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Human anti-Siglec-15 antibody and application thereof

An antibody and sequence technology, applied in the field of human anti-Siglec-15 antibody, can solve the problems of unclear immune function and achieve the effect of increasing diversity, high binding activity and long half-life

Active Publication Date: 2021-12-17
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pro-inflammatory function of Siglec-15 in humans and mice has also been confirmed. In addition, it also plays an important role in osteoclast differentiation and bone remodeling. The loss of Siglec-15 in mice can lead to osteoporosis, But its immune function is still unclear

Method used

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  • Human anti-Siglec-15 antibody and application thereof
  • Human anti-Siglec-15 antibody and application thereof
  • Human anti-Siglec-15 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Screening of Siglec-15 fully human phage antibody library

[0039] Dissolve 10 μg of Siglec-15 protein in 500 μL of PBS and add to the immunotube to coat overnight at 4°C, and set a control tube without antigen. Pour off the coated immunotube liquid and seal it with 4% milk at room temperature for 1h, and at the same time, put 5×1012 A natural phage library was diluted in 1 mL containing 4% milk and blocked for 1 h at room temperature. The blocking solution in the immunotube was discarded, and 500 μL of the blocked phage library solution was added to the antigen-coated immunotube and the control tube respectively, and incubated at room temperature for 1 h. Use PBST (0.1% Tween 20 added to PBS) to wash the immunotube 25 times to wash away unbound and non-specifically bound phages, and elute specifically bound phages with glycine-hydrochloric acid (100mM, pH1.7), Tris base After neutralization (pH 8.0), add to TG1 in the logarithmic growth phase and incubate at...

Embodiment 2E

[0040] Embodiment 2 ELISA method identifies positive clone binding activity

[0041] Dilute siglec-15 protein with coating solution (0.1M sodium carbonate-sodium bicarbonate buffer (pH9.6)) to 1 μg / mL, add 100 μL per well to the ELISA strip, overnight at 4°C; wash the plate with PBST washing solution 3 Add 200 μL 4% skimmed milk powder to each well, block for 1 hour at 37°C, wash the plate 3 times; centrifuge the phage supernatant collected above at 1800r / min 4°C for 15 minutes, add 100 μL to each well into the ELISA plate, and react for 1 hour at 37°C ; wash the plate 3 times; add 100 μL anti-M13 / HRP to each well, and react in the dark at room temperature for 45 minutes; wash the plate 4 times; add 100 μL TMB substrate chromogenic solution to each well, and react in the dark at room temperature for 3 minutes; 2 SO 4 The reaction was terminated, and the OD450 value was detected with a microplate reader; the results showed that most of the clones had good binding activity, and...

Embodiment 3

[0042] Example 3 Antibody expression and identification

[0043] The antibody heavy chain and light chain variable regions and human Fc constant region gene sequences obtained by screening were cloned into the commercial vector pCDNA3.4, the correctness of the clones was identified by enzyme digestion, and the plasmids were extracted using a plasmid extraction kit. Resuscitate 293T cells in advance, when the cells are in the logarithmic growth phase, use JetPRIME transfection reagent to transfect the antibody expression vector into 293T cells, change the complete medium for 4 hours and continue to culture for 12 hours, change to OPM-293CD05 medium and continue to culture After 72 hours, the supernatant was collected by centrifugation, filtered with a 0.22 μM filter element, purified with a Protein A chromatography column, and identified by SDS-PAGE and SEC-HPLC. The antibody molecular weight was about 60Kda under SDS-PAGE reducing conditions, and the antibody purity was > 95%,...

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PUM

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Abstract

The invention discloses a human anti-Siglec-15 antibody and an application thereof. The human anti-Siglec-15 antibody screened through a phage antibody library technology can be combined with cells expressing human Siglec-15 antigens in a high-specificity mode, the preparation method of the targeted human Siglec-15 antibody is simple, the use of a hybridoma technology is avoided, and the obtained anti-human Siglec-15 antibody can well detect Siglec-15 protein expressed on tumor cells. The targeted human Siglec-15 antibody disclosed by the invention is high in specificity and has a very good prospect in application to treatment and / or prevention or diagnosis of Siglec-15 related diseases such as tumors.

Description

technical field [0001] The invention relates to the technical field of immunotherapy biomedicine, in particular to a human anti-Siglec-15 antibody, its preparation method and application. Background technique [0002] An important advance in tumor immunology in recent years is the existence of adaptive resistance mechanisms in the TME to prevent the execution of tumor immunity. For example, specific monoclonal antibodies to B7-H1 or PD-1 block the B7-H1 / PD-1 pathway (anti-PD therapy) to make the immune response "normal" in the tumor microenvironment by releasing tumor cells from suppressing effector T cells. It can achieve the effect of eliminating tumor cells and treating cancer. It can not only reduce side effects, but also obtain anti-tumor immunity, which opens a new door for tumor treatment. In addition to anti-PD therapy, the Siglec receptor family is considered an important target for tumor sialoglycans to modulate many immune effects of tumor-infiltrating immune cel...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00G01N33/574G01N33/68
CPCC07K16/2803C07K16/005A61P35/00G01N33/57484G01N33/6893C07K2317/56C07K2317/622C07K2317/76G01N2333/70503
Inventor 罗龙龙吴佳果王志宏胡乃静时宁周阳怡华张丁木全瑰琪吴缘宇冯健男沈倍奋
Owner ACADEMY OF MILITARY MEDICAL SCI
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