CN113227148A

CN113227148A - anti-GPC 3 antibody, antigen-binding fragment thereof, and medicinal use thereof

Info

Publication number: CN113227148A
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: 2021-08-06
Anticipated expiration: 2040-12-03
Also published as: WO2021110095A1; TW202134286A; CN113227148B

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 medicinal use thereof

The present application claims priority from a chinese patent application (application No. 201911236102.2) filed on 12/05/2019 and a chinese patent application (application No. 202010910640.1) filed on 09/02/2020.

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 cytostatic agent and an anticancer drug, and for detecting or diagnosing tumors.

Background

Glypican 3(Glypican3, GPC3) is an approximately 70kd membrane protein, belongs to the family of Glypicans, and exerts a cell adhesion effect as an extracellular matrix protein in organ formation or a receptor of a cell growth factor. After expression of GPC3, it is cleaved by furin enzyme to generate an N-terminal approximately 40kDa soluble portion and an approximately 30kDa portion, which is anchored to the C-terminal of the cell membrane by 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), Carcinoembryonic antigen (CEA), and the like. That is, 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-conjugated 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 part of the hepatocellular carcinoma development process.

The current macromolecular GPC 3-targeted drug that enters the clinical stage and discloses clinical outcomes is Codrituzumab developed by roche (cotrutuzumab; alias GC33), 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 induced antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and was shown to have significant tumor growth-inhibiting effects in a HuH-7 tumor pharmacodynamic mouse model. GC33 is in clinical stage II (NCT01507168), and has indications of 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: 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: 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 variable region of the antibody light chain comprises at least 1 LCDR2 selected from the following sequences: SEQ ID NO: 17, SEQ ID NO: 18,

and the variable region of the antibody light chain comprises at least 1 LCDR3 selected from the following sequences: 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:

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

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

Or as shown in SEQ ID NO: 8. SEQ ID NO: 11 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 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 shown at 19;

or as shown in SEQ ID NO: 16. SEQ ID NO: 18 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 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: respectively shown in SEQ ID NO: 7. SEQ ID NO: 9 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 12; the light chain variable region comprises: respectively shown in SEQ ID NO: 15SEQ ID NO: 17 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 19.

The present application also relates to an 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 sequence identical to SEQ ID NO: 7 compared with HCDR1 with 1-3 amino acid mutations,

SEQ ID NO: 9 or a sequence identical to SEQ ID NO: 9 HCDR2 with 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;

the light chain variable region comprises:

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

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

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

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 shown at 19.

the heavy chain variable region comprises:

SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10 compared to HCDR2 with 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:

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

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 HCDR2 having 1-3 amino acid mutations compared to SEQ ID NO: 14 or a sequence identical to SEQ ID NO: HCDR3 with 1-3 amino acid mutations compared to 14;

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

the heavy chain variable region comprises: SEQ ID NO: 7 or a sequence identical to SEQ ID NO: 7 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; 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: 7. SEQ ID NO: 10 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13; 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 shown at 20.

the heavy chain variable region comprises: SEQ ID NO: 7 or a sequence identical to SEQ ID NO: 7 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: respectively shown in SEQ ID NO: 15. SEQ ID NO: 17 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 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 HCDR2 having 1-3 amino acid mutations compared to SEQ ID NO: 14 or a sequence identical to SEQ ID NO: HCDR3 with 1-3 amino acid mutations compared to 14; the light chain variable region comprises: SEQ ID NO: 15 or a variant of SEQ ID NO: 15 compared to LCDR1, 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 antigen-binding fragment thereof as described above, 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.

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 IgG 4.

In particular 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 a sequence set forth as 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 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 particular 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 antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or 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 a light chain constant region as set forth in SEQ ID NO: 42.

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 variable region selected from the group consisting of seq id nos: 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 antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the sequence: 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 values.

