of SEQ ID NO:37, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and SEQ ID NO:28, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

SEQ ID NO:37, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and SEQ ID NO:31, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

SEQ ID NO:38, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and SEQ ID NO:28, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or, alternatively,

the amino acid sequence of SEQ ID NO:38, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and the heavy chain of SEQ ID NO:31, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

SEQ ID NO:39, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and the heavy chain of SEQ ID NO:28, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto; or the like, or a combination thereof,

SEQ ID NO:39, or a heavy chain having at least 80%,85%,90%,95% or 99% identity thereto, and the heavy chain of SEQ ID NO:31, or a light chain having at least 80%,85%,90%,95%, or 99% identity thereto.

The present application also relates to some embodiments, providing an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody comprises:

the heavy chain is SEQ ID NO:36 and the light chain of SEQ ID NO:28;

the heavy chain is SEQ ID NO:36 and the light chain of SEQ ID NO:31;

the heavy chain is SEQ ID NO:37 and the light chain of SEQ ID NO:28;

the heavy chain is SEQ ID NO:37 and the light chain of SEQ ID NO:31;

the heavy chain is SEQ ID NO:38 and the light chain of SEQ ID NO:28;

the heavy chain is SEQ ID NO:38 and the light chain is SEQ ID NO:31;

the heavy chain is SEQ ID NO:39 and the light chain of SEQ ID NO:28; or

The heavy chain is SEQ ID NO:39 and the light chain of SEQ ID NO:31.

the present application further provides a polynucleotide encoding an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above.

The present application further provides an expression vector comprising a polynucleotide as described above.

The present application further provides a host cell introduced or containing an expression vector as described above.

In a particular embodiment of the present application, a host cell as described above, wherein said host cell is a bacterium, in particular E.coli.

In a specific embodiment of the present application, a host cell as described above, wherein said host cell is a yeast, in particular pichia pastoris.

In a specific embodiment of the present application, a host cell as described above, wherein said host cell is a mammalian cell, in particular a CHO cell or a HEK293 cell.

The present application further provides a method of producing an anti-GPC 3 antibody or an antigen-binding fragment thereof, comprising: culturing the above-described host cells (e.g., HEK293 cells); isolating the antibody from the culture (e.g., isolating the antibody from the cell culture fluid); and purifying the antibody (e.g., purifying the antibody by chromatography).

The present application further provides a pharmaceutical composition comprising an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, and a pharmaceutically acceptable excipient, diluent, or carrier.

In a further specific embodiment of the present application, there is provided a detection or diagnostic kit comprising an anti-GPC 3 antibody or an antigen-binding fragment thereof described herein, and one or more agents capable of detecting the binding of the anti-GPC 3 antibody or the antigen-binding fragment thereof to GPC3 (or an epitope thereof).

In a specific embodiment of the present application, there is provided a detection or diagnostic kit comprising an anti-GPC 3 antibody or an antigen-binding fragment thereof described herein, or a pharmaceutically acceptable salt thereof, and a labeled secondary antibody, buffer, and substrate useful for detection or diagnosis.

In a particular embodiment of the present application, there is provided the use of an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above or a composition as described above in the manufacture of a medicament for the treatment or prevention of a GPC 3-mediated disease or condition.

In a particular embodiment of the present application, an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above or a composition as described above is used in the manufacture of a medicament for treating or preventing a GPC 3-mediated disease or condition.

In a particular embodiment of the present application, an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above, or a composition as described above, is used in the preparation of a reagent or kit for detecting, diagnosing, prognosing a GPC 3-mediated disease or condition.

In a particular embodiment of the present application, an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above or a composition as described above is used for the detection, diagnosis, prognosis of a GPC 3-mediated disease.

The present application still further provides a method of treating or preventing a GPC 3-mediated disease, comprising the steps of: providing to the subject a therapeutically effective amount or a prophylactically effective amount of an anti-GPC 3 antibody or an antigen-binding fragment thereof as described above; or providing a therapeutically effective amount or a prophylactically effective amount of a pharmaceutical composition as described previously to the subject.

The disease or disorder described herein is cancer; in particular, the disease or condition is GPC 3-mediated cancer; further, the cancer is selected from: breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, or melanoma.

Provided is an anti-GPC 3 antibody or antigen-binding fragment which can specifically bind to a GPC3 antigen (or an epitope thereof) and a GPC 3-expressing cell, has a significant CDC activity, and has a significant tumor-killing effect.

In addition, the anti-GPC 3 antibody or the antigen-binding fragment thereof of the present application has good endocytosis and is suitable for drug conjugation. The anti-GPC 3 antibody or antigen-binding fragment of the application has lower immunogenicity and higher stability on the basis of high antibody activity, and is significantly better than Codrituzumab. The anti-GPC 3 antibody or antigen binding fragment has better potential as an anti-cancer drug, and ensures the drug safety.

Drawings

FIG. 1: the heavy chain variable region of the humanized antibody Ab9 and the heavy chain variable region of the GC33 antibody correspond to the sequences of the human germline templates (note: the amino acids in which the antibody does not correspond to the corresponding human germline template are marked in grey shading).

FIG. 2: the humanized antibody Ab9 light chain variable region, GC33 antibody light chain variable region and their human germline template sequences correspond (note: grey shading identifies amino acids in the antibody that are inconsistent with the corresponding human germline template).

Detailed Description

Term(s)

In order that the present application may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Amino acid three letter codes and one letter codes as used herein are as described in j.biol.chem,243, p3558 (1968).

The term "antibody" as used herein refers to an immunoglobulin, which is a tetrapeptide chain structure of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus in their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., igM, igD, igG, igA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of Ig can be divided into different subclasses according to the differences of amino acid composition of the hinge region and the number and position of disulfide bonds of heavy chains, for example, igG can be divided into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. In the five classes of igs, the second class of igs can have either kappa chains or lambda chains.

In the present application, the antibody light chain described herein may further comprise a light chain constant region derived from a kappa, lambda chain of human or murine origin or variants thereof.

In the present application, the antibody heavy chain described herein may further comprise a heavy chain constant region derived from IgG1, igG2, igG3, igG4 of human or murine origin, or a variant thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, as are the variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the VL region and VH region of the antibodies or antigen-binding fragments described herein conform in number and position to the rules of the known Kabat or Chothia or ABM definitions.

In this application, the numbering of the amino acids in the variable regions is by the Kabat numbering system, unless otherwise indicated. The correspondence of a particular amino acid position in different numbering systems can be determined by one skilled in the art based on common knowledge.

The term "GPC3" includes any variant or isoform of GPC3 that is naturally expressed by a cell. The antibodies (or antigen-binding fragments thereof) of the present application can cross-react with GPC3 of a non-human species. Alternatively, the antibody may be specific for human GPC3 and may not exhibit cross-reactivity with other species. GPC3, or any variant or isoform thereof, may be isolated from cells or tissues or produced by recombinant techniques using techniques common in the art and those described herein. Specifically, the anti-GPC 3 antibody targets human GPC3 (or an epitope thereof) with a normal glycosylation pattern.

The term "murine antibody" is used herein to refer to a monoclonal antibody directed against human GPC3 (or an epitope thereof) prepared according to the knowledge and skill in the art. The preparation is carried out by injecting the test subject with the GPC3 antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional properties. In a specific embodiment of the present application, the murine GPC3 antibody or the antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, igG2, igG3, or IgG4 or variant thereof.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present application can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include "humanized antibodies".

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting non-human CDR sequences into the framework of the human antibody variable regions. Humanized antibodies help to overcome the disadvantage of strong immune response induced by chimeric antibodies due to their non-human protein component. To avoid reduced activity due to reduced immunogenicity, the variable region of the humanized antibody may be minimally back-mutated to retain activity.

