TW202134286A - An anti-gpc3 antibody, antigen-binding fragment and the pharmaceutical use thereof - Google Patents

Abstract

This application relates to an anti-GPC3 antibody, antigen-binding fragment and the pharmaceutical use thereof. Further, this application relates to chimeric antibody and humanized antibody containing the CDR regions of the anti-GPC3 antibody; and pharmaceutical composition containing the anti-GPC3 antibody or antigen-binding fragment thereof; and the use thereof as anti-cancer medicament. In particular, this application relates to a humanized anti-GPC3 antibody and the use in the preparation of medicament for the treatment of GPC3-mediated diseases or disorders, and the use as cell growth inhibitor, and the use in detection and diagnosis of tumor.

Description

Anti-GPC3 antibody, its antigen binding fragment and its medical use

This application claims the priority of the Chinese patent application (application number 201911236102.2) filed on December 5, 2019 and the Chinese patent application (application number 202010910640.1) filed on September 2, 2020.

This application relates to an anti-GPC3 antibody that is specifically immunoreactive to human GPC3, an antigen-binding fragment thereof, a chimeric antibody containing the CDR region of the anti-GPC3 antibody, a humanized antibody, and a human anti-GPC3 antibody and its The pharmaceutical composition of the antigen-binding fragment, and its use as a cell growth inhibitor and anti-cancer drug, and its use in detecting or diagnosing tumors.

Glypican 3 (Glypican 3, GPC3) is a membrane protein of approximately 70 kd, belonging to the Glypicans family. It acts as an extracellular matrix protein in organ formation and plays a role in cell adhesion or as a receptor for cell growth factors. . After GPC3 is expressed, it will be cleaved by furin enzyme to produce a soluble part of about 40kDa and a part of about 30kDa at the N-terminal, which is anchored to the C-terminal of the cell membrane by GPI molecules.

GPC3 is expressed in embryonic tissues (especially liver and kidney) and is an extracellular matrix protein related to organ formation. In adult tissues, GPC3 expression is not observed outside the placenta, but It is expressed in various cancer tissues such as hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma. It can be seen that, like alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and other proteins, GPC3 is also a protein expressed in embryonic tissues, so it is classified as an embryonic cancer antigen. That is, GPC3 is characterized in that it is not expressed in normal tissue cells but specifically expressed in cancer cells, so it can be used as a target molecule and tumor marker for cancer therapy.

In addition, genomics and functional studies have shown that GPC3 plays an important role in maintaining the activation of the Wnt pathway and the Hedgehogs pathway. For example, GPC3-conjugated acetoheparin sulfate molecules can enhance the binding of Wnts to their receptors, thereby playing an important role in maintaining the Wnt pathway. GPC3 is expressed in the brain, digestive tract, bladder, gonads and skin, and is highly expressed on the surface of hepatocellular carcinoma; Wnt pathway plays an important role in the development of hepatocellular carcinoma, such as 20% hepatocellular carcinoma β-Catenin pathway mutation and Frizzled-7 receptor Overexpression, so GPC3 may play a role in promoting the occurrence of some hepatocellular carcinomas.

The GPC3 macromolecular drug that currently enters the clinical stage and publishes clinical results is Codrituzumab (coltrastuzumab; alias GC33) developed by Roche, patent number: US20100248359, this antibody drug targets the C-terminal 524 of human GPC3 -563 amino acid, belonging to human IgG1, kappa subtype. GC33 can induce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and has a significant inhibitory effect on tumor growth in the HuH-7 tumor pharmacodynamic mouse model. GC33 is in clinical phase II (NCT01507168), and its indications include hepatocellular carcinoma (HCC), metastatic hepatocellular carcinoma, liver cancer, metastatic liver cancer, etc. However, due to poor clinical efficacy, Roche stopped the development of Codrituzumab.

The stability of the antibody is closely related to the drug effect, metabolism and safety in the human body. For example, the better the stability of the antibody, the less immunogenicity it produces.

The application provides an anti-GPC3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region.

In some embodiments, the anti-GPC3 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 one HCDR1 selected from the following sequences: SEQ ID NO: 7, SEQ ID NO: 8,

And the antibody heavy chain variable region includes at least one HCDR2 selected from the following sequences: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,

And the antibody heavy chain variable region comprises at least one HCDR3 selected from the following sequences: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14; and

The antibody light chain variable region comprises at least one LCDR1 selected from the following sequence: SEQ ID NO: 15, SEQ ID NO: 16,

And the antibody light chain variable region includes at least one LCDR2 selected from the following sequences: SEQ ID NO: 17, SEQ ID NO: 18,

And the antibody light chain variable region includes at least one LCDR3 selected from the following sequences: SEQ ID NO: 19 and SEQ ID NO: 20.

In some embodiments of the present application, the CDR sequence of the antibody or antigen-binding fragment thereof may have 1-3 amino acid mutations, where 1-3 amino acid mutations refer to any 1-3 amino acid mutations. The insertion, deletion or substitution of one amino acid; specifically, the mutation of 1-3 amino acid is an insertion or deletion of 1-3 amino acid that can optimize antibody activity, antibody stability or reduce immunogenicity Or replace.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the heavy chain variable region of the antibody comprises:

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

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

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises:

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

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises: The HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 12; the light chain variable region includes: SEQ ID NO: 15 SEQ ID NO: 17 and SEQ ID NO, respectively : LCDR1, LCDR2 and LCDR3 shown in 19.

This application also relates to a specific solution, an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region contains:

SEQ ID NO: 7 or HCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 7

SEQ ID NO: 9 or HCDR2 with 1-3 amino acid mutations compared with SEQ ID NO: 9 and

SEQ ID NO: 12 or HCDR3 with 1-3 amino acid mutations compared with SEQ ID NO: 12;

The light chain variable region contains:

SEQ ID NO: 15 or LCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 15

SEQ ID NO:17 or LCDR2 with 1-3 amino acid mutations compared with SEQ ID NO:17 and

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

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

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region contains:

SEQ ID NO: 7 or HCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 7

SEQ ID NO: 10 or HCDR2 with 1-3 amino acid mutations compared with SEQ ID NO: 10 and

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

The variable region of the antibody light chain contains:

SEQ ID NO: 15 or LCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 15

SEQ ID NO:17 or LCDR2 with 1-3 amino acid mutations compared with SEQ ID NO:17 and

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region contains:

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

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region comprises: SEQ ID NO: 8 or HCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 8, SEQ ID NO: 11 or 1 compared with SEQ ID NO: 11 -HCDR2 with 3 amino acid mutations and SEQ ID NO: 14 or HCDR3 with 1-3 amino acid mutations compared with SEQ ID NO: 14;

