Anti-human PD-1 humanized monoclonal antibody and application thereof

A monoclonal antibody, PD-L1 technology, applied in the field of biomedicine, can solve the problem of undetectable

Active Publication Date: 2016-10-12
REYOUNG SUZHOU BIOLOGY SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PD-L1 mRNA is abundant in nonlymphoid tissues such as placenta, heart, lung, and skeletal muscle, but PD...

Method used

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  • Anti-human PD-1 humanized monoclonal antibody and application thereof
  • Anti-human PD-1 humanized monoclonal antibody and application thereof
  • Anti-human PD-1 humanized monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061]Example 1 Murine Antibody Screening

[0062] 1.1 Animal immunity

[0063] Using the classic immunization schedule, BALB / c mice were immunized, and the immunogen was hPD-1 (human PD-1) protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), so that the animals could produce anti-hPD-1 Antibodies, the specific scheme is shown in Table 1:

[0064] Table 1 hPD-1 protein animal immunization scheme

[0065]

[0066]

[0067] 1.2 Cell fusion and screening of hybridoma cells

[0068] Adjust the state of mouse myeloma SP2 / 0 before fusion to ensure that its growth density does not exceed 1.0×10 6 cells, the final immunization was performed 3 days in advance, and the final immunization was by tail vein injection. The feeder cells were prepared one day in advance, and the number of plates was 2.0×10 4 cells / well. Through PEG fusion, ensure that the ratio of splenocytes to SP2 / 0 cells is between 10:1 and 5:1, and the number of splenocytes per well should ...

Embodiment 2

[0091] Example 2 Humanization and affinity maturation of murine antibody

[0092] 2.1 Acquisition of mouse antibody gene

[0093] Use Purelink RNA Micro kit to extract mouse anti-PD-1 hybridoma total RNA, and then use PrimeScript TM II 1st Strand cDNA Synthesis Kit reverse transcribed total RNA to prepare cDNA. The heavy chain and light chain variable regions of the antibody were amplified with Leader primer respectively, and the reaction system and PCR conditions are shown in Table 2 and Table 3, respectively.

[0094] Table 2 Mouse antibody gene cDNA PCR reaction system

[0095] Reagent name

add volume

10×Buffer

5μL

10μM dNTP Mix

1μL

50mM MgSO4

2μL

Upstream and downstream primers

1μL each

cDNA template

1μL

Taq

0.2 μL

ddH2O

up to 50μL

[0096] Table 3 PCR reaction conditions of murine antibody gene cDNA

[0097]

[0098] To analyze the PCR results by electrophoresis, add 0...

Embodiment 3

[0143] Example 3 Construction of Humanized Antibody Expression Plasmid

[0144] Using P3.1GS-hup01-HC and P3.1GS-hup01-LC as templates, the full-length antibody heavy chain fragment and light chain fragment were amplified by PCR to construct humanized antibody expression plasmids.

[0145] The upstream and downstream primers, reaction systems and PCR conditions of the light chain and heavy chain are shown in Table 5, Table 6 and Table 7.

[0146] Table 5 The upstream and downstream primers of humanized antibody light chain and heavy chain PCR reaction

[0147]

[0148] Table 6 Humanized antibody light chain and heavy chain PCR reaction system

[0149] Reagent name

add volume

heavy chain / light chain template

1μL

5×Buffer

10 μL

2.5μM dNTP Mix

4μL

Upstream and downstream primers (10μM)

1μL each

Taq

0.5μL

wxya 2 o

up to 50μL

[0150] Table 7 PCR reaction conditions of humanized antibody li...

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Abstract

The invention relates to the field of biological medicine and particularly relates to an anti-human PD-1 humanized monoclonal antibody and application thereof. By virtue of sieving, the anti-human PD-1 humanized monoclonal antibody with favorable specificity and relatively high compatibility and stability is obtained, can be specifically combined with human PD-1, but is not combined with other members of the CD28 family, is capable of blocking combination of PD-L1 and PD-1 and partially restoring functions of T cells, and has an obvious inhibiting effect on tumor growth.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to an anti-human PD-1 humanized monoclonal antibody and its application. Background technique [0002] T cell activation requires two signals. The first signal comes from the combination of T cell antigen receptor (TCR) and antigen peptide-MHC complex, which is antigen-specific; The receptor of the molecule binds to the corresponding ligand on the antigen-presenting cell (APC), which is antigen-nonspecific. The second signal plays an important role in the activation of T cells. If there is no co-stimulatory molecule to provide the second signal, T cells will be in an unresponsive state or apoptosis after recognizing the antigen. The combination of CD28 / CTLA-4 and its ligands B7-1 and B7-2 is a co-stimulatory pathway necessary for T cell activation and participates in the body's antigen-specific humoral and cellular immunity. New members of the CD28-B7 family include: ICOS (induc...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61K47/48A61P35/00A61P37/00A61P31/12A61P31/00
CPCA61K39/00C07K16/2818C07K2317/24C07K2317/565C07K2317/567C07K2317/73C07K2317/92A61P31/00A61P31/04A61P31/12A61P35/00A61P35/02A61P37/00A61P37/04C07K2317/76C07K2317/94A61K39/3955A61K47/6849C07K2317/30C07K2317/56C07K2319/30
Inventor 李戈郭树华张佳春朱一翔
Owner REYOUNG SUZHOU BIOLOGY SCI & TECH CO LTD
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