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 light chain variable region selected from the group consisting of seq id nos: 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 antigen-binding fragment thereof as described above, wherein the light chain variable region of the anti-GPC 3 antibody or 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, 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: 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, 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: 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: 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, alternatively,

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 antigen-binding fragment thereof as described above, wherein the anti-GPC 3 antibody or 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 compared to the 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 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 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:

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,

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,

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,

SEQ ID NO: 32, 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: 32, 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.

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 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: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, alternatively,

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, alternatively,

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, alternatively,

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,

SEQ ID NO:38, 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,

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, alternatively,

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 is 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 the anti-GPC 3 antibody or 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 specific 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 broth); and purifying the antibody (e.g., purifying the antibody by chromatography).

The present application further provides a pharmaceutical composition comprising the anti-GPC 3 antibody or an antigen-binding fragment thereof 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 reagents capable of detecting the binding of the anti-GPC 3 antibody or antigen-binding fragment thereof to GPC3 (or an epitope thereof).

In a particular 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 or a pharmaceutically acceptable salt thereof as described herein, 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 disorder.

In a particular embodiment of the present application, the use of an anti-GPC 3 antibody or 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 preparation of a reagent or kit for detecting, diagnosing, prognosing a GPC 3-mediated disease or disorder.

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 for detecting, diagnosing, prognosing 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 a therapeutically effective amount, or a prophylactically effective amount, of an anti-GPC 3 antibody, or an antigen-binding fragment thereof, as described above, to a subject; 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.

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

In addition, the anti-GPC 3 antibody or antigen-binding fragment thereof of the present application has good endocytosis and is suitable for conjugation with a drug. The anti-GPC 3 antibody or antigen-binding fragment disclosed by 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 humanized antibody Ab9 heavy chain variable region and the GC33 antibody heavy chain variable region correspond to the human germline template sequences (note: amino acids in which the antibody does not correspond to the corresponding human germline template are marked in gray shading).

FIG. 2: the humanized antibody Ab9 light chain variable region and the GC33 antibody light chain variable region correspond to the human germline template sequences (note: amino acids in which the antibody does not correspond to the corresponding human germline template are marked in gray shading).

Detailed Description

Term(s) for

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 formed by 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, 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 igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. 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 human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being 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, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments described herein are in numbers and positions conforming to 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 "GPC 3" 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 also 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 a cell or tissue 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. Preparation is accomplished by injecting the test subject with the GPC3 antigen and then isolating the hybridomas that express the antibodies with the desired sequence or functional properties. In a specific embodiment of the present application, the murine GPC3 antibody or 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 is capable of reducing an 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 IgG1, IgG2, IgG3, or IgG4 of a second species, or a variant thereof; specifically, 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')₂Fv fragments, linear antibodies (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.

A "Fab fragment" consists of one light chain, one heavy chain CH1 and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

"Fab' fragments"A portion comprising one light chain and one heavy chain (comprising 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 that contains 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 GPC 3" 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 the 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 typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after denaturing solvent treatment. Epitopes typically 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 those 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) is present at about less than 10 as measured by Surface Plasmon Resonance (SPR) techniques in the instrument^-7M or even smaller equilibrium dissociation constant (K)_D) Binding to a predetermined antigen; and, the antibody (or antigen-binding fragment) binds to the predetermined antigen with at least twice its affinity to a non-specific antigen (e.g., 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 GPC 3. 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 anti-GPC 3 antibody compared to ligand not contacted with anti-GPC 3 antibody.

The term "inhibit growth" (e.g., in relation to a cell) is intended to include any measurable decrease in cell growth.

The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induction" with respect to induction of CDC or ADCC refers to 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 modification of the Fc-domain 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 test technical guide of cold spring harbor, chapters 5-8 and 15. For example, mice can be immunized with human GPC3 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using 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 facesBook 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 either 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, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques.

"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 "treating" may 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, for example, 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 useful 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 may 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 context.

"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 conventional reagents are 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, Gln 25 to His 559 fragment), with a His-tag at the C-terminus, human GPC3His protein, purchased from Acro biosystems (Cat # GP3-H52H 4).

