Polypeptides, antibody variable domains and antagonistsmethods for
a technology of antibody variable domain and polypeptide, which is applied in the field of polypeptides, antibody variable domain and antagonistsmethod, can solve the problems of limited efficacy of agents that have potential in vivo use (e.g., treating, diagnosing or preventing disease), and generally not effective antagonists of tnfr1
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Lead Selection & Characterisation of Domain Antibodies to Human TNFR1
[0364]Domain antibodies generated were derived from phage libraries. Both soluble selections and panning to passively absorbed human TNFR1 were performed according to the relevant standard methods. Human TNFR1 was purchased as a soluble recombinant protein either from R&D systems (Cat No 636-R1-025 / CF) or Peprotech (Cat no. 310-07) and either used directly (in the case of passive selections) or after biotinylation using coupling via primary amines followed by quality control of its activity in a biological assay and analysis of its MW and extent of biotinylation by mass spectrometry. Typically 3 rounds of selection were performed utilising decreasing levels of antigen in every next round.
Outputs from selections were screened by phage ELISA for the presence of anti-TNFR1 binding clones. DNA was isolated from these phage selections and subcloned into a expression vector for expression of soluble dAb fragments. Solubl...
example 1
Phage Vector pDOM13
[0382]A filamentous phage (fd) display vector, pDOM13 was used. This vector produces fusion proteins with phage coat protein III. The multiple cloning site of pDOM13 is illustrated in FIG. 1. The genes encoding dAbs were cloned as SalI / NotI fragments.
example 2
Test Protease Selections on Phage-Displayed Domain Antibodies (dAbs) with a Range of Resistance to Trypsin
[0383]The genes encoding dAbs DOM4-130-54 which binds IL-1R1, DOM1h-131-511 which binds TNFR1, and DOM15-10, DOM15-26 and DOM15-26-501, which bind VEGFA, were cloned in pDOM13 and phages displaying these dAbs were produced according to standard techniques. Phages were purified by PEG precipitation, resuspended in PBS and titered.
[0384]The above dAbs displayed a range of ability to resist degradation by trypsin when tested as isolated proteins. Resistance to degradation was assessed as follows: dAb (1 mg / ml) in PBS was incubated with trypsin at 40 μg / ml at 30° C., resulting in a molecular ratio of 25:1 dAb:trypsin. Samples (30 μl) were taken immediately before addition of trypsin, and then at T=1 hour, 3 hours, and 24 hours. Protease activity was neutralized by addition of Roche Complete Protease Inhibitors (2×) followed by immersion in liquid nitrogen and storage on dry ice. 15 ...
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