Novel tnfr agonists and uses thereof
A kind of technology of agonist and use, applied in the field of use
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Embodiment 1
[0121] Production and screening of mouse anti-human OX40 antibody
[0122] To generate recombinant human OX40-his protein, the extracellular region of human TNFRSF4 (amino acids 1-214 shown in SEQ ID NO: 1) was amplified by PCR and a 3'GSG-6xHis linker and restriction sites were added for cloning. The PCR product was subsequently cloned into the modified pcDNA3.1(-) plasmid described above. This recombinant plasmid allows expression of human OX40-his protein in mammalian cells and secretion into the cell culture medium driven by the native signal peptide of human TNFRSF4. For protein production, use jetPEI TM Transfection reagent (Polyplus-transfection S.A., Strasbourg, France; Distributor: Brunschwig, Basel, Switzerland) transfects the recombinant vector into suspension-adapted HEK 293 cells (ATCC No. CRL 1573). Cell culture supernatants were collected 5 days after transfection and used in Ni operated on an FPLC system (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) ...
Embodiment 2
[0132] Cloning and sequencing of the VH and VL chains of anti-OX40 antibodies from hybridoma cells
[0133] For each positively selected hybridoma, total RNA was prepared, reverse transcribed into cDNA, and the VH and VL genes were separately amplified by PCR. Ligation of these PCR products into a rescue-vector (pDrive vector; QIAGEN AG, Hombrechtikon, Switzerland; Cat.No. 231124) allows DNA sequencing of individual PCR products and determination of the monoclonal or polyclonal nature of selected hybridomas. sex. This vector allows blue / white selection on LB-agar plates containing IPTG and X-gal (colony without insert is blue because LacZa-peptide degrades X-gal). Recombinant plasmids from positive (white) bacterial clones were prepared and sequenced using standard DNA sequencing primers specific to the vector backbone (M13rev, M13fwd, T7 or SP6). Finally, the DNA sequence is subcloned into an expression vector for recombinant expression of the target antibody in mammalian c...
Embodiment 3
[0166] Biological characteristics of anti-human OX40 antibody
[0167] OX40 specific antibody detection ELISA
[0168] Antibody titer, specificity, and production of hybridoma and recombinant antibody candidates were determined by direct ELISA. Briefly, 96-well microtiter plates (Costar USA, distributor VWR AG, Nyon, Switzerland) were coated with 100 μl of recombinant human OX40-his at 2 μg / ml in PBS (see Example 1 for the production of OX40 -his protein). Plates were incubated overnight at 4°C and then blocked with PBS 2% BSA (Bovine Serum Albumine, PAA Laboratories, Pasching, Austria) for 1 hour at room temperature (RT). Blocking solution was removed and hybridoma supernatant or purified antibody was added. Plates were incubated at room temperature for 30 min and then washed with PBS 0.01% Tween-20 (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and horseradish peroxidase (HRP)-labeled goat anti-mouse H+L-9 Second-rate. Detection antibodies (Sigma-Aldrich Chemie GmbH, B...
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