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Anti-PD-1 antibody and application thereof

A PD-1 and antibody technology, applied in the field of pharmaceutical compositions containing them, can solve the problems of weakening the clinical therapeutic effect and universality of PD-1 antibodies, toxic and side effects, therapeutic doses, limitations, etc., to improve blocking activity And the effect of inducing T cell activation, high degree of humanization, and significant clinical value

Pending Publication Date: 2021-12-31
XIAMEN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although PD-1 antibody tumor immunotherapy has achieved certain results in recent years, an analysis of PD-1 antibodies that have entered the domestic clinical research on tumor treatment found that due to different amino acid sequences of PD-1 antibodies, especially the sequence of the CDR region There is heterogeneity, so their characteristics and functions and the effect of treating tumors are not the same
In general, the existing PD-1 antibodies have some defects such as certain toxic and side effects and the obvious limitation of their therapeutic dose in some populations, which greatly weakens the clinical therapeutic effect and universality of PD-1 antibodies, and Leading to certain security risks (Michot J M, Bigenwald C, et al. Immune-related adverse events with immune checkpoint blockade: a comprehensive review [J]. Eur J Cancer, 2016,54:139-48)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0459] Example 1: Production of mouse-derived anti-PD-1 monoclonal antibody

[0460] In this example, the anti-human PD-1 monoclonal antibody was obtained by conventional hybridoma fusion technology; the monoclonal antibody with high binding activity was screened by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) ; Use chemiluminescent enzyme-linked immunoassay (CLEIA) to screen monoclonal antibodies that can highly block the interaction between PD-1 and PD-L1.

[0461] 1.1 Construction of PD-1 expression plasmid

[0462] The human full-length PD-1 sequence (genbank: NM_005018.2) was amplified by PCR using the primers shown in Table 2, and ligated into the plv vector (Addgene Company) to construct a plasmid expressing PD-1, named for plv-PD-1.

[0463] In addition, a plasmid expressing human full-length PD-L1 (named plv-PD-L1) was obtained by the same method. For the sequence of the human full-length PD-L1, see genbank: NM_014143.3.

[0464] Table 2: Pri...

Embodiment 2

[0487] Example 2: Characterization of murine anti-PD-1 monoclonal antibody

[0488] 2.1 Detection of PD-1 antibody binding activity by FACS method

[0489] The principle of FACS method to detect the binding activity of PD-1 antibody is as follows: figure 1 Shown in B.

[0490] The U-2OS / PD-1 cells to be tested were digested and blown to make a single cell suspension, which was resuspended in PBS containing 3% fetal bovine serum after centrifugation. The cell suspension was filtered through a 200-mesh screen for testing, and each cell sample was collected about 1×10 5 cells. Dilute the antibody to be tested to 1 μg / mL and incubate with the above cells, use FITC-labeled goat anti-mouse IgG antibody (Sigma Company, product number F9006) and use flow cytometry in a flow cytometer (BD Company, FACS Aria II ) to detect the binding activity of the PD-1 antibody. The abscissa represents the fluorescence value of the detected cells.

[0491] The result is as image 3 As show...

Embodiment 3

[0496] Embodiment 3: surface plasmon resonance method (SPR) detects the antigen affinity activity of antibody

[0497] Antigen affinity determination of antibodies was carried out using BIAcoreTM instrument (GE Healthcare company, model BIACORE3000). Anti-mouse Fc CM5 biosensor chips (GE Healthcare) were generated using standard primary amine coupling procedures. The murine antibody was captured on the anti-mouse Fc surface for 1 min at a flow rate of 10 μl per min. Serial dilutions of PD-1 / Fc from 3.3 nM to 120 nM were injected on the antibody-binding surface at a flow rate of 30 μl per minute for 3 minutes, followed by a 10-minute dissociation phase. Use the EIA evaluation software (GE Healthcare company) one-to-one Langmuir (Langmuir) binding model to calculate binding rate (K a or K on ) and dissociation rate (K d or K off ). at a rate of K off / K on to calculate the equilibrium constant (K D ). The results are shown in the table below. The results showed that 1...

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Abstract

The invention relates to the field of disease treatment and immunology, in particular to an anti-PD-1 antibody or an antigen-binding fragment thereof, a nucleic acid molecule for encoding the anti-PD-1 antibody or the antigen-binding fragment thereof, a method for preparing the anti-PD-1 antibody or the antigen-binding fragment thereof, and a pharmaceutical composition containing the anti-PD-1 antibody or the antigen-binding fragment thereof. The invention further relates to an application of the above antibody ( especially the humanized antibody) or the antigen-binding fragment thereof in preparation of a medicine, wherein the medicine is used for improving immune cell activity and enhancing immune response or is used for preventing and / or treating tumors or infections.

Description

[0001] This application is a divisional application of the invention application with the application number 201910654839.X, the application date is July 19, 2019, and the invention title is "anti-PD-1 antibody and its use". technical field [0002] The present invention relates to the fields of disease treatment and immunology, in particular, the present invention relates to anti-PD-1 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding them, methods for preparing them, and pharmaceutical compositions containing them. The present invention further relates to the use of the antibody (especially a humanized antibody) or an antigen-binding fragment thereof in the preparation of a drug, the drug is used to improve the activity of immune cells, enhance the immune response, or to prevent and / or treat tumors or infection. Background technique [0003] The activation process of T cells requires two signals: one is the antigenic peptide bound by the major...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61K45/06A61P35/00A61P31/00
CPCC07K16/2818A61K39/39533A61K39/39558A61K45/06A61P35/00A61P31/00C07K2317/565C07K2317/56A61K2039/505A61K2300/00
Inventor 黄承浩熊丹林超龙游敏罗文新张军夏宁邵
Owner XIAMEN UNIV
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