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

(2) Construction of cell lines

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 full-length DNA sequence of human or monkey GPC3 synthesized in vitro (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 (three reagents were purchased from GIBCO) containing 8. mu.g/ml puromycin and 10% FBS, the expression level of GPC3 was detected by FACS, and then a high-expression monoclonal cell strain was obtained by gradient dilution.

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) the immunogen and the immunologic adjuvant are fully mixed and emulsified in a ratio of 1:1 by using Sigma Complete Freund's Adjuvant (CFA) and Sigma Incomplete Freund's Adjuvant (IFA) to prepare a stable water-in-oil liquid.

(2) Aluminum salt adjuvant with Invivo Gen ()

2 percent of adjuvant), mixing the antigen and the adjuvant according to the ratio of 1:1 to form a water-soluble solution, and then immunizing.

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 example 4(2) FACS test for the ability to bind cell surface antigens, control titer test (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 mice, and obtaining hybridomas after spleen cells and SP2/0 myeloma cells are fused; screening target hybridomas by 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 collected, RNA extracted using Trizol (ThermoFisher, 15596-Asca 026) and reverse transcribed (PrimeScript)^TMFirst strand cDNA synthesis kit, Takara # 6110A). The cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-primer set (Sigma #69831) and then sequenced, and the sequences of murine antibodies M1, M2 and M3 were 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 a fragment of "NGN" sequence, which is 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" and thus 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-H52H4) 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 (pH7.4PBS containing 0.05% Tween-20), 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, 100nM initial, 5-fold gradient diluted, 11 doses, 100. mu.L/well to the microplate, and incubated at 37 ℃ for 1 hour. After the incubation was completed, the plate was washed 3 times with PBST, 200. mu.L/well of an HRP-labeled secondary goat-anti-mouse antibody (Jackson ImmunoResearch Laboratories, cat # 115-. 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: murine antibodies have good affinity for the human GPC3 antigen.

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

Murine antibodies	ELISA，EC ₅₀(nM)
M1	0.05
M2	0.03
M3	0.07

Example 4: mouse antibody chimerization assay

And (3) carrying out chimerization on the positive murine antibody obtained by screening, 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 the heavy chain constant region of human IgG1 and the constant region of the human kappa chain.

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 plasmids that were verified to be acceptable were transfected into HEK293 cells (see kyoha) with TF1 and cultured continuously. HEK293 cells were cultured to logarithmic growth phase with serum-free CD medium (Cat # SMM 293-TI, yokehou, seiko) for cell transfection. Mu.g of light chain plasmid and 23.6. mu.g of heavy chain plasmid were dissolved in 10mL Reduced Serum Medium (GIBCO, 31985-. Cell culture conditions: 5% CO2, 37 ℃, 125 rpm/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 antibodies 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 (10mM sodium acetate, pH 5.0), using approximately 100. mu.L mouse anti-human IgG (Fc) antibody per channel on the chip, and adding approximately 10. mu.L mouse anti-human IgG (Fc) antibody using 190. mu.L immobilized reagent for both channels. First, the surface of the CM5 chip was activated with 400mM EDC and 100mM NHS at a flow rate of 10. mu.L/min for 420 s. Next, 25. mu.g/mL of a mouse anti-human IgG (Fc) antibody was injected into the experimental channel (FC4) at a flow rate of 10. mu.L/min for about 420s, at a fixed amount of about 9000 to 14000 RU. Finally, the chip was blocked with 1M ethanolamine at 10. mu.L/min for 420 s. The reference channel (FC3) was subjected to the same procedure as the test channel (FC 4).

Capturing a ligand: the antibody stock was diluted to 4. mu.g/mL with running reagents, respectively, and injected into the assay channel (FC4) at a flow rate of 10. mu.L/min to capture approximately 200 RU. The reference channel (FC3) did 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 injected into the experimental channel and the reference channel at flow rates of 30 μ L/min in order of corresponding 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 (FC3) 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 showed that the chimeric antibody had good affinity to human GPC3 antigen.

