Conjugate of anti-human DLL4 humanized antibody and dolastatin derivative MMAE as well as preparation method and application of conjugate

A technology of humanized antibody and dolastatin, which is applied in the direction of non-active ingredient medical preparations, drug combinations, anti-tumor drugs, etc., can solve the problem of wrong intra-chain or inter-chain disulfide bonds, coupling drug linker molecule inconsistency, etc. Stability, difficult to dissociate and other issues

Active Publication Date: 2020-08-18
CHINA PHARM UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] ADC drugs currently have traditional ADC product heterogeneity, drug / antibody ratios (drug / antibody ratios, DAR) are not uniform; the design of the mutation site is not appropriate, if the site is not exposed on the surface of the antibody, small molecules cannot reach or even Close to the coupling site, if the site reacts with a certain thiol group in the chain or between the chains, it will form a wrong intra-chain or inter-chain disulfide bond, and the free thiol group at the coupling site It will also be reduced to disulfide bonds, etc., which will lead to a decrease in the coupling rate and the problem of DAR inhomogeneity; the drug coupling site is not exposed on the antibody surface, and wrong chains are formed between the coupling sites Intra-chain or inter-chain disulfide bonds, conjugated drug linker molecules are unstable, conjugated drugs are easy to fall off, dissociate in blood, and cannot be released in target cells (off-target), specifically due to the spatial structure of macromolecular antibodies and small molecule compounds There may be structural interaction, such as affecting the binding of macromolecular antibodies to receptors, antibodies may wrap small molecular compounds and are difficult to release; small molecules and linkers are too firmly bound, and it is difficult to dissociate under the action of enzymes or acid-base conditions
Antibodies bind to the cell surface, and small molecules are released in advance, unable to enter the cell interior, making it difficult to play the role of small molecules

Method used

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  • Conjugate of anti-human DLL4 humanized antibody and dolastatin derivative MMAE as well as preparation method and application of conjugate
  • Conjugate of anti-human DLL4 humanized antibody and dolastatin derivative MMAE as well as preparation method and application of conjugate
  • Conjugate of anti-human DLL4 humanized antibody and dolastatin derivative MMAE as well as preparation method and application of conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Using molecular biology technology to prepare the engineered anti-human DLL4 humanized antibody THL4 (including the anti-human DLL4 humanized antibody H3L2 (naked antibody) preparation method obtained by site-directed mutagenesis of the anti-human DLL4 humanized antibody H3L2, as in the reference literature [ 1].): (1) According to the nucleic acid sequences of the light chain and heavy chain of the humanized antibody H3L2, determine the corresponding antibody light chain 207 (valine) and heavy chain 121 (lysine), and design primers (8 items in total), mutation into the nucleotide corresponding to cysteine ​​by Overlap-PCR; (2) TA cloning, transforming the target fragment into E. coli DH5α host strain, and picking a single clone for DNA sequencing , The sequence of the correct sequence was digested and linked to the two expression plasmids pMH3 (neomycin resistance) and pCA-puro (puromycin resistance). After DNA sequencing was correct, 4 engineering plasmids were obtained...

Embodiment 2

[0067] The coupling between MMAE and the engineered anti-human DLL4 humanized antibody THL4 was detected by HPLC.

[0068] Agilent 1200HPLC analyzes the coupling of antibody-drug conjugate HLvM4. The sample detection conditions are as follows: (1) Mobile phase A: 20mmol / LPBS(pH 7.0)+1.5mol / L ammonium sulfate; (2) Mobile phase B: 20mmol / LPBS(pH7.0) / isopropanol=7.5 / 2.5 (3) Elution gradient: 10-100% B; (4) Elution time: 20 min; (5) Flow rate: 0.60 mL / min; (6) Injection volume: 10 uL; (7) Detection wavelength: 280 nm. According to the number of peaks and the area of ​​each peak, the antibody-drug coupling ratio (DAR) is calculated proportionally.

[0069] See the results of the experiment figure 2 Compared with H3L2, which has the main peak at 9.min, HLvM4 also peaks at 11min, 13.5min, 17min and 19min, respectively. The number of MMAE small molecules corresponding to the coupling is 2, 4, 6 and 8 and the corresponding peak area ratio

Embodiment 3

[0071] Affinity test of antibody drug conjugate HLvM4 and human DLL4 antigen: using ELISA method, add 1μg / mL DLL4 antigen to 96-well microtiter plate at 100μL per well, and coat overnight at 4℃; after washing the plate three times with PBS, it will be implemented The conjugates HLvM4 and H3L2 of Example 1, according to the blank group, the concentration gradients 0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 nM were added to a 96-well plate, 37 ℃, incubated for 2 hours; After washing the plate three times with PBS, add goat anti-mouse IgG antibody coupled with horseradish peroxidase (HRP) at 37°C for 1 hour; after washing the plate three times with PBS, add TMB color developing solution at room temperature The reaction was kept in the dark for 20 minutes, and finally the stop solution was added to terminate the reaction. The microplate reader detected OD450-OD630.

[0072] See the results of the experiment image 3 Compared with H3L2, HLvM4 has a slightly lower affinity for ...

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Abstract

The invention discloses a conjugate of an anti-human DLL4 humanized antibody and a dolastatin derivative MMAE as well as a preparation method and an application of the conjugate, and relates to the technical field of biological pharmacy. The conjugate of the novel anti-human DLL4 humanized antibody THL4 and the dolastatin derivative MMAE is obtained. The preparation method is characterized in thata small molecular toxin aplysiatoxin derivative MMAE is coupled to an engineered anti-human DLL4 humanized antibody THL4 at a fixed point to synthesize a conjugate, and the conjugate is used in the preparation method. The conjugate HLvM4 is used for promoting the tumor targeting of toxin molecules MMAE, reducing the toxic action on normal cells of a body and achieving a better treatment effect.

Description

Technical field [0001] The present invention is in the field of antibody-conjugated drugs. Specifically, the present invention relates to site-directed mutagenesis of the anti-human DLL4 humanized antibody H3L2 to obtain an engineered antibody, which is a conjugate of a small toxin molecule that is a dopetoxin derivative MMAE. Background technique [0002] DLL4 (Delta like 4) is an important ligand of Notch receptor in the evolutionarily conserved Notch signaling pathway. DLL4 ligand is involved in regulating the proliferation, apoptosis, and differentiation of adjacent cells by binding to Notch receptors on the surface of adjacent cells. And the biological processes of stem cell proliferation and renewal. Not only that, DLL4 is overexpressed in human tumor tissues and participates in the development of tumor blood vessels. So far, researchers have observed DLL4 overexpression in tumor blood vessels such as kidney cancer, bladder cancer, colon cancer, brain tumor and breast can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/68A61K38/07A61P35/00
CPCA61K38/07A61K47/6843A61P35/00
Inventor 吴旻王旻王世静费文仪赵玉红温慧冯宇琪匡璐
Owner CHINA PHARM UNIV
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