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Design and application of mutant of antibody resisting to human CD24

A CD24, mutant technology, applied in the field of bioengineering, can solve problems such as hindered recognition and loose substrates

Inactive Publication Date: 2017-04-26
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Farias et al. inserted the C-terminals of HC and LC in the anti-M1S1 antibody into LLQGA and GGLLQGA sequences, respectively, and coupled with MMAE based on the catalytic effect of TG. The average DAR of the obtained ADCs was 1.9 and 1.8, respectively, and the monomer rate was 99%. and 98.5%, but about 1.3% of the antibody in the resulting ADC formed an additional conjugation site at the HC-Q295 residue, which may be due to the fact that TG is not strict with the substrate and will deglycosylate the antibody The upper HC-Q295 site is regarded as its substrate, while the normal antibody will hinder the recognition of the nearby HC-Q295 site by TG due to glycosylation modification at the HC-N297 site

Method used

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  • Design and application of mutant of antibody resisting to human CD24
  • Design and application of mutant of antibody resisting to human CD24
  • Design and application of mutant of antibody resisting to human CD24

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of anti-human CD24 antibody mutant cG7Q

[0031] Firstly, the plasmid containing the cG7 heavy chain gene was extracted from the constructed anti-human CD24 chimeric antibody. Using the extracted heavy chain gene as a template, add 2 pairs of synthetic primers containing mutation sites. The primer sequences are Primer1: 5'-AGTGCTAGCGCCTCCACCAAGGGCCCATCGGTC-3'; Primer2: 5'-CGCTGACCACACGGTACGTGCTTTGGTACTGCT-3'; Primer3: 5'- AGCAGTACCAAAAGCACGTACCGTGTGGTCAGCG-3'; Primer4: 5'-TTGCGGCCGCAATCTAGAGCTTACTATTTACCC-3'. Overlap PCR technology was used for gene mutation and splicing, PCR products were detected by 1% agarose gel electrophoresis, and target gene fragments were recovered with an agarose gel recovery kit. The target gene and pMH3 / pCApuro were digested separately, and the target gene and plasmid fragments were recovered after digestion. T4 ligase was ligated overnight at 16°C to obtain two recombinant plasmids (HT-pMH3, HT-PCApuro) containing th...

Embodiment 2

[0032] Example 2 Expression and purification of anti-human CD24 antibody mutant cG7Q

[0033] Firstly, the heavy chain recombinant plasmid vectors HT-pMH3, HT-PCApuro and the light chain vector L-pMH3, L-PCApuro were introduced into CHO- s cells, and two rounds of pressurized screening (Dot Blot semi-quantitative screening) were performed to obtain cell lines stably expressing cG7Q. The screened high-expression cell lines were expanded step by step, the cell culture fluid was collected, and the sample was filtered through a 0.22 μm filter membrane, followed by protein A column affinity chromatography purification, and finally a large amount of the target protein was obtained. 8% SDS-PAGE and 12% SDS-PAGE protein electrophoresis and Western blot for preliminary verification of the target protein.

Embodiment 3

[0034] Example 3 Surface plasmon resonance affinity analysis for cG7Q

[0035] In this experiment, Biacorx100 was used as the detection instrument to analyze the interaction between antibody mutant cG7Q and CD24. Coupled with appropriate concentration of antibody cG7Q or cG7 on the CM5 chip. The serially diluted CD24-GST antigen was injected at a constant flow rate, allowing the antibody conjugated on the chip to bind to the CD24-GST in the sample, and the change in response value (RU) was recorded. Theoretically, the antibody and CD24-GST protein are combined at a ratio of 1:2, so a bivalent model was selected for fitting. The binding constant ka and dissociation constant kd were analyzed with Biacore X100 software, and the equilibrium dissociation constant KD (kd / ka) was calculated.

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Abstract

The invention belongs to the technical field of genetically engineered antibodies, and particularly relates to a preparation method and application of a mutant of an antibody resisting to the human CD24. According to the preparation method, by the adoption of a genetic engineering technology, asparagine at the conserved glycosylation site of the 297th position in a heavy chain CH2 region of the chimeric antibody resisting to the human CD24 is mutated into glutamine; cG7Q is stably expressed by a mammal eukaryotic expression system; the mutant cG7Q of the antibody resisting to the human CD24 basically retains the high affinity of the maternal monoclonal antibody cG7 and can be specifically bound with CD24 molecules on the surfaces of tumor cells, and the ADCC effect of the mutated antibody is slightly decreased, so the nonspecific toxicity of the antibody and conjugates thereof can be easily reduced; the mutated antibody exposes alternative glutamine at position 295, so that micromolecular toxic substances can be conjugated to the mutated antibody in a site-directed mode by means of a primary amine-containing linker under the catalysis of glutamine transaminase, and therefore an antibody-drug conjugate with the conjugation ratio of drug to antibody being 4 is prepared.

Description

Technical field [0001] The invention belongs to the field of bioengineering, and specifically involves chimeric transformation of the mouse anti-human CD24 monoclonal antibody G7mAb (Patent Publication No.: CN103819561A) independently screened by our laboratory through hybridoma technology to reduce its immunogenicity, and thereby Site-directed mutagenesis was performed based on the chimeric antibody cG7. This new antibody mutant cG7Q has a mutation in the conserved glycosylation site at position 297 of the CH2 region of the antibody constant region. It exposes the natural coupling site glutamine at position 295 of the CH2 region and introduces it for antibody-drug site-directed coupling. Two additional glutamine sites are linked while maintaining the high-affinity targeting and effector activity of the parent antibody. Background technique [0002] Antibody-drug conjugate (ADC) is composed of a monoclonal antibody and a potent toxic drug coupled through a bioactive linker....

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K16/28C12N15/85A61K47/68A61K45/00A61P35/00
Inventor 马招兄袁敏讷徐瑶
Owner CHINA PHARM UNIV
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