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Compositions and methods of use for binding molecules to dickkopf-1 or dickkopf-4 or both

A technology of DKK1, a composition, applied in the field of expression-related disorders or conditions, capable of solving problems such as poor characterization

Inactive Publication Date: 2012-04-18
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Dickkopf-4 (DKK4) is poorly characterized but also a secreted inhibitor of the Wnt pathway

Method used

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  • Compositions and methods of use for binding molecules to dickkopf-1 or dickkopf-4 or both
  • Compositions and methods of use for binding molecules to dickkopf-1 or dickkopf-4 or both
  • Compositions and methods of use for binding molecules to dickkopf-1 or dickkopf-4 or both

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0313] Example 1: From HuCAL GOLD Preparation of human DKK1-specific antibody in the library

[0314] Therapeutic antibodies against human DKK1 protein can be obtained by using the commercially available phage display library MorphoSys HuCAL GOLD as a source of antibody variant protein library to select clones with high binding affinity for generation. HuCAL GOLD is a Fab library (Knappik et al., 2000 J. Mol. Biol. 296:57-86; Krebs et al., 2001 J Immunol. Methods 254:67-84; Rauchenberger et al., 2003 J Biol Chem.278(40):38194 -38205), in which all six CDRs were diversified by appropriate mutations, and CysDisplay TM Techniques link Fab fragments to the surface of phage (WO 01 / 05950 Lδhning 2001).

[0315] Routine procedures: phagemid recovery, phage amplification, and purification

[0316] HuCAL GOLD Libraries were amplified in standard rich bacterial medium (2xYT) containing 34 μg / ml chloramphenicol and 1% glucose (2xYT-CG). in OD 600nm When the temperature was 0....

Embodiment 2

[0345] Example 2: DKK1 specific HuCAL Antibody identification

[0346] The BEL extracts of each Escherichia coli clone selected by the above panning strategy were analyzed by microvolume fluorescence assay technique (FMAT TM , 8200 Cellular Detection System analyzer, Applied Biosystems, Foster City, Calif) analysis to identify clones encoding DKK1-specific Fab. FM AT TM The 8100 HTS system is a confocal high-throughput fluorescence screening instrument that automatically detects the mixing and reading of living cells or beads, and is a non-radioactive assay (Miraglia, J. Biomol. Screening (1999), 4(4) 193-204 ).

[0347] A microvolume-based fluorescence assay technique for detecting DKK1-bound Fabs from bacterial lysates (FMAT) binding assay

[0348] For detection of DKK1-bound Fab antibodies from E. coli lysates (BEL extracts), binding was analyzed using a FMAT 8200 cell detection system (Applied Biosystems). To bind His-Strep-tagged DKK1 to M-450Expoxy beads (Dyna...

Embodiment 3

[0358] Example 3: Identification of anti-human DKK1 Fab candidates that inhibit the Wnt antagonistic activity of DKK1

[0359] Use obtained from HuCAL GOLD A library of 57 different DKK1-specific antibodies was obtained to obtain purified antibodies, which were then used to test the ability to inhibit the Wnt antagonistic activity of human DKK1. Seventeen of these antibody candidates were functionally active.

[0360] Detection of each HuCAL using a luciferase reporter assay Functional activity of the Fab. Twelve binding sites for TCF / Lef were cloned upstream of a luciferase reporter gene that exhibited a luciferase gene TCF / Lef response. Regular canonical Wnt proteins lead to the stabilization of β-catenin, which activates the transcription of TCF / Lef and produces the luciferase protein. Addition of the DKK1 protein blocks Wnt activity and thus also transcription of the luciferase gene. As a result, the level of luciferase produced by individual cells is expected to co...

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Abstract

Methods of using binding molecules and fragments thereof that bind to the protein target Dickkopf -1 (DKK1), Dickkopf-4 (DKK4) or both (wherein specificity to DKK1 or DKK4 or both is herein denoted as DKK1 / 4) are provided.

Description

Background of the invention [0001] The Wnt signaling pathway is involved in the control of embryonic development and tumorigenic processes. Extracellular Wnt proteins are responsible for the growth and differentiation of multiple cell types during embryogenesis and contribute to the development of many cancers. [0002] There are at least two protein families that inhibit Wnt signaling, the secretory frizzled-related family and the Dickkopf (DKK) family. The DKK family currently contains four family members, namely DKK1 (human DNA accession number: NM_012242; PRT accession number: O94907), DKK2 (human accession number: NM_014421; PRT accession number: NP_055236), DKK3 (human accession number: NM_015881; PRT accession number: AAQ88744), and DKK4 (human accession number: NM_014420; PRT accession number: NP_055235). [0003] Dickkopf-1 (DKK1) is a secreted inhibitor of the Wnt / β-catenin signaling pathway. See eg PCT Publication Nos. WO9922000 by Niehrs; WO9846755 by McCarthy; ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18A61P35/00A61P3/10
CPCC07K16/18C07K2317/565C07K2317/92C07K2317/21A61K2039/505C07K2316/96C07K2317/33C07K2317/70C07K2317/73C07K2317/76A61P1/00A61P1/04A61P1/16A61P1/18A61P19/00A61P19/08A61P19/10A61P21/00A61P25/00A61P29/00A61P3/00A61P3/04A61P31/00A61P35/00A61P3/06A61P3/08A61P43/00A61P7/00A61P9/00A61P9/02A61P9/04A61P9/10A61P9/12A61P3/10A61K39/395A61K38/19A61K39/39533A61K45/06
Inventor E·布伦德勒P·雷S·M·兰瓦拉D·R·斯托弗A·泰勒
Owner NOVARTIS AG
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