Tumor necrosis factor-like ligand 1a specific antibodies and compositions and uses thereof
A tumor necrosis factor-like ligand technology, applied in the field of full-length antibodies and their antigen-binding fragments, can solve problems such as short bowel syndrome
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Embodiment 1
[1001] Example 1: Production of Anti-TL1A Antibodies
[1002] Recombinant soluble human and mouse TL1A proteins were transiently expressed in HEK293 cells. TL1A protein was purified by HitrapNTA, HitrapQ and Sephacryl-200 (all purchased from GE healthcare). The resulting purified protein solution was concentrated and stored below -80°C. Purity was confirmed by SDS-PAGE and analytical SEC.
[1003] Recombinant soluble human and mouse TL1A proteins were used to immunize Medarex KM and Hco mice. Some mice received alternating human and mouse TL1A, while others received only human TL1A. In some instances, 3x25 μg recombinant human TL1A + 1x25 μg recombinant mouse TL1A in Ribi adjuvant were administered weekly intraperitoneally and subcutaneously to mice. Hybridomas generated by the E-fusion protocol were prepared from mice showing reactivity against TL1A by serum titer analysis. Subsequent hybridomas were screened for production of antibodies that bind TL1A but not TNF[alpha]...
Embodiment 2
[1004] Example 2: Epitope grouping of anti-TL1A antibodies by SPR
[1005] A pair-binding strategy was used to characterize anti-TL1A antibodies by epitope grouping using surface plasmon resonance. One antibody was immobilized directly to the carboxymethylated dextran sensor chip surface (on CM5) by amine coupling using a Biacore 2000 or 3000 instrument. Subsequently, inject in 8.1mM Na at a flow rate of 10μl / min 2 HPO 4 , 1.47mMKH 2 PO 4 Recombinant soluble human TL1A or murine TL1A diluted to 10 nM in (pH 7.2), 237mM NaCl, 2.7mM KCl, 3.4mM EDTA and 0.01% tween20 (PBS-NET) for about 1 minute to achieve on immobilized antibody or antigen-binding fragment thereof Binding levels of at least 100 response units (RU). Then, the same antibody immobilized on the chip was injected at 30 nM for 5 minutes to saturate all potential binding sites on trimeric TL1A. Repeat injections of antibody were performed to confirm this saturation. Finally, secondary antibody in PBS-NET or PBS-...
Embodiment 3
[1008] Example 3: Characterization of TL1A binding kinetics of neutralizing antibodies
[1009] To characterize the binding kinetics of anti-TL1A antibodies against TL1A by surface plasmon resonance, each anti-TL1A antibody was captured on a carboxymethylated dextran sensor chip by directly immobilized anti-human IgG (GE Healthcare) using a Biacore T100 or T200 instrument on the surface (CM5). Anti-human IgG was immobilized by amine coupling to a density of approximately 4,000 to 13,000 response units (RU). at 8.1mM Na 2 HPO 4 , 1.47mMKH 2 PO 4 Each anti-TL1A antibody was diluted to 0.075-0.15 μg / ml in (pH 7.2), 237 mM NaCl, 2.7 mM KCl, 3.4 mM EDTA and 0.01% tween20 (PBS-NET) and injected on the anti-hIgG surface at a flow rate of 5 μl / min. About 1 to 2 minutes to achieve low capture levels as low as 30RU. After capture, the flow rate was increased to 100 μl / min and various concentrations of recombinant soluble human TL1A, cynomolgus TL1A or murine TL1A were injected in ...
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