The light chain variable region comprises: SEQ ID NO: 16 or LCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 16, SEQ ID NO: 18 or 1 compared with SEQ ID NO: 18 -LCDR2 with 3 amino acid mutations and SEQ ID NO: 20 or LCDR3 with 1-3 amino acid mutations compared to SEQ ID NO: 20.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region comprises: SEQ ID NO: 7 or HCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 7, SEQ ID NO: 9 or 1 compared with SEQ ID NO: 9 -HCDR2 with 3 amino acid mutations and SEQ ID NO: 12 or HCDR3 with 1-3 amino acid mutations compared with SEQ ID NO: 12; the light chain variable region comprises: SEQ ID NO: 16 or LCDR1 with 1-3 amino acid mutations compared with SEQ ID NO: 16, SEQ ID NO: 18 or LCDR2 with 1-3 amino acid mutations compared with SEQ ID NO: 18 and SEQ ID NO: 20 or LCDR3 with 1-3 amino acid mutations compared with SEQ ID NO:20.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises a heavy chain variable region and a light chain variable region, wherein:

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the antibody is a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, a human antibody or a fragment thereof, and a human Chemical antibodies or fragments thereof.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant derived from human IgG1, IgG2, IgG3 or IgG4 Zone or its variants.

In a specific embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4.

In a specific embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1.

In a specific embodiment, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO:41.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a mutated IgG1 heavy chain constant region, which is compared with the parent Amino acids have enhanced ADCC toxicity. In a specific embodiment, the mutated IgG1 heavy chain constant region is shown in SEQ ID NO:40.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, a lambda chain, or Its mutants;

Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain;

More preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:42.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequences: SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26.

This application also relates to a preferred solution, an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof contains at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical heavy chain variable region: SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 26.

In the context of this 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 the range between any two values.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the following sequences: SEQ ID NO: 22 or SEQ ID NO: 25.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the anti-GPC3 antibody or antigen-binding fragment thereof comprises at least 70% compared with the following sequence , 75%, 80%, 85%, 90%, 95%, or 99% identical light chain variable region: SEQ ID NO: 22 or SEQ ID NO: 25.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises:

The heavy chain variable region shown in SEQ ID NO: 21, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity therewith, and SEQ ID NO: the light chain variable region shown by 22, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to it; or,

The heavy chain variable region shown in SEQ ID NO: 21, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity therewith, and SEQ ID NO: the light chain variable region shown by 25, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to it; or,

The heavy chain variable region shown in SEQ ID NO: 23, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity therewith, and SEQ ID NO: the light chain variable region shown by 22, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to it; or,

The heavy chain variable region shown in SEQ ID NO: 23, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity therewith, and SEQ ID NO: the light chain variable region shown by 25, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to it; or,

The heavy chain variable region shown in SEQ ID NO: 24, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity therewith, and SEQ ID NO: the light chain variable region shown by 22, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to it; or,

The heavy chain variable region shown in SEQ ID NO: 24, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity therewith, and SEQ ID NO: the light chain variable region shown by 25, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to it; or,

The heavy chain variable region shown in SEQ ID NO: 26, or a heavy chain variable region having at least 70%, 5%, 80%, 85%, 90%, 95%, or 99% identity therewith, and SEQ ID NO: the light chain variable region shown by 22, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to it; or,

The heavy chain variable region shown in SEQ ID NO: 26, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity therewith, and SEQ ID The light chain variable region shown by NO: 25, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to it.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises:

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

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

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

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

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

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

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

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises at least 80%, 85%, 90% compared with the following sequence: , 95%, 99% or 100% identical heavy chain: 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-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises at least 80%, 85%, 90% compared with the following sequence: , 95%, 99% or 100% identical light chain: SEQ ID NO: 28 or SEQ ID NO: 31.

In some other embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises:

The heavy chain shown in SEQ ID NO: 27, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 27, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 29, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 29, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 30, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 30, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 32, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity therewith, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 32, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain that is at least 80%, 85%, 90%, 95%, or 99% identical.

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody comprises:

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

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

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

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

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

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

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

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

In some embodiments of the present application, there is provided an anti-GPC3 antibody or antigen-binding fragment thereof as described above, which comprises:

The heavy chain shown in SEQ ID NO: 36, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 36, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 37, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 37, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 38, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 38, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 39, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 28, or a heavy chain with A light chain with at least 80%, 85%, 90%, 95%, or 99% identity; or,

The heavy chain shown in SEQ ID NO: 39, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the heavy chain, and the light chain shown in SEQ ID NO: 31, or a heavy chain with A light chain that is at least 80%, 85%, 90%, 95%, or 99% identical.

This application also relates to some embodiments, providing an anti-GPC3 antibody or antigen-binding fragment thereof as described above, wherein the anti-GPC3 antibody comprises:

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

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

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

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

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

The application further provides a polynucleotide encoding the anti-GPC3 antibody or antigen-binding fragment thereof as described above.

The application further provides an expression vector containing the polynucleotide as described above.

The application further provides a host cell, which is introduced or contains the expression vector as described above.

In a specific embodiment of the present application, a host cell as described above, wherein the host cell is bacteria, specifically Escherichia coli.

In a specific embodiment of the present application, a host cell as described above, wherein the host cell is yeast, specifically Pichia pastoris.

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

This application further provides a method for producing an anti-GPC3 antibody or an antigen-binding fragment thereof, comprising: culturing the above-mentioned host cell (for example, HEK293 cell); separating the antibody from the culture (for example, separating the antibody from the cell culture fluid); and The antibody is purified (for example, the antibody is purified by chromatography).

The application further provides a medical composition, which contains the aforementioned anti-GPC3 antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable excipient, diluent or carrier.

In a further specific embodiment of the present application, a detection or diagnosis kit is provided, which contains the anti-GPC3 antibody or antigen-binding fragment thereof described in the present application, and one or more types capable of detecting the anti-GPC3 antibody or antigen A reagent that binds the fragment to GPC3 (or its epitope).

In a specific embodiment of the present application, a detection or diagnosis kit is provided, which contains the anti-GPC3 antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof described in the present application, and can be used for detection or diagnosis The labeled secondary antibody, buffer and substrate.

In a specific embodiment of the present application, there is provided a use of the anti-GPC3 antibody or its antigen-binding fragment or composition as described above in the preparation of a medicament for the treatment or prevention of GPC3-mediated Disease or condition.

In a specific embodiment of the present application, an anti-GPC3 antibody or antigen-binding fragment thereof or the composition as described above is used in the preparation of a medicament for the treatment or prevention of GPC3-mediated diseases or disorders.

In a specific embodiment of the present application, an anti-GPC3 antibody or antigen-binding fragment thereof or the composition as described above is used in the preparation of reagents or kits, wherein the reagents or kits can be used for detection and diagnosis , Prognosis of diseases or conditions mediated by GPC3.

In a specific embodiment of the present application, an anti-GPC3 antibody or antigen-binding fragment thereof or the composition as described above is used for the detection, diagnosis, and prognosis of GPC3-mediated diseases.

