Endoplasmic reticulum Golgi apparatus targeting micromolecule, conjugate and application of endoplasmic reticulum Golgi apparatus targeting micromolecule

A technology of Golgi apparatus and endoplasmic reticulum, which is applied in the direction of antibody medical ingredients, medical preparations containing active ingredients, carrier-bound antigen/hapten ingredients, etc. Effects of inducing dendritic cells, etc.

Active Publication Date: 2022-03-11
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]Using immunoadjuvant mixed injection administration, it is easy to cause systemic diffusion of immune adjuvant, reduce the effect of inducing dendritic cells, and trigger systemic inflammatory response
If a targeting polypeptide is used, the endoplasmic reticulum targeting polypeptide as a foreign antigen is likely to induce the body to produce an immune response against it, and these immune responses will clear the entire vaccine together when the targeting polypeptide is used for the second time, making the same system unable to use many times

Method used

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  • Endoplasmic reticulum Golgi apparatus targeting micromolecule, conjugate and application of endoplasmic reticulum Golgi apparatus targeting micromolecule
  • Endoplasmic reticulum Golgi apparatus targeting micromolecule, conjugate and application of endoplasmic reticulum Golgi apparatus targeting micromolecule
  • Endoplasmic reticulum Golgi apparatus targeting micromolecule, conjugate and application of endoplasmic reticulum Golgi apparatus targeting micromolecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Synthesize diABZI and diABZI-MAL respectively according to the synthetic route shown below.

[0039]

[0040] Reactions and Conditions:

[0041] 1) saturated ammonia water, 25°C, 24h and then 50°C, 4h;

[0042] II) (4-aminobut-2-en-1-yl) tert-butyl carbamate, N, N-diisopropylethylamine (DIPEA), dimethylformamide (DMF), 120°C, 16h, Then trifluoroacetic acid (TFA), dichloromethane (DCM), 25°C, 2h;

[0043] III) boron bromide, DCM, 25°C, 16h;

[0044] IV) 4a: N-(3-chloropropyl)morpholine, DIPEA, DMF, 70°C, 16h or 4b: 4-(3-(tosyloxy)propyl)piperazine-1-carboxylic acid tert Butyl ester, DIPEA, DMF, 80℃, 4h;

[0045] V) DIPEA, isopropanol (IPA) 120 ° C, 16 ~ 24h;

[0046] VI) sodium dithionite, MeOH: water = 4: 3, 25°C, 15min;

[0047] VII) 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate, DMF / 1,4-dioxane, 0°C, 30min, then 1-(3-dimethyl Aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), DIPEA, 25°C, 6~14h;

[0048] VIII) 1-ethyl-3-methyl-1H-pyrazole-5-carb...

Embodiment 2

[0079] Example 2: Preparation of diABZI-MAL and polypeptide COVA-i conjugates

[0080]It should be noted that the present invention only provides the preparation of conjugates of diABZI-MAL and polypeptide COVA-i as a schematic example, but the antigens referred to herein that can form conjugates with diABZI-MAL are not limited to The polypeptide, and can be any other antigen known in the art. It should be understood that those skilled in the art can couple other antigens with diABZI-MAL in a similar way to form diABZI-MAL-antigen conjugates.

[0081] For antigens containing cysteine: dissolve the diABZI derivative in dimethylformamide (DMF), dissolve the antigen in a weak alkaline buffer solution, mix in a 1:1 molar ratio, incubate at room temperature for 24 hours, use HPLC Or FPLC separation and purification, concentration and lyophilization into powder.

[0082] Determination of the linked diABZI-antigen conjugate: the product was dissolved in a suitable solvent, and the ...

Embodiment 3

[0087] Example 3: diABZI-COVA-i activation assay and cross-presentation assay

[0088] 1. Preparation of BMDC cells (mouse bone marrow-derived dendritic cells)

[0089] The tibial bone marrow of 4-6-week-old mice (purchased from Guangdong Provincial Animal Center) was dispersed into single cells through a 40 μm cell mesh, and red blood cells were removed with ACK lysate. followed by 1×10 6 / ml bone marrow cells were cultured in RPMI1640 (containing 10% v / v FBS) medium containing 20ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) for 2 days, the suspended cells were removed, and new medium was replaced (RPMI1640 (containing 10% v / v FBS) medium containing 20ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF)) continued to culture for 2 days, then removed the suspended cells and replaced with a new medium (containing 20ng / ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) in RPMI1640 (containing 10% v / v FBS) medium), after continuing to ...

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Abstract

The invention discloses an endoplasmic reticulum Golgi apparatus targeting micromolecule. The structural formula of the endoplasmic reticulum Golgi apparatus targeting micromolecule is as shown in formula I, the invention also provides an endoplasmic reticulum Golgi apparatus targeting micromolecule conjugate, and the structural formula of the conjugate is as shown in the formula III. The invention also provides an application of the endoplasmic reticulum Golgi apparatus targeting micromolecule and the conjugate in preparation of an STING protein agonist, or in improvement of an antigen cross presentation level, or in improvement and enhancement of antigen targeting endoplasmic reticulum Golgi apparatus, or in preparation of a drug for inducing CD8 + T immune response, or in preparation of an immunotherapy drug. According to the endoplasmic reticulum Golgi apparatus targeting micromolecule and the conjugate provided by the invention, good connection between the endoplasmic reticulum Golgi apparatus targeting micromolecule and an antigen can be realized, the binding force of a bis-benzopyrimidine skeleton to STING protein is maintained, systemic inflammatory response caused by excessive diffusion of an STING agonist diABZI is reduced, the antigen targeting endoplasmic reticulum Golgi apparatus at a subcellular level is enhanced, and the immunogenicity of the antigen is improved. The antigen cross presentation level is improved, DC cell maturation can be promoted, and CD8 + T immune response is efficiently induced.

Description

technical field [0001] The invention belongs to the field of biomolecular chemistry, and in particular relates to an endoplasmic reticulum-Golgi target small molecule, a conjugate and an application thereof. Background technique [0002] The induction of CD8+ T cell response is the key to virus preventive vaccines to deal with virus mutation, and also the core of tumor therapeutic vaccines. In order to improve the response of CD8+ T cells, it is key to enhance the cross-presentation of antigen-presenting cells (especially dendritic cells), but under natural conditions, the antigen presentation of dendritic cells is affected by the efficiency of antigens entering cells, and antigens are in the The sublocalization in the cell and the degree of activation of dendritic cells and other factors show a lower efficiency. [0003] In order to improve the antigen presentation efficiency of dendritic cells, it is necessary to start from two aspects: 1) the maturation of DC cells; 2) t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D403/14C07K7/08A61K39/385A61P37/02
CPCC07D403/14C07K7/08A61K39/385A61P37/02A61K2039/60A61K2039/5154
Inventor 王骥王夏峰黄章屏尚丽茹蒋娟邓蔡冠曦
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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