US2020165322A1PendingUtilityA1
Immunoglobulin frameworks which demonstrate enhanced stability in the intracellular environment and methods of identifying same
Est. expiryMay 22, 2022(expired)· nominal 20-yr term from priority
C07K 2317/82C07K 16/00C07K 2317/55A61K 48/00C07K 2317/567C07K 2317/94G01N 33/6857C07K 2317/622C07K 2317/21C07K 2317/80A61P 43/00
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Claims
Abstract
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.
Claims
exact text as granted — not AI-modified1 . 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 AH, BH, CH, DH, EH, FH, GH, AI, BI, CI, DI, EI, FI, GI, AJ, BJ, CJ, DJ, EJ, FJ, GJ, AK, BK, CK, DK, EK, FK, or GK wherein A is the amino acid sequence (Seq. Id. No. 1)
EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIY
KASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFG
QGTKLTVLG;
B is the amino acid sequence (Seq. Id. No. 2)
EIVLTQSPSSLSASVGDRVILTCRASQGIRNELAWYQQRPGKAPKRLIY
AGSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMF
GQGTKVDIKR;
C is the amino acid sequence (Seq. Id. No. 3)
EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPR
LLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHW
PPWTFGQGTKVEIKR;
D is the amino acid sequence (Seq. Id. No. 4)
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLI
YDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSG
VVFGGGTKLTVLG;
E is the amino acid sequence (Seq. Id. No. 5)
EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPR
LLIYDTSKRATGVPARFSGSGSGTDFILTISSLEPEDSALYYCQQRNSW
PHTFGGGTKLEIKR;
F is the amino acid sequence (Seq. Id. No. 6)
SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVL
VVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSS
DHNVFGSGTKVEIKR;
G is the amino acid sequence (Seq. Id. No. 7)
LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVL
VVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSS
DYVVFGGGTKLTVLG;
H is the amino acid sequence (Seq. Id. No. 8)
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHV
LRFLEWLPDAFDIWGQGTLVTVSS;
I is the amino acid sequence (Seq. Id. No. 9)
EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPK
LLIYAASSSQSGVPSRFRGSESGTDFILTISNLQPEDFATYYCQQSYRTP
FTFGPGTKVEIKR;
J is the amino acid sequence (Seq. Id. No. 10)
VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRI
NPDSGDTIYAQKFQDRVILTRDTSIGTVYMELTSLTSDDTAVYYCARVPR
GTYLDPWDYFDYWGQGTLVTVSS;
and
K is the amino acid sequence (Seq. Id. No. 11)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL
EWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY
CAKDAGIAVAGTGFDYWGQGTLVTVSSS.
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 1 wherein the VL framework is of the kappa1, lambda 1 or 3 type.
5 . The single chain antibody according to claim 2 , wherein the second protein provides a read-out for intracellular assays.
6 . 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:
AH, BH, CH, DH, EH, FH, GH, AI, BI, CI, DI, EI, FI, GI, AJ, BJ, CJ, DJ, EJ, FJ, GJ, 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
LLIYDTSKRATGVPARFSGSGSGTDFILTISSLEPEDSALYYCQQRNSWP
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;
H is the amino acid sequence (Seq. Id. No. 8)
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHV
LRFLEWLPDAFDIWGQGTLVTVSS;
I is the amino acid sequence (Seq. Id. No. 9)
EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPK
LLIYAASSSQSGVPSRFRGSESGTDFILTISNLQPEDFATYYCQQSYRTP
FTFGPGTKVEIKR;
J is the amino acid sequence (Seq. Id. No. 10)
VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRI
NPDSGDTIYAQKFQDRVILTRDTSIGTVYMELTSLTSDDTAVYYCARVPR
GTYLDPWDYFDYWGQGTLVTVSS;
and
K is the amino acid sequence (Seq. Id. No. 11)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL
EWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY
CAKDAGIAVAGTGFDYWGQGTLVTVSSS.
7 . A single chain antibody selected from the group consisting of variants of the single chain antibody according to claim 6 .
8 . A single chain antibody selected from the group consisting of derivatives of the single chain antibody according to claim 6 .
9 . A method of using the single chain antibody according to claim 6 in target validation, diagnostic applications, library construction or therapeutic applications.
10 . A method of using at least two framework sequences of claim 6 in the identification of a conserved framework residue class.
11 . The method according to claim 10 , wherein the conserved framework residue class is selected from the group consisting of:
polar but uncharged R groups; positively charged R groups; negatively charged R groups; hydrophobic R groups; and special amino acids.
12 . A method of using at least two framework sequences of claim 6 in the identification of at least one conserved framework sequence.
13 . The method according to claim 12 , wherein the conserved framework sequence is 2-5 residues.
14 . The method according to claim 12 , wherein the conserved framework sequence is 5-10 residues.
15 . The method according to claim 12 , wherein the conserved framework sequence is 10-25 residues.
16 . The method according to claim 12 , wherein the conserved framework sequence has gaps.
17 . An antibody comprising the VL or the VH or both from the single chain framework according to claim 6 .
18 . An antibody fragment comprising the VL or the VH or both from the single chain framework according to claim 6 .
19 . A nucleic acid capable of encoding the single chain antibody according to claim 1 .
20 . A vector comprising the nucleic acid according to claim 19 .
21 . A host cell comprising the nucleic acid according to claim 19 .Join the waitlist — get patent alerts
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