The term "chimeric antibody" is an antibody that is a fusion of the variable region of an antibody of a first species and the constant region of an antibody of a second species, and that reduces the immune response induced by the antibody of the first species. As a non-limiting example, to create a chimeric antibody, a hybridoma is now created that secretes a monoclonal antibody of a first species, then variable region genes are cloned from the hybridoma cells, then constant region genes of an antibody of a second species are cloned as needed, the variable region genes and the constant region genes are ligated into a chimeric gene and inserted into a vector, and finally the chimeric antibody molecule is expressed in an industrial system. The constant region may be selected from a heavy chain constant region of an IgG1, igG2, igG3, or IgG4 from a second species, or a variant thereof; in particular, an IgG1, igG2, or IgG4 heavy chain constant region of the second species, or an IgG1 heavy chain constant region with enhanced ADCC toxicity, is included.

The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. Antibody fragments retain at least some of the binding activity of the parent antibody. Typically, an antigen-binding fragment retains at least 10% of maternal binding activity when expressed on a molar basis. In particular, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%,95%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to: fab, fab ', F (ab') 2, fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson,2005, nat. Biotechnol.23: 1126-1136.

An "Fab fragment" consists of one light chain, one heavy chain CH1 and the variable region. The heavy chain of a Fab molecule is unable to form a disulfide bond with another heavy chain molecule.

"Fab' fragments" contain portions of one light chain and one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab 'fragments to form F (ab')₂A molecule.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitopic specificity; an antibody having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, diabodies (diabodies), bispecific diabodies and triabodies (triabodies), antibody fragments that have been covalently or non-covalently linked together, and the like.

The term "single-chain antibody" is a single-chain recombinant protein formed by connecting a heavy chain variable region VH and a light chain variable region VL of an antibody via a linker peptide, and is the smallest antibody fragment having a complete antigen-binding site.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only heavy chain variable regions or light chain variable regions. As a non-limiting example, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment may target the same or different antigens.

The term "binding to GPC3" in the present application refers to the ability to interact with human GPC3 or an epitope thereof.

The term "antigen binding site" in the present application refers to a linear or three-dimensional spatial site recognized by an antibody or antigen binding fragment of the present application.

The term "epitope" refers to a site on an antigen to which an antibody (or antigen-binding fragment) binds. Epitopes can be formed by adjacent amino acids, or amino acids that are close but not adjacent by tertiary folding of the peptide chain. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after denaturing solvent treatment. Epitopes usually comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope binds to a given antibody are well known in the art and include immunoblot and immunoprecipitation detection assays, and the like. Methods of determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

The term "specifically binds" as used herein refers to the binding of an antibody (or antigen-binding fragment) to an epitope on a predetermined antigen. Typically, when human GPC3 is used as the analyte and an antibody is used as the ligand, the antibody (or antigen-binding fragment) binds to the predetermined antigen with an equilibrium dissociation constant (KD) of about less than 10 "7M or even less, as determined by Surface Plasmon Resonance (SPR) techniques in the instrument; and, the antibody (or antigen-binding fragment) binds to the predetermined antigen with at least twice the affinity as it binds to a non-specific antigen, such as BSA.

The term "cross-reactive" refers to the ability of an antibody (or antigen-binding fragment) of the present application to bind GPC3 (or an epitope thereof) from a different species. Cross-reactivity is measured by detecting specific reactivity with an antigen in binding assays (e.g., SPR and ELISA), or binding or functional interactions with cells that physiologically express GPC3. Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Preferably, inhibition/blocking of the ligand is capable of reducing or altering the normal level or type of activity that occurs upon ligand binding without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-GPC 3 antibody, as compared to a ligand not contacted with an anti-GPC 3 antibody.

The term "inhibiting growth" (e.g., referring to a cell) is intended to include any measurable reduction in cell growth.

The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to the stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induction" with respect to inducing CDC or ADCC refers to the stimulation of a specific direct cell killing mechanism.

"ADCC", i.e., antibody-dependent cell-mediated cytotoxicity, as used herein, refers to the direct killing of antibody-coated target cells by Fc receptor-expressing cells by recognition of the Fc domain of the antibody. The ADCC effector function of an antibody may be enhanced or reduced or eliminated by modifying the Fc portion of the IgG. The modification refers to mutation in the heavy chain constant region of the antibody.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the antibody Experimental technical guidance of Cold spring harbor, chapters 5-8 and 15. For example, mice can be immunized with human GPC3 or a fragment thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced by conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. Antibodies or antigen-binding fragments described herein are genetically engineered to incorporate one or more human FR regions in CDR regions of non-human origin. Human FR germline sequences can be obtained from The database ImmunoGeneTiCs (IMGT) or from The Immunoglulin facebook journal (Academic Press, 2001ISBN 012441351).

The engineered antibodies or antigen binding fragments of the present application can be prepared and purified using conventional methods. As a non-limiting example, cDNA sequences of the antibodies may be cloned and recombined into an expression vector, which may stably transfect host cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones were obtained by expressing antibodies that specifically bind to the antigen. Positive clones were expanded in a bioreactor to produce antibodies. The culture broth can be purified and collected by conventional techniques. The concentration by filtration can be carried out by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is immediately frozen, e.g., -70 ℃, or lyophilized.

A monoclonal antibody (mAb), as used herein, refers to an antibody obtained from a single clonal cell line, not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other techniques known in the art.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering," "administering," and "treating" also mean treating, e.g., a cell in vitro and ex vivo, by an agent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering an internal or external therapeutic agent, such as comprising any of the antibodies of the present application, to a subject having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population being treated, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce a desired therapeutic effect in the subject. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present application (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in each patient, they should alleviate the symptoms of the target disease in a statistically significant number of subjects, as determined according to any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, kruskal-Wallis test (H-test), jonckhere-Terpsra test, and Wilcoxon test.

The term "consisting essentially of or variants thereof as used throughout the specification and claims is meant to encompass all such elements or groups of elements, and optionally other elements of similar or different nature than the elements, which other elements do not materially alter the basic or novel characteristics of a given dosing regimen, method or composition.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a medical condition or symptom thereof. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on the following factors: such as the disease to be treated, the general health of the subject, the method and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"exogenous" refers to a substance that is to be produced outside an organism, cell, or human body by background.

"endogenous" refers to a substance produced in an organism, cell, or human body according to background.

"homology" or "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny thereof. Thus, "transformants" and "transformed cells" include the primary test cells and cultures derived therefrom, regardless of the number of passages. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"pharmaceutical composition" means a composition containing one or more antibodies or antigen-binding fragments thereof described herein, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The present application is further described below with reference to examples, but these examples do not limit the scope of the present application. The experimental method not specified in the examples of the present application is generally carried out under conventional conditions such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and are conventional reagents purchased in the market.

Examples

Example 1: protein antigen and cell line preparation

(1) Protein antigen preparation

The protein antigen was human GPC3 recombinant protein (UniProt # P51654, gln25 to His559 fragment) with a His-tag at the C-terminus, i.e. human GPC3His protein, purchased from AcroBiosystems (Cat # GP3-H52H 4).

TABLE 1 human GPC3 (Gln 25-His 559) amino acid sequence

(2) Cell line construction

A monoclonal stable cell strain stably expressing the full-length human or monkey (Macaca mulatta) GPC3 protein was constructed using CHO-K1 as a host cell, and named CHO-K1-hGPC3 and CHO-K1-cynoGPC3, respectively. The cell strain is constructed as follows: the in vitro synthesized full-length human or monkey GPC3 DNA sequence (see Table 2 below) was cloned into a lentiviral vector carrying a puromycin resistance gene, CHO-K1 cells were transfected, the resulting cell pool was subjected to selection culture using F12K medium (all three reagents purchased from GIBCO) containing 8. Mu.g/ml puromycin, 10% FBS, FACS-detected the expression level of GPC3, and then the gradient dilution method to obtain a highly expressed monoclonal cell strain.

