US 12,186,409 B2
Methods of screening for sortilin binding antagonists
Arnon Rosenthal, Woodside, CA (US); and Tina Schwabe, San Francisco, CA (US)
Assigned to ALECTOR LLC, South San Francisco, CA (US)
Filed by Alector LLC, South San Francisco, