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Novel unnatural amino acid marked antibody-drug conjugate and preparation thereof

A technology of unnatural amino acids and amino acids, which is applied in the field of fixed-point coupling of small molecule drugs, can solve the problems of increasing the immunogenicity of antibody drugs, reducing the half-life of antibodies, and affecting the binding of antibody antigens, achieving simple and easy coupling reactions, reducing The possibility of off-target, the effect of reducing immunogenicity

Active Publication Date: 2016-11-23
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are about 80 lysines and about 30 cysteines on the surface of the antibody, so the traditional modification method is non-specific and non-quantitative
However, the conjugates of antibody small molecules lack effective separation means, so the traditional coupling method is not suitable for quality control of large-scale production preparation
There are many functional regions on the surface of the antibody, and even destroy the disulfide bond of the antibody. Random coupling may affect the binding of the antibody antigen, reduce the half-life of the antibody in the body, and affect antibody-mediated cell killing and complement-mediated cell killing. Killing effect, coupling too many small molecules will also increase the immunogenicity of antibody drugs

Method used

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  • Novel unnatural amino acid marked antibody-drug conjugate and preparation thereof
  • Novel unnatural amino acid marked antibody-drug conjugate and preparation thereof
  • Novel unnatural amino acid marked antibody-drug conjugate and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1: Construction of the gene vector comprising the rituximab of site-directed mutation

[0099] (1) Construction and acquisition of auxiliary plasmids

[0100] Through the optimization and experimentation of the conditions, it was determined that the tandem expression of the four tRNAs from the 7sk promoter (its sequence is shown in SEQ ID NO: 6) was optimal, and pXH-N was constructed and obtained 3 Auxiliary vector: the vector uses pUC19 as a template, and introduces four tandem 7sk promoters and tRNA by using the BamHI restriction site; then introduces tRNA synthetase and its poly A termination signal under the control of the CMV strong promoter; uses EcorI Restriction sites are introduced into eukaryotic replication originals f1 ori and SV40; the plasmid can express tRNA and tRNA synthetase that specifically recognize the unnatural amino acid NAEK.

[0101] (2) Obtaining the plasmid containing rituximab

[0102] The genes of rituximab (SEQ ID NO: 1 and ...

Embodiment 2

[0111] Example 2: Expression and purification of rituximab for site-directed mutation

[0112] Construct pXH-N in the present invention 3 The plasmid contains tRNA (tRNA Pyl ) and pyrrolysyl-tRNA synthetase (pheRS), in expressing cells, the amber stop codon (TAG) is used as the sense codon, which can make the non-natural amino acid NAEK incorporated into the protein, thus causing the site-specific expression of rituximab mutation. Next, the inventors examined the incorporation possibility of NAEK and the production performance of the mutant protein.

[0113] 1: Synthesis and identification of unnatural amino acid NAEK

[0114] The chemical synthesis reaction formula of unnatural amino acid Lys-azido is as follows

[0115]

[0116] As described in the above formula, 2.3 mL of raw material 1 (2-bromoethanol) was dissolved in a mixed solution of 90 mL of acetone and 15 mL of water, and 3.12 g of NaN3 was added, and heated to reflux in an oil bath at 60°C for 20 h. Cool ...

Embodiment 3

[0131] Example 3: Site-directed coupling of mutants to a bifunctional tether (DIBO-DOTA)

[0132] 1: Synthesis and identification of bifunctional linker DIBO-DOTA:

[0133]

[0134] Compound 1 (2.88g, 14.0mmol) was dissolved in 20mL of anhydrous CH2Cl2, under N2 protection, BF3 OEt2 (2.59mL, 21.0mmol) was slowly added, then the system was moved to a cold well at -10°C, and slowly added dropwise under stirring A CH2Cl2 solution of trimethylsilylated diazomethane (2.0 mol / Lin hexanes) (10.5 mL of trimethylsilylated diazomethane dissolved in 20 mL of anhydrous CH2Cl2) was added dropwise within 1 hour. After continuing to react at -10°C for 2-4 hours, the reaction solution was poured into 50 mL of ice water to quench the reaction, the organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (2×50 mL). The organic phases were combined and washed with saturated brine (2×40mL), dried over anhydrous sodium sulfate, concentrated by filtration, and separated on...

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Abstract

The invention provides a site-directed mutated rituximab containing unnatural amino acid markers and a method for conjugating the rituximab by site-directed mutation and site-directed micro-molecules. The method includes the steps: leading unnatural amino acid into rituximab genes by genetic codon extension technology in a site-directed manner; performing site-directed connection by the aid of the unnatural amino acid and modifiers such as poly-difunctional connecting arms DIBO-DOTA; further realizing site-directed conjugating of the rituximab and radioactive isotope such as Cu64 through DOBO-DOTA. The invention further relates to an application of site-directed conjugated micro-molecule rituximab as development, tracing and therapeutic radio-immunity conjugate.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a method for point-fixed insertion of unnatural amino acids in proteins and a point-point coupling method for small molecule drugs. The method includes site-directed introduction of unnatural amino acids into proteins based on gene codon expansion technology, and site-specific linking of unnatural amino acids with effector small molecules. Background technique [0002] The following takes rituximab as an example to illustrate the background technology of the present invention, but the following content can not be understood as an acknowledgment of the prior art in any case, and it can not be considered that the present invention is only applicable to rituximab monoclonal antibody. [0003] (1) Rituximab [0004] The B lymphocyte antigen CD20 molecule is expressed on the surface of all B cells from late pre-B cells to mature B cells (Loken et al., 1987, Blood 70, 1316-1324). It i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C07K19/00C12N15/13C12N15/62C12N1/21C12N5/10A61K39/395A61K47/48A61K51/10A61K31/704A61K38/07A61P35/00A61P35/02
Inventor 周德敏杨志吴一鸣张博朱华徐欢刘菲
Owner PEKING UNIV
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