TABLE 2 DNA sequences of human GPC3 and monkey GPC3

Example 2: mouse hybridoma and obtaining of antibody sequence

The immunogen is human GPC3-His antigen protein and/or CHO-K1-hGPC3 cell. 10 Balb/C and 5 SJL mice, and 5C 57 mice, female, 10 weeks old.

Two immunological adjuvants:

(1) Using Sigma Complete Freund's Adjuvant (CFA) and Sigma Incomplete Freund's Adjuvant (IFA), the immunogen and the immunoadjuvant were mixed thoroughly in a ratio of 1: 1 and emulsified to prepare a stable "water-in-oil" liquid.

(2) Aluminum salt adjuvant with Invivo Gen ()

adjuvant 2%), mixed with the antigen and adjuvant 1: 1 to form a water-soluble solution for immunization.

The immunization protocol and immunization program are shown in tables 3, 4 and 5 below:

TABLE 3 immunization protocol

TABLE 4 immunization procedure (protein immunization)

TABLE 5 immunization procedure (protein and cell combination immunization)

Serum titers were assessed using the indirect ELISA method described in example 3 below on immune mouse sera and the ability of example 4 (2) to detect binding to cell surface antigens by FACS, with control titer detection (greater than 10 ten thousand dilutions) determining initiation of cell fusion.

Selecting an immune mouse with strong serum titer, affinity and FACS combination for primary final immunization; killing the mouse, taking splenocytes and fusing the splenocytes with SP2/0 myeloma cells to obtain hybridoma; screening target hybridomas through indirect ELISA and FACS; the strain is established into a monoclonal cell strain by a limiting dilution method. The resulting positive cell lines were further screened using indirect ELISA and FACS to select hybridomas that bind the recombinant protein. The logarithmic growth phase hybridoma cells were harvested, RNA extracted using Trizol (ThermoFisher, 15596-026) and reverse transcribed (PrimeScript)^TMFirst strand cDNA synthesis kit, takara # 6110A). The cDNA obtained by reverse transcription is subjected to PCR amplification by adopting a mouse Ig-primer group (Sigma # 69831) and then sequenced, and the sequences of murine antibodies M1, M2 and M3 are obtained by sequencing.

TABLE 6 heavy and light chain variable region sequences of murine mAbs

By analyzing the CDR regions of M1, M2 and M3, LCDR1 of M1, M2 and M3 is found to contain "NGN" sequence fragments, which are presumed to have an effect on the stability of the antibody. In the subsequent chimeric and humanized antibodies, the "NGN" sequence of LCDR1 was mutated to "NRN" so the CDR region sequences in the subsequent studies are shown in the following table.

TABLE 7 CDR sequences of the heavy and light chain variable regions

Example 3: method for detecting in-vitro binding activity of mouse antibody

In vitro indirect ELISA binding experiments:

GPC3His protein (Acrobiosystems, cat # GP3-H52H 4) was diluted to a concentration of 0.5. Mu.g/ml with PBS pH7.4, added to a 96-well high affinity microplate at a volume of 100. Mu.L/well, and incubated overnight (16-20 hours) at 4 ℃ in a refrigerator. After washing the plate 3 times with PBST (0.05% Tween-20 in PBS, pH7.4), 200. Mu.L/well of 1% Bovine Serum Albumin (BSA) blocking solution diluted with PBST was added, and the plate was incubated at 37 ℃ for 0.5 hour for blocking. After blocking was complete, the blocking solution was discarded and the plate was washed 1 time with PBST buffer.

The test antibody was diluted with 1% BSA in PBST, starting at 100nM, diluted in a 5-fold gradient, 11 doses, added to the ELISA plate at 100. Mu.L/well, and incubated at 37 ℃ for 1 hour. After the incubation was completed, the plate was washed 3 times with PBST, and 200. Mu.L/well of an HRP-labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, cat # 115-035-071) or an HRP-labeled goat anti-human secondary antibody (Rockland, cat # 609-103-123) diluted with 1% BSA in PBST was added, and incubated at 37 ℃ for 0.5 hour. After washing the plate 5 times with PBST, 100. Mu.l/well of TMB chromogenic substrate (cat # S0025, suzhou subfamily chemical reagents Co., ltd.) was added, incubated at 25 ℃ for 8-15 minutes in the dark, the reaction was stopped by adding 50. Mu.l/well of 1M HCl, and the absorbance was read at 450nm with a microplate reader (Thermo, ascent) to analyze the data.

The results of the concentration-signal value curve analysis are shown in the following table. The results show that: the affinity of the murine antibody to the human GPC3 antigen is good.

TABLE 8 affinity (EC) of murine antibodies for human GPC3 antigen₅₀Value)

Murine antibodies	ELISA，EC₅₀(nM)
		M1	0.05
M2	0.03
		M3	0.07

Example 4: mouse antibody chimerization assay

And (3) carrying out chimerization on the screened positive murine antibody, cloning the variable region of the murine antibody in the constant region of the human antibody to obtain the chimeric antibody, wherein the constant region of the chimeric antibody is selected from a human IgG1 heavy chain constant region and a human kappa chain constant region.

The heavy chain vector was designed as follows: signal peptide + heavy chain variable region sequence + human IgG1 constant region sequence.

The light chain vector was designed as follows: signal peptide + light chain variable region sequence + human Kappa constant region sequence.

The sequences were inserted into pCEP4 vectors, and expression vectors were synthesized according to the design described above. After obtaining the vector plasmid, extracting the plasmid, and sending the plasmid to sequencing verification. The plasmid that was verified to be acceptable was transfected into HEK293 cells (qian shenzhou) with TF1 and cultured continuously. HEK293 cells were cultured to logarithmic growth phase with serum-free CD medium (Cat # SMM 293-TI, yoyo). Mu.g of light chain plasmid and 23.6. Mu.g of heavy chain plasmid were dissolved in 10mL Reduced Serum Medium (GIBCO, 31985-070), mixed, then added to 200ug TF1, mixed, incubated at RT for 15min, and added to 50mL cells. Cell culture conditions: 5% C02, 37 ℃,125rpm/min. During the culture period, feeding materials (Cat # M293-SUPI-100, chinesia, yi) were added on

days

1, 3 and 5 until the cell viability was less than 70%, and cell supernatants were collected and centrifuged. Loading the cell culture solution after centrifugal filtration to a Protein-A affinity chromatography column, washing the column by phosphate buffer, eluting by glycine-hydrochloric acid buffer solution (pH2.70.1M Gly-HCl), neutralizing by 2M Tris-hydrochloric acid pH 9.0, and dialyzing by phosphate buffer solution to finally obtain each purified chimeric antibody.

(1) In vitro protein binding experiments:

the affinity of the chimeric antibody to human GPC3 antigen was tested according to the in vitro indirect ELISA binding experiment of example 3 and the Biacore method as shown below:

chip preparation: mouse anti-human IgG (Fc) antibody was diluted to 25. Mu.g/mL with immobilized reagent (10 mM sodium acetate, pH 5.0) using approximately 100. Mu.L mouse anti-human IgG (Fc) antibody per channel on the chip and 190. Mu.L immobilized reagent was added to 10. Mu.L mouse anti-human IgG (Fc) antibody for both channels. First, the surface of the CM5 chip was activated for 420s with 400mM MDC and 100mM NHS at a flow rate of 10. Mu.L/min. Next, 25. Mu.g/mL of a mouse anti-human IgG (Fc) antibody was injected into the experimental channel (FC 4) at a flow rate of 10. Mu.L/min for about 420s, at a fixed amount of about 9000 to 14000RU. Finally, the chip was blocked with 1M ethanolamine at 10. Mu.L/min for 420 s. The reference channel (FC 3) performs the same operation as the test channel (FC 4).

