Anti-PCSK9 antibodies and methods of use
An antibody, antibody fragment technology, applied in the direction of antibodies, chemical instruments and methods, anti-enzyme immunoglobulin, etc., can solve problems such as increase, increase susceptibility to coronary artery disease, and decrease
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Embodiment 1
[0286] Example 1: Production of anti-PCSK9 antibodies
[0287] Residue numbering is according to Kabat (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).
[0288] Library sorting and screening to identify anti-PCSK9 antibodies
[0289] Biotinylated human PCSK9 produced in-house was used as antigen for library sorting. Five rounds of phage library sorting were performed against biotinylated PCSK9 in solution phase. For the first round of sorting, 20 μg / mL of biotinylated PCSK9 was added to phage blocking buffer PBST (phosphate-buffered saline (PBS) with 1% (w / v) bovine serum albumin (BSA) and 0.05 % (v / v) Tween20) pre-blocked antibody phage library VH (see, for example, Lee et al., J. Immunol. Meth. 284:119-132, 2004) and VH / VL (see Liang et al., JMB.366: 815-829, 2007), and incubated overnight at room temperature. The next day, add 120 μl of pre-absorbed PBST / BSA to each libra...
Embodiment 2
[0301] Example 2: Characterization of anti-PCSK9 antibodies by BIAcore
[0302] Binding affinities of anti-PCSK9 antibodies by surface plasmon resonance (SPR) using BIAcore TM -3000 instruments to measure. Anti-PCSK9 human antibody was captured by mouse anti-human Fc antibody (Cat. No. BR-1008-39, GE Healthcare, Piscataway, NJ) coated on a CM5 biosensor chip to obtain approximately 200 response units (RU). For kinetic measurements, human, mouse, rhesus and cynomolgus PCSK9 (Genentech, South San Francisco, CA) were injected twice at 25°C at a flow rate of 30 μl / min in PBT buffer (PBS containing 0.05% Tween20). Serial dilutions (500 nM to 0.245 nM). Use a simple one-to-one Langmuir binding model (BIAcoreEvaluationSoftware3.2 version) to calculate the binding rate (k on ) and dissociation rate (k off ). Equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on . See Figure 3.
Embodiment 3
[0303] Example 3: LDLR-PCSK9 Binding Assay
[0304] A competition binding ELISA was performed to investigate the activity of anti-PCSK9 antibodies in blocking human PCSK9 binding to LDLR. Briefly, 1 μg / mL of soluble human LDLR extracellular domain (R&D Systems, Minneapolis, MN) was coated overnight at 4°C in 384-well MaxiSorp plates. NUNC TM International, Rochester, NY). Then 0.25 μg / mL of biotinylated human PCSK9 premixed with different concentrations of anti-PCSK9 antibody and control antibody was added to the plate and incubated at room temperature for 2 hours. PCSK9 and encapsulation were detected by adding streptavidin-HRP (GE Healthcare) and the substrate 3,3′,5,5′-tetramethylbenzidine (TMBE-1000, Moss, Inc., Pasadena, MD). LDLR binding. Binding results (OD) were plotted against antibody concentration and IC was generated using KaleidaGraph software 50 value. See Figure 4 .
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