Engineered polypeptides
A technology of constructs and sequences, applied in the direction of peptides, hybrid peptides, specific peptides, etc., can solve problems such as limiting the therapeutic use of macromolecules
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Embodiment 1
[0409] Example 1. Generation of TfR targets
[0410] DNA encoding the transferrin receptor (TfR) extracellular domain (ECD) (residues 121-760 of human (SEQ ID NO: 235) or cynomolgus monkey (SEQ ID NO: 300) TfR) was cloned into the In mammalian expression vectors with C-terminal cleaved His and Avi tags. The plasmid was transfected into HEK293 cells and allowed to express. The extracellular domain was purified from the collected supernatant using Ni-NTA chromatography followed by size exclusion chromatography to remove any aggregated protein. The yield was about 5 mg per liter of culture. Store protein in 10 mM K 3 PO 4 (pH 6.7), 100 mM KCl, 100 mM NaCl, and 20% glycerol and frozen at -20°C.
[0411] DNA encoding the permuted TfR apical domain (SEQ ID NO: 301) (residues 326-379 and 194-296 of human or cynomolgus TfR) was cloned with an N-terminal His-tag for purification and for in vitro Biotinylated Avi-tagged pET28 vector. The plasmid was co-transformed into BL21(DE3) ...
Embodiment 2
[0414] Example 2. Design and Characterization of Engineered Transferrin Receptor Binding Polypeptides
[0415] This example describes the design, generation and characterization of polypeptides of the invention. For purposes of this example and comparing amino acids that are identical in cloned sequences, "conservative" mutations are considered mutations present in all clones identified (non-conservative amino acid substitutions), while "semi-conservative" mutations are those present in >50% Mutations present in clones.
[0416] Unless otherwise indicated, the positions of amino acid residues in this section are based on the numbering of SEQ ID NO: 1 , the human IgGl wild-type Fc region has three residues from the hinge, PCP, at the amino terminus.
[0417] Design of Peptide Fc Region Library
[0418]By selecting certain solvent-exposed surface patches for modification, constructing surface-display libraries in which the amino acid composition of the selected patches is al...
Embodiment 3
[0575] Example 3. Using Biacore TM Binding characteristics of other CH3C variants identified
[0576] Use Biacore TM T200 instrument, determination of affinity of clonal variants to recombinant TfR apical domain by surface plasmon resonance. Biacore TM S-series CM5 sensor chips were immobilized with anti-human Fab (Human Fab Capture Kit from GE Healthcare). 5 μg / mL of polypeptide-Fab fusions were captured on each flow cell for 1 min and injected into serial 3-fold dilutions of human or cynomolgus apical domains at a flow rate of 30 μL / min at room temperature. Each sample was assayed with 45 minutes of association and 3 minutes of dissociation. After each injection, the chip was regenerated using 10 mM glycine hydrochloride (pH 2.1). Binding responses were corrected for by subtracting RU from the flow cell capturing irrelevant IgG at a similar density. By using Biacore TM T200 evaluation software v3.1, for the response of concentration fitting equilibrium to obtain the...
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