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CTLA-4 antibody and preparation method thereof

A technology of antibodies and monoclonal antibodies, applied in botany equipment and methods, biochemical equipment and methods, antibodies, etc.

Pending Publication Date: 2021-06-25
BIO THERA SOLUTIONS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, generalization of this mechanism of action to humans has provided limited clinical benefit in only a small fraction of treated patients

Method used

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  • CTLA-4 antibody and preparation method thereof
  • CTLA-4 antibody and preparation method thereof
  • CTLA-4 antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1. Preparation of Antibody 1 and Antibody 2

[0085] Prepare CHO-BAT (CHO-BAT cells are CHO-K1 cell lines (for example, can come from ATCC CCL-61) Chinese hamster ovary cells adapted to suspension growth through culture, invention patent CN109096399A has disclosed CHO-BAT cells) and CHO-BAT -KF (CHO-BAT-KF cell is a Chinese hamster ovary cell line adapted to suspension growth and knocked out α-(1,6)-fucosyltransferase, which is preserved in the China Center for Type Culture Collection. Number: CCTCC NO: C2017127) cells. The linearized plasmid containing the nucleotide sequence encoding the light chain / heavy chain of the anti-CTLA-4 antibody was transfected into two cell lines by electroporation, and the heavy chain CDR1 of the anti-CTLA-4 antibody was SEQ ID NO: 12. The heavy chain CDR2 is SEQ ID NO: 13, the heavy chain CDR3 is SEQ ID NO: 14, the light chain CDR1 is SEQ ID NO: 15, the light chain CDR2 is SEQ ID NO: 16, and the light chain CDR3 is SEQ ID NO: ...

Embodiment 2

[0091] Example 2. Binding Activity of Antibody 2 Protein and CTLA-4 Antigen in Vitro

[0092] The plasmid used to express the extracellular region of CTLA-4 antigen and the sequence of Fc segment of human IgG1 antibody heavy chain (SEQ ID NO: 6, its amino acid sequence is SEQ ID NO: 5) was transiently transfected into 293F cells.

[0093] 293F cells were cultured with CD 293 TGE Medium (BPM Cell Culture, product number: CM-1156), and when the viability of the cells was above 95%, the cells were passaged to 80-100×10 with fresh medium. 5 Cells / mL, the cell density is about 150-200×10 after 24 hours 5 cells / mL, ready for transfection. Put the plasmid in a water bath at 65°C for 30 minutes, every 100×10 5 1 μg of plasmid was used for cells, and 3 μL of PEI (Polysciences, catalog number: 24765-2) was used for each 1 μg of plasmid. Dilute the plasmid and PEI with culture medium (the sum of the volumes of the two solutions is 5% of the total volume), and let stand at room tempe...

Embodiment 3

[0100] Example 3. Biological activity of antibody 2 and antibody 1 proteins in vitro

[0101] The schematic diagram of the in vitro biological activity model of anti-CTLA-4 antibody is as follows: Figure 4 .

[0102] Construction of pCMV2-OKT3 (wherein the OKT3 part is the ScFv form of the antibody, its nucleotide sequence is shown in SEQ ID NO: 7, the heavy chain amino acid sequence of OKT3 is shown in SEQ ID NO: 8, and the light chain amino acid sequence is SEQ ID NO: 9) plasmid, the plasmid map is as follows Figure 5 .

[0103] By electroporation, pCMV2-OKT3 was transfected into Raji cells, screened with hygromycin under pressure, mixed with Jurkat-IL2-luciferase cells and stimulated for 6 hours, adding luciferase substrate and using a microplate reader In the method of detecting autofluorescence, positive single clones capable of stimulating Jurkat-IL2-luciferase cells (Promega, JA3005) to produce autofluorescence were screened out and named as Raji-OKT3 cells.

[...

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Abstract

The invention discloses a CTLA-4 monoclonal antibody and preparation and application of the monoclonal antibody. The CTLA-4 monoclonal antibody has the advantages of lower toxicity and better activity. In addition, the CTLA-4 antibody also has low-content high mannose glycoforms and / or low-content sialylated glycoforms so that the half-life period of the antibody is prolonged and / or the immunogenicity is reduced.

Description

technical field [0001] The invention relates to a CTLA-4 monoclonal antibody, its preparation and application. Background technique [0002] The immune system is an important defense against cancer development and progression. Immunotherapy, such as IL-2 and adoptive transfer of autologous tumor-infiltrating lymphocytes, can induce sustained tumor responses in patient subgroups, resulting in long-term survival of patients with poor prognosis (Atkins MB, et al. (1999), J Clin Oncol .17(7):2105-2116). With an improved understanding of the importance of the initiation and effector phases of antitumor immunity, checkpoint blockade-based therapeutic approaches have recently begun to provide durable benefit to patients and appear to be applicable across a wide range of malignancies. [0003] The immune system is tightly regulated to respond to appropriate antigens rather than to self (Bretscher P, Cohn M. (1970), Science. 169(3950): 1042-1049). T cells are one of the key mediat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K45/06A61P35/00
CPCA61K45/06A61K2039/505A61P35/00C07K16/2818C07K2317/41C07K2317/565C07K2317/622C07K2317/92
Inventor 苏紫琪秦超俞金泉
Owner BIO THERA SOLUTIONS LTD
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