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Humanized fabs and humanized antibodies against complement factor d and their uses

A humanized antibody and anti-complement technology, applied in the direction of antibodies, antibody medical components, chemical instruments and methods, etc., can solve problems such as macular degeneration

Active Publication Date: 2022-01-04
CHENGDU KANGHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Drusen is also seen in the elderly with normal vision, but after various pathological changes secondary to it, it leads to macular degeneration

Method used

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  • Humanized fabs and humanized antibodies against complement factor d and their uses
  • Humanized fabs and humanized antibodies against complement factor d and their uses
  • Humanized fabs and humanized antibodies against complement factor d and their uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1, expression of antigen-binding fragment Fab

[0110] (1) Obtaining light and heavy chains

[0111] The humanized antibody Fab light chain and heavy chain sequences were synthesized by artificial gene synthesis (Suzhou Jinweizhi Biological Co., Ltd.). Each sequence is composed of a variable region and a constant region sequence, wherein: the synthetic light chain variable region sequence is sequence 63, the heavy chain variable region sequence is sequence 34, the light chain constant region is sequence 42, and the heavy chain The constant region is sequence 40.

[0112] (2) Construction of humanized Fab vector

[0113] Use the PCR site-directed mutagenesis method to construct the humanized Fab expression vector. The specific steps are: using the synthetic light and heavy chain sequence (plasmid) or the obtained mutant Fab plasmid as a template, the light and heavy chains of each Fab expression vector are constructed with 4 primers were divided into 3 times f...

Embodiment 2

[0128] Expression of embodiment 2Fab periplasmic protein

[0129] The Fab expression vectors constructed above were transformed into Escherichia coli competent cells BL21 (DE3) (Tiangen) respectively, and coated with ampicillin-resistant 2-YT plates (peptone 1.6%, yeast extract 1%, NaCl 0.5%, agar powder 1.5%) ). On the second day, select a plate with a suitable colony density to pick a monoclonal colony and inoculate 5 ml of ampicillin-resistant 2-YT liquid medium, and cultivate overnight. The conditions are: 37°C, 220rpm, 16h. On the next day, inoculate 5ml of bacterial liquid into 500ml of ampicillin-resistant 2-YT liquid medium, and cultivate to OD at 33°C and 220rmp 600is 0.6. IPTG was added to the medium to a final concentration of 50 μM to induce the expression of different Fab proteins for 3 hours. Centrifuge the culture medium after induction expression at 4500rpm and 10°C for 15min, discard the supernatant, and use 40ml pre-cooled TES solution (Tris24.23g, sucrose...

Embodiment 3

[0130] Embodiment 3 Expression of different full-length antibodies

[0131] The full-length antibody protein expression process includes the following steps: transfect CHO cells with the recombinant IgG expression vector constructed above; utilize Puromycin (Invitrogen Company) and MTX (Sigma Company) to jointly screen engineered cell lines that highly express anti-complement factor D protein, The cell line is expanded and cultivated, and finally the full-length antibody is isolated and purified from the cell culture medium. Specifically include:

[0132] (1) Transfection of expression vector

[0133] The host cell CHO-S cell was selected, and the linearized expression vector plasmid constructed above was used for electrotransfection according to the Cell lineNucleofector kit V transfection reagent (Lonza Company). After transfection, place the 6-well plate at 37°C, 8% CO 2 Shaker culture 48h.

[0134] (2) Establishment of CHO-S engineering cell line

[0135] The transfec...

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Abstract

The present invention discloses an anti-complement factor D humanized Fab and humanized antibody and application thereof, the humanized Fab and humanized antibody contain light chain variable region and heavy chain variable region, the use Human complement factor D-related diseases are diseases caused by elevated human complement factor D.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-complement factor D humanized Fab and humanized antibody and their application. Background technique [0002] Age-related macular degeneration, also known as age-related macular degeneration (AMD), is a disease caused by the degeneration of cells or tissues in the central area of ​​the retina and the proliferation of blood vessels. It can be divided into dry AMD and wet AMD. Age-related macular degeneration is mainly manifested as a decrease in the ability of retinal pigment epithelial cells to phagocytize and digest the outer segmental disc membrane of the visual cell, resulting in the retention of incompletely digested disc membrane residual bodies in the basal cell plasma and excreted out of the cells. Deposited on Bruch's membrane, forming drusen. Due to the special structure and function of the macula, this change is more obvious. Drusen is also seen in the elderly wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C12N15/13A61K39/395A61P27/02A61P19/02A61P29/00A61P9/10A61P41/00G01N33/573
CPCC07K16/40C07K2317/70C07K2317/76C07K2317/92C07K2317/24C07K2317/55A61K39/00A61K39/395C07K16/18G01N33/577
Inventor 柯潇雷刚
Owner CHENGDU KANGHONG BIOTECH
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