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Development and application of novel tumor adapter therapeutic drug

A technology of sequence and antigen, applied in the field of development and application of novel tumor-connector therapeutic drugs

Pending Publication Date: 2022-06-03
CHENGDU CONMED BIOSCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Codrituzumab is the first therapeutic monoclonal antibody targeting GPC3, which showed significant tumor inhibitory activity in mouse tumor models, but in a phase II clinical trial (NCT01507168), Codrituzumab was free of disease progression and overall survival compared with the control group No significant difference (Abou-Alfa GK 2017), studies have shown that Codrituzumab may bring clinical benefits to patients with tumors with high GPC3 expression or CD16 high affinity mutations (Chen G 2018)

Method used

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  • Development and application of novel tumor adapter therapeutic drug
  • Development and application of novel tumor adapter therapeutic drug
  • Development and application of novel tumor adapter therapeutic drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0248] Example 1: Antigen expression and construction of stably transfected cells

[0249] Using human GPC3 (Sino biological, HG10088) as a template, amplify the extracellular region of human GPC3 (25-563), transfect HEK293E cells, and express the recombinant human GPC3 protein (SEQ ID NO. 1), or the near-membrane sequence ( aa 524-563) was inserted into the EcoRI site of pGEX3X vector by Gibson assembly method, the plasmid was transfected into Escherichia coli BL21 (DE3), and IPTG induced expression and purification to obtain GST-human GPC3 stem recombinant protein (SEQ ID NO. 2). The GST-cynomolgus monkey GPC3 stem recombinant protein (SEQ ID NO. 3) and the GST-mouse GPC3 stem recombinant protein (SEQ ID NO. 4) were expressed in the same way. figure 1 Non-reducing SDS-PAGE results of GPC3 recombinant protein are shown.

[0250] The Lenti vector and packaging plasmid (Genecoepia) containing the full-length sequence of human and rhesus monkey GPC3 were co-transfected into HEK...

Embodiment 2

[0252] Example 2. Animal immunization and preparation of GPC3 antibody

[0253] 1. GPC3 antibody screening

[0254] Select 4-6-week-old female Balb / C mice, use the full-length or near-membrane sequence (aa 524-563) of the extracellular region of human GPC3 to construct a transient expression plasmid, and immunize the mice with DNA by gene gun. 20 μg plasmid / mouse, once a week, after 8 times of immunization, the serum titer was detected, and the cells were stably transfected with CHO-hGPC3 (5 × 10 6 cells / only) for shock immunization. After 3 days, the mice were sacrificed by cervical dislocation, the spleen and peripheral lymph nodes of the mice were collected, and the total RNA was extracted after grinding and centrifugation. region cDNA library. Using phage antibody library display technology, a GPC3 immune library based on filamentous phage M13 was constructed for subsequent phage panning.

[0255] Using the classical panning method, the recombinant human GPC3 protein w...

Embodiment 3

[0515] Example 3: Construction of GPC3×CD3 Bispecific Antibody

[0516] 1. Construction of CD3 humanized antibody

[0517] Murine hybridoma CD3 antibody (EMBO J. 1985. 4(2): 337-344; J. Immunol. 1986, 137(4): 1097-100; J. Exp. Med. 1991, 174: 319-326; J. Immunol. 1991, 147(9):3047-52) recognizes human and monkey CD3 receptors, the sequence of which is as follows:

[0518] Anti-CD3 mouse mAb light chain amino acid sequence:

[0519] QAVVTQESALTTSPGETVTLTCR SSTGAVTTSNYAN WVQQKPDHLFTGLIG GTNKRAP GVPARFSGSLIGDKAALTITGAQTEDEAIYFCA LWYSNLWV FGGGTKLTVL

[0520] Anti-CD3 mouse mAb heavy chain amino acid sequence:

[0521] EVQLVESGGGLVQPKGSLKLSCAASGFTFN TYAMN WVRQAPGKGLEWVAR IRSKYNNYATYYADS VKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR HGNFGNSYVSWFAY WGQGTLVTVSS

[0522]Anti-CD3 mouse monoclonal antibody was humanized, the human germline gene IMGT_hVL7-43 with the highest homology was selected for light chain CDR transplantation, human IGLJ3*02 was selected for FM4; human IMGT_hV...

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Abstract

The invention relates to development and application of a novel tumor adapter therapeutic drug. The therapeutic drug comprises a bispecific antibody that binds to GPC3 and CD3, the bispecific antibody comprising a first binding domain that binds to GPC3 on the surface of a target cell and a second binding domain that binds to CD3 on the surface of a T cell. The bispecific antibody can inhibit tumor growth at an extremely low dosage, and effectively inhibit the growth of transplanted tumor of an immune reconstruction mouse; the toxicological research in cynomolgus monkeys also shows that animals have good tolerance to the bispecific antibody, and the curative effect and the safety of the bispecific antibody are superior to those of similar antibodies.

Description

technical field [0001] The present disclosure relates to a bispecific antibody and its application, in particular to the development and application of a novel tumor-connector therapeutic drug. Background technique [0002] Glypican-3 (Glypican-3, GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) is a glypican proteoglycan belonging to the acetyl sulfate The heparan sulfateproteoglycans (HSPGs) family is anchored to the outer surface of the cell membrane by the glycosylphosphatidylinositol (Glycosyl phosphatidyl inositol, GPI). The core of GPC3 protein consists of 580 amino acids with a molecular weight of about 65kDa. The protease Furin is cleaved between 358Arg-359Ser (355R-356Q-357Y-358R-359S) to form two subunits, and two heparin sulfate polysaccharide chains are connected to the membrane near the The C-terminal subunit is an important part of the extracellular matrix and may be involved in the activation of cells by multiple signaling pathways su...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13A61K47/68A61K45/00A61K51/10A61P35/00
CPCC07K16/303C07K16/2809A61K47/6849A61K47/6859A61K45/00A61K51/1042A61K51/1057A61P35/00C07K2317/31C07K2317/565C07K2317/56C07K2317/51C07K2317/515C07K2317/92C07K2317/24C07K2317/33C07K2317/732C07K2317/77C07K2317/74C07K16/30A61K47/6879A61K47/6843C07K16/468C07K2317/52C12N15/63G01N33/574
Inventor 徐刚陈博宋芹
Owner CHENGDU CONMED BIOSCI CO LTD
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