(2) In vitro cell binding experiments:

GPC3 high expression cells including CHO-K1 cell (ATCC, Cat # CCL-61) overexpressing human or monkey GPC3, and human hepatoma cell JHH-7 (Nanjing Kobai, Cat # CBP60204), HepG2 (Capt # SCSP-510) expressing GPC3, trypsinized, centrifuged to collect the cells, adjusted in cell density with FACS buffer (1 XPBS containing 2% FBS), and plated in 96-well U-bottom plates at 1X 10/well⁵To 2X 10⁵And (4) cells. Centrifuging: 1200g, 5min, discard the supernatant, add 100. mu.L of antibody solution that has been diluted with a FACS buffer gradient (antibody initial working concentration 100nM, 5 fold dilution, 7 concentration points, and set 0nM points), 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 100. mu.L per well, 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 cells expressing GPC 3.

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) 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

The humanized antibody Ab9 heavy chain variable region had a sequence identity of 81.7% and a sequence similarity of 87.0% 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 humanized antibody Ab9 and the heavy chain variable region of 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 had 86.6% sequence identity and 89.3% similarity to its human germline template (germline 3). The Ab9 light chain variable region corresponded to its human germline template (germline 2) and the GC33 light chain variable region to its human germline template (germline 3) as 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 enhanced ADCC after amino acid mutation or the natural human IgG1 heavy chain constant region with the sequences as 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 to 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 cells expressing GPC 3.

Example 7: endocytosis of humanized antibodies

Whether the antibody of the present application can be engulfed into cells together with human GPC3 after binding to GPC3 was examined, and evaluated by the stable transformant CHO-K1-human GPC 3.

Cell useTrypsinizing, collecting cells and resuspending with pre-cooled FACS buffer to adjust cell concentration to 1X 10⁶one/mL. Taking an EP tube, adding 1mL of cell suspension, centrifuging at 1500rpm for 5 minutes, then discarding the supernatant, adding 1mL of prepared antibody to be detected for resuspension of cells, wherein the final concentration of the antibody is 20 mu g/mL, incubating for 1 hour at 4 ℃ in a shaking table, centrifuging, discarding the supernatant (4 ℃, 1500rpm multiplied by 5 minutes), washing twice by FACS buffer solution, and discarding the supernatant. mu.L of a fluorescently labeled secondary antibody working solution PE anti-human IgG Fc antibody (Biolegend, Cat # 409304) or FITC anti-mouse IgG antibody (Biolegend, Cat # 406001) was added to each tube, the cells were resuspended, incubated at 4 ℃ for 30 minutes in a shaker, centrifuged to discard the supernatant (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. To all treatment groups, except the 0-minute group, 250. mu.L of strip buffer was added to the EP tubes, incubated at room temperature for 8 minutes, centrifuged to discard the supernatant (4 ℃ C., 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, 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 transfused cell strain CHO-K1-human GPC 3.

Example 8: ADCC assay of humanized antibodies

The effector cell of the experiment is NK92MI (NK 92 cell over expressing IL-2 gene, purchased from Nanjing Kebai), and the target cell is human hepatoma cell HepG 2. The experimental medium was RPMI1640 with 10% FBS for cell and antibody resuspension or dilution. HepG2 cells were trypsinized, harvested by centrifugation, and resuspended at 1X 10 in experimental medium⁵Each 100. mu.L of each antibody 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, and incubated at 4 ℃ for 15 minutes. NK92MI cells were collected and resuspended at 1X 10 in experimental medium⁶mu.L/ml, 50. mu.L was added to the above reaction so that the ratio of effector cells to target cells was 5:1, and the incubations were carried out at 37 ℃ for 3-4 hours. 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) × 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 of the experiment is a stable cell strain CHO-K1-hGPC3 over-expressing human GPC 3. The experimental medium was FBS-free cell culture medium F12K (GIBCO) for cell and antibody resuspension or dilution.

