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Bispecific antibody aiming at phosphatidylinositols protein polysaccharide-3 and T cell antigen

A bispecific antibody, phosphatidylinositol technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, cells modified by introducing foreign genetic material, etc. Antigen binding ability, cannot be guaranteed, cannot guarantee binding ability, etc.

Inactive Publication Date: 2014-06-04
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, even if each antibody can correctly bind to its antigen alone, there is no guarantee that it can retain its original binding ability after forming a bispecific antibody, and due to steric hindrance, it cannot be guaranteed that it can successfully bind to its antigen after forming a bispecific antibody. form synergy
The problem of steric hindrance can be partially solved by the design of flexible linkers, but the design of bispecific antibody construction cannot guarantee the correct folding and antigen-binding ability of the antigen-binding site in advance.

Method used

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  • Bispecific antibody aiming at phosphatidylinositols protein polysaccharide-3 and T cell antigen
  • Bispecific antibody aiming at phosphatidylinositols protein polysaccharide-3 and T cell antigen
  • Bispecific antibody aiming at phosphatidylinositols protein polysaccharide-3 and T cell antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1 Preparation of GPC3 / CD3 bispecific antibody

[0098] 1) Construction of an expression vector containing a nucleic acid encoding a GPC3 / CD3 bispecific antibody

[0099] VL GPC3 and VH GPC3 The nucleotide fragments are respectively based on the gene sequences of the corresponding VL part (the '086 patent SEQ ID NO: 91) and the VH part (the '086 patent SEQ ID NO: 81) of the GC33 antibody published in the patent document US7919086B2 (hereinafter referred to as the '086 patent) get. The sequence of nucleotides encoding the GPC3 single chain antibody fragment is passed in VL GPC3 and VH GPC3 The introduction of coding between nucleotide fragments (Gly 4 Ser) 3 (Abbreviated as (G 4 S) 3 ) The nucleotide composition of the amino acid sequence linker.

[0100] VL CD3 and VH CD3 The gene fragments were respectively obtained according to the 857-1585th nucleotide sequence of SEQ ID NO: 9 published in the patent document US7112324B1, wherein the nucleotide sequ...

Embodiment 2

[0115] Example 2. Analysis of binding specificity of GPC3 / CD3 bispecific antibody to target cells

[0116] 2.1 Experimental materials:

[0117] cell line

name

Purchased (obtained) from

Huh-7

human hepatoma cell line

Japan RIKEN Cell Bank (Tsukuba, Japan)

HepG2

human hepatoma cell line

American ATCC

CHO-K1

Chinese hamster ovary cells

American ATCC

PBMC*

human peripheral blood mononuclear cells

Shanghai Blood Center

Jurkat cells

Human Peripheral Blood Leukemia T Cells

Chinese Academy of Sciences Cell Bank

[0118] *Isolated from healthy human donor blood using Ficoll (from Biochrom) density gradient centrifugation method following standard procedures. PBMCs contain T cells.

[0119] 2.2 Experimental method:

[0120] The binding ability of the bispecific antibody GPC3 / CD3 was analyzed by a fluorescence-activated cell sorter (FACS, also commonly referred to as a flow cytometer FACS...

Embodiment 3

[0136] Example 3. Cytotoxic killing effect on tumor cells mediated by GPC3 / CD3 bispecific antibody

[0137] 3.1 Experimental materials:

[0138] Two GPC3-related tumor cells (Huh-7 and HepG2 cell lines) and a control cell not expressing GPC3 (CHO-K1 cell line) were used to analyze the GPC3 / CD3 bispecific-mediated T cell pairing, respectively. tumor cell killing ability.

[0139] 3.2 Experimental method:

[0140] Peripheral blood mononuclear cells (PBMC) were isolated from blood of healthy human donors using Ficoll (from Biochrom) density gradient centrifugation method following standard procedures. After centrifugation, the cells were washed with phosphate buffered saline (PBS) at a concentration of 0.1M and then resuspended in RPMI 1640 complete medium (Gibco) to adjust the cell concentration to 7×10 5 / mL. PBMCs were used as effector cells in cytotoxicity experiments. Different tumor cells are used as target cells. Adjust the concentration of target cells to 7 × 10 wit...

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Abstract

The first aspect of the invention relates to a bispecific antibody, which comprises a first functional domain for specific identification of phosphatidylinositol protein polysaccharide-3, a second domain for specific identification of human T cell antigen CD3, and a connection for connecting the functional domains. The second aspect of the invention relates to a nucleotide sequence encoding the above antibody. The third aspect of the invention relates to a carrier containing the above nucleotide sequence, and includes an expressive vector. The fourth aspect of the invention relates to a eukaryotic or prokaryotic expression system containing the above carrier. The fifth aspect of the invention relates to application of the above antibody to preparation of medicament for treating or preventing tumor.

Description

technical field [0001] The present invention relates to tumor-associated antibodies, more specifically, the field of genetically engineered antibodies. Background technique [0002] Glypican-3 (Glypican-3, GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) is a cell surface protein belonging to the heparan sulfate proteoglycan family. The GPC3 gene encodes a 70-kDa precursor core protein, which can be cleaved by furin to produce a 40-kDa soluble amino-terminal peptide that can be secreted into the blood, and a 30-kDa protein containing 2 A membrane-bound carboxy-terminal peptide of a heparan sulfate (HS) sugar chain. The GPC3 protein is attached to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. [0003] GPC3 is highly expressed in fetal liver, but not in normal adult liver tissue. However, the restoration of expression in hepatocellular carcinoma is closely related to the occurrence and development of liver cancer. Not only i...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N5/10C12N1/21A61K39/395A61P35/00
Inventor 李宗海王华茂蒋华石必枝王红阳杨胜利顾健人
Owner SHANGHAI INST OF ONCOLOGY
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