The application further provides a method for treating or preventing GPC3-mediated diseases, including the steps of: providing a subject with a therapeutically effective amount or a preventively effective amount of the anti-GPC3 antibody or antigen-binding fragment thereof as described above; or The person provides a therapeutically effective amount or a preventive effective amount of the aforementioned pharmaceutical composition.

The disease or disorder described in this application is cancer; specifically, the disease or disorder is GPC3-mediated cancer; further, the cancer is selected from the group consisting of 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 or melanoma.

The application provides an anti-GPC3 antibody or antigen-binding fragment, which can specifically bind to GPC3 antigen (or epitope) and GPC3-expressing cells, has significant CDC activity, and has a significant killing effect on tumors.

In addition, the anti-GPC3 antibody or antigen-binding fragment thereof of the present application has good endocytosis and is suitable for coupling with drugs. On the basis of high antibody activity, the anti-GPC3 antibody or antigen-binding fragment of the present application has lower immunogenicity and higher stability, and is significantly better than Codrituzumab. The anti-GPC3 antibody or antigen-binding fragment of the present application has better potential as an anti-cancer drug and ensures the safety of medication.

Figure 1 shows the corresponding relationship between the humanized antibody Ab9 heavy chain variable region, the GC33 antibody heavy chain variable region and its human germline template sequence (Note: The gray shading marks the amines that are inconsistent with the corresponding human germline template. Base acid).

Figure 2 shows the corresponding relationship between the humanized antibody Ab9 light chain variable region, the GC33 antibody light chain variable region and its human germline template sequence (Note: the gray shading marks the amines that are inconsistent with the corresponding human germline template. Base acid).

the term

In order to make it easier to understand this application, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this application belongs.

The three-letter codes and one-letter codes of amino acids used in this application are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" as used in this application refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, γ chain, Alpha chain and epsilon chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of its hinge area and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is classified into a kappa chain or a lambda chain by the difference in the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain.

In this application, the antibody light chain described in this application may further comprise a light chain constant region derived from human or murine κ, λ chains or variants thereof.

In this application, the antibody heavy chain described in this application may further comprise a heavy chain constant region derived from human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, arranged in sequence from the amino end to the carboxyl end. The sequence is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues of the VL region and VH region of the antibody or antigen-binding fragment described in this application comply with the known Kabat or Chothia or ABM definition rules.

In this application, unless otherwise specified, the amino acid numbering of the variable region adopts the Kabat numbering system. Those skilled in the art can determine the corresponding relationship of a specific amino acid position in different numbering systems based on common knowledge.

The term "GPC3" includes any variant or isoform of GPC3 that is naturally expressed by the cell. The antibody (or antigen-binding fragment thereof) of the present application can cross-react with GPC3 of non-human species. As another option, the antibody may also be specific for human GPC3, and may not show cross-reactivity with other species. GPC3 or any variants or isoforms thereof can be isolated from cells or tissues, or can be produced by recombinant techniques using techniques commonly used in the art and those herein. Specifically, the anti-GPC3 antibody targets human GPC3 (or its epitope) with a normal glycosylation pattern.

The term "murine antibody" in this application refers to a monoclonal antibody against human GPC3 (or its epitope) prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with GPC3 antigen, and then the fusion tumor expressing the antibody with the desired sequence or functional characteristics is isolated. In a specific embodiment of the present application, the murine GPC3 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1, IgG2, and IgG3. Or the heavy chain constant region of IgG4 or its variants.

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

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting non-human CDR sequences into the framework of a human antibody variable region. Humanized antibodies help overcome the shortcomings of strong immune responses induced by chimeric antibodies that carry non-human protein components. In order to avoid the decrease of immunogenicity and the decrease of activity, the variable region of the humanized antibody can be subjected to minimal back mutation to maintain activity.

The term "chimeric antibody" is an antibody formed by fusing the variable region of an antibody of the first species with the constant region of an antibody of the second species, which can alleviate the immune response induced by the antibody of the first species. As a non-limiting example, in order to establish a chimeric antibody, a fusion tumor that secretes the monoclonal antibody of the first species is established, and then the variable region genes are selected from the fusion tumor cells, and then the constant region genes of the second species antibody are selected as needed. , The variable region gene and the constant region gene are connected to form a chimeric gene and then inserted into the vector, and finally the chimeric antibody molecule is expressed in an industrial system. The constant region may be selected from the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 of the second species or variants thereof; specifically, it comprises the heavy chain constant region of IgG1, IgG2 or IgG4 of the second species, or has enhanced ADCC toxicity IgG1 heavy chain constant region.

The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody. The antibody fragment retains at least some of the binding activity of the parent antibody. Generally, when the activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab’, F(ab’)2, Fv fragments, linear antibodies, single-chain antibodies, nanoantibodies Body, domain antibodies and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.

"Fab fragments" consist of one light chain, one heavy chain CH1 and variable regions. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

"Fab'fragment" contains a light chain and a heavy chain part (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, the two heavy chains of the two Fab' fragments An interchain disulfide bond is formed between them to form F(ab') ₂ molecules.

The "Fv region" contains 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 with polyepitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH regions The antibody, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable domains, each single variable domain with a different target Or binding to different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together, etc. .

The term "single-chain antibody" is a single-chain recombinant protein formed by connecting the VH and VL of the heavy chain variable region of an antibody with a linking peptide. It is the smallest antibody fragment with a complete antigen-binding site.

The term "domain antibody fragment" is an immunoglobulin fragment with immunological functions that only contains a heavy chain variable region or a light chain variable region chain. 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 can target the same or different antigens.

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

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

The term "epitope" refers to a site on an antigen that binds to an antibody (or antigen-binding fragment). 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 usually maintained after exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost after treatment with denaturing solvents. Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope binds to a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.

The term "specific binding" as used in this application refers to the binding of an antibody (or antigen-binding fragment) to an epitope on a predetermined antigen. Generally, when human GPC3 is used as an analyte and an antibody is used as a ligand, the surface plasmon resonance (SPR) technique is used in the instrument to determine that the antibody (or antigen-binding fragment) is approximately lower than 10 ^-7 M or even more. The small equilibrium dissociation constant (K _D ) binds to the predetermined antigen; and the affinity of the antibody (or antigen-binding fragment) to the predetermined antigen is at least twice that of the non-specific antigen (such as BSA).

The term "cross-reactivity" refers to the ability of the antibody (or antigen-binding fragment) of the present application to bind to GPC3 (or its epitope) from different species. Cross-reactivity is determined by detecting specific reactivity with antigens in binding assays (such as SPR and ELISA), or with cells that physiologically express GPC3 Measured by binding or functional interaction. Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

The terms "inhibition" or "blocking" are used interchangeably and encompass both partial and complete inhibition/blocking. Preferably, the inhibition/blocking of the ligand can reduce or change the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with anti-GPC3 antibody compared to ligand not contacted with anti-GPC3 antibody.

