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Anti-human ErbB2 antibody-ansamitocin conjugate and applications thereof

A technology of maytansinoid and antibody, which is applied in the field of biomedicine, can solve the problems of decreased stability, increased toxicity, and reduced drug efficacy of conjugates, achieving the effect of improving homogeneity and eliminating influence

Active Publication Date: 2015-02-18
SHANGHAI JIAOLIAN MEDICINE RES & DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally, antibodies with high drug loading have stronger in vitro biological activity, however, increased drug loading will also lead to increased polymer composition, decreased stability, increased toxicity, high immunogenicity, and faster clearance in vivo , short half-life and low actual therapeutic index, etc.
In addition, antibody complementary determinants (complementarity-determining regions, CDR) or its nearby lysine coupling will also affect the binding of the antibody to the antigen, thus reducing the efficacy of the conjugate

Method used

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  • Anti-human ErbB2 antibody-ansamitocin conjugate and applications thereof
  • Anti-human ErbB2 antibody-ansamitocin conjugate and applications thereof
  • Anti-human ErbB2 antibody-ansamitocin conjugate and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Construction and expression of anti-Her2 antibody Trastuzumab

[0045] DNA fragments encoding the heavy and light chains of Trastuzumab were synthesized from the whole gene and cloned into the antibody heavy and light chain expression vector PL101 respectively (Liu Yanjun et al., Chinese patent CN200610117245.8, the invention name is "anti-Her2 / ErbB2 antigen monoclonal antibody and its preparation Methods and Pharmaceutical Compositions", the announcement number is CN101165068B), the operations of enzyme cleavage and ligation were carried out according to the instructions of the kit provided by the business.

[0046] Trastuzumab antibody heavy chain amino acid sequence 1-449 is shown in SEQ ID NO: 1;

[0047] Trastuzumab antibody heavy chain amino acid sequence 1-214 is shown in SEQ ID NO:2.

[0048] The Trastuzumab heavy chain and light chain expression vectors constructed above were transformed into Escherichia coli DH5α, and positive clones were picked an...

Embodiment 2

[0051] Example 2: Trastuzumab mutant heavy chain K30R (JL-01)

[0052] Site-directed mutagenesis was performed on the DNA fragment encoding the heavy chain of Trastuzumab synthesized by conventional molecular biology techniques, and the heavy chain K30R of the Trastuzumab mutant was cloned into the expression vector of the antibody heavy chain, and the operation of enzyme digestion and ligation was carried out according to the instructions of the commercially provided kit. .

[0053] Trastuzumab mutant heavy chain K30R (JL-01) amino acid sequence 1-449 is shown in SEQ ID NO: 3;

[0054] The above constructed Trastuzumab mutant heavy chain K30R expression vector was transformed into Escherichia coli DH5α, and positive clones were picked and inoculated in 500ml LB medium for amplification. DNA was extracted and purified using Qiagen's Ultrapure Plasmid DNA Purification Kit (Ultrapure Plasmid DNA Purification Kit) according to the manufacturer's instructions. The above-mentione...

Embodiment 3

[0056] Example 3: Trastuzumab mutant heavy chain K65D (JL-02)

[0057] Using conventional molecular biology techniques, site-directed mutagenesis was performed on the DNA fragment encoding the heavy chain of Trastuzumab synthesized from the whole gene, and the heavy chain K65D of Trastuzumab mutant was cloned into the expression vector of the antibody heavy chain, and the operations of enzyme digestion and ligation were carried out according to the instructions of the commercially provided kit. .

[0058] Trastuzumab mutant heavy chain K65D (JL-02) amino acid sequence 1-449 is shown in SEQ ID NO:4;

[0059] The above constructed Trastuzumab mutant heavy chain K65D expression vector was transformed into Escherichia coli DH5α, and positive clones were picked and inoculated in 500ml LB medium for amplification. DNA was extracted and purified using Qiagen's Ultrapure Plasmid DNA Purification Kit (Ultrapure Plasmid DNA Purification Kit) according to the manufacturer's instructions...

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PUM

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Abstract

The invention belongs to a biomedical technology field. The invention provides an anti-human ErbB2 antibody and ansamitocin conjugate, comprising: substituting lysine at 30 or / and 65 position in a heavy chain variable region of Trastuzumab with a hydrophilic amino acid, and then coupling with ansamitocin (DM1). On basis of maintaining coupling Pharmaceutical activity, a proportion of ansamitocin in a coupling medicament is reduced, thereby reducing toxic and side effect of the coupling medicament. The invention further provides applications of the anti-human ErbB2 antibody and ansamitocin conjugate for preparing antitumor drugs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an anti-human ErbB2 antibody-maytansinoid conjugate and its application in medicine. Background technique [0002] ErbB receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. This family includes 4 distinct members: commodity growth factor receptor (EGFR or ErbB1), Her2 (ErbB2), Her3 (ErbB3) and Her4 (ErbB4). [0003] Hudziak et al. in Molecular Cell Biology 9(3): 1165-1172 (1989) describe the generation of a panel of anti-ErbB2 antibodies characterized by the use of human breast cancer cells SK-BR-3. In SK-BR 72 hours after the -3 cells were exposed to the antibody, the relative proliferation rate of the cells was determined by staining the cell monolayer with crystal violet. Using this test, up to one came from an antibody called 4D5, which kept 56 percent of the cells proliferating. Trastuzumab was obtained through th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61K31/537A61K47/48A61P35/00
Inventor 刘彦君
Owner SHANGHAI JIAOLIAN MEDICINE RES & DEV CO LTD
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