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anti-lrp6 antibody

A technology of antibodies and human antibodies, applied in the direction of antibodies, antibody medical components, antibody mimics/scaffolds, etc., can solve the problem that the role of LRP5/6 or FZD homodimerization has not been clearly defined

Inactive Publication Date: 2016-04-06
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

While ligand-induced receptor-coreceptor heterodimerization is required for canonical Wnt signaling, the role of LRP5 / 6 or FZD homodimerization has not been clearly defined

Method used

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Experimental program
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Embodiment 1

[0329] Experimental protocol

[0330] Cell Culture and Cell Assays

[0331]Cell lines EKVX and M14 were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine; JHH-1 cells were cultured in Williams' medium E with the same supplements. All other cell lines were obtained from the American Type Culture Collection (ATCC) and maintained as recommended.

[0332] Cells were transfected with FuGENE6 transfection reagent (Roche) in 24-well plates according to the manufacturer's recommendations. For the luciferase reporter assay, a mix of transfected expression plasmid DNA: 7.5 ng TOPglow (Upstate) or TOPbrite (Zhang et al., 2009) firefly luciferase Wnt reporter, 0.5 ng pRL-SV40 Renilla luciferase (Promega ), and 1ngLEF1. Cells were treated with antibodies for 16-20 h, starting 24 h after transfection. Wnt3a protein (purified according to X, or purchased from R&D Systems) was added to the cells, starting 1 h after initiation of antibody treatment. ...

Embodiment 2

[0351] Isolation of Wnt-antagonistic and potentiating LRP6 monoclonal antibodies

[0352] To develop candidate therapeutic molecules to manipulate Wnt signaling, antibodies capable of inhibiting or enhancing signaling induced by Wnt3a protein were generated. Recombinant LRP6.E1-E2-Fc (SEQ ID NO: 30) and LRP6.E3-E4-Fc (SEQ ID NO: 31 ) proteins were used to screen a human synthetic Fab phage display library and to confirm binding of LRP6 from isolated phage clones by ELISA. Twenty-four unique antibody heavy chain clones against LRP6.E1-E2 and 22 clones against LRP6.E3-E4 were isolated, reformatted and expressed human IgG1 antibodies. Six LRP6.E3-E4 antibodies inhibited Wnt luciferase reporter activity in HEK293 cells induced with 0.1 mg / ml purified Wnt3a in a concentration-dependent manner ( Figure 1A . Error bars for this and all other graphs represent the standard deviation of at least 3 replicates unless otherwise indicated). These antibodies were named YW211.03, YW211.0...

Embodiment 3

[0356] Effect of LRP6 monoclonal antibody on autocrine Wnt signaling

[0357] The ability of LRP6 antibodies to antagonize or potentiate endogenous or autocrine Wnt signaling was determined using various tumor cell lines (Bafico et al., 2004; DeAlmeida et al., 2007; Akiri et al., 2009). In teratoma cell lines PA-1 and NTERA-2, the YW211.31 antibody inhibited reporter activity induced by autocrine Wnt signaling with potency similar to that observed for exogenous Wnt3a ( Figure 2A , showing concentration-dependent inhibition of autocrine Wnt signaling in PA-1 teratoma carcinoma cells transfected with a luciferase reporter and treated with LRP6 antibody alone or in combination, or Fzd8CRD-Fc protein (positive control) and strengthen).

[0358] In PA1 cells, inhibition of Wnt signaling by the YW211.31 antibody was also observed for the expression of endogenous Wnt target genes ( Figure 2B ). Figure 2B Shown with or without 0.3mg / mlWnt3a protein treatment, and with 10mg / mlY...

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Abstract

The present invention provides anti-LRP6 antibodies and methods of use thereof. A particular aspect of the invention provides bispecific anti-LRP6 antibodies that inhibit signaling of multiple Wnt isoforms.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application No. 61 / 317,137, filed March 24, 2010, and U.S. Provisional Application No. 61 / 394,836, filed October 20, 2010, the entire disclosures of which are incorporated herein by reference . field of invention [0003] The present invention relates to anti-LRP6 antibodies and methods of using anti-LRP6 antibodies to treat cancer or bone disorders. Background of the invention [0004] Similar to most other morphogen and growth factor signaling pathways, mammalian Wnt signaling is mobilized multiple times during development and tissue homeostasis through the use of 19 different ligands, 10 receptors and multiple co-receptors. body, including LRP5 / 6, Ror1 / 2, and Ryk (van Amerongen and Nusse, 2009). In addition, different secretory antagonists that bind Wnt (such as SFRP1 / 2 / 3 / 4 / 5 and WIF1) or LRP5 / 6 (including DKK1 / 2 / 4 and SOST) modulate the interaction between l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCA61K2039/505A61K2039/507C07K2317/21C07K2317/31C07K2317/34C07K2317/526C07K2317/55C07K2317/75C07K2317/76C07K2317/92C07K2319/00C07K16/2863A61P19/00A61P19/08A61P19/10A61P35/00A61P43/00A61K38/17A61K38/18A61K39/395A61K47/42A61K49/16C07K14/435C07K14/475C07K16/18C07K16/22C07K16/24C07K16/28A61K39/39533A61K39/3955
Inventor E.博里斯R.卡拉诺A.科克伦M.科斯塔V.德阿尔梅达J.厄恩斯特巩岩R.汉努什P.波拉基斯B.鲁宾菲尔德M.索罗韦吴雁T.C.曹
Owner F HOFFMANN LA ROCHE & CO AG
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