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

The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation (ie, passive or adaptive) of the immune response to a specific antigen. The term "induction" for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.

The “ADCC” mentioned in this application, namely antibody-dependent cell-mediated cytotoxicity, refers to the cells expressing Fc receptors, which directly kill the affected cells by recognizing the Fc segment of the antibody. Antibody-coated target cells. The Fc segment on IgG can be modified to enhance or reduce or eliminate the ADCC effect of antibodies. This modification refers to mutations in the constant region of the heavy chain of the antibody.

The methods of producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human GPC3 or fragments thereof, and the obtained antibodies can be coated and purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described in this application are genetically engineered to add one or more human FR regions to the CDR regions of non-human origin. Human FR germline sequence The columns can be obtained from the database ImmunoGeneTics (IMGT), or from The Immunoglobulin FactsBook magazine (, Academic Press, 2001 ISBN012441351).

The engineered antibody or antigen-binding fragment of the application can be prepared and purified by conventional methods. As a non-limiting example, the cDNA sequence of the antibody can be cloned and recombined into an expression vector, which can be stably transfected into host cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable pure strains are obtained by expressing antibodies that specifically bind to the antigen. The positive pure strains are expanded in the bioreactor to produce antibodies. The culture solution can be purified and collected by conventional techniques. It can be filtered and concentrated by conventional methods. Common methods can also be used to remove soluble mixtures and polymers, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

Monoclonal antibody (mAb) in this application refers to an antibody obtained from a single pure cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage pure cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, fusion tumor technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.

"Administration", "administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact. "Administration", "administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes the contact of the reagent with the cell, and the contact of the reagent with the fluid, where the fluid is in contact with the cell. "Administration", "administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent to a subject, such as comprising any one of the antibodies of the present application, the subject has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population to be treated, whether by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measured extent. The amount of the therapeutic agent effective to alleviate the symptoms of any specific disease (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the subject's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the subject . By any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated. Although the embodiments of the present application (such as treatment methods or products) may not be effective in alleviating the symptoms of the target disease in each patient, according to any statistical test methods known in the art such as Student t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.

The term "essentially composed of" or its variants used throughout the specification and claims means that it includes all the elements or element groups, and optionally includes other elements with similar or different properties to the elements, and the other elements are not significant To change the basic or new properties of a prescribed dosing regimen, method, or composition.

"Effective amount" includes an amount sufficient to improve or prevent a medical disease or its symptoms. 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 factors such as the disease to be treated, the general health of the subject, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.

"Exogenous" refers to substances that are produced outside organisms, cells, or humans according to the background.

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

"Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecule is homologous at that position of. 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 × 100%. For example, in the optimal sequence alignment, if 6 of the 10 positions in the two sequences match or are homologous, then the two sequences will be 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Therefore, "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.

"Optional" or "optionally" means that the event or environment described later can but does not have to occur, and the description includes the occasion where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.

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

The following examples are used to further describe the application, but these examples do not limit the scope of the application. The experimental methods that do not specify specific conditions in the examples of this application usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular selection manual; or according to the conditions recommended by the raw material or commodity manufacturer. The reagents without specific sources are the conventional reagents purchased on the market.

Example

Example 1: Protein antigen and cell line preparation

(1) Protein antigen preparation

The protein antigen is human GPC3 recombinant protein (UniProt #P51654, Gln 25 to His 559 fragment), C-terminal with a His tag, that is, human GPC3 His protein, purchased from AcroBiosystems (Cat # GP3-H52H4).

Table 1. Human GPC3 (Gln 25-His 559) amino acid sequence

(2) Cell line construction

Using CHO-K1 as the host cell, a single stable cell line stably expressing the full-length human or monkey (Macaca mulatta) GPC3 protein was constructed, named CHO-K1-hGPC3 and CHO-K1-cynoGPC3, respectively. The cell line construction method is as follows: The human or monkey GPC3 full-length DNA sequence (see Table 2 below) synthesized in vitro is cloned into a lentiviral vector with puromycin resistance gene, and CHO-K1 cells are transfected to obtain a cell pool. , 10% FBS in F12K media (three reagents were purchased from GIBCO) carried out with 8 μ g / ml puromycin containing screening culture, FACS analysis of GPC3 expression and then dilution method to obtain a high gradient plant expressing cell lines.

Table 2. DNA sequences of human GPC3 and monkey GPC3

Example 2: Obtaining mouse fusion tumor and antibody sequence

The immunogens were human GPC3-His antigen protein and/or CHO-K1-hGPC3 cells. 10 Balb/c and 5 SJL mice, and 5 C57 mice, female, 10 weeks old.

Two immune adjuvants:

(1) Using Sigma Complete Freund's Adjuvant (CFA) and Sigma Incomplete Freund's Adjuvant (IFA), the immunogen and immune adjuvant are fully mixed and emulsified at a ratio of 1:1 to make a stable "water-in-oil" liquid .

(2) Use Invivo Gen's aluminum adjuvant (Alhydrogel® adjuvant 2%), mix the antigen and adjuvant 1:1 to form a water-soluble solution and then immunize.

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

Table 3. Immunization program

Table 4. Immunization Program (Protein Immunization)

Table 5. Immune program (combined protein and cell immunization)

The immunized mouse serum was used in Example 3, the indirect ELISA method to evaluate the serum titer and Example 4 (2) FACS to detect the ability to bind to cell surface antigens, compared with the titer detection (greater than 100,000 times dilution) to determine the starting cell Fusion.

Select immunized mice with strong serum titer, affinity and FACS binding for a final immunization; sacrifice the mice, take spleen cells and SP2/0 myeloma cells to obtain fusion tumors; screen the target fusion tumors by indirect ELISA and FACS ; Establish a single cell line by the limiting dilution method. The obtained positive cell lines were further screened using indirect ELISA and FACS to select fusion tumors that bind the recombinant protein. The logarithmic growth phase fusion tumor cells were collected, and RNA was extracted with Trizol (ThermoFisher, 15596-026) and reverse transcribed (PrimeScript ^TM first-strand cDNA synthesis kit, Takara #6110A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-primer set (Sigma #69831) and then sequenced. The sequences of the murine antibodies M1, M2, and M3 were obtained by sequencing.

Table 6. Heavy chain and light chain variable region sequences of murine monoclonal antibodies

By analyzing the CDR regions of M1, M2, and M3, it is found that LCDR1 of M1, M2, and M3 all contain "NGN" sequence fragments, which may affect the stability of the antibody. In the subsequent chimeric antibody and humanized antibody, the "NGN" sequence of LCDR1 was mutated to "NRN", so the CDR region sequence in the subsequent study is shown in the following table.

