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Anti-GPC3 fully-humanized antibody, and chimeric antigen receptor cell and application thereof

A fully humanized and antibody-based technology, applied in anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, application, genetically modified cells, etc., can solve problems such as high recurrence rate and insensitivity to chemotherapy drugs , to achieve the effect of strong specificity and high affinity

Active Publication Date: 2018-12-18
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most patients are already in the advanced stage of cancer when they are diagnosed, they are not sensitive to chemotherapy drugs, and the recurrence rate after surgical resection is high [2,3]

Method used

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  • Anti-GPC3 fully-humanized antibody, and chimeric antigen receptor cell and application thereof
  • Anti-GPC3 fully-humanized antibody, and chimeric antigen receptor cell and application thereof
  • Anti-GPC3 fully-humanized antibody, and chimeric antigen receptor cell and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1, screening anti-GPC3 fully humanized antibodies

[0044] Phage display technology was used to screen human GPC3 (Q25-S550) protein as GPC3 antigen.

[0045] Wherein, the preparation process of the GPC3 antigen is as follows: construct the pFUSE-hGPC3(Q25-S550)-Fc eukaryotic cell expression vector; the expression vector replaces the signal peptide sequence of GPC3 with the signal peptide sequence of IL-2, and guides GPC3-Fc The fusion protein is secreted into the medium; using Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA) the above expression vector was transfected into HEK293T cells, the supernatant was collected, and GPC3-Fc fusion protein was separated and purified by ProteinA Argrose (GE Healthcare, Piscataway, NJ) affinity column to obtain the GPC3 antigen.

[0046] The specific process of phage display is as follows: coat the immune plate with 10 μg / ml GPC3 antigen obtained above at 4°C overnight; block the immune plate with PBS solution containing 5%...

Embodiment 2

[0058] Example 2, Expression and Purification of Antibody 32A9

[0059] The heavy chain sequence and light chain sequence of antibody 32A9 were cloned into expression vectors pFUSE-CHIg-hg1 and pFUSE-CLIg-hk (Invivigen, San Diego, CA), respectively, to prepare plasmids. Plant 5 million HEK293T cells in a cell culture dish with DMEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin, placed in 5% CO 2 , cultured in a 37°C incubator. When the cell density reaches 60-80%, add 5μg pFUSE-32A9VH plasmid and 5μg pFUSE-32A9VL plasmid to 1ml opti-MEM medium and let stand for 5 minutes; add 30μg PEI to the opti-MEM medium mixed with plasmid , stand still for 20 minutes, and during the standstill, replace HEK293T cells with fresh DMEM medium supplemented with 10% fetal bovine serum, 100U / ml penicillin, and 0.1mg / ml streptomycin; after standing still for 20 minutes, replace the mixed opti- Add MEM medium to HEK293T cell culture dish; recover...

Embodiment 3

[0060] Example 3, Specificity Analysis of Antibody 32A9

[0061] (1) Antigen protein binding specificity

[0062] Coat immunoplates with 5 μg / ml of GPC3-his protein and control GPC5-his protein at 4°C overnight (GPC3-his protein and GPC5-his fusion protein were purchased from R&D respectively); use 3% skimmed milk powder, 0.05% The PBS solution of Tween-20 was used to block the immune plate at room temperature for 1 hour; the 32A9IgG recombinant protein was diluted to 5 μg / ml with PBS solution containing 3% skimmed milk powder and 0.05% Tween-20, and added to the blocked immune plate (50 μl / well) , incubate at room temperature for 1 hour; wash the immunoplate 3 times with 0.05% Tween-20 in PBS solution (340 μl / well); mix goat anti-human FcγHRP (Iackson ImmunoResearch) with 3% skimmed milk powder, 0.05% Tween-20 at a ratio of 1:2000 Mix 20% PBS solution, add to the washed immune plate (50μl / well), incubate at room temperature for 1 hour; wash the immune plate with 0.05% Tween-...

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Abstract

The invention relates to an anti-GPC3 fully-humanized antibody, and a chimeric antigen receptor cell and application thereof. The antibody at least has one selected from a group consisting of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3. The antibody can be used for preparing a cell or conjugate, a diagnostic kit and a drug or pharmaceutical composition; a nucleic acid in the invention is used for preparing the anti-GPC3 fully-humanized antibody, the drug or the pharmaceutical composition; the cell or conjugate is used for preparing the drug or the pharmaceutical composition; the drug or the pharmaceutical composition has anti-tumor effect against a GPC3-positive tumor. According to the invention, the anti-GPC3 fully-humanized antibody is screened out by using a phage display technology, and the screened antibody has good specificity and high affinity; and cells using the antibody, e.g., chimeric antigen receptor T-cells,have effective killing effect on GPC3-positive tumors.

Description

technical field [0001] The invention relates to an anti-GPC3 fully humanized antibody, a chimeric antigen receptor cell and application thereof, and belongs to the field of biotechnology. Background technique [0002] As far as the inventors know, hepatocellular carcinoma has a wide incidence and a high mortality rate, and lacks effective treatment methods. The late survival rate of patients is only 3% [1] . Most patients are already in the advanced stage of cancer when they are diagnosed, they are not sensitive to chemotherapy drugs, and the recurrence rate after surgical resection is high [2,3] . Glypican-3 (GPC3) is a heparan sulfate proteoglycan anchored on the cell membrane surface by GPI. GPC3 is expressed in the early stage of embryonic development and participates in the regulation of cell proliferation by regulating key signaling pathways during development (such as Wnt signaling). After ontogeny matures, GPC3 expression is switched off. A large number of stud...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N15/13C12N5/10A61K35/17A61K47/68A61P35/00
CPCA61K35/17A61K47/6859A61P35/00C07K16/303C07K2317/24C07K2317/33C07K2317/92C12N2510/00
Inventor 高威刘晓宇魏丽雯苟黎明李娜
Owner NANJING MEDICAL UNIV
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