Capture ligand: the antibody stock was diluted to 4. Mu.g/mL with running reagents, respectively, and injected into the assay channel (FC 4) at a flow rate of 10. Mu.L/min to capture approximately 200RU. The reference channel (FC 3) does not require capture of the ligand.

Analyte multi-cycle analysis: human GPC3 protein was diluted 2-fold with the running reagent, and the diluted samples were sequentially injected into the experimental channel and the reference channel at a flow rate of 30. Mu.L/min for the respective binding time and dissociation time. After each concentration analysis, the chip was regenerated with 3M magnesium chloride at a flow rate of 20. Mu.L/min for 30s, washing off the ligand and the undissociated analyte. For the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.

And (3) data analysis: KD values for each antibody were calculated using Biacore T200 analysis software, and the reference channel (FC 3) was used for background subtraction.

The results of the in vitro indirect ELISA binding assay and Biacore assay are shown in Table 9:

TABLE 9 affinity of chimeric antibodies to human GPC3 antigen.

ELISA and Biacore results show that the chimeric antibody has good affinity to human GPC3 antigen.

(2) In vitro cell binding experiments:

GPC3 high expression cells including CHO-K1 cells (ATCC, cat # CCL-61) overexpressing human or monkey GPC3, and human hepatoma cells JHH-7 (Nanjing Kebai, cat # CBP 60204), hepG2 (cell bank of Chinese academy of sciences, cat # SCSP-510) expressing GPC3, were trypsinized, harvested by centrifugation, adjusted in cell density with FACS buffer (1 XPBS containing 2% FBS), and then plated onto 96-well U-plates at 1X 10/well⁵To 2X 10⁵And (4) cells. Centrifuging: 1200g, 5min, discard the supernatant, add 100. Mu.L of antibody solution (initial working concentration of antibody 100nM, 5-fold dilution, 7 concentration points, and set up 0nM points) that has been diluted with a FACS buffer gradient, incubate for 1 h at 4 ℃. Centrifuging: 1200g, 5min, discard the supernatant, wash the cells 2 times with PBS, add the fluorescence labeled secondary working solution PE anti-human IgG Fc antibody (Biolegged, cat # 409304) or FITC anti-mouse IgG antibody (Biolegged, cat # 406001) in FACS buffer, resuspend the cells at 100. Mu.L per well, and incubate for 1 h at 4 ℃. Centrifuging: 1200g, 5min, discard the supernatant. After washing the cells 2 times with PBS, resuspend in PBS, detect the fluorescence signal using a flow cytometer DxFlex, and profile the EC of antibody-bound cells₅₀And (4) concentration.

TABLE 10 affinity (EC) of chimeric antibodies for GPC 3-expressing cells₅₀Value)

The results show that: the chimeric antibody has high affinity with GPC3 expressing cells.

Example 5: mouse antibody humanization experiments

Humanization of murine anti-human GPC3 monoclonal antibodies was performed as disclosed in the literature in the art. Briefly, murine antibodies M1, M2 and M3 were humanized using human constant domains instead of the parent (murine antibody) constant domains, and human antibody sequences were selected based on the homology of the murine and human antibodies.

Based on the obtained typical structure of VH/VL CDR of the murine antibody, the variable region sequences of the heavy and light chains are compared with the germline database of the human antibody to obtain a human germline template with high homology.

The CDR regions of the murine antibody were grafted onto the corresponding humanized template that was selected. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, residues directly interacting with the CDR region, and residues having important influence on the conformation of VL and VH are subjected to back mutation, the CDR region is optimized for chemically unstable amino acid residues, and the humanized heavy chain variable region HCVR and light chain variable region LCVR sequences are designed to be combined to form the antibody through expression test and back mutation quantity comparison, wherein the sequences are as follows:

TABLE 11 CDR regions of humanized antibodies

TABLE 12 heavy and light chain variable region sequences of humanized antibodies

Among them, the humanized antibody Ab9 heavy chain variable region has 81.7% sequence identity and 87.0% sequence similarity to its human germline template (germline 1) sequence. The GC33 heavy chain variable region has the same sequence similarity and identity ratio as Ab9 compared to its human germline template (germline 1) sequence. The corresponding relationship between the heavy chain variable region of the humanized antibody Ab9 and the heavy chain variable region of the GC33 antibody and the human germline template sequence is shown in FIG. 1.

The Ab9 light chain variable region has 88.4% sequence identity and 97.3% similarity to its human germline template (germline 2). The GC33 light chain variable region has 86.6% sequence identity and 89.3% similarity to its human germline template (germline 3). The correspondence between Ab9 light chain variable regions and their human germline templates (germline 2) and GC33 light chain variable regions and their human germline templates (germline 3) is shown in fig. 2.

The designed heavy and light chain variable region sequences are linked to the heavy and light chain constant region sequences, respectively, of a human antibody. Illustratively, the antibody heavy chain constant region is selected from: the human IgG1 heavy chain constant region with ADCC enhancement after amino acid mutation or the natural human IgG1 heavy chain constant region has the sequences shown in SEQ ID NO: 40. SEQ ID NO:41 is shown; the light chain constant region is selected from the group consisting of SEQ ID NO:42, constant region of human kappa chain. The resulting heavy and light chain sequences are shown in table 13, and the antibody constant region sequences are shown in table 14.

TABLE 13 heavy and light chain sequences of humanized antibodies

TABLE 14 constant region sequence numbering

TABLE 15 sequence numbering of antibodies and their heavy, light, variable regions

Humanized antibody numbering	HCVR	LCVR	HC	LC
					Ab1	SEQ ID NO：21	SEQ ID NO：22	SEQ ID NO：27	SEQ ID NO：28
Ab2	SEQ ID NO：23	SEQ ID NO：22	SEQ ID NO：29	SEQ ID NO：28
					Ab3	SEQ ID NO：24	SEQ ID NO：25	SEQ ID NO：30	SEQ ID NO：31
Ab4	SEQ ID NO：21	SEQ ID NO：25	SEQ ID NO：27	SEQ ID NO：31
					Ab5	SEQ ID NO：23	SEQ ID NO：25	SEQ ID NO：29	SEQ ID NO：31
Ab6	SEQ ID NO：24	SEQ ID NO：22	SEQ ID NO：30	SEQ ID NO：28
					Ab7	SEQ ID NO：26	SEQ ID NO：22	SEQ ID NO：32	SEQ ID NO：28
Ab8	SEQ ID NO：21	SEQ ID NO：22	SEQ ID NO：36	SEQ ID NO：28
					Ab9	SEQ ID NO：23	SEQ ID NO：22	SEQ ID NO：37	SEQ ID NO：28
Ab10	SEQ ID NO：24	SEQ ID NO：25	SEQ ID NO：38	SEQ ID NO：31
					Ab11	SEQ ID NO：21	SEQ ID NO：25	SEQ ID NO：36	SEQ ID NO：31
Ab12	SEQ ID NO：23	SEQ ID NO：25	SEQ ID NO：37	SEQ ID NO：31
					Ab13	SEQ ID NO：24	SEQ ID NO：22	SEQ ID NO：38	SEQ ID NO：28
Ab14	SEQ ID NO：26	SEQ ID NO：22	SEQ ID NO：39	SEQ ID NO：28

Example 6: in vitro binding Activity assay for humanized antibodies

The affinity of each humanized antibody for human GPC3 antigen was determined using the in vitro indirect ELISA binding assay described in example 3 and the Biacore method described in example 4 (1), and the results are shown in table 16:

TABLE 16 affinity of humanized antibodies to human GPC3 antigen

The results show that: the humanized antibody has good affinity with human GPC3 antigen.

The affinity (EC) of each humanized antibody for GPC 3-expressing cells was determined using the in vitro cell binding assay described in example 4 (2)₅₀) The results are shown in Table 17:

TABLE 17 affinity (EC) of humanized antibodies for GPC3 expressing cells₅₀Value)

The results show that: the humanized antibody has good affinity with GPC3 expressing cells.