Table 7. CDR sequences of heavy and light chain variable regions

Example 3: In vitro binding activity detection method of mouse antibody

In vitro indirect ELISA binding experiment:

The GPC3 His protein (AcroBiosystems, Cat # GP3-H52H4 ) was diluted with PBS pH7.4 to 0.5 μ g / ml concentration, in a volume of 100 μ L / well added to 96-well microtiter plates in high affinity, at 4 ℃ Incubate overnight (16-20 hours). After (pH7.4 PBS containing 0.05% Tween-20) plates were washed three times with PBST, diluted in PBST 1% bovine serum albumin (BSA) blocking solution 200 μ L / well and incubated for 37 ℃ 0.5 hour closed. After blocking, discard the blocking solution, and wash the plate with PBST buffer once.

Test antibody was diluted in PBST containing 1% BSA in, starting 10OnM, 5-fold serial dilutions, 11 doses to 100 μ L / hole was added to the microtiter plate, placed in 37 ℃ for 1 hour. HRP-labeled goat end of the incubation the plate was washed three times with PBST, was added 200 μ L / well dilution in PBST containing 1% BSA of anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, cat # 115-035-071 ) or HRP-labeled goat anti Human secondary antibody (Rockland, cat#609-103-123), incubated at 37°C for 0.5 hours. After washing the plate 5 times with PBST, add 100μl/well TMB chromogenic substrate (Suzhou Yake Chemical Reagent Co., Ltd., cat# S0025), incubate at 25°C in the dark for 8-15 minutes, add 50μl/well 1M HCl to stop For the reaction, read the absorbance value at 450nm with a microplate reader (Thermo, Ascent), and analyze the data.

Analyze the results of the concentration-signal value curve, as shown in the following table. The results show that the mouse antibody has a good affinity for the human GPC3 antigen.

Table 8. Affinity of mouse antibodies to human GPC3 antigen (EC ₅₀ value)

Example 4: Mouse antibody chimerization experiment

The positive mouse antibodies obtained from the screening are chimerized, and the variable regions of the murine antibodies are cloned in the constant regions of human antibodies to obtain chimeric antibodies. The constant regions of the chimeric antibodies of the present application are selected from the constant regions of human IgG1 heavy chains and humans. Constant region of kappa chain.

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

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

The above sequences were inserted into the pCEP4 vector, and the expression vector was synthesized according to the above design. After obtaining the vector plasmid, extract the plasmid and send the plasmid to sequencing for verification. The qualified plasmid was transfected into HEK293 cells (Yiqiao Shenzhou) with TF1 and cultured continuously. HEK293 cells were cultured with serum-free CD medium (Yiqiao Shenzhou, Cat#SMM 293-TI) to the logarithmic growth phase for cell transfection. 21.4 μ g The light chain plasmid and 23.6 μ g heavy chain plasmid was dissolved in 10mL Reduced Serum Medium (GIBCO, 31985-070 ) and mix well, then add 200ug TF1, mixing, RT incubated for 15min, 50mL added to the cells. Cell culture conditions: 5% CO2, 37°C, 125rpm/min. During the culture period, supplements (Yiqiao Shenzhou, Cat# M293-SUPI-100) were added on the first 1, 3, and 5 days until the cell viability was less than 70%, and the cell supernatant was collected and centrifuged. Load the cell culture fluid after centrifugation and filtration on the Protein-A affinity chromatography column, wash the column with phosphate buffer, wash with glycine acid buffer (pH 2.7 0.1M Gly-HCl), and extract with 2M Tris hydrochloric acid. 9.0 Neutralization and phosphate buffer dialysis to finally obtain purified chimeric antibodies.

(1) In vitro protein binding experiment:

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

Chip Preparation: The mouse anti-human IgG (Fc) antibody was diluted with fixation reagent (10mM sodium acetate, pH 5.0) to 25 μ g / mL, chips per channel to about 100 μ L using mouse anti-human IgG (Fc) antibody, fixed two channels using approximately 190 μ L were added 10 μ L fixation reagent mouse anti-human IgG (Fe) antibody. First, the surface of the chip and 400mM EDC CM5 100mM NHS at a flow rate of 10 μ L / min of activation of 420s. Next, 25 μ g / mL of mouse anti-human IgG (Fc) antibody at a flow rate of 10 μ L / min was injected into the test channel (FC4) of about 420s, a fixed amount of about 9000 to 14000RU. Finally, the chip 10 μ L / min for 420s blocked with 1M ethanolamine. The reference channel (FC3) and the test channel (FC4) perform the same operation.

Capture Ligand: stock solution was diluted with the antibody reagent were run to 4 μ g / mL, and a flow rate of 10 μ L / min was injected into the test channel (FC4) capture approximately 200RU. The reference channel (FC3) does not require ligand capture.

Analyte multi-cycle analysis: The human GPC3 protein 2-fold serial dilution with running reagent, the sample was diluted successively with 30 μ L / min flow rate and dissociation time is injected into the test channel and the reference channel according to the corresponding bonding time. After each concentration analysis, the chip requires 3M magnesium chloride with a flow rate of 20 μ L / min regeneration 30s, washed ligand and analyte undissociated. For the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.

Data analysis: Biacore T200 analysis software was used to calculate the KD value of each antibody, and the reference channel (FC3) was used for background subtraction.

In vitro indirect ELISA binding experiment and Biacore method determination results are shown in Table 9:

Table 9. Affinity of chimeric antibodies to human GPC3 antigen.

The results of ELISA and Biacore showed that the chimeric antibody has good affinity with human GPC3 antigen.

(2) In vitro cell binding experiment:

GPC3 high-expressing cells, including CHO-K1 cells (ATCC, Cat# CCL-61) overexpressing human or monkey GPC3, and human liver cancer cells JHH-7 (Nanjing Kebai, Cat# CBP60204), HepG2 (China) expressing GPC3 Cell Bank of the Academy of Sciences, Cat# SCSP-510). After trypsinization, the cells were collected by centrifugation, and the cell density was adjusted with FACS buffer (1×PBS containing 2% FBS) and spread on a 96-well U bottom plate, 1 per well ×10 ⁵ to 2 ×10 ⁵ cells. Centrifugation: 1200g, 5 minutes, discard the supernatant, was added 100 μ L of serially diluted antibody solution with FACS buffer (100nM, 5-fold diluted antibody working concentration of starting seven concentration points, and set point 0nM), 4 Incubate for 1 hour at °C. Centrifugation: 1200g for 5 minutes, discard the supernatant, wash the cells twice with PBS, add the fluorescent-labeled secondary antibody working solution prepared by FACS buffer PE anti-human IgG Fc antibody (Biolegend, Cat# 409304) or FITC anti-mouse IgG antibody (Biolegend, Cat # 406001), 100 μ L per well of cells were resuspended, incubated for 1 hour 4 ℃. Centrifugation: 1200g, 5 minutes, discard the supernatant. After washing the cells twice with PBS, they were resuspended in PBS, the fluorescence signal was detected by the flow cytometer DxFlex, and the EC ₅₀ concentration of the antibody-bound cells was analyzed by a curve.

