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Human CD19 and CD3 binding bispecific antibody

A bispecific antibody, specific technology, applied in the direction of antibodies, specific peptides, anti-inflammatory agents, etc., can solve problems such as increasing the proportion of bifunctional antibodies

Active Publication Date: 2018-04-13
BEIJING LUZHU BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technical feature described in this patented patents relates to combining both immunogloblin protein and scavenger receptor proteins into one single entity called monoantisin or biotype 1 fusion polynucleotides. These agents allow multiple targets like cancerous ones to attach themselves together effectively killing them even if they don't require these treatments. They also work well alone but show promise because their activity depends solely upon the presence of certain parts of the agent itself rather than just its own unique properties such as being able to recognize different types of tissue surfaces. Overall, it offers improved efficiency and accuracy in identifying and developing therapeutic drugs specifically targeting those areas where there may exist unmatched levels of HLA class I molecules associated with autoimmune diseases.

Problems solved by technology

Technologies described in the technical problem addressed in this patented text include developing smaller versions of bioactive compounds called bimultibisceptanbs without compromised properties like immunosuppression capacity, reduced risk of adverse events associated with current methods of administering them, reducing costs involved in producing them, increasing safety during administration, providing flexibility in different ways to optimize the production process, delivering them safely over time, etc., finding suitable formats for preparations based on desired targets, optimizing the ratio between active ingredients while minimizing unwanted interactions.

Method used

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  • Human CD19 and CD3 binding bispecific antibody
  • Human CD19 and CD3 binding bispecific antibody
  • Human CD19 and CD3 binding bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Embodiment 1, the construction of plasmid expression vector:

[0089] Construction of plasmid LZ19HT (pMD19-T Vector+CD19VH+hIgG1CH1) and LZ19VkT (pMD19-TVector+CD19Vk+hIgG1Ck)

[0090] Using primers H-F1, LZ19H-F2 and LZ19H-R1, the fragment CD19VH+hIgG1CH1 was amplified from the heavy chain plasmid PTY5-KJ2-h containing humanized anti-CD19 monoclonal antibody, and the KOZAK sequence and heavy chain signal peptide sequence, Linker ((G4S)3) and restriction site, then add A tail and connect with pMD19-T Vector to obtain plasmid LZ19HT;

[0091] Use primers P71-F1, LZ19Vk-F2 and LZ19Vk-R1 to amplify the CD19Vk+hCk gene fragment from the plasmid PTY5-KJ2-1 containing humanized anti-CD19 monoclonal antibody light chain and introduce KOZAK sequence and light chain signal peptide sequence and Restriction site, then add A tail and connect with pMD19-T Vector to obtain plasmid LZ19VkT;

[0092] The different clones of LZ19HT and LZ19VkT were sent to the sequencing company (Bei...

Embodiment 2

[0099] Embodiment 2, establishment and screening of stable clones

[0100] In a sterile laminar flow workbench, set the perforation voltage of the gene pulse generator Xcell (Bio-Rad) to 300V, 900μF, and exponential pulse, take out a disposable electric shock cup with a gap of 4mm, and add 40μg of linearized plasmid DNA ( 100 μl) and 0.7ml CHO K1 cell (GS KO) suspension, set the resistance of the electroporation instrument to infinity, and directly transfect the linearized plasmid 193HVkP- into CHO K1 cells by electroporation, and electroporate Transfer the cells in the cup to the triangular culture flask, add CD CHO medium, and store at 36-37°C, 5% CO 2 Culture on a shaker at 135 rpm. After 24 hours of culture, collect cells by low-speed centrifugation, replace with CD CHO medium (without glutamine) containing 50 μM MSX, obtain monoclonal cell lines by limiting dilution method, and then pass ELISA The method (mouse anti-human κ chain monoclonal antibody + expression product ...

Embodiment 3

[0101] Example 3. Purification of the expression product of CD19-CD3 bispecific antibody

[0102] Inoculate the obtained 41C11 clone (K193) into a 2L Erlenmeyer flask containing 500ml of CD CHO culture medium, tighten the cap of the ventilated bottle, and store at 36-37°C, 5% CO 2 Cultivate on a shaker at 135 rpm. After 7 days of culture, when the viable cell density drops to 60% to 70%, centrifuge at 12000r / min to remove cells and cell debris, collect the supernatant of the cell culture, and then rinse with pure water. Or 20mM citric acid phosphate buffer solution with pH5.0-6.0 for limited dilution, diluted until the conductivity of the solution does not exceed 4mSimens / cm, and then flows through the chromatographic column filled with strong cation exchange gel Eshmuno S (XK26*20cm, GEHealthCare), under this condition, Eshmuno S gel can adsorb CD19-CD3 bispecific antibody. After loading the sample, wash it with citrate phosphate buffer to the baseline, increase the concentra...

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Abstract

The invention relates to a human CD19 and CD3 binding bispecific antibody. The human CD19 and CD3 binding bispecific antibody comprises Fab fragments for specifically recognizing cell membrane antigens and single-chain antibodies for recognizing CD3 molecules. The single-chain antibodies for recognizing the CD3 molecules are connected with C tail ends of CH1 region peptide fragments of the Fab fragments by hydrophilic connecting peptides-linker; the Fab fragments for specifically recognizing the cell membrane antigens are of Fab structures with specific human CD19 recognizing antigens, the structure of the human CD19 and CD3 binding bispecific antibody is shown, and the connecting peptides-linker comprises 8-20 hydrophilic amino acid.

Description

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Claims

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Application Information

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Owner BEIJING LUZHU BIOTECH
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