Example 7: endocytosis of humanized antibodies

The antibodies of the present application were tested for their ability to engulf cells with human GPC3 after binding to GPC3 and evaluated using the stable cell line CHO-K1-human GPC3.

Cells were digested with pancreatin, collected and resuspended in pre-cooled FACS buffer to a cell concentration of 1X 10⁶One per mL. Taking an EP tube, adding 1mL of cell suspension, centrifuging at 1500rpm for 5 minutes, then removing the supernatant, adding 1mL of prepared antibody to be detected to resuspend cells, performing shake-table incubation at 4 ℃ for 1 hour, centrifuging, removing the supernatant (4 ℃,1500rpm and 5 minutes), washing twice with FACS buffer solution, and removing the supernatant. mu.L of a fluorescently labeled secondary antibody working solution PE anti-human IgG Fc antibody (Biolegged, cat # 409304) or FITC anti-mouse IgG antibody (Biolegged, cat # 406001) was added to each tube, the cells were resuspended, incubated on a shaker at 4 ℃ for 30 minutes, the supernatant was discarded by centrifugation (4 ℃,1500 rpm. Times.5 minutes), washed twice with FACS buffer, and discarded. 1mL of preheated cell culture medium is added into each tube to resuspend the cells and mix evenly, the mixture is divided into 4 tubes, 200 mu L of each tube is respectively a 0-minute group, a blank group, a 30-minute group and a 2-hour group, the 0-minute group and the blank group are taken out and placed on ice, the rest are placed in an incubator at 37 ℃, the endocytosis is carried out for 30 minutes and 2 hours respectively, an EP tube is taken out at a corresponding time point and placed on ice for precooling for 5 minutes, all treatment groups are centrifuged to discard the supernatant (4 ℃,1500rpm multiplied by 5 minutes), the supernatant is washed once by FACS buffer solution, and the supernatant is discarded. In all treatment groups except the 0-minute group, 250. Mu.L strip buffer was added to the EP tubes, incubated at room temperature for 8 minutes, centrifuged to discard the supernatant (4 ℃,1500 rpm. Times.5 minutes), washed twice with FACS buffer, and discarded. All treatment groups were resuspended in 100. Mu.L PBS and examined by flow cytometry DxFlex.

Percent endocytosis (%) of antibody (= fluorescence intensity value at each time point-average fluorescence intensity value of blank)/(average fluorescence lightness value at zero-average fluorescence intensity value of blank) × 100, the results are shown in table 18:

TABLE 18 endocytosis of humanized antibodies in cells

The results from table 18 show: the humanized antibody has good endocytosis in the stable cell strain CHO-K1-human GPC3.

Example 8: ADCC assay of humanized antibodies

The effector cells of this experiment were NK92MI (NK 92 cells over-expressing IL-2 gene, purchased from Nanjing Kebai), and the target cells were human hepatoma cells HepG2. The experimental medium was RPMI1640 containing 10% FBS for cell and antibody resuspension or dilution. HepG2 cells were trypsinized, collected by centrifugation and resuspended at 1X 10 in experimental medium⁵Each 100. Mu.L of the suspension was plated on a white 96-well plate (Corning, 3610), incubated at 37 ℃ for 2 days, then 50. Mu.L of 4X working concentration antibody was added thereto, and incubated at 4 ℃ for 15 minutes. NK92MI cells were collected and resuspended to 1X 10 with experimental medium⁶mu.L/ml, 50. Mu.L were added to the above reaction so that the ratio of effector cells to target cells was 5: 1, and the cells were incubated at 37 ℃ for 3-4 hours in an incubator. Finally, 100. Mu.L of Cell Titer-Glo (Promega, cat # G7573) was added, mixed well and reacted for 10 minutes at room temperature in the dark, and read with a multifunctional microplate reader (Thermofish, lux).

Percent antibody killing (%) = (E-S)/(E-M). Times.100

E is the number of wells without antibody, i.e. effector cells + tumor cells;

s is the value of the sample well, namely effector cells, tumor cells and antibodies;

m is the number of medium wells.

The results show that: the humanized antibody has remarkable ADCC effect.

Example 9: CDC experiments with humanized antibodies

The target cell in this experiment is a stable cell line CHO-K1-hGPC3 over-expressing human GPC3. The experimental medium was FBS-free cell culture medium F12K (GIBCO) for cell and antibody resuspension or dilution.

CHO-K1-hGPC3 cells were trypsinized, collected by centrifugation, and resuspended at 1X 10 in experimental medium⁵50 μ L/ml were plated in white 96-well plates (Corning, 3610), 25 μ L of 4 Xworking concentration antibody (initial working concentration of antibody was 20nM, 5-fold dilution, 10 concentration points, and OnM points were set up), incubated at 37 ℃ for 30 minutes, and 25 μ L of antibody was addedL80% human serum (GemCell)^TMUS, cat # 100-512), incubated in an incubator at 37 ℃ for 24 hours. Add 50. Mu.L of CellTiter-Glo (Promega, cat # G7573), mix well and react for 10 minutes at room temperature in the dark, read on a multifunctional microplate reader (Thermofeisher, lux).

Percent antibody killing (%) = (E-S)/(E-M). Times.100

E is the value of wells without antibody, i.e.cells + medium + human serum;

s is the numerical value of the sample well, namely cell + antibody + human serum;

m is the number of medium + human serum wells.

TABLE 19 CDC Effect of humanized antibodies

Humanized antibodies	Cells	IC₅₀(nM)
			Ab9	CHO-K1-hGPC3	0.308

The results show that: the humanized antibody Ab9 has significant CDC effect.

Example 10: charge heterogeneous stability experiments for humanized antibodies

The stability of the humanized antibodies was examined by detecting and comparing the charge heteroplasmon purity of the humanized antibodies at the initial 0 point and at 25 ℃ and 40 ℃ for one month by means of panoramic isoelectric focusing (iCIEF).

When a DC voltage is applied to two ends of the capillary, the carrier ampholyte can form a certain range of pH gradient in the capillary, the components of the sample can be electrophoresed to the cathode or the anode according to the charged electricity, the pH value in the capillary is the same as the isoelectric point (pI) of the components, the net charge of solute molecules is zero, macroscopically the components are gathered at the point and do not further migrate, and the aim of separating the components in the complex sample is fulfilled. And after the atlas is collected, obtaining the pI value and the peak ratio (main peak, acid peak and alkali peak) of the sample according to the linear relation between the MarkerpI value and the migration time of the chromatographic peak. The method mainly comprises the following steps:

system adaptive sample preparation: the adaptive sample tube in the Maurice cIEF System reliability Kit (Protein Simple, cat # 046-044) was taken out, 40. Mu.l of deionized water and 160. Mu.l of System reliability Test Mix were added, mixed well and transferred to a 1.5ml centrifuge tube, vortexed, centrifuged, and 160. Mu.l of the supernatant was transferred to a 96-well sample plate for use.

Preparing a cIEF Master mixed solution: contains 37. Mu.l of ultrapure water, 35. Mu.l of 1% MC (Protein Simple, cat # 101876), 4. Mu.l of Pharmalyte pH 3-10 (Protein Simple, cat # 17-0456-01), 2. Mu.l of 500mM argine (Protein Simple, cat # 042-691), and correspondingly two pI markers 6.14 (Protein Simple, cat: 046-031) and 9.99 (Protein Simple, cat: 046-034) each in a total volume of 80. Mu.l.

