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Anti-pd-l1 antibodies and methods of making and using thereof

A technology of PD-L1 and antibody, applied in the field of preparation of anti-PD-L1 antibody

Active Publication Date: 2020-02-21
SYSTIMMUNE INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Negative secondary signaling can lead to T cell suppression and tolerance

Method used

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  • Anti-pd-l1 antibodies and methods of making and using thereof
  • Anti-pd-l1 antibodies and methods of making and using thereof
  • Anti-pd-l1 antibodies and methods of making and using thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Production of anti-PD-L1 antibodies

[0085] A monoclonal antibody against human PD-L1 was developed by immunizing New Zealand white rabbits. Initial immunizations were performed subcutaneously with 100 μg recombinant human PD-L1 ectodomain mixed 1:1 v / v in complete Freund's adjuvant. This was followed by booster immunizations at weeks 3, 6, 9 and 10 with 50 μg of antigen in incomplete Freund's adjuvant. In addition to antigen, at weeks 6, 9 and 10, 1×10 6 Animals were boosted with HEK-293 cells that were transiently transfected to express full-length human PD-L1.

[0086] At weeks 9 and 12, sera from animals were tested for anti-PD-L1 titers by ELISA. 96-well plates were passively adsorbed overnight at 4°C with goat anti-rabbit IgG antibody (Jackson ImmunoResearch). The coated wells were washed and blocked with 1% milk for 1 hour at room temperature, and then incubated with human PD-L1 extracellular domain-human Fc domain fusion protein for 1 hour at r...

Embodiment 2

[0097] Example 2: Binding affinity of anti-PD-L1 antibodies

[0098] Binding kinetics of selected anti-PD-L1 antibodies AB1-AB5 and AB1HU-AB5HU were determined by biolayer interferometry on a Fortebio Octbe Red 96 instrument. First, purified antibodies were produced by protein A chromatography using antibody supernatants transiently transfected with HEK-293. The assay was performed by immobilizing purified antibodies onto anti-human Fc biosensors. The association and dissociation of PD-L1 to the biosensor was then observed at various concentrations of PD-L1. Specifically, eight anti-human Fc biosensors were placed in wells containing the same purified antibody for 5 minutes. The biosensor was equilibrated for 1 min in kinetic buffer (Pall Fortebio) to establish a baseline. PD-L1 association was observed by placing the biosensor in wells containing various concentrations of human PD-L1 ectodomain for 5 min. Dissociation was measured after transferring the biosensor to kin...

Embodiment 3

[0102] Example 3: Antibodies that bind to PD-L1 expressed on the cell surface

[0103] Human lung cancer cell line H441 was labeled with FVS520 reactive dye (BD Biosciences, diluted 1:2000) for 15 min at room temperature. Labeled cells were then diluted in FACS buffer (2% FBS in PBS), added to V-bottom 96-well plates (50,000 cells per well) and incubated with 0-100 nM anti-huPDL1 or negative control isotype Matched or positive control isotype-matched control antibodies were incubated on ice for 30 min. Cells were washed to remove excess primary antibody and then labeled with Alex Fluor647-conjugated goat anti-human Fc secondary antibody (Jackson ImmunoResearch, 1:1600 dilution). Cells were incubated on ice for 20 min and then washed with FACS buffer before acquisition on a FACSCalibur flow cytometer equipped with a Cytek AMS plate loading system. Figure 5 Median fluorescence intensity (MFI) on the AF647 channel after gating on live cells is shown. Error bars represent SE...

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Abstract

The present application provides anti-PD-L1 monoclonal antibodies, antigen binding portions thereof, therapeutic compositions thereof and / or nucleic acids encoding the same, and their use to upregulate T cell function in the treatment of cancer and other T cell dysfunctions to enhance cell-mediated immune responses.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application No. 62524553, filed June 25, 2017, which is expressly incorporated herein by reference in its entirety. technical field [0003] The present disclosure generally relates to the technical field of antibodies, and more specifically relates to the preparation and use of anti-PD-L1 antibodies. Background technique [0004] Cancer is a major health problem worldwide. In the United States alone, an estimated 1,685,210 new cancer cases were diagnosed and 595,690 deaths occurred in 2016 (http: / / www.cancer.gov). Therefore, any agent that reduces cancer severity or mortality is desirable. [0005] In the immune system, resting T cells can be activated in response to antigens by primary signals delivered by foreign antigenic peptides presented by antigen-presenting cells (APCs) through T-cell receptors (TCRs). In addition to this primary signal, there a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K47/68A61P35/00
CPCC07K16/2827A61K45/06A61P35/00C07K2317/565C07K2317/56C07K2317/24C07K2317/51C07K2317/515C07K2317/92C07K2317/73C07K2317/76A61K39/39541C07K2317/31
Inventor 布莱恩·科瓦切维奇夏冬安妮·詹森乔恩·法莱布莱尔·伦肖杰弗里·B·阿达莫菲尔·谭高泽人朱义
Owner SYSTIMMUNE INC
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