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 nucleic acid capable of encoding a single chain antibody comprising a human variable light chain framework (VL) and a human variable heavy chain framework (VH) 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
AK, BK, CK, DK, EK, FK, or GK:
wherein A is the amino acid sequence (Seq. Id. No. 1)
EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFGQG
TKLTVLG;
B is the amino acid sequence (Seq. Id. No. 2)
EIVLTQSPSSLSASVGDRVILTCRASQGIRNELAWYQQRPGKAPKRLIYA
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;
and
K is the amino acid sequence (Seq. Id. No. 11)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDA
GIAVAGTGFDYWGQGTLVTVSS.
2. A vector comprising the nucleic acid according to claim 1 .
3. A host cell comprising the nucleic acid according to claim 1 .
4. The nucleic acid of claim 1 , wherein orientation of the VH and VL regions of the single chain antibody is reversed.
5. The nucleic acid of claim 1 , wherein the VL domain has the framework sequence of SEQ ID NO: 1 and the VH domain has the framework sequence of SEQ ID NO: 11.
6. A vector comprising the nucleic acid according to claim 5 .
7. A host cell comprising the nucleic acid according to claim 5 .
8. The nucleic acid of claim 1 , wherein the single-chain antibody comprises a variant of the VL framework sequence of SEQ ID NO: 1 and a variant of the VH framework sequence of SEQ ID NO: 11.
9. A vector comprising the nucleic acid according to claim 8 .
10. A host cell comprising the nucleic acid according to claim 8 .