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Anti-human PD-L1 antibody with high affinity, high specificity and multiple antigen recognition epitopes and having higher functionality

A PD-L1, expression vector technology, applied in the field of tumor immunotherapy and molecular immunology, can solve problems such as side effects

Active Publication Date: 2018-07-03
NANJING LEGEND BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there is only one PD-L1 monoclonal antibody drug currently on the market, and immune checkpoint monoclonal antibodies also have varying degrees of side effects in the human body, including inducing immunogenicity in some patients and excessive inhibition of checkpoints. Signaling may cause autoimmune diseases (Claire et al, JAMA Oncol, 2(10):1346-1353, 2016), and different PD-L1 monoclonal antibodies will have different degrees of developability

Method used

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  • Anti-human PD-L1 antibody with high affinity, high specificity and multiple antigen recognition epitopes and having higher functionality
  • Anti-human PD-L1 antibody with high affinity, high specificity and multiple antigen recognition epitopes and having higher functionality
  • Anti-human PD-L1 antibody with high affinity, high specificity and multiple antigen recognition epitopes and having higher functionality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Obtaining of human PD-L1 hybridoma cell line and preparation of monoclonal antibody

[0052] 1) Animal immunity

[0053] As the antigen, the recombinant protein PD-L1-Fc (GenScript, Z03371) fused to the human PD-L1 extracellular domain of the human IgG1 Fc fragment was used. Female Balb / c and C57bl / 6 mice were immunized subcutaneously with a 1:1 emulsion containing 50 μg PD-L1-Fc fusion protein in 200 μl Freund's complete adjuvant (Sigma-Aldrich). Subsequently, mice were boosted with alternating intraperitoneal / subcutaneous injections of 25 μg PD-L1-Fc in a 1:1 emulsion in Freund's incomplete adjuvant (Sigma-Aldrich) every two weeks up to 3 times. Serum titers of 10 mice all reached 10 after three immunizations 5 above. 4 days before myeloma fusion, showing the highest antibody titer ( figure 1 ) of two mice (No. 848 and No. 853) received an intraperitoneal booster immunization with 25 μg of PD-L1-Fc (without adjuvant).

[0054] 2) Hybridoma fusion and s...

Embodiment 2

[0061] Example 2: Variable region sequencing of monoclonal antibodies and antibody recombinant production

[0062] After using the fast ELISA mouse antibody subtype identification kit (Clonotyping System-HRP, SouthernBiotech) to identify the subtype of the monoclonal antibody, 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 TM1stStrandcDNA 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 18B7F4G8, 29A8H8C7, 51F3D2G4, 42G2D7C3, 53C1F3D4 were obtained.

[0063] Sequence information:

[0064] 18B7F4G8 heavy chain variabl...

Embodiment 3

[0087] Example 3: Binding of monoclonal antibodies to human PD-L1 recombinant protein

[0088] Indirect ELISA was used to evaluate the binding ability of purified antibodies to PD-L1-Fc. ELISA plates (Nunc) were coated with 100 μl / well of 0.5 μg / ml recombinant PD-L1-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...

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Abstract

The invention discloses an anti-human PD-L1 antibody with high affinity, high specificity and multiple antigen recognition epitopes and having higher functionality. The PD-L1 monoclonal antibody can be specifically combined with PD-1, and can effectively block combination of PD-L1 and PD-1 protein, specifically relieve immune negative regulation of PD-L1, and activate T cell secretory cell factors. All the functions can reach a currently unique level of PD-L1 targeted drug Tecentriq (MPDL3280A), and partial antibodies have greater diversity by being different from epitope of Tecentriq. One PD-L1 antibody can promote combination of PD-L1 with PD-1 protein, but can still relieve immune negative regulation of PD-L1, and activate T cell secretory cell factors.

Description

technical field [0001] The invention belongs to the field of tumor immunotherapy and molecular immunology, and in particular relates to an anti-human PD-L1 antibody with high affinity, high specificity, multiple antigen recognition epitopes and higher functionality. Background technique [0002] The human acquired immune system resists external infection and internal disease through humoral immunity (mediated by B cells) and cellular immunity (mediated by T cells). As an important member of the immune system, T cells play a huge role in tumor immunotherapy. From the traditional cytokine drugs to the recently emerging immune checkpoint inhibitor therapy, the goal of tumor elimination is achieved by directly or indirectly activating T cells (Esther et al, physiol, 25(2): 85-101, 2010 ; Drew, Nature Reviews Cancer 12:252-264, 2012). [0003] T cell activation requires two levels of signaling. The first level signal, also known as the primary stimulatory signal, is achieved t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12P21/08A61K39/395A61P35/00
CPCA61K39/00C07K16/2827C07K2317/21C07K2317/56C07K2317/76C07K2317/92
Inventor 殷刘松张贵斌蒋忻坡林峰覃喜建赵涛
Owner NANJING LEGEND BIOTECH CO LTD
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