_{Table 10. Affinity (EC 50} value) of chimeric antibodies to cells expressing GPC3

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

Example 5: Mouse antibody humanization experiment

The humanization of the murine anti-human GPC3 monoclonal antibody was carried out according to the method disclosed in the literature in this field. In short, human constant domains are used to replace the parental (murine antibody) constant domains, and human antibody sequences are selected based on the homology of the murine antibody and the human antibody. This application combines the murine antibodies M1, M2, and M3. Humanized.

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

The CDR region of the mouse antibody is transplanted to the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH are backmutated, and the CDR region Chemically labile amino acid residues were optimized. After expression testing and comparison of the number of back mutations, an antibody was selected that was designed with a combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences. The sequence is as follows:

Table 11. CDR regions of humanized antibodies

Table 12. Humanized antibody heavy and light chain variable region sequences

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

Compared with its human germline template (germline 2), the variable region of the Ab9 light chain has a sequence identity of 88.4% and a similarity of 97.3%. Compared with its human germline template (germline 3), the GC33 light chain variable region has a sequence identity of 86.6% and a similarity of 89.3%. Refer to Figure 2 for the correspondence between the Ab9 light chain variable region and its human germline template (germline 2), and the GC33 light chain variable region and its human germline template (germline 3).

The designed heavy chain and light chain variable region sequences are respectively connected with the heavy chain constant region and light chain constant region sequences of the human antibody. Exemplarily, the antibody heavy chain constant region is selected from: a human IgG1 heavy chain constant region with enhanced ADCC after amino acid mutation, or a natural human IgG1 heavy chain constant region, the sequences of which are as SEQ ID NO: 40 and SEQ, respectively ID NO: 41; the light chain constant region is selected from the constant region of the human kappa chain whose sequence is shown in SEQ ID NO: 42. The obtained heavy chain 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 numbers

Table 15. Sequence numbers of antibodies and their heavy chains, light chains and variable regions

Example 6: In vitro binding activity experiment of humanized antibody

The in vitro indirect ELISA binding experiment in Example 3 and the Biacore method in (1) in Example 4 were used to determine the affinity of each humanized antibody to the human GPC3 antigen. 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 a good affinity with the human GPC3 antigen.

The in vitro cell binding experiment in Example 4 (2) was used to determine the affinity (EC ₅₀ ) of each humanized antibody to cells expressing GPC3. The results are shown in Table 17:

_{Table 17. Affinity (EC 50} value) of humanized antibodies to cells expressing GPC3

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

Example 7: Endocytosis of humanized antibody

To test whether the antibody of the present application can co-endoculate into cells with human GPC3 after binding to GPC3, the stable transfected cell line CHO-K1-human GPC3 is used for evaluation.

The cells were digested with trypsin, and the cells were collected and resuspended in pre-cooled FACS buffer to adjust the cell concentration to 1×10 ⁶ cells/mL. Take the EP tube, add 1 mL of cell suspension, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 1 mL of the prepared antibody to be tested to resuspend the cells, the final concentration of the antibody is 20 μ g/ml, incubate at 4°C on a shaker 1 After hours, centrifuge and discard the supernatant (4°C, 1500 rpm×5 minutes), wash twice with FACS buffer, and discard the supernatant. Each tube was added 100 μ L fluorescent labeled secondary antibody was anti-PE antibodies into IgG Fc (Biolegend, Cat # 409304) or FITC anti-mouse IgG antibody (Biolegend, Cat # 406001), cells were resuspended, 4 ℃ incubated shaker After 30 minutes, centrifuge and discard the supernatant (4°C, 1500 rpm×5 minutes), wash twice with FACS buffer, and discard the supernatant. 1mL of cell culture medium was added to each tube of resuspended cells were pre-mixed and aliquoted to 4, each tube 200 μ L, 0 min group, the control group, 30 minutes and 2 hours groups groups groups and taken 0 minutes The blank group was placed on ice, the rest were placed in a 37°C incubator, and the EP tube was taken out at the corresponding time point for 30 minutes and 2 hours, and placed on ice to pre-cool for 5 minutes. All treatment groups were centrifuged to discard the supernatant (4 ℃, 1500rpm×5 minutes), washed once with FACS buffer, and discarded the supernatant. In addition to the group 0 minutes, all treatment groups EP tube was added 250 μ L strip buffer, incubate at room temperature for 8 minutes, the supernatant was discarded by centrifugation (4 ℃, 1500rpm × 5 min), washed twice with FACS buffer, the supernatant was discarded . All treatment groups was added 100 μ L PBS resuspended cells were detected by flow cytometry DxFlex.

Percentage of antibody endocytosis (%)=(fluorescence intensity value at each time point-average fluorescence intensity value of blank group)/(average fluorescence intensity value at zero point-average fluorescence intensity value of blank group)×100 , The results are shown in Table 18:

Table 18. Endocytosis of humanized antibodies in cells

The results from Table 18 show that the humanized antibody has good endocytosis in the stable transfected cell line CHO-K1-human GPC3.

Example 8: ADCC experiment of humanized antibody

The effector cell in this experiment is NK92MI (NK92 cell overexpressing IL-2 gene, purchased from Nanjing Kebai), and the target cell is human liver cancer cell HepG2. The experimental medium is RPMI1640 with 10% FBS for resuspension or dilution of cells and antibodies. HepG2 cells were trypsinized, collected by centrifugation, resuspended in assay medium to th 1 × 10 ⁵ / ml, 100 μ L were plated in white 96 well plate (Corning, 3610), 37 ℃ incubator for 2 days, added 50 μ L 4× working concentration of antibody, incubate at 4°C for 15 minutes. NK92MI proportion of cells were collected, resuspended in assay medium to 1 × 10 ⁶ / ml, and 50 μ L was added to the above reaction, so that the effector cells and target cells was 5: 1,37 ℃ incubator incubated for 3-4 hours. Finally, add 100 μ L Cell Titer-Glo ( Promega, Cat # G7573), mixing at room temperature in the dark for 10 minutes, multifunctional microplate reader (Thermofisher, Lux) readings.

Antibody kill percentage (%)=(E-S)/(E-M)×100

E is the value of the hole without antibody, that is, effector cell + tumor cell;

S is the value of the sample well, that is, effector cell + tumor cell + antibody;

M is the value of the medium hole.

The results showed that: the humanized antibody has a significant ADCC effect.

Example 9: CDC experiment of humanized antibody

The target cell of this experiment is the stable cell line CHO-K1-hGPC3 that overexpresses human GPC3. The experimental medium is FBS-free cell culture medium F12K (GIBCO) for resuspension or dilution of cells and antibodies.

