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A highly functional anti-human CTLA4 antibody with high affinity, high specificity, and multiple antigen recognition epitopes

A monoclonal antibody, variable region technology, applied in the field of tumor immunotherapy and molecular immunology, can solve problems such as side effects and achieve large diversity effects

Active Publication Date: 2019-08-20
NANJING GENSCRIPT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is only one CTLA4 monoclonal antibody currently on the market, and CTLA4 monoclonal antibodies also have varying degrees of side effects, including the induction of immunogenicity in some patients, excessive inhibition of CTLA4 signaling may cause autoimmune diseases, and different CTLA4 monoclonal antibodies. Monoclonal antibodies have varying degrees of developability

Method used

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  • A highly functional anti-human CTLA4 antibody with high affinity, high specificity, and multiple antigen recognition epitopes
  • A highly functional anti-human CTLA4 antibody with high affinity, high specificity, and multiple antigen recognition epitopes
  • A highly functional anti-human CTLA4 antibody with high affinity, high specificity, and multiple antigen recognition epitopes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: Obtaining of human CTLA4 hybridoma cell line

[0046] 1) Animal immunity

[0047] As the antigen, a recombinant protein CTLA4-Fc (GenScript, Z03373) fused to the human CTLA4 extracellular domain of the human IgG1 Fc fragment was used. Female Balb / c and C57bl / 6 mice were immunized subcutaneously with 50 μg CTLA4-Fc fusion protein in 200 μl Freund's complete adjuvant (Sigma-Aldrich) in a 1:1 emulsion. Mice were then boosted with alternating ip / sc injections of 25 μg CTLA4-Fc in a 1:1 emulsion in Freund's incomplete adjuvant (Sigma-Aldrich) every two weeks up to 3 times. 4 days before myeloma fusion, showing the highest antibody titer ( figure 1 ) received an intraperitoneal boost of 25ug CTLA4-Fc (without adjuvant).

[0048] 2) Hybridoma fusion and screening

[0049] Spleens were extracted and homogenized to generate single cell suspensions, while myeloma cell (SP2 / 0) single cell suspensions were prepared. The 8.9×10 7 splenocytes with 4.1×10 7 SP2 / 0 ...

Embodiment 2

[0055] Example 2: Variable region sequencing of monoclonal antibodies and antibody recombinant production

[0056] After using the rapid ELISA mouse antibody subtype identification kit (Clonotyping System-HRP, SouthernBiotech) to identify the subtype of the antibody in the hybridoma cell culture supernatant, use TRIzol (Ambion) from 3 × 10 6 -5×10 6 Total RNA was extracted from hybridoma cells, and antibody subtype-specific primers and universal primers (PrimeScript TM 1stStrand cDNA Synthesis Kit, Takara) to reverse transcribe it into cDNA. Murine immunoglobulin heavy and light chain V-region fragments were subsequently amplified by RACE PCR (GenScript), and the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara) and inserted using vector-specific primer pairs Fragments are sequenced. Finally, unique V-region nucleotide / protein sequences of clones 26A12E8, 24H2C4B4, 42B11G12D3, 41B6F9C8, 42F8A6 were obtained.

[0057] Amino acid sequence of 26A...

Embodiment 3

[0080] Example 3: Binding of monoclonal antibodies to human CTLA4 recombinant protein

[0081] Indirect ELISA was used to assess the binding ability of purified antibodies to CTLA4-Fc. ELISA plates (Nunc) were coated with 100 μl / well of 0.5 μg / ml recombinant CTLA4-Fc or human IgG1 in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween), and blocked with 200 μl / well of 1% BSA in PBST at 37° C. for 0.5 hours. Then discard the blocking solution, add 100 μl of 10 μg / ml purified antibody to the first well, and dilute according to 3-fold gradient, a total of 11 test concentration gradients. Then incubate for 1 hour at room temperature. Plates were washed three times with PBST and incubated with 100 μl / well horseradish peroxidase-conjugated goat anti-mouse IgG (Fab-specific) (GenScript) for 0.5 hours at 37°C. Plates were washed five times with PBST, then TMB Chromogenic Solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark. The r...

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Abstract

The invention discloses an anti-human CTLA4 antibody with high affinity, high specificity, multiple antigen recognition epitopes and higher functionality. The CTLA4 monoclonal antibody of the present invention can specifically combine with CTLA4, and can effectively block the combination of CTLA4 and B7 protein, specifically remove the immune negative regulation of CTLA4, and activate T cells to secrete cytokines. The above functions have reached or exceeded the level of Yervoy, the only CTLA4 targeting drug currently on the market, and some antibodies are different from the epitopes of Yervoy, and have greater diversity.

Description

technical field [0001] The invention belongs to the field of tumor immunotherapy and molecular immunology, and specifically relates to an anti-human CTLA4 antibody with high affinity, high specificity, multiple antigen recognition epitopes and higher functionality. Background technique [0002] The immune system of vertebrates is a multi-level, functional system composed of various organs, tissues, cells and molecules, which is used to protect the body from foreign infections and maintain homeostasis (Janeway et al., Immunology: TheImmune System in Health and Disease. New York: Garland Science, 2005). It includes innate immune system and acquired immune system. Among them, the acquired immune system is composed of highly professional T cells and B cells, as well as a variety of effector molecules, which can specifically recognize pathogens including bacteria, fungi, viruses, etc. and eliminate them. The adaptive immune system consists of humoral immunity (B cell-mediated) ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12P21/08A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2818C07K2317/56C07K2317/92C12N15/85
Inventor 殷刘松张贵斌蒋忻坡陈黎徐飞覃喜建
Owner NANJING GENSCRIPT BIOTECH CO LTD
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