Antibody aiming at human complement protein C5 and application thereof

A technology of antibody and protein, applied in application, antibody, anti-animal/human immunoglobulin, etc., can solve problems such as non-response

Pending Publication Date: 2021-10-29
SHANGHAI PUREMAB BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, although the above two drugs can effectively treat most patients with PNH and aHUS, due to the polymorphism of C5 (Arg885 mutant type) in some patients, the patients do not respond to the two drugs.

Method used

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  • Antibody aiming at human complement protein C5 and application thereof
  • Antibody aiming at human complement protein C5 and application thereof
  • Antibody aiming at human complement protein C5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Immunization of Balb / c mice with human complement protein C5

[0094] Antigen Recombinant Human Complement Protein C5 (Gln19-Cys1676) was purchased from Sino Biological (Product No. 13416-H18H). This protein was recombinantly expressed by human 293 cells (HEK293), with a histidine tag at the C-terminus and a Flag tag at the N-terminus.

[0095] Referring to Antibodies: A Laboratory Manual, Second Edition (Edward A. Greenfield 2012), 8-week-old Balb / c mice were immunized for a total of 42 days at intervals of 14 days. Human C5 protein was emulsified in complete or incomplete Freund's adjuvant, and it was injected unilaterally into the subcutaneous tissue of the back of the neck, the root of the tail, and the groin of the mouse, and into the peritoneal cavity. Blood was collected from the tail vein on the 35th day of immunization, and after the antibody titer was detected by ELISA, spleen cells of immunized mice were fused with myeloma cells.

Embodiment 2

[0096] Example 2 Screening and identification of anti-human C5 monoclonal antibody hybridoma cell line

[0097] Human C5 protein was used to immunize the spleen cells of Balb / c mice, and fused with myeloma cells using PEG or electrofusion method, and the fused hybridoma cells were 1x10 7 Cells / well were inoculated in HAT-containing medium and placed in a 384-well plate. After 5 days of culture, the medium was replaced with HT-containing medium to continue culturing for 2-3 days to screen hybridoma cells. After culturing in 384-well plates for 7-10 days, the supernatant of the cells was taken for ELISA experiments to screen hybridoma mother clones capable of secreting anti-human C5 antibodies.

[0098] The method for screening human C5 binding agents by ELISA is as follows: Dilute human C5 protein to 1 μg / ml with PBS buffer, coat 100 μl per well on a 96-well plate (Microwell 96F 167008, Thermo) and incubate overnight at 4°C; take out the next day The 96-well plate was washed...

Embodiment 3

[0100] Example 3 Sequence determination of anti-human C5 monoclonal antibody

[0101] Add the hybridoma mother clones that secrete anti-human C5 antibody and have the activity of inhibiting complement-dependent hemolysis into 96-well plate by limiting dilution method, observe and label the monoclonal cells under the microscope after 2-3 days, and pass ELISA experiment and Monoclonal hybridoma cells secreting anti-human C5 monoclonal antibody were screened by hemolysis assay.

[0102] After expanding the culture of the screened monoclonal hybridoma cells, the total RNA of the cells was extracted according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); the total RNA of the hybridoma cells was reversed using 5×PrimeScript RT Master Mix (Takara) Record into cDNA; use degenerate primers and Extaq PCR reagent (Takara) to amplify antibody light chain variable region IgVL (κ) and heavy chain variable region VH sequence; use PCR clean-up Gel extr...

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Abstract

The invention discloses an antibody aiming at human complement protein C5 and application thereof. The antibody or an antigen binding fragment thereof is bound with human complement protein C5 with high affinity, and the C5 protein or an R885 mutant thereof is prevented from being split into C5a and C5b. The antibody can be used for treating diseases such as rheumatoid arthritis, paroxysmal sleep hemoglobin urine, atypical hemolytic uremia syndrome, myasthenia gravis, neuromyelitis optica or macular degeneration, so that patients are benefited.

Description

technical field [0001] The invention relates to the field of antibody medicine, in particular, the invention relates to an antibody against human complement protein C5 and its use for preparing medicine. Background technique [0002] It is known that the complement system is composed of dozens of proteins (C1 (C1q, C1r, C1s), C2, C3, C4, C5, C6, C7, C8, C9, etc.), and is a complex immune monitoring system that acts as a defense against other biological Infection's first host defense system functions and also distinguishes healthy host tissues from cellular debris, apoptotic and necrotic cells; in addition, participates in clearing immune complexes, regulating adaptive immune responses, promoting tissue regeneration, Angiogenesis, stem cell mobilization, and development of the central nervous system. [0003] Complement protein C5 is a major component of the complement system, a key part of the innate immune system. C5 is a glycoprotein of 190kDa, consisting of two polypept...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P19/02A61P29/00A61P21/04A61P27/02A61P13/12
CPCC07K16/18A61P19/02A61P29/00A61P21/04A61P27/02A61P13/12C07K2317/56C07K2317/565C07K2317/92C07K2317/734A61K2039/505A61K39/395C07K16/00G01N33/53C12N5/10
Inventor 王雪徐晓红高攀陶春艳邓小芳吴建李祥烽毕建军王骊淳任红媛林鉴
Owner SHANGHAI PUREMAB BIO TECH CO LTD
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