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Pd-1 antibodies and uses thereof

A technology of PD-1 and antibody, which is applied in the field of immunotherapy for the treatment of human diseases, and can solve problems such as the lack of MYPPPY motifs

Active Publication Date: 2018-08-03
SHANGHAI ZHANGJIANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although structurally similar to CTLA-4, PD-1 lacks the MYPPPY motif critical for B7-1 and B7-2 binding

Method used

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  • Pd-1 antibodies and uses thereof
  • Pd-1 antibodies and uses thereof
  • Pd-1 antibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Example 1. Construction and expression of PD-1-Fc and PD-L1-Fc

[0143]A DNA (SEQ ID NO.11) containing cDNA encoding the extracellular region of human PD-1 fused to human Fc with a Hind III site at its 5' end and an EcoRI site at its 3' end was synthesized. This DNA was digested with Hind III and EcoRI and then cloned into the pcDNA3.1(+) vector (Invitrogen) which had been digested with the same restriction enzymes, resulting in the expression vector pcDNA3.1(+)-PD-1-Fc. The appropriate pcDNA3.1(+)-PD-1-Fc expression vector was transfected into CHO-K1 cells (ATCC) using Lipofectamine 2000 reagent (Invitrogen). Stable transfectants were isolated by limiting dilution in the presence of 500 μg / ml G418. The clone producing the highest amount of PD-1-Fc protein was selected and grown in serum-free medium. Finally, PD-1-Fc protein was purified from serum-free culture supernatant by protein A affinity chromatography. Protein concentration was determined by absorbance at 280...

Embodiment 2

[0145] Example 2. Characterization of PD-L1-Fc binding to PD-1 expressed on CHO cells

[0146] A Chinese hamster ovary (CHO) cell line expressing recombinant human PD-1 on the cell surface was developed and used to determine the binding of PD-L1 to PD-1 by flow cytometry. A DNA (SEQ ID NO. 13) containing a cDNA encoding full-length human PD-1 with a Hind III site at its 5' end and an EcoRI site at its 3' end was synthesized. This DNA was digested with Hind III and EcoRI and then cloned into the pcDNA3.1(+) vector (Invitrogen) which had been digested with the same restriction enzymes, resulting in the expression vector pcDNA3.1(+)-PD-1. The appropriate pcDNA3.1(+)-PD-1 expression vector was transfected into CHO-K1 cells (ATCC) using Lipofectamine 2000 reagent (Invitrogen). Stable transfectants were isolated by limiting dilution in the presence of 500 μg / ml G418. The amino acid sequence of full length human PD-1 is shown in SEQ ID NO:14. The PD-L1-Fc recombinant protein was l...

Embodiment 3

[0147] Example 3. Characterization of PD-1-Fc binding to PD-L1-Fc

[0148] The PD-1-Fc recombinant protein was labeled with biotin using the Biotin protein labeling kit (Roche). Different concentrations of biotin-labeled PD-1-Fc (PD-1-Fc-biotin) were added to the 96-well plate coated with PD-L1-Fc, followed by incubation at room temperature for 1 h. After washing, avidin-HRP was added and the plates were further incubated for 1 h. Finally, 3,3',5,5'-tetramethylbenzidine (TMB) was added as a substrate for horseradish peroxidase (HRP), and the absorbance was measured at 450 nm. figure 2 The results shown in indicate that biotin-PD-1-Fc can effectively bind PD-L1-Fc.

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Abstract

The present invention provides monoclonal antibodies that specifically bind to human PD-1 with high affinity. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, hostcells and methods for expressing the antibodies of the invention are also provided. The PD-1 antibodies of the present invention inhibit the binding of PD-L1 to PD-1, thereby modulating immune responses in general, and those mediated by TcR and CD28, in particular. The invention also provides methods for treating various diseases, including infectious diseases and cancer using the PD-1 antibodiesof the present invention.

Description

technical field [0001] The present invention relates generally to immunotherapy for the treatment of human disease. More specifically, the present invention relates to the use of anti-PD-1 antibodies in the treatment of cancer. Background technique [0002] The adaptive immune response involves the activation, selection and clonal expansion of two major classes of lymphocytes called T cells and B cells. Upon encountering an antigen, T cells proliferate and differentiate into antigen-specific effector cells, while B cells proliferate and differentiate into antibody-secreting cells. [0003] T cell activation is a multistep process requiring several signaling events between T cells and antigen presenting cells (APCs). In order for T cell activation to occur, two types of signals must be delivered to dormant T cells. The first type is mediated by antigen-specific T cell receptors (TcRs) and confers specificity to the immune response. The second (co-stimulatory) type modulat...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/395A61P31/00A61P35/00
CPCC07K2317/24C07K2317/76C07K2317/92C07K2317/94C07K2317/33C07K16/2818A61P31/00A61P35/00A61P37/02A61K47/6803A61K39/39558A61K39/395C07K14/70503C07K16/46C07K19/00C07K2317/73
Inventor 顾娜娜邵珂
Owner SHANGHAI ZHANGJIANG BIOTECH
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