Immunoglobulin frameworks which demonstrate enhanced stability in the intracellular environment and methods of identifying same

摘要

Compositions are provided, which can be used as frameworks for the creation of very stable and soluble single-chain Fv antibody fragments. These frameworks have been selected for intracellular performance and are thus ideally suited for the creation of scFv antibody fragments or scFv antibody libraries for applications where stability and solubility are limiting factors for the performance of antibody fragments, such as in the reducing environment of a cell. Such frameworks can also be used to identify highly conserved residues and consensus sequences which demonstrate enhanced solubility and stability.

主权项

1. A single-chain antibody comprising a variable light chain framework VL and a variable heavy chain framework VH of a human and naturally occurring antibody which is highly stable and soluble under reducing conditions having the general structure:
NH2-VL-linker-VH-COOH; or NH2-VH-linker-VL-COOH wherein the single-chain antibody has the VH framework and the VL framework of AJ, BJ, CJ, DJ, EJ, FJ, or GJ; wherein A is the amino acid sequence (Seq. Id. No. 1)EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFGQGTKLTVLG; B is the amino acid sequence (Seq. Id. No. 2)EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYAGSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQGTKVDIKR; C is the amino acid sequence (Seq. Id. No. 3)EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPRLLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWPPWTFGQGTKVEIKR; D is the amino acid sequence (Seq. Id. No. 4)QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVVFGGGTKILTVLG; E is the amino acid sequence (Seq. Id. No. 5)EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPRLLTYDTSKRATGVPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWPHTFGGGTKLEIKR; F is the amino acid sequence (Seq. Id. No. 6)SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSDHNVFGSGTKVEIKR; G is the amino acid sequence (Seq. Id. No. 7)LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVLVVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVVFGGGTKLTVLG; H is the amino acid sequence (Seq. Id. No. 8)QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHVLRFLEWLPDAFDIWGQGTLVTVSS; I is the amino acid sequence (Seq. Id. No. 9)EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSSQSGVPSRFRGSESGTDFTLTISNLQPEDFATYYCQQSYRTPFTFGPGTKVETKR; and J is the amino acid sequence (Seq. Id. No. 10)VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRINPDSGDTIYAQKFQDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARVPRGTYLDPWDYFDYWGQGTLVTVSS.