A gallium 68-labeled affimer protein pet imaging agent and its application

A technology of PET imaging agent and affimer, which is applied in the direction of in vivo radioactive preparations, radioactive carriers, preparations for in vivo experiments, etc., and can solve problems such as insufficient stability

Active Publication Date: 2022-04-22
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] existing at the same time 68 In the Ga-PDGFR-β target affimer report, monomeric and dimeric affimer molecules were used, and the affinity constant of the monomeric affimer and PDGFR-β was about 249.2nM; while the dimeric affimer had a better affinity constant (32.6nM), and because its dimeric structure depends on the disulfide bond between two monomers, the structure is not stable enough in vivo

Method used

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  • A gallium 68-labeled affimer protein pet imaging agent and its application
  • A gallium 68-labeled affimer protein pet imaging agent and its application
  • A gallium 68-labeled affimer protein pet imaging agent and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Preparation of PDGFR-β Targeting Trimeric Affimer Protein (Z-tri)

[0031] 1.1 Sequence of Affibody Z-tri

[0032] The amino acid sequence of the Affibody Z-tri protein is shown in SEQ ID NO.1.

[0033] 1.2 Preparation of Affibody Z-tri Gene Expression Plasmid

[0034] The expression plasmid pQE30-Z-tri of Affibody Z-tri was designed and constructed by the laboratory, and stored in the TOP10 strain (TOP10-pQE30-Z-tri). Take the TOP10-pQE30-Z-tri bacterial liquid and inoculate it in LB liquid medium containing ampicillin (Amp, 100 μg / ml), and cultivate overnight at 37°C with shaking at 220 rpm until the bacterial liquid A 600 is 1. Take part of the bacterial liquid to extract the pQE30-Z-tri plasmid according to the operation method of the plasmid extraction kit, do agarose gel electrophoresis to test the size of the extracted plasmid, and send it to the company for sequencing to verify whether it is pQE30-Z-tri, and the correct pQE30- Z-tri plasmids were stored at 2...

Embodiment 2

[0054] 68 Preparation of Ga-Z-tri

[0055] image 3 Shows 68 Schematic diagram of the Ga-Z-tri affimer probe, wherein the far right is the trimeric affimer probe of the present invention 68 Ga-Z-tri, its preparation method is as follows:

[0056] 3.1 Coupling of Z-tri with bifunctional chelating agents

[0057] After 0.5 mg of Z-tri protein was dissolved in 50 mM sodium bicarbonate solution (pH 8.2-8.5), it was mixed with bifunctional chelating agent 2-[(4-isothiocyanatophenyl)methyl]-1, 4, 7, 10 -Tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA, referred to as DOTA, the same hereinafter) was mixed with 20 μL (5 molL), and incubated at 37° C. for 2 hours. Use PD-10 Desalting column from GE Company, use 0.5M sodium acetate solution (pH 8.8-9.0) as the mobile phase, remove unbound DOTA, and store the successfully coupled DOTA-Z-tri protein in 0.5M Sodium acetate solution (pH 8.8-9.0) is used for later use.

[0058] 3.2Z-tri 68 Ga label

[0059] Obtained by...

Embodiment 3

[0062] 68 PET imaging effectiveness test of Ga-Z-tri

[0063] 1) use 68 The Ga-Z-tri affinity body protein was used for PET imaging of the subcutaneous tumor-bearing nude mouse animal model to verify its effectiveness.

[0064] Construct subcutaneous tumor-bearing nude mouse animal models by subcutaneously inoculating human tumor cell lines respectively, and test malignant glioma U-87MG, colon cancer LS-174T, lung cancer A549, osteosarcoma Saos2, breast cancer MDA-MB-231, Subcutaneous tumor-bearing nude mouse models of liver cancer Huh7, a total of 6 malignant tumors.

[0065] PET imaging was performed after the tumor diameter reached 5-10 mm. The specific steps are: inject 3.7MBq of 68 Ga-Z-tri Affimer protein solution (the protein amount is about 1.5 μg), and use microPET-CT to develop after 1 hour, as shown in Figure 5 As shown, where the arrow points to the tumor, it can be clearly seen from the figure that most of the drugs are metabolized into the body through the ...

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PUM

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Abstract

The invention discloses a gallium 68-labeled affimer protein PET imaging agent and its application, including 68 Ga‑Z‑tri, the 68 Ga-Z-tri is obtained by labeling the affibody protein with gallium 68. The affibody protein includes PDGFR-β targeting trimeric affibody protein Z-tri. The amino acid sequence of the affibody Z-tri protein is SEQ ID NO.1 shown. The present invention utilizes a trimeric PDGFR-β targeting affibody with a unique amino acid sequence, which has the characteristics of high affinity and high stability compared with monomer and dimeric affibody, can greatly improve the labeling rate of nuclides, and realize The effectiveness of its use as a PET imaging probe.

Description

technical field [0001] The invention relates to the field of nuclear medicine PET imaging agents, in particular to a gallium 68-labeled affimer protein PET imaging agent and its application. Background technique [0002] Positron emission tomography (PET) is a kind of fluorine-18( 18 F), Gallium 68 ( 68 Ga), copper 64 ( 64 Cu) and other radioactive positron nuclide drugs, imaging technology for non-invasive in vivo diagnosis; PET probes constructed by using positron nuclide with different targeting molecular imaging agents can complete specificity for different diseases and organs Functional imaging is of great value in the diagnosis of malignant tumors, neuropsychiatric diseases, and cardiovascular and cerebrovascular diseases. [0003] Platelet derived growth factor receptor-β (Platelet derived growth factor receptor-beta, PDGFR-β) is an important neovascular growth factor receptor, which has a significant role in tumors, organic fibrosis and other diseases with abnorma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K51/08A61K51/04
CPCA61K51/08A61K51/0482
Inventor 蔡华伟卢晓风杨浩李林田蓉程惊秋
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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