Preparing a test article: the sample was set up for 3 conditions: starting at 0 point; standing in a stability test chamber (Memmer, model HPP 1060) at a humidity of 65% and a temperature of 25 deg.C for 1 month; the plates were placed in a stability test chamber (Memmer, model HPP 1060) at a humidity of 65% and a temperature of 40 ℃ for 1 month. 20 μ l of the corresponding sample was added to the EP tube containing 80 μ l of cIEF MasterMix solution in step 2), vortexed, mixed and centrifuged, 80ul of the supernatant was transferred to a 96-well sample plate and centrifuged for use.

And (3) computer detection: opening a capillary electrophoresis apparatus (Protein Simple, male) and software, performing self-inspection of the apparatus according to the operation steps of the apparatus, installing a capillary cartridge (Protein Simple, cat # PS-MC 02-C), placing a 96-well sample plate in the corresponding position of the apparatus, and performing cIEF analysis.

TABLE 20 iCIEF detection of humanized antibodies

The results show that: compared with the initial 0 point, ab9 has little change of the proportion of the main peak after being placed at 25 ℃ for one month and has good stability. When the antibody is placed for one month at the temperature of 40 ℃ under the forced degradation condition, the expected degradation phenomenon of the antibody appears, which is reflected in that the proportion of the main peak is reduced to 26.3 percent; while the control antibody GC33 showed a 2.5% decrease in the proportion of the main peak after a month at 40 ℃.

Example 11: molecular variant stability experiments of humanized antibodies

This experiment examined and compared the purity of the humanized antibody at the initial 0 point, and the molecular variants at 25 ℃ and 40 ℃ for one month, respectively, by capillary electrophoresis (CE-SDS). CE-SDS is based on the migration of protein samples in gel electrophoresis under denaturing conditions, and separation is completed according to the difference of migration time of proteins with different molecular weights, so as to obtain the detection results of the purity of the sample after non-reduction and reduction treatment.

The method mainly comprises the following steps:

sample preparation: sampling the sample at the initial 0 point; placing in a stability test chamber (Memmer, model HPP 1060) at a humidity of 65% and a temperature of 25 deg.C for 1 month, and sampling; the samples were taken for 1 month under conditions of a stability test chamber (Memmer, model HPP 1060) humidity of 65% and temperature of 40 ℃.

Non-reduced CE sample treatment: samples were added to the EP tube at 50. Mu.g Protein loading per sample, 1. Mu.L of 10kD internal standard (Protein Simple, cat # 046-144), 2.5. Mu.L of 250mM IAM (Sigma, cat # I1149-5G), and 1 Xsample buffer (Protein Simple, cat # 046-567) to a final volume of 50. Mu.L.

Treatment of reduced CE samples: samples were added to the EP tube at 50. Mu.g of Protein per sample, 1. Mu.L of 10kD internal standard (Protein Simple, cat # 046-144), 2.5. Mu.L of beta-mercaptoethanol (Sigma, cat # M3148-25 ML), and 1 Xsample buffer (Protein Simple, cat # 046-567) to a final volume of 50. Mu.L.

After shaking, the mixture was incubated at 70 ℃ for 10min in a dry thermostat (MK 20001, oshend, hangzhou Osheng apparatus Co., ltd.), then the mixture was taken out, incubated on ice for 5min, cooled and centrifuged at 12000rpm for 5min. After centrifugation, 35. Mu.L of the supernatant was transferred to 96 wells matched to the instrument, followed by centrifugation at 1000rpm for 5min.

Sample detection: putting the 96-hole sample plate into a capillary electrophoresis apparatus (Protein Simple, maurice), opening the apparatus and software, performing self-checking on the apparatus according to the operation steps of the apparatus, installing a capillary cartridge (Protein Simple, cat # 090-157), and preparing corresponding reagents to be put into corresponding positions of the apparatus. And setting corresponding parameters according to the operating steps of the instrument, and carrying out reduction or non-reduction CE analysis.

Data processing: and after the sample is detected, setting corresponding integral parameters, and performing calculation analysis through software carried by the instrument to obtain the purity of the sample.

TABLE 21 CE-SDS detection of humanized antibodies

The non-reduced CE results showed that the humanized antibody Ab9 showed a similar proportion of the main peak at 25 ℃ for one month as the 0 point, and the main peak was reduced at 40 ℃ for one month. In the reduced CE, the sum of the proportion of heavy and light chains of the Ab9 antibody was less changed and the stability was good compared with that at point 0 even when the antibody was left at 25 ℃ or 40 ℃ for one month.

Sequence listing

<110> Shanghai Hansen Bio-medical technology, inc

Jiangsu Haosen Pharmaceutical Group Co., Ltd.

<120> anti-GPC 3 antibody, antigen-binding fragment thereof, and medical use thereof

<140> CN 202080006983.3

<141> 2020-12-03

<150> 201911236102 .2

<151> 2019-12-05

<150> 202010910640 .1

<151> 2020-09-02

<160> 42

<170> SIPOSequenceListing 1.0

<210> 1

<211> 115

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(115)

<223> HCVR of murine mab M1

<400> 1

Gln Ile Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asp Pro Lys Thr Gly Ser Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Ile Leu Thr Ala Asp Ile Ser Ser Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ala His Tyr Cys

85 90 95

Thr Arg Tyr Phe Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 2

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(112)

<223> LCVR of murine mab M1

<400> 2

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Phe Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Ile His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 3

<211> 115

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(115)

<223> HCVR of murine mab M2

<400> 3

Gln Ile Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Arg Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ala His Tyr Cys

85 90 95

Thr Arg Tyr Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 4

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(112)

<223> LCVR of murine mab M2

<400> 4

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Ile His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 5

<211> 115

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(115)

<223> HCVR of murine mab M3

<400> 5

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr

20 25 30

Glu Ile His Trp Met Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asp Pro Lys Thr Asp Ser Thr Ala Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 6

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(112)

<223> LCVR of murine mab M3

<400> 6

Asp Val Val Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Phe Lys Val Ser Asn Arg Leu Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Ala Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 7

<211> 5

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(5)

<223> HCDR1 of murine mab M1,M2

<400> 7

Asp Tyr Glu Met His

1 5

<210> 8

<211> 5

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(5)

<223> HCDR1 of murine mab M3

<400> 8

Ala Tyr Glu Ile His

1 5

<210> 9

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(17)

<223> HCDR2 of murine mab M1

<400> 9

Gly Ile Asp Pro Lys Thr Gly Ser Thr Ala Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 10

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(17)

<223> HCDR2 of murine mab M2

<400> 10

Ala Ile Asp Pro Lys Thr Gly Arg Thr Ala Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 11

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(17)

<223> HCDR2 of murine mab M3

<400> 11

Gly Ile Asp Pro Lys Thr Asp Ser Thr Ala Tyr Asn Gln Asn Phe Lys

1 5 10 15

Gly

<210> 12

<211> 6

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(6)

<223> HCDR3 of murine mab M1

<400> 12

Tyr Phe Ser Tyr Ala Tyr

1 5

<210> 13

<211> 6

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(6)

<223> HCDR3 of murine mab M2

<400> 13

Tyr Tyr Ser Tyr Ala Tyr

1 5

<210> 14

<211> 6

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(6)

<223> HCDR3 of murine mab M3

<400> 14

Phe Tyr Ser Phe Thr Tyr

1 5

<210> 15

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(16)

<223> LCDR1 of murine mab M1,M2

<400> 15

Arg Ser Ser Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu Gln

1 5 10 15

<210> 16

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(16)

<223> LCDR1 of murine mab M3

<400> 16

Arg Ser Ser Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His

1 5 10 15

<210> 17

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(7)

<223> LCDR2 of murine mab M1,M2

<400> 17

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 18

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(7)

<223> LCDR2 of murine mab M3

<400> 18

Lys Val Ser Asn Arg Leu Ser

1 5

<210> 19

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(9)

<223> LCDR3 of murine mab M1,M2

<400> 19

Ser Gln Ser Ile His Val Pro Tyr Thr

1 5

<210> 20

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> DOMAIN

<222> (1)..(9)