CHO-K1-hGPC3 cells were trypsinized, harvested 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 20nM, 5-fold dilution, 10 concentration points, and 0nM point set up) was added, incubation was carried out at 37 ℃ for 30 min, and 25 μ L of 80% human serum (GemCell) was added^TMUS, Cat # 100-. Add 50. mu.L CellTiter-Glo (Pr)omega, Cat # G7573), mixed well, and reacted for 10 minutes at room temperature in the dark, and read by a multifunctional microplate reader (Thermofeisher, Lux).

Percent (%) antibody killing (E-S)/(E-M) × 100

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

s is the value of the sample well, i.e. cell + antibody + human serum;

m is the number of medium + human serum wells.

TABLE 19 CDC Effect of humanized antibodies

Humanized antibodies	Cells	IC ₅₀(nM)
Ab9	CHO-K1-hGPC3	0.308

The results show that: the humanized antibody Ab9 had significant CDC effects.

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).

Injecting a mixture of a sample with amphoteric groups, an ampholyte, a buffering agent and an auxiliary additive into a capillary, wherein when a direct-current voltage is applied to two ends of the capillary, the ampholyte as a carrier can form a pH gradient in a certain range in the capillary, sample components can migrate to a cathode or an anode according to the charged electricity of the sample components, 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, and the components can be gathered at the point on a macroscopic scale and can not further migrate, so that the aim of separating the components in the complex sample is fulfilled. And after the atlas is collected, the pI value and the peak ratio (main peak, acid peak and alkali peak) of the sample are obtained according to the linear relation between the Marker pI value and the migration time of the chromatographic peak. The method mainly comprises the following steps:

system adaptive sample preparation: taking out the adaptive sample tube in the Maurice cIEF System reliability Kit (Protein Simple, Cat #046-044), adding 40 mu l of deionized water and 160 mu l of System reliability Test Mix, uniformly mixing, transferring into a 1.5ml centrifuge tube, performing vortex oscillation, centrifuging, taking 160ul of supernatant, and transferring to a 96-well sample plate for later 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 1. mu.l each of the corresponding two pI markers 6.14(Protein Simple, Cat:046-031) and 9.99(Protein Simple, Cat:046-034), in a total volume of 80. mu.l.

Preparing a test sample: the sample was set up for 3 conditions: starting at 0 point; standing in a stability test chamber (Memmer, type HPP 1060) at a humidity of 65% and a temperature of 25 deg.C for 1 month; the plates were kept for 1 month in a stability chamber (Memmer, model HPP 1060) at a humidity of 65% and a temperature of 40 ℃. Taking 20 ul of corresponding sample, adding into the EP tube containing 80ul of cIEF Master Mix solution in the step 2), vortexing, shaking, mixing, centrifuging, taking 80ul of supernatant, transferring to a 96-well sample plate, and centrifuging for later 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-MC02-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: the Ab9 showed less change in the proportion of the main peak after one month at 25 ℃ and better stability than the initial 0 point. 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 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 result of the purity of the sample after non-reduction and reduction treatment.

The method mainly comprises the following steps:

sample preparation: the sample was sampled at the initial 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, and sampling; the samples were taken after 1 month in a stability chamber (Memmer, model HPP 1060) at a humidity of 65% and a temperature of 40 ℃.

Non-reduced CE sample treatment: samples were added to the EP tubes at a Protein loading of 50. mu.g per sample, 1. mu.L of 10kD internal standard (Protein Simple, Cat # 046-.

Treatment of reduced CE samples: samples were added to the EP tubes at a Protein loading of 50. mu.g per sample, 1. mu.L of 10kD internal standard (Protein Simple, Cat # 046-.

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

Sample detection: putting a 96-well sample plate into a capillary electrophoresis apparatus (Protein Simple, Maurice), opening the apparatus and software, carrying out self-inspection of the apparatus according to the operation steps of the apparatus, installing a capillary cartridge (Protein Simple, Cat # 090-. 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 show that the humanized antibody Ab9 has a similar proportion of the main peak at 25 ℃ for one month as the 0 point, and the main peak is 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.

