Polypeptide that binds aberrant cells and induces apoptosis
a polypeptide and aberrant cell technology, applied in the field of biotherapeutics, can solve the problems of affecting the therapeutic application of these anti-mhc antibodies, lack of target cell specificity, and cancer development, and achieve the effects of improving the ability to reverse thermal denaturation, improving affinity and stability, and improving the ability to bind aberrant cells
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example 1
Selection of Human Antibody Fragments Specific for HLA-A0201 / Multi-MAGE-A
[0106]To obtain human antibody fragments specific for the HLA-A0201-presented multi-MAGE-A epitope Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:11) or Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:23), a Human Fab phage display library was constructed according to the procedure previously described by de Haard et al.(2) and used for selections essentially as described by Chames et al.(3) Alternatively, a human VhCh library was constructed and used for selections. Human Fab / VhCh phages (1013 colony-forming units) were first pre-incubated for 1 hour at room temperature in PBS containing 2% non-fat dry milk (PB SM). In parallel, 200 μl Streptavidin-coated beads (Dynal™) were equilibrated for 1 hour in PBSM. For subsequent rounds, 100 μl beads were used. To deplete for pan-MHC binders, each selection round, 200 nM of biotinylated MHC class I-peptide (MHC-p) complexes containing an irrelevant peptide (Sanquin, the Netherlands) were added to the pha...
example 2
Production of Monovalent and Multivalent AH5-Apoptin Polypeptides and Monovalent and Multivalent AH5-Cath-Apoptin Polypeptides
Design of Genes for Production of Tetrameric AH5 VH-Apoptin and AH5 Vh-Cath-Apoptin
[0110]Human antibody germline gene VH3 demonstrates high homology to llama single domains VHH. Exchange of amino-acids 44, 45 and 47 in the human VH3 genes by amino-acids present in llama VHH at these positions has shown to enhance stability and expression of the human VH3 genes. All substitutions described to have an effect on protein stability and / or solubility include: E6A, A33C, V37F, G44E, L45R, W47G, S74A, R83K, A84P or L108Q.
[0111]The AH5 VH demonstrates a low homology to germline gene VH3-33*01 (71% as determined by IMGT homology search); however, its expression and stability might benefit from the exchange of amino-acids 6, 44, 45 and 47 and 108 by llama VHH amino-acid residues, a process called camelization. In addition, a gene was compiled that upon expression, compr...
example 3
Cell Binding and Internalization of Tetra-AH5-Cath-Apoptin
[0114]Binding capacity of tetra-AH5-cath-apoptin was analyzed by flow-cytometry. HLA-A0201 / multi-MAGE-A-positive tumor cells (Daju, MDN and mel 624) and HLA-A0201 / multi-MAGE-A-negative cells (BSM, G43 and 293) were incubated on ice with purified protein and detected by addition of fluorescently labeled anti-His antibodies. Cells bound by the proteins were quantified and visualized by flow cytometry. Internalization of tetra-AH5-cath-apoptin was analyzed by confocal microscopy. To this end, cells were incubated with the proteins, kept on ice for 30 minutes to allow binding but no internalization. Next, fluorescently labeled anti-His antibodies were added. To induce internalization, cells were transferred to 37° C. and fixed with 1% PFA after 5, 10 and 15 minutes.
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