Preparation and application of anti-human programmed death factor 1 (PD-1) monoclonal antibody

A monoclonal antibody, PD-1 technology, applied in the field of biomedicine, can solve problems such as structural differences and deficiencies, and achieve high affinity and wide application prospects

Active Publication Date: 2016-10-26
大庆东竺明生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PD-1 protein lacks the MYPPPY sequence that mediates the combination of CD28/CTLA-4 and B7.1/B7.2, and the FDPPPF sequence that med

Method used

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  • Preparation and application of anti-human programmed death factor 1 (PD-1) monoclonal antibody
  • Preparation and application of anti-human programmed death factor 1 (PD-1) monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Stable membrane expression of hPD-1.

[0026]In order to obtain a cell line expressing hPD-1 protein on the cell membrane surface as an immunogen, a cDNA clone encoding the full-length open reading frame of human PD-1 was purchased (Beijing Sino Biological Technology Co., Ltd. HG10377-CF, Genbank accession number NM-005018.2) , insert it into the pCDNA3.1 (+) (Invitrogen Company) vector, and sequence it to confirm that the coding frame of hPD-1 gene is correct. The plasmid was electrotransformed into CHO-DG44 cells, pressurized screening, and cloned to obtain CHO-DG44 cells stably expressing hPD-1. Named hPD-1 / DG44.

Embodiment 2

[0028] Animals are immune.

[0029] Five A / J mice aged 6-8 weeks were selected and immunized five times in total, with an interval of 14 days between each immunization. 10 per immunity 7 hPD-1 / DG44 cells, subcutaneous and intraperitoneal multipoint immunization. For the first immunization, an equal volume of Freund's complete adjuvant was mixed with cell suspension, and for the remaining 4 immunizations, Freund's incomplete adjuvant was mixed with cell suspension.

[0030] Cell culture medium configuration.

[0031] MD6 serum-free medium was used for culturing sp2 / 0 cells. After fusion, the cells were cultured in HAT medium, and the specific components were as follows: 10% fetal bovine serum and HAT were added to MD6 serum-free medium.

[0032] Cell fusion.

[0033] Three days after the final immunization, three mice with high titers were selected for cell fusion. Aseptically remove the mouse spleen and the pre-prepared sp2 / 0 cells, count the cells, mix them at a ratio o...

example 3

[0041] Functional characterization of anti-hPD1 monoclonal antibody.

[0042] Affinity identification.

[0043] The 96-well ELISA plate was coated with hPD-L1-Fc (RD); the blocking antibody was diluted 3 times as the primary antibody, and a total of 12 gradients were added to the 96-well ELISA plate coated with hPD-L1-Fc. Add goat anti-mouse IgG-Fc-HRP (SANT CruzBIotechnology) as the secondary antibody, add the chromogenic solution, and read the OD450 value after termination. EC50 concentrations were generated using Graphpad software. The EC50 of this antibody is 1.0667nM, and the EC50 of the positive control is 0.5344nM. It can be seen from the results that the screened antibody has obvious affinity for PD-1 and is similar to the positive control (see figure 1 ).

[0044] Functional characterization of antibodies blocking hPD-1 binding to hPD-L1.

[0045] A 96-well ELISA plate was coated with hPD-L1-Fc (RD); the purified hPD-1 antibody was diluted 4 times, and a total of ...

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Abstract

The invention discloses preparation, a variable-region sequence and application of an anti-human programmed death factor 1 (PD-1) monoclonal antibody. The invention provides the anti-human PD-1 monoclonal antibody which comprises gene sequences of heavy-chain variable regions of the monoclonal antibody and gene sequences of light-chain variable regions of the monoclonal antibody. The amino acid sequences of the heavy-chain variable regions are as shown in site 20th to site 134th of SEQ ID NO.3; the amino acid sequences of the light-chain variable regions are shown in site 20th to site 131th of SEQ ID NO.4. The invention discloses the monoclonal antibody 189-H-1 capable of blocking a human PD-1 function and a coding gene thereof. The monoclonal antibody 189-H-1 can be specifically combined with human PD-1 antigen, has median effective concentration EC50 being 1.0667 nM, can specifically block a PD-1/PD-L inhibition signal, and therefore, the monoclonal antibody 189-H-1 can be used as a blocker of a PD-1 access, so that the anti-human PD-1 monoclonal antibody becomes a novel drug for tumor immune therapy, chronic virus infective disease treatment and autoimmune disease treatment.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to the preparation, variable region sequence and application of anti-human PD-1 monoclonal antibody. Background technique [0002] The PD-1 gene was first discovered and cloned by Tasuku Honjo and colleagues in 1992. Its extracellular region has an IgV-like region, which has 23% homology with CTLA-4 and consists of 288 amino acids. The globulin superfamily type I transmembrane glycoprotein was originally thought to be related to apoptosis and was named programmed death-1 (PD-1). (Ishida, Y. et al., Induced expression of PD-1, a novel member of the immunoglobulinene superfamily, upon programmed cell death. EMBO J, 1992, 11:3887). However, PD-1 protein lacks the MYPPPY sequence that mediates the combination of CD28 / CTLA-4 and B7.1 / B7.2, and the FDPPPF sequence that mediates the combination of ICOS and ICOS-L, so it is structurally related to CD28, CTLA -4 and ICOS are si...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395G01N33/68G01N33/577A61P35/00A61P31/00A61P37/02
CPCA61K39/00C07K16/2818C07K2317/56C07K2317/565
Inventor 苏庆杨晓明刘云成杨岚哈卓王桂芬汤炜
Owner 大庆东竺明生物技术有限公司
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