<223> LCDR3 of murine mab M3

<400> 20

Ser Gln Asn Ser His Val Pro Tyr Thr

1 5

<210> 21

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(115)

<223> HCVR of Ab1,Ab8

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asp Pro Lys Thr Gly Ser Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Phe Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 22

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(112)

<223> LCVR of Ab1,Ab8

<400> 22

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Pro

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Ile His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 23

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(115)

<223> HCVR of Ab2,Ab9

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Arg Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 24

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(115)

<223> HCVR of Ab3,Ab10

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asp Pro Lys Thr Asp Ser Thr Ala Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 25

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(112)

<223> LCVR of Ab3,Ab10

<400> 25

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 26

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(115)

<223> HCVR of Ab7,Ab14

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asp Pro Lys Thr Gly Ser Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Phe Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 27

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(445)

<223> HC of Ab1

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asp Pro Lys Thr Gly Ser Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Phe Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 28

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(219)

<223> LC of Ab1

<400> 28

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Pro

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Ile His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 29

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(445)

<223> HC of Ab2

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Arg Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 30

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(445)

<223> HC of Ab3

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asp Pro Lys Thr Asp Ser Thr Ala Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 31

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(219)

<223> LC of Ab3

<400> 31

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 32

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (11)..(445)

<223> HC of Ab7

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asp Pro Lys Thr Gly Ser Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Phe Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 33

<211> 535

<212> PRT

<213> Homo sapiens

<220>

<221> DOMAIN

<222> (1)..(535)

<223> human GPC3 (Gln 25-His 559)

