# US 9,518,108 B2 · Immunoglobulin frameworks which demonstrate enhanced stability in the intracellular environment and methods of identifying same

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- Number: US 9,518,108 B2
- Document ID: US9518108B2
- Status: Granted
- Kind: B2
- Application number: 14/469,276
- Filed: 2014-08-26
- Published: 2015-02-26
- Granted: 2016-12-13
- Earliest claimed: 2002-05-22
- Assignees: ESBATech, an Alcon Biomedical Research Unit LLC
- Applicants: ESBATech, an Alcon Biomedical Research Unit LLC
- Inventors: Kathrin Tissot; Stefan Ewert; Adrian Auf Der Maur; Alcide Barberis; Dominik Escher
- Web page: https://patentgrep.com/patent/US9518108B2

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- G01N 33/6857: {Antibody fragments}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C07K 2317/21: from primates, e.g. man
- C07K 2317/55: Fab or Fab'
- C07K 2317/567: Framework region [FR]
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/80: remaining in the (producing) cell, i.e. intracellular antibodies or intrabodies
- C07K 2317/82: functional in the cytoplasm, the inner aspect of the cell membrane, the nucleus or the mitochondria
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## 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

### Claim 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:
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
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.

### Claim 2 (depends on 1)

A single-chain antibody of claim 1 fused to a second protein moiety to yield a fusion construct of the general structure:
NH2—VL-linker-VH-second protein-COOH; or
NH2-second protein-VL-linker-VH—COOH.

### Claim 3 (depends on 1)

The single chain antibody of claim 1, wherein orientation of the VH and VL regions is reversed.

### Claim 4 (depends on 2)

The single chain antibody according to claim 2, wherein the second protein provides a read-out for intracellular assays.

### Claim 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.

### Claim 6 (depends on 5)

A single chain antibody framework selected from the group consisting of variants of the single chain antibody frameworks according to claim 5.

### Claim 7 (depends on 5)

An antibody comprising the VL or the VH or both from the single chain antibody framework according to claim 5.

### Claim 8 (depends on 5)

An antibody fragment comprising the VL or the VH or both from the single chain antibody framework according to claim 5.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

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.