Anti-HER2 antibodies and compositions
An antibody composition and composition technology, applied in the direction of antibodies, drug combinations, antibody medical components, etc., can solve problems such as large room for improvement
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example 1
[0633] Example 1: Cloning of Anti-HER2 Antibody
[0634] immunity
[0635] Female BALB / c (strain A) or C57B16 mice (8-10 weeks old) were immunized by injecting purified different proteins and HER2 overexpressing cells.
[0636] Commercially available HER2 protein (R&D Systems cat. #1129-ER) was used for immunization. Human breast cancer cell line AU565 (ATCC, CRL-2351) was used for cellular immunization. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P / S). Before each immunization, cells were washed with PBS, digested with TrypLE, and resuspended in growth medium. The cell suspension was then washed twice with PBS, centrifuged at 250 g for 5 minutes, then removed and resuspended in 15 ml sterile PBS.
[0637] Cells or antigen were diluted in PBS and mixed 1:1 with Freund's adjuvant. Freund's adjuvant enhances and modulates the immune response. For the first immunization, complete Freund's adjuvan...
example 2
[0672] Example 2: Functional characterization of selected anti-HER2 antibodies
[0673] Using a viability assay, 41 unique antibodies were selected for functional testing. Cell damage inevitably leads to the cell's ability to maintain and provide energy for metabolic cell function and growth. Metabolic activity assays based on this premise typically measure mitochondrial activity. Cell Proliferation Reagent WST-1 (Roche Cat. No. 11 644 807 001) is a ready-to-use substrate for measuring metabolic activity in living cells. It is hypothesized that metabolic activity correlates with the number of viable cells. In this example, the WST-1 assay was used to measure the number of metabolically active cells treated with 2 μg / ml of different anti-HER2 antibodies for 96 hours.
[0674] The cancer cell lines SKBR-3 (ATCC cat.#HTB-30), BT-474 (ATCC cat.#HTB-20), NCI-N87 (ATCC cat.#CRL-5822) and MDA-453 (ATCC cat. #HTB-130) was inoculated into a 96-well plate at a concentration of 1000 ...
example 3
[0680] Example 3: Determining overlapping epitopes
[0681] In order to select compounds containing anti-HER2 antibodies that exert a synergistic effect in combination, the antibodies in each mixture should bind to non-overlapping epitopes. Therefore, in order to investigate the degree of overlap among anti-HER2 antibodies, epitope pooling was performed using surface plasmon resonance (SPR) technology. Available at ProteOn TM SPR analysis was performed with the XPR 36 protein interaction array system (Biorad Laboratories). The system can measure 6 interactions in two directions (defined as L and A), simultaneously generating a total of 36 possible interactions.
[0682] set up
[0683] ProteOn GLC sensor chips (BioRad) were conjugated to anti-Fc antibodies (Biacore, GE Healthcare) at 3600-3620 resonance units (RU) that could be injected into flow cells L1 to L6 using the ProteOn Amino Conjugation Kit according to the manufacturer's instructions (Biorad). Using a flow rat...
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