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 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, and 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)
EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYA
GSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ
GTKVDIKR;
C is the amino acid sequence
(Seq. Id. No. 3)
EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPR
LLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWP
PWTFGQGTKVEIKR;
D is the amino acid sequence
(Seq. Id. No. 4)
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIY
DNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVV
FGGGTKLTVLG;
E is the amino acid sequence
(Seq. Id. No. 5)
EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPR
LLIYDTSKRATGVPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWP
HTFGGGTKLEIKR;
F is the amino acid sequence
(Seq. Id. No. 6)
SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVL
VVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSD
HNVFGSGTKVEIKR;
G is the amino acid sequence
(Seq. Id. No. 7)
LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVL
VVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSD
YVVFGGGTKLTVLG; and
K is the amino acid sequence
(Seq. Id. No. 11)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL
EWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY
CAKDAGIAVAGTGFDYWGQGTLVTVSS.
2. A single-chain antibody of claim 1 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.
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 2 , wherein the second protein provides a read-out for intracellular assays.
5. A single chain antibody framework comprising a human variable light chain framework (VL) and a human variable heavy chain framework (VH), the single chain antibody framework being selected from the group consisting of:
AK, BK, CK, DK, EK, FK, and 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)
EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYA
GSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ
GTKVDIKR;
C is the amino acid sequence
(Seq. Id. No. 3)
EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPR
LLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWP
PWTFGQGTKVEIKR;
D is the amino acid sequence
(Seq. Id. No. 4)
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIY
DNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVV
FGGGTKLTVLG;
E is the amino acid sequence
(Seq. Id. No. 5)
EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPR
LLIYDTSKRATGVPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWP
HTFGGGTKLEIKR;
F is the amino acid sequence
(Seq. Id. No. 6)
SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVL
VVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSD
HNVFGSGTKVEIKR;
G is the amino acid sequence
(Seq. Id. No. 7)
LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVL
VVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSD
YVVFGGGTKLTVLG; and
K is the amino acid sequence
(Seq. Id. No. 11)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL
EWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY
CAKDAGIAVAGTGFDYWGQGTLVTVSS.
6. A single chain antibody framework selected from the group consisting of variants of the single chain antibody frameworks according to claim 5 .
7. An antibody comprising the VL or the VH or both from the single chain antibody framework according to claim 5 .
8. An antibody fragment comprising the VL or the VH or both from the single chain antibody framework according to claim 5 .
9. The single-chain antibody 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.
10. The single-chain antibody of claim 1 , comprising 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.