Claims

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 at least 1 HCDR1 selected from the group consisting of seq id nos: SEQ ID NO: 7, SEQ ID NO: 8; at least 1 HCDR2 selected from the group consisting of seq id nos: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; and at least 1 HCDR3 selected from the group consisting of seq id nos: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14; and is

The light chain variable region comprises at least 1 LCDR1 selected from the group consisting of seq id nos: SEQ ID NO: 15, SEQ ID NO: 16; at least 1 LCDR2 selected from the group consisting of those represented by the following sequences: SEQ ID NO: 17, SEQ ID NO: 18; and at least 1 LCDR3 selected from the group consisting of those shown in seq id no: SEQ ID NO: 19, SEQ ID NO: 20.
the anti-GPC 3 antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises:

respectively shown in SEQ ID NO: 7. SEQ ID NO: 9 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 12; or the like, or, alternatively,

respectively shown in SEQ ID NO: 7. SEQ ID NO: 10 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13; or the like, or, alternatively,

respectively shown in SEQ ID NO: 8. SEQ ID NO: 11 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 14.
The anti-GPC 3 antibody or antigen-binding fragment thereof of claim 1, wherein 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 shown at 19; or the like, or, alternatively,

respectively shown in SEQ ID NO: 16. SEQ ID NO: 18 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 20.
The anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1-3, wherein:

the heavy chain variable region comprises: respectively shown in SEQ ID NO: 7. SEQ ID NO: 9 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 12; 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 shown at 19, or,

the heavy chain variable region comprises: respectively shown in SEQ ID NO: 7. SEQ ID NO: 10 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13; 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 shown at 19, or,

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

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

the heavy chain variable region comprises: respectively shown in SEQ ID NO: 7. SEQ ID NO: 10 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13; 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 shown at 20, or,

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 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 shown at 19.
The anti-GPC 3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, 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.
The anti-GPC 3 antibody or antigen-binding fragment thereof of any one of claims 1-5, 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;

preferably, the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, or IgG 4;

more preferably, the anti-GPC 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG 1;

further preferably, 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 as set forth in SEQ ID NO: 40, or a heavy chain constant region variant thereof.
The anti-GPC 3 antibody or antigen-binding fragment thereof of any one of claims 1-6, 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;

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 a light chain constant region as set forth in SEQ ID NO: 42.
The anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1-7, 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: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26;

and/or is selected from the group consisting of the light chain variable region set forth in 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 or SEQ ID NO: 25.
the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1-8, comprising:

SEQ ID NO: 21 and the heavy chain variable region of SEQ ID NO: 22; or the like, or, alternatively,

SEQ ID NO: 21 and the heavy chain variable region of SEQ ID NO: 25; or the like, or, alternatively,

SEQ ID NO: 23 and SEQ ID NO: 22; or the like, or, alternatively,

SEQ ID NO: 23 and SEQ ID NO: 25; or the like, or, alternatively,

SEQ ID NO: 24 and SEQ ID NO: 22; or the like, or, alternatively,

SEQ ID NO: 24 and SEQ ID NO: 25; or the like, or, alternatively,

SEQ ID NO: 26 and the heavy chain variable region of SEQ ID NO: 22; or the like, or, alternatively,

SEQ ID NO: 26 and the heavy chain variable region of SEQ ID NO: 25, or a light chain variable region as shown.
The anti-GPC 3 antibody or antigen-binding fragment thereof of claim 8 or 9, wherein the anti-GPC 3 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of those set forth in seq id nos, or comprises a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity as compared to seq id nos: 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.
the anti-GPC 3 antibody or antigen-binding fragment thereof of claim 8 or 9, 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 having at least 80%, 85%, 90%, 95%, or 99% identity as compared to seq id nos: SEQ ID NO: 28 or SEQ ID NO: 31.
the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1-11, comprising:

SEQ ID NO: 27, and the heavy chain of SEQ ID NO: 28, a light chain; or the like, or, alternatively,

SEQ ID NO: 27, and the heavy chain of SEQ ID NO: 31, a light chain; or the like, or, alternatively,

SEQ ID NO: 29, and the heavy chain of SEQ ID NO: 28, a light chain; or the like, or, alternatively,

SEQ ID NO: 29, and the heavy chain of SEQ ID NO: 31, a light chain; or the like, or, alternatively,

SEQ ID NO: 30, and SEQ ID NO: 28, a light chain; or the like, or, alternatively,

SEQ ID NO: 30, and SEQ ID NO: 31, a light chain; or the like, or, alternatively,

SEQ ID NO: 32, and SEQ ID NO: 28, a light chain; or the like, or, alternatively,

SEQ ID NO: 32, and SEQ ID NO: 31, a light chain; or the like, or, alternatively,

SEQ ID NO:36, and SEQ ID NO: 28, a light chain; or the like, or, alternatively,

SEQ ID NO:36, and SEQ ID NO: 31, a light chain; or the like, or, alternatively,

SEQ ID NO: 37, and SEQ ID NO: 28, a light chain; or the like, or, alternatively,

SEQ ID NO: 37, and SEQ ID NO: 31, a light chain; or the like, or, alternatively,

38, and the heavy chain of SEQ ID NO: 28, a light chain; or the like, or, alternatively,

SEQ ID NO:38, and SEQ ID NO: 31, a light chain; or the like, or, alternatively,

SEQ ID NO: 39, and the heavy chain of SEQ ID NO: 28, a light chain; or the like, or, alternatively,

SEQ ID NO: 39, and the heavy chain of SEQ ID NO: 31, or a light chain as shown in figure 31.
A polynucleotide encoding the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1-12.
An expression vector comprising the polynucleotide of claim 13.
A host cell into which the expression vector of claim 14 is introduced or which contains;

preferably, the host cell is a bacterial, yeast or mammalian cell; preferably E.coli, Pichia, CHO cells or HEK293 cells.
A method of producing an anti-GPC 3 antibody or an antigen-binding fragment thereof, comprising:

culturing the host cell of claim 15, preferably a HEK293 cell;

isolating the antibody from the culture, preferably from the cell culture broth; and

the antibody is purified, preferably chromatographically.
A pharmaceutical composition comprising:

the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1 to 12, and

a pharmaceutically acceptable excipient, diluent or carrier.
A detection or diagnostic kit comprising:

the anti-GPC 3 antibody or antigen-binding fragment thereof of any one of claims 1-12,

optionally, further comprising one or more agents capable of detecting the binding of said anti-GPC 3 or antigen-binding fragment thereof to GPC3 or an epitope thereof.
Use of any one of the following in the manufacture of a medicament for the treatment or prevention of a GPC 3-mediated disease or condition:

the anti-GPC 3 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 17.
Use of any one of the following in the preparation of a kit:

the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1-12, the pharmaceutical composition of claim 17;

wherein the kit is used for detecting, diagnosing and prognosing a GPC 3-mediated disease or condition.
The use according to claim 19 or 20, wherein:

the disease or disorder is cancer;

preferably, the disease or disorder is cancer expressing GPC 3;

more preferably, the cancer is selected from the group consisting of 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.
The anti-GPC 3 antibody or an antigen-binding fragment thereof according to any one of claims 1-12 or the pharmaceutical composition of claim 17 for use in detecting, diagnosing, prognosing a GPC 3-mediated disease; preferably, the 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.
A method of treating or preventing a GPC 3-mediated disease, comprising the steps of:

providing to a subject a therapeutically effective amount or a prophylactically effective amount of the anti-GPC 3 antibody or an antigen-binding fragment thereof of any one of claims 1-12; or

Providing a therapeutically effective amount or a prophylactically effective amount of the pharmaceutical composition of claim 17 to a subject;

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