Immunoglobulin frameworks which demonstrate enhanced stability in the intracellular environment and methods of identifying same
View Patent ↗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.
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:
NH 2 -VL-linker-VH-COOH; or
NH 2 -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)
EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFGQG
TKLTVLG;
B is the amino acid sequence (Seq. Id. No. 2)
EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYA
GSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ
GTKVDIKR;
C is the amino acid sequence (Seq. Id. No. 3)
EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPRLLIYG
ATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWPPWTFG
QGTKVEIKR;
D is the amino acid sequence (Seq. Id. No. 4)
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIY
DNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVV
FGGGTKILTVLG;
E is the amino acid sequence (Seq. Id. No. 5)
EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPRLLTYD
TSKRATGVPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWPHTFGG
GTKLEIKR;
F is the amino acid sequence (Seq. Id. No. 6)
SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSDHNVFG
SGTKVEIKR;
G is the amino acid sequence (Seq. Id. No. 7)
LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVLVVSDD
SVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVVFG
GGTKLTVLG;
H is the amino acid sequence (Seq. Id. No. 8)
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHV
LRFLEWLPDAFDIWGQGTLVTVSS;
I is the amino acid sequence (Seq. Id. No. 9)
EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSSQSGVPSRFRGSESGTDFTLTISNLQPEDFATYYCQQSYRTPFTFGP
GTKVETKR; and
J is the amino acid sequence (Seq. Id. No. 10)
VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRI
NPDSGDTIYAQKFQDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARVPR
GTYLDPWDYFDYWGQGTLVTVSS.
2. A single-chain framework fused to a second protein moiety to yield a fusion construct of the general structure:
NH 2 -VL-linker-VH-second protein-COOH; or
NH 2 -second protein-VL-linker-VH-COOH
wherein the single-chain framework 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)
EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFGQG
TKLTVLG;
B is the amino acid sequence (Seq. Id. No. 2)
EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYA
GSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ
GTKVDIKR;
C is the amino acid sequence (Seq. Id. No. 3)
EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPRLLIYG
ATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWPPWTFG
QGTKVEIKR;
D is the amino acid sequence (Seq. Id. No. 4)
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIY
DNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVV
FGGGTKLTVLG;
E is the amino acid sequence (Seq. Id. No. 5)
EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPRLLIYD
TSKRATGVPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWPHTFGG
GTKLEIKR;
F is the amino acid sequence (Seq. Id. No. 6)
SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSDHNVFG
SGTKVEIKR;
G is the amino acid sequence (Seq. Id. No. 7)
LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVLVVSDD
SVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVVFG
GGTKLTVLG;
H is the amino acid sequence (Seq. Id. No. 8)
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHV
LRFLEWLPDAFDIWGQGTLVTVSS;
I is the amino acid sequence (Seq. Id. No. 9)
EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSSQSGVPSRFRGSESGTDFTLTISNLQPEDFATYYCQQSYRTPFTFGP
GTKVEIKR; and
J is the amino acid sequence (Seq. Id. No. 10)
VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRI
NPDSGDTIYAQKFQDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARVPR
GTYLDPWDYFDYWGQGTLVTVSS.
3. The single chain antibody of claim 1 , wherein orientation of the VH and VL regions is reversed.
4. The single chain antibody according to claim 1 wherein the VL framework is of the kappa1, lambda 1 or 3 type.
5. The single chain framework according to claim 2 , wherein the second protein provides a read-out for intracellular assays.
6. A single chain antibody selected from the group consisting of variants of the single chain frameworks according to claim 1 or 2 .
7. A single chain antibody selected from the group consisting of derivatives of the single chain frameworks according to claim 1 or 2 .
8. An antibody comprising the VL or the VH or both from the single chain framework according to claim 1 .
9. An antibody fragment comprising at least one variable domain of an immunoglobulin and comprising the VL or the VH or both from the single chain framework according to claim 1 .