CHO-K1-hGPC3 cells were trypsinized, collected by centrifugation, resuspended in assay medium to th 1 × 10 ⁵ / ml, 50 μ L were plated in white 96 well plate (Corning, 3610), was added 25 μ L 4 × work antibody concentration (concentration of the initial operation antibody 20nM, 5 fold diluted, 10 concentration points and set points 0nM), incubated for 30 min 37 ℃, was added 25 μ L 80% human serum (GemCell ^TM US, Cat # 100- 512), incubate for 24 hours in a 37°C incubator. Add 50 μ L CellTiter-Glo (Promega , Cat # G7573), mixing at room temperature in the dark for 10 minutes, multifunctional microplate reader (Thermofisher, Lux) readings.

Antibody kill percentage (%)=(E-S)/(E-M)×100

E is the value of the well without antibody, namely cell + culture medium + human serum;

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

M is the value of medium + human serum well.

Table 19. CDC effects of humanized antibodies

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

Example 10: Charge heterogeneity stability experiment of humanized antibody

In this experiment, the panoramic isoelectric focusing (iCIEF) method was used to detect and compare the purity of the humanized antibody at the starting 0 point and the charge heterogeneity of the humanized antibody at 25℃ and 40℃ for one month, so as to investigate the antibody The stability.

A mixture of sample with amphoteric groups, amphoteric electrolyte, buffer and auxiliary additives is injected into the capillary. When a DC voltage is applied to both ends of the capillary, the carrier amphoteric electrolyte can form a certain range of pH gradient in the tube. The sample group When the DH value in the capillary is the same as the isoelectric point (pI) of the component, the net charge of the solute molecule is zero, and the component will aggregate at this point in a macroscopic view. Migration, to achieve the purpose of separating each component in a complex sample. After collecting the spectrum, according to the linear relationship between the Marker pI value and the migration time of the chromatographic peak, the pI value and peak ratio (main peak, acid peak, and peak peak) of the test product can be obtained. The main steps are as follows:

System suitability sample preparation: Take out the suitability sample tube in the Maurice cIEF System Suitability Kit (Protein Simple, Cat#046-044), add 40 μl deionized water and 160 μl System Suitability Test Mix, mix well and transfer Transfer to a 1.5ml centrifuge tube, vortex and shake, then centrifuge, and transfer 160ul of supernatant to a 96-well sample plate for later use.

Prepare eIEF Master mixed solution: contains 37 μl ultrapure water, 35 μl 1% MC (Protein Simple, Cat#101876), 4 μl Pharmalyte pH 3-10 (Protein Simple, Cat#17-0456-01) , 2 μl 500mM arginine (Protein Simple, Cat#042-691), and the corresponding two pI Marker 6.14 (protein simple, cat: 046-031) and 9.99 (protein simple, cat: 046-034) each 1 μ l, the total volume is 80 μl .

Preparation of the test product: The sample is set with 3 conditions: starting at 0 o'clock; placed in a stability test chamber (Memmer, model HPP 1060) with a humidity of 65% and a temperature of 25°C for 1 month; in the stability test chamber (Memmer, model HPP 1060) placed for 1 month at a humidity of 65% and a temperature of 40°C. Take 20 μl of the corresponding sample and add it to the EP tube containing 80 μl of cIEF Master Mix solution in step 2), vortex to mix and centrifuge, transfer 80 μl of the supernatant to a 96-well sample plate, and centrifuge for later use.

On-board testing: open the capillary electrophoresis instrument (Protein Simple, maurice) and software, follow the instrument operation steps, perform the instrument self-test, install the capillary cartridge (Protein Simple, Cat# PS-MC02-C), and place the 96-well sample plate Enter the corresponding position of the instrument for cIEF analysis.

Table 20. iCIEF detection of humanized antibodies

The results showed that compared with the initial 0 point, the ratio of the main peak of Ab9 after being placed at 25°C for one month did not change much, and the stability was good. Placed at 40°C under the forced degradation condition for one month, the antibody appeared as expected The degradation phenomenon is reflected in the reduction of the main peak ratio to 26.3%; while the control antibody GC33 is placed at 40°C for one month and the main peak ratio drops to 2.5%.

Example 11: Stability experiment of molecular variants of humanized antibodies

In this experiment, the method of capillary electrophoresis (CE-SDS) was used to detect and compare the purity of the molecular variants of the humanized antibody at the starting 0 point and at 25°C and 40°C for one month. CE-SDS is based on the migration of protein samples in gel electrophoresis under denaturing conditions, and completes the separation according to the difference in the migration time of proteins of different molecular weights, so as to obtain the detection results of the purity of the samples after non-reduction and reduction treatments.

The main steps are as follows:

Sample preparation: samples are taken at the starting point 0; samples are placed in a stability test chamber (Memmer, model HPP 1060) at a humidity of 65% and a temperature of 25°C for 1 month; in a stability test chamber (Memmer, model HPP 1060) HPP 1060) The humidity is 65% and the temperature is 40℃ and placed for 1 month to sample.

Non-reducing sample treatment CE: EP tube was added to the sample, each protein sample was taken in an amount of 50 μ g, 1 μ L was added 10kD internal standard (Protein Simple, Cat # 046-144) , was added 2.5 μ L of 250mM IAM (Sigma, Cat # I1149-5G) , was added 1 × sample buffer (Protein Simple, Cat # 046-567) to a final volume of 50 μ L.

CE reducing sample processing: EP tube was added to the sample, each protein sample was taken in an amount of 50 μ g, 1 μ L was added 10kD internal standard (Protein Simple, Cat # 046-144) , was added 2.5 μ L of β- mercaptoethanol (Sigma, Cat # M3148-25ML), was added 1 × sample buffer (Protein Simple, Cat # 046-567) to a final volume of 50 μ L.

After shaking and mixing, heat and incubate at 70°C for 10 minutes in a dry thermostat (Hangzhou Aosheng Instrument Co., Ltd., model MK20001), then take it out, incubate on ice for 5 minutes, and centrifuge at 12000 rpm for 5 minutes after cooling. After centrifugation, 35 μ L supernatant was transferred to 96-well matching the instrument, then centrifuged at 1000rpm 5min.

Sample detection: Put the 96-well sample plate into the capillary electrophoresis instrument (Protein Simple, Maurice), turn on the instrument and software, follow the instrument operation steps, perform the instrument self-test, and install the capillary cartridge (Protein Simple, Cat#090-157) , Prepare the corresponding reagent and put it into the corresponding position of the instrument. According to the operation steps of the instrument, set the corresponding parameters to perform reduction or non-reduction CE analysis.

Data processing: After the sample detection is completed, set the corresponding integration parameters, and calculate and analyze the sample purity by the instrument's built-in software.

Table 21. CE-SDS detection of humanized antibodies

The non-reducing CE results showed that the main peak ratio of humanized antibody Ab9 stored at 25°C for one month was similar to that at 0 o'clock, while the main peak at 40°C for one month decreased. In reduced CE, compared with 0 o'clock, whether it is stored at 25°C or 40°C for one month, the sum of the heavy and light chain ratios of Ab9 antibody does not change much, and the stability is good.