<400> 33

Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser

1 5 10 15

Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro

20 25 30

Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys

35 40 45

Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn

50 55 60

Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile

65 70 75 80

Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg

85 90 95

His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser

100 105 110

Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val

115 120 125

Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn

130 135 140

Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn

145 150 155 160

Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly

165 170 175

Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met

180 185 190

Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala

195 200 205

Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe

210 215 220

Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr

225 230 235 240

Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val

245 250 255

Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp

260 265 270

Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg

275 280 285

Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His

290 295 300

Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr

305 310 315 320

Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala

325 330 335

Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala

340 345 350

His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile

355 360 365

Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr

370 375 380

Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn

385 390 395 400

Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly

405 410 415

Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu

420 425 430

Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu

435 440 445

Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu

450 455 460

Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu

465 470 475 480

Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu

485 490 495

Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro

500 505 510

Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe

515 520 525

His Asn Leu Gly Asn Val His

530 535

<210> 34

<211> 1743

<212> DNA

<213> Homo sapiens

<400> 34

atggccggga ccgtgcgcac cgcgtgcttg gtggtggcga tgctgctcag cttggacttc 60

ccgggacagg cgcagccccc gccgccgccg ccggacgcca cctgtcacca agtccgctcc 120

ttcttccaga gactgcagcc cggactcaag tgggtgccag aaactcccgt gccaggatca 180

gatttgcaag tatgtctccc taagggccca acatgctgct caagaaagat ggaagaaaaa 240

taccaactaa cagcacgatt gaacatggaa cagctgcttc agtctgcaag tatggagctc 300

aagttcttaa ttattcagaa tgctgcggtt ttccaagagg cctttgaaat tgttgttcgc 360

catgccaaga actacaccaa tgccatgttc aagaacaact acccaagcct gactccacaa 420

gcttttgagt ttgtgggtga atttttcaca gatgtgtctc tctacatctt gggttctgac 480

atcaatgtag atgacatggt caatgaattg tttgacagcc tgtttccagt catctatacc 540

cagctaatga acccaggcct gcctgattca gccttggaca tcaatgagtg cctccgagga 600

gcaagacgtg acctgaaagt atttgggaat ttccccaagc ttattatgac ccaggtttcc 660

aagtcactgc aagtcactag gatcttcctt caggctctga atcttggaat tgaagtgatc 720

aacacaactg atcacctgaa gttcagtaag gactgtggcc gaatgctcac cagaatgtgg 780

tactgctctt actgccaggg actgatgatg gttaaaccct gtggcggtta ctgcaatgtg 840

gtcatgcaag gctgtatggc aggtgtggtg gagattgaca agtactggag agaatacatt 900

ctgtcccttg aagaacttgt gaatggcatg tacagaatct atgacatgga gaacgtactg 960

cttggtctct tttcaacaat ccatgattct atccagtatg tccagaagaa tgcaggaaag 1020

ctgaccacca ctattggcaa gttatgtgcc cattctcaac aacgccaata tagatctgct 1080

tattatcctg aagatctctt tattgacaag aaagtattaa aagttgctca tgtagaacat 1140

gaagaaacct tatccagccg aagaagggaa ctaattcaga agttgaagtc tttcatcagc 1200

ttctatagtg ctttgcctgg ctacatctgc agccatagcc ctgtggcgga aaacgacacc 1260

ctttgctgga atggacaaga actcgtggag agatacagcc aaaaggcagc aaggaatgga 1320

atgaaaaacc agttcaatct ccatgagctg aaaatgaagg gccctgagcc agtggtcagt 1380

caaattattg acaaactgaa gcacattaac cagctcctga gaaccatgtc tatgcccaaa 1440

ggtagagttc tggataaaaa cctggatgag gaagggtttg aaagtggaga ctgcggtgat 1500

gatgaagatg agtgcattgg aggctctggt gatggaatga taaaagtgaa gaatcagctc 1560

cgcttccttg cagaactggc ctatgatctg gatgtggatg atgcgcctgg aaacagtcag 1620

caggcaactc cgaaggacaa cgagataagc acctttcaca acctcgggaa cgttcattcc 1680

ccgctgaagc ttctcaccag catggccatc tcggtggtgt gcttcttctt cctggtgcac 1740

tga 1743

<210> 35

<211> 1743

<212> DNA

<213> Macaca mulatta

<400> 35

atggctggca ctgtcagaac tgcttgtctg gtggtcgcta tgctgctgtc actggatttc 60

cccggtcagg ctcagccccc tcctcctcca cctgacgcta cctgccacca agtgaggtct 120

ttctttcaga gactgcagcc cggcctgaag tgggtgcctg agacaccagt gccaggaagc 180

gatctgcaag tgtgtctgcc caagggccct acctgctgtt ctcgcaagat ggaggagaag 240

taccagctga cagccaggct gaacatggag cagctgctgc agtctgcttc catggagctg 300

aagttcctga tcatccagaa tgccgctgtg ttccaggagg cctttgagat cgtcgtgagg 360

catgccaaga actacacaaa cgctatgttc aagaacaact acccttccct gaccccacag 420

gctttcgagt ttgtgggcga gttctttaca gacgtgagcc tgtacatcct gggctctgat 480

atcaacgtgg acgatatggt gaatgagctg ttcgacagcc tgtttcccgt gatctatacc 540

cagctgatga acccaggcct gcccgactct gccctggata tcaatgagtg cctgagggga 600

gctaggcggg atctgaaggt gttcggcaac tttcctaagc tgatcatgac acaggtgagc 660

aagtctctgc aggtgaccag gatcttcctg caggccctga acctgggcat cgaggtgatc 720

aataccacag accacctgaa gttttccaag gattgcggcc gcatgctgac caggatgtgg 780

tactgcagct attgtcaggg cctgatgatg gtgaagccat gcggcggcta ctgtaacgtg 840

gtgatgcagg gctgtatggc tggcgtggtg gagatcgaca agtactggcg ggagtatatc 900

ctgagcctgg aggagctggt gaacggcatg tacagaatct atgacatgga gaatgtgctg 960

ctgggcctgt tctctacaat ccacgattcc atccagtacg tgcagaagaa tgccggcaag 1020

ctgaccacaa ccatcggcaa gctgtgtgcc cattcccagc agcggcagta tagaagcgct 1080

tactatcccg aggacctgtt tatcgataag aaggtgctga aggtggctca cgtggagcat 1140

gaggagacac tgtccagcag acgcagggag ctgatccaga agctgaagtc tttcatctcc 1200

ttttacagcg ccctgccagg ctatatctgc tcccatagcc ccgtggctga gaacgacaca 1260

ctgtgttgga atggccagga gctggtggag cgctactctc agaaggccgc taggaacggc 1320

atgaagaacc agttcaatct gcacgagctg aagatgaagg gccctgagcc agtggtgtcc 1380

cagatcatcg ataagctgaa gcatatcaac cagctgctgc ggaccatgag cgtgcccaag 1440

ggcagagtgc tggacaagaa tctggatgag gagggcttcg agtctggcga ctgcggcgac 1500

gatgaggatg agtgtatcgg cggctccggc gacggcatga tgaaggtgaa gaaccagctg 1560

cggtttctgg ccgagctggc ttatgacctg gatgtggacg atgtgcctgg caacaatcag 1620

caggctacac caaaggataa tgagatctct acctttcaca acctgggcaa tgtgcattcc 1680

cctctgaaac tgctgacaag tatggcaatc tccgtcgtct gcttcttctt cctggtccac 1740

taa 1743

<210> 36

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(445)

<223> HC of Ab8

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asp Pro Lys Thr Gly Ser Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Phe Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 37

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(445)

<223> HC of Ab9

<400> 37

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Arg Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 38

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(445)

<223> HC of Ab10

<400> 38

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr

20 25 30

Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asp Pro Lys Thr Asp Ser Thr Ala Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 39

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(445)

<223> HC of Ab14

<400> 39

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asp Pro Lys Thr Gly Ser Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Phe Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 40

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(330)

<223> Heavy chain constant region of IgG1 variant

<400> 40

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 41

<211> 330

<212> PRT

<213> Homo sapiens

<220>

<221> DOMAIN

<222> (1)..(330)

<223> Heavy chain constant region of IgG1

<400> 41

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 42

<211> 107

<212> PRT

<213> Homo sapiens

<220>

<221> DOMAIN

<222> (1)..(107)

<223> Constant region of κ chain

<400> 42

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims

1. An anti-GPC 3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises: respectively shown in SEQ ID NO: 7. the amino acid sequence of SEQ ID NO:10 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3; the light chain variable region comprises: respectively shown in SEQ ID NO: 15. the amino acid sequence of SEQ ID NO:17 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in 19.

2. The anti-GPC 3 antibody or the antigen-binding fragment thereof of claim 1, wherein the antibody is a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, and a humanized antibody or fragment thereof.

3. The anti-GPC 3 antibody or antigen-binding fragment thereof of any of claims 1-2, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, igG3, or IgG4, or a variant thereof.

4. The anti-GPC 3 antibody or antigen-binding fragment thereof of claim 3, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, or IgG4.

5. The anti-GPC 3 antibody or antigen-binding fragment thereof of claim 4, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from a human IgG 1.

6. The anti-GPC 3 antibody or antigen-binding fragment thereof of claim 5, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises an amino acid sequence set forth in SEQ ID NO:41, or a heavy chain constant region as set forth in SEQ ID NO:40, or a heavy chain constant region variant thereof.

7. The anti-GPC 3 antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, lambda chain, or a variant thereof.

8. The anti-GPC 3 antibody or an antigen-binding fragment thereof of claim 7, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain.

9. The anti-GPC 3 antibody or an antigen-binding fragment thereof of claim 8, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a light chain constant region as set forth in SEQ ID No. 42.

10. The anti-GPC 3 antibody or the antigen-binding fragment thereof of any one of claims 1 to 9, comprising:

selected from the group consisting of the heavy chain variable regions represented by seq id nos, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95% or 99% identity compared to seq id no: SEQ ID NO:23;

and/or is selected from the group consisting of the light chain variable region represented by seq id no, or a light chain variable region having at least 70%,75%,80%,85%,90%,95% or 99% identity compared to seq id no: SEQ ID NO:22.

11. the anti-GPC 3 antibody or the antigen-binding fragment thereof of any one of claims 1 to 10, comprising: SEQ ID NO:23 and SEQ ID NO:22, or a light chain variable region as shown in fig.

12. The anti-GPC 3 antibody or antigen-binding fragment thereof of claim 10 or 11, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of those shown in seq id nos, or comprises a heavy chain having at least 80%,85%,90%,95%, or 99% identity compared to seq id nos: SEQ ID NO:29 or SEQ ID NO:37.

13. the anti-GPC 3 antibody or antigen-binding fragment thereof of claim 10 or 11, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof comprises a light chain selected from the group consisting of those set forth in seq id nos, or comprises a light chain that is at least 80%,85%,90%,95%, or 99% identical compared to the seq id nos: SEQ ID NO:28.

14. the anti-GPC 3 antibody or the antigen-binding fragment thereof of any one of claims 1-13, comprising:

SEQ ID NO:29, and the heavy chain set forth in SEQ ID NO:28, a light chain; or the like, or a combination thereof,

SEQ ID NO:37, and SEQ ID NO:28, or a light chain as shown.

15. A polynucleotide encoding the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1 to 14.

16. An expression vector comprising the polynucleotide of claim 15.

17. A host cell into which the expression vector of claim 16 is introduced or which contains the expression vector.

18. The host cell of claim 17, which is a bacterial, yeast or mammalian cell.

19. The host cell of claim 18, which is an escherichia coli, pichia pastoris, CHO cell, or HEK293 cell.

20. A method of producing an anti-GPC 3 antibody or an antigen-binding fragment thereof, comprising:

culturing the host cell of any one of claims 17-19;

isolating the antibody from the culture; and

purifying the antibody.

21. The method of claim 20, comprising:

culturing HEK293 cells;

isolating the antibody from the cell culture fluid; and

the antibody is purified by chromatographic methods.

22. A pharmaceutical composition comprising:

the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1 to 14, and

a pharmaceutically acceptable excipient, diluent or carrier.

23. A detection or diagnostic kit comprising:

the anti-GPC 3 antibody or antigen-binding fragment thereof of any one of claims 1-14, one or more agents capable of detecting binding of the anti-GPC 3 antibody or antigen-binding fragment thereof to GPC3.

24. Use of any one of the following in the manufacture of a medicament for treating or preventing a GPC 3-mediated disease or condition:

the anti-GPC 3 antibody or the antigen-binding fragment thereof according to any one of claims 1 to 14, the pharmaceutical composition according to claim 22.

25. Use of any one of the following in the preparation of a kit:

the anti-GPC 3 antibody or the antigen-binding fragment thereof of any one of claims 1 to 14, the pharmaceutical composition of claim 22;

wherein the kit is used for detecting, diagnosing and prognosing a GPC 3-mediated disease or condition.

26. The use according to claim 24 or 25, wherein:

the disease or disorder is cancer.

27. The use according to claim 26, wherein:

the disease or disorder is a GPC 3-expressing cancer.

28. The use according to claim 27, wherein:

the cancer is selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma.

29. An anti-GPC 3 antibody or an antigen-binding fragment thereof according to any of claims 1 to 14 or a pharmaceutical composition according to claim 22 for use in the detection, diagnosis, prognosis of GPC 3-mediated diseases.

30. The anti-GPC 3 antibody or an antigen-binding fragment thereof or the pharmaceutical composition according to claim 29, for detecting, diagnosing, prognosing a GPC 3-mediated disease selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gall bladder cancer, glioblastoma, and melanoma.

31. Use of any one of the following in the manufacture of a medicament for treating or preventing a GPC 3-mediated disease:

the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1-14; or the pharmaceutical composition of claim 22;

wherein said prevention and/or treatment is embodied by providing a therapeutically effective amount or a prophylactically effective amount of the medicament to the subject.

32. The use according to claim 31, wherein:

the GPC 3-mediated disease is selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma.