Anti-cd25 for tumour specific cell depletion
An acd25-a-686-hcdr3, antibody technology, applied in anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, etc., can solve problems such as blocking signal transduction
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Embodiment 1
[0169] Example 1: Production of CD25-binding antibodies in vitro
[0170] Materials and methods
[0171] CD25 antigen preparation
[0172]Mouse CD25-HIS, human CD25-Fc and untagged recombinant protein were purchased from R&D Systems Biotechne. Cynomolgus monkey CD25-Fc and CD25-HIS recombinant proteins were purchased from Sino Biological. Biotinylation of protein reagents was performed using the EZ-Link Sulfo-NHS-Biotinylation kit (Thermo Scientific, Cat# 21425). The CD25 antigen was concentrated to approximately 1 mg / mL and the buffer was exchanged into PBS prior to the addition of a 1:7.5 molar ratio biotinylation reagent (EZ-LinkSulfo-NHS-Biotinylation Kit, Thermo Scientific, Cat# 21425). The mixture was kept at 4°C overnight, after which another buffer exchange was performed to remove free biotin in solution. Biotinylation was confirmed by streptavidin sensor binding of the labeled protein on the ForteBio.
[0173] Library interrogation and selection methods for iso...
Embodiment 2
[0251] Example 2: Cell-Based Models for Validation of CD25 Modulating Antibody Agents
[0252] Materials and methods
[0253] In vitro IL-2 signaling assayed by STAT5 phosphorylation
[0254]IL-2 blockade was characterized using a STAT5 phosphorylation assay that detects IL-2 signaling. Previously frozen PBMCs (Stemcell Technologies) were incubated in 96-well U-bottom plates in the presence of 10 μg / ml anti-CD25 antibody for 30 minutes, followed by incubation with 10% FBS (Sigma), 2 mM L-glutamine (Life Technologies) and 10,000U / ml Pen-Strep (Sigma) in RPMI 1640 (Life Technologies) with different concentrations of 10U / ml or 0.25U / ml, 0.74U / ml, 2.22U / ml, 6.66U / ml or 20U / ml IL-2 (Peprotech) for 10 min. When fixing cells and using eBioscience TM IL-2-induced STAT5 phosphorylation was terminated when Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen) was permeabilized and treated with BD Phosflow Perm Buffer III (BD Biosciences). Cells were then simultaneously lab...
Embodiment 3
[0273] Example 3: Preparation of Variants of CD25 Modulating Antibody Agents
[0274] aCD25-a-686 was subjected to further affinity maturation. Optimization was performed by introducing diversity into the heavy chain variable region. Recombine the CDRH3 of the antibody into a pre-made 1×10 8 Libraries of diverse CDRH1 and CDRH2 variants were selected by one round of MACS and four rounds of FACS as described in Initial Discovery. In FACS rounds, libraries are viewed for PSR binding, species cross-reactivity, antigen cross-reactivity, and affinity pressure, and sorted to obtain populations with desired characteristics. For these selections, either by titrating down biotinylated monomeric antigen or by pre-incubating biotinylated antigen with parental Fab or IgG for 30 min and then applying the pre-complexed mixture to the yeast library for a period of time to allow selection to reach equilibrium. Apply affinity pressure. Higher affinity antibodies can then be sorted out.
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