<110> Shanghai Hansoh BioMedical Co.,Ltd. Jiangsu Hansoh Pharmaceutical Group Co.,Ltd.

<120> Anti-GPC3 antibody, its antigen-binding fragment and its medical use

<130> 702114CPCT

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<141> 2020-12-04

<150> CN201911236102.2

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Claims (23)

An anti-GPC3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises at least one HCDR1 selected from the following sequences: SEQ ID NO: 7, SEQ ID NO: 8; at least one HCDR2 selected from the following sequences: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; and at least one HCDR3 selected from the following sequences: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14; and The light chain variable region includes at least one LCDR1 selected from the following sequence: SEQ ID NO: 15, SEQ ID NO: 16; at least one selected from LCDR2: SEQ ID NO: 17, SEQ ID NO: 18; and at least one LCDR3 selected from the following sequences: SEQ ID NO: 19, SEQ ID NO: 20. The anti-GPC3 antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 12 respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 13, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 11 and SEQ ID NO: 14, respectively. The anti-GPC3 antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19 respectively; or, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20, respectively. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The heavy chain variable region includes: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 12, respectively; the light chain variable region includes: respectively as SEQ ID NO: 15. LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 17 and SEQ ID NO: 19, or, The heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 13, respectively; the light chain variable region comprises: respectively as SEQ ID NO: 15. LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 17 and SEQ ID NO: 19, or, The heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 11 and SEQ ID NO: 14, respectively; the light chain variable region comprises: respectively as SEQ ID NO: 16. LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 18 and SEQ ID NO: 20, or, The heavy chain variable region includes: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 12, respectively; the light chain variable region includes: respectively as SEQ ID NO: 16 LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 18 and SEQ ID NO: 20, or, The heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 13, respectively; the light chain variable region comprises: respectively as SEQ ID NO: 16. LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 18 and SEQ ID NO: 20, or, The heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 11 and SEQ ID NO: 14, respectively; the antibody light chain variable region comprises: respectively as SEQ ID NO : 15, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:17 and SEQ ID NO:19. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, a human antibody or a fragment thereof, and a human化 Antibody or fragments thereof. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or Its variants Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4; More preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1; More preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 41, or a heavy chain constant region variant as shown in SEQ ID NO: 40. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, a lambda chain or a variant thereof body; Preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain; More preferably, the anti-GPC3 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:42. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which comprises: It is selected from the heavy chain variable region shown in the following sequence, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26; And/or selected from the light chain variable region shown in the following sequence, or a light chain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity compared with the following sequence Variable region: SEQ ID NO:22 or SEQ ID NO:25. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which comprises: The heavy chain variable region shown in SEQ ID NO: 21 and the light chain variable region shown in SEQ ID NO: 22; or, The heavy chain variable region shown in SEQ ID NO: 21 and the light chain variable region shown in SEQ ID NO: 25; or, The heavy chain variable region shown in SEQ ID NO: 23 and the light chain variable region shown in SEQ ID NO: 22; or, The heavy chain variable region shown in SEQ ID NO: 23 and the light chain variable region shown in SEQ ID NO: 25; or, The heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 22; or, The heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 25; or, The heavy chain variable region shown in SEQ ID NO: 26 and the light chain variable region shown in SEQ ID NO: 22; or, The heavy chain variable region shown in SEQ ID NO:26 and the light chain variable region shown in SEQ ID NO:25. The anti-GPC3 antibody or antigen-binding fragment thereof according to claim 8 or 9, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the following sequences, or comprises at least 80% compared with the following sequences , 85%, 90%, 95%, or 99% identical heavy chains: 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-GPC3 antibody or antigen-binding fragment thereof according to claim 8 or 9, wherein the anti-GPC3 antibody or antigen-binding fragment thereof comprises a light chain selected from the following sequences, or comprises at least 80% compared with the following sequences , 85%, 90%, 95%, or 99% identical light chain: SEQ ID NO: 28 or SEQ ID NO: 31. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, which comprises: The heavy chain shown in SEQ ID NO: 27, and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 27, and the light chain shown in SEQ ID NO: 31; or, The heavy chain shown in SEQ ID NO: 29, and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 29, and the light chain shown in SEQ ID NO: 31; or, The heavy chain shown in SEQ ID NO: 30, and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 30, and the light chain shown in SEQ ID NO: 31; or, The heavy chain shown in SEQ ID NO: 32, and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 32, and the light chain shown in SEQ ID NO: 31; or, The heavy chain shown in SEQ ID NO: 36, and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 36, and the light chain shown in SEQ ID NO: 31; or, The heavy chain shown in SEQ ID NO: 37, and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 37, and the light chain shown in SEQ ID NO: 31; or, The heavy chain shown in SEQ ID NO: 38, and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 38, and the light chain shown in SEQ ID NO: 31; or, The heavy chain shown in SEQ ID NO: 39, and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 39, and the light chain shown in SEQ ID NO: 31. A polynucleotide encoding the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12. An expression vector containing the polynucleotide according to claim 13. A host cell which has been introduced into or contains the expression vector as described in claim 14; Preferably, the host cell is bacteria, yeast or mammalian cells; preferably Escherichia coli, Pichia pastoris, CHO cells or HEK293 cells. A method for producing anti-GPC3 antibodies or antigen-binding fragments thereof, including: Culturing the host cell as described in claim 15, preferably HEK293 cell; Isolate the antibody from the culture, preferably from the cell culture fluid; and The antibody is purified, preferably, the antibody is purified by chromatography. A medical composition containing: The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and a pharmaceutically acceptable excipient, diluent or carrier. A test or diagnostic kit, which contains: The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, Optionally, it further comprises one or more reagents capable of detecting the binding of the anti-GPC3 or its antigen-binding fragment to GPC3 or its epitope. Use of any one selected from the following in the preparation of a medicament for the treatment or prevention of GPC3-mediated diseases or disorders: The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and the pharmaceutical composition according to claim 17. Use of any one selected from the following in the preparation of a kit: The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and the pharmaceutical composition according to claim 17; The kit is used for detecting, diagnosing, and prognosing diseases or disorders mediated by GPC3. The use described in claim 19 or 20, wherein: The disease or condition is cancer; Preferably, the disease or condition is a cancer expressing GPC3; More preferably, the cancer 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 melanin tumor. The anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 17, which is used for the detection, diagnosis, and prognosis of GPC3-mediated diseases; preferably The 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. A method for treating or preventing GPC3-mediated diseases, including the following steps: Provide a subject with a therapeutically effective amount or a prophylactically effective amount of the anti-GPC3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12; or Provide a subject with a therapeutically effective amount or a preventively effective amount of the pharmaceutical composition according to claim 17; Preferably, the GPC3-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 Cell tumors and melanomas.

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