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Antibody-macromolecular conjugates, fluorescent derivatives thereof and their preparation methods

A technology of polymer combination and polymer monomer, applied in the field of biomedicine, can solve the problems of difficult separation of products, uncertain coupling sites, poor quality control, etc., and achieve the effect of simple steps.

Active Publication Date: 2018-05-29
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional synthesis method of antibody-polymer conjugates is to link pre-prepared polymers with antibodies, which often has uncertain coupling sites, low efficiency, poor yield, difficult separation of products, poor quality control, and difficult activity. Keeping (the reaction site is close to the antibody active site) and many other problems

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  • Antibody-macromolecular conjugates, fluorescent derivatives thereof and their preparation methods
  • Antibody-macromolecular conjugates, fluorescent derivatives thereof and their preparation methods
  • Antibody-macromolecular conjugates, fluorescent derivatives thereof and their preparation methods

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preparation example Construction

[0070]Specifically, the preparation method may include two methods: the first is an in-situ polymerization method, and the method includes: a1) preparing an antibody-initiator conjugate, wherein the elicitor is bound to the disulfide of the antibody bond or free sulfhydryl group; b1) the antibody-initiator conjugate is mixed with a polymer monomer or a polymer monomer and a fluorescent monomer in a buffer, and triggers the polymerization of the polymer monomer under the action of a catalyst And the antibody-macromolecule conjugate or the fluorescent derivative of the antibody-polymer conjugate is prepared. The second is the grafting method. The grafting method pre-prepared the polymer, and then coupled with the specific site of the antibody to prepare the antibody-polymer conjugate and its fluorescent derivatives. The method includes: a2) the initiator is placed in the catalyst Initiate the polymerization of polymer monomers or polymer monomers and fluorescent monomers under t...

Embodiment 1

[0095] 1. Preparation of ATRP initiator DBEB (chemical formula I-2).

[0096]

[0097] Preparation of 3,4-dibromo-1-((2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)methyl)-1H-pyrrole-2,5-dione (chemical formula I-1): Dibromomaleimide (1.02g, 4mmol) was dissolved in 20ml of dry tetrahydrofuran, and triphenylphosphine (1.05g, 4mmol) and diisopropyl azodicarboxylate were slowly added at room temperature (DIAD, 790 μL, 4 mmol) and tetraethylene glycol (1.4 ml, 8 mmol) and stirred for 20 hours. Use a rotary evaporator to remove the solvent, dry loading, and flash column chromatography to obtain the compound (yellow oil, 398mg, yield rate 23%) described in the chemical formula I-1 provided by the invention, and reclaimed 50% of the dibromo Substitute maleimide.

[0098] The compound of chemical formula I-1 is a yellow oil. 1 H NMR (CDCl 3 , 400MHz) δ3.59-3.71(m, 14H), 3.81-3.84(m, 2H). 13 C NMR (CDCl 3 , 100MHz) δ39.0, 61.8, 67.7, 70.1, 70.4, 70.6, 70.8, 72.6, 129.5, 164.0. MS (IES) ...

Embodiment 2

[0126] 1. Preparation of rhodamine B derivative fluorescent monomer (formula I-6)

[0127]

[0128] 1) Synthesis of 3-bromopropyl methacrylate (chemical formula I-3).

[0129] Methacryloyl chloride (1.04g, 10mmol) and 3ml of triethylamine were slowly added dropwise to a mixed solution of 3-bromopropanol (1.39g, 10mmol) in dichloromethane (20ml) and DFM (1ml). After reacting at room temperature for 12 hours, 0.1M dilute hydrochloric acid was slowly added dropwise to the reaction solution until pH<7.0, extracted with ethyl acetate (3×25 mL), the organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated. Flash column chromatography obtained the compound of chemical formula I-3 provided by the present invention (colorless oil, 1.13 g, 55%).

[0130] The compound of formula I-3 is a yellow oil. 1 H NMR (400MHz, CDCl 3 )δ1.95(s, 3H), 2.21-2.27(m, 2H), 3.49(t, 2H, J=6.4), 4.29(t, 2H, J=6.4), 5.58(s, 1H), 6.11( s, 1H). MS (ESI) m / z: 229 [M+Na] + .

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Abstract

Antibody-macromolecular conjugates, fluorescent derivatives thereof and their preparation methods. The antibody contains at least one disulfide bond or free sulfhydryl group, and the macromolecule is bound to the antibody through the disulfide bond or free sulfhydryl group attached to the antibody. The method is to modify the specific site of the antibody and grow the polymer and its fluorescent derivatives in situ; or prepare the polymer and its fluorescent derivatives in advance, and then couple with the specific site of the antibody to prepare the antibody-polymer Conjugates and their fluorescent derivatives. The method of the present invention is simple and efficient, and the prepared product not only retains the original biological activity of the antibody but also improves the water solubility, stability, pharmacokinetics, biodistribution and therapeutic efficacy of the antibody, and reduces its immunogenicity. In addition, the present invention Compared with antibody-fluorescent markers prepared by traditional methods, the prepared antibody-polymer fluorescent derivatives have stronger fluorescent signals and detection signal intensity.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a preparation method of an antibody-macromolecule conjugate and a fluorescent derivative thereof. Background technique [0002] As the most important immune protein in the body, antibodies have been widely used in many fields such as biomedical imaging, targeted therapy, and clinical diagnosis. Antibodies used alone have problems such as short half-life and poor solubility. Linking polymers to antibodies to prepare antibody-polymer conjugates can effectively improve the solubility, stability, pharmacokinetics and therapeutic efficacy of antibodies and reduce their immunogenicity. The traditional synthesis method of antibody-polymer conjugates is to link pre-prepared polymers with antibodies, which often has uncertain coupling sites, low efficiency, poor yield, difficult separation of products, poor quality control, and difficult activity. Keeping (the reaction site is close to the an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/68A61K39/395A61K49/00C08F120/28C08F220/28C08F220/36C09K11/06G01N21/64G01N33/531
CPCA61K49/0058A61K47/60C07K16/32C09B11/24C09B69/103C09K11/06C08F220/286G01N21/6428G01N33/52G01N2021/6439
Inventor 高卫平张立彬
Owner TSINGHUA UNIV
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