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Anti-pd-1 antibodies and methods of making and using thereof

A PD-1 and monoclonal antibody technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc.

Active Publication Date: 2020-02-14
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-1 antibodies and methods of making and using thereof
  • Anti-pd-1 antibodies and methods of making and using thereof
  • Anti-pd-1 antibodies and methods of making and using thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Production of anti-PD-1 antibodies

[0092] Development of monoclonal antibodies against human PD-1 by immunizing New Zealand white rabbits. Initial immunizations were performed subcutaneously with 100 μg recombinant human PD-1 ectodomain mixed 1:1 v / v in complete Freund's adjuvant. This was followed by booster immunizations at 3, 6, 9 and 8 weeks with 50 μg of antigen in incomplete Freund's adjuvant. In addition to antigen, at weeks 3, 6 and 8, 1X10 6 Animals were boosted with HEK-293 cells that were transiently transfected to express full-length human PD-1.

[0093] At week 8, sera from animals were tested for anti-PD-1 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-1 extracellular domain-human Fc domain fusion protein for 1 hour at room temperature...

Embodiment 2

[0101] Example 2): Binding affinity of anti-PD-1 antibodies

[0102] Binding kinetics of selected anti-PD-1 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.

[0103] The assay was performed by immobilizing purified antibodies onto anti-human Fc biosensors. The association and dissociation of PD-1 to the biosensor was then observed at various concentrations of PD-1. 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-1 association was observed by placing the biosensor in wells containing various concentrations of human PD-1 ectodomain for 5 min. Dissociation was measured after transferring the biosensor to...

Embodiment 3

[0107] Example 3: Antibodies that bind to PD-1 expressed on the cell surface

[0108] The human PD-1 gene was subcloned into the pcDNA3.1 mammalian expression vector and transfected into the Jurkat human T cell line. Clonal cell populations expressing high levels of huPD-1 were generated by G418 drug selection and single cell sorting. The Jurkat / huPD-1 clonal cell line was labeled with FVS520 viability dye (BD Biosciences, diluted 1:2000) for 15 minutes at room temperature. Labeled cells were then diluted in FACS buffer (2% FBS in PBS), plated into V-bottom 96-well plates (50,000 cells per well) and incubated with 0-25 nM anti-huPDL1 or isotype control antibody Incubate on ice for 30 minutes. Cells were washed to remove excess primary antibody and then labeled with Alexa Luor647-conjugated goat anti-human Fc secondary antibody (Jackson ImmunoResearch, 1:1600 dilution) for 20 minutes on ice. Labeled cells were then washed with FACS buffer before harvesting on a BDFACSCalib...

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Abstract

The present application provides anti-PD-1 monoclonal antibodies, antigen binding portions thereof, therapeutic compositions thereof and / or nucleic acids encoding the same, and their use to upregulateT 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 U.S. Provisional Patent Application No. 62545603, filed August 15, 2017, U.S. Provisional Patent Application No. 62524553, filed June 25, 2017, U.S. Provisional Patent Application No. 62524554, filed June 25, 2017 , U.S. Provisional Patent Application No. 62524557, filed June 25, 2017, and U.S. Provisional Patent Application No. 62524558, filed June 25, 2017, which are expressly incorporated herein by reference in their entirety. technical field [0003] This application generally relates to the field of antibody technology, and more specifically relates to the preparation and use of anti-PD-1 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61K47/68A61P35/00
CPCC07K16/2818C07K2317/24C07K2317/76C07K2317/92A61P35/00
Inventor 布莱恩·科瓦切维奇夏冬安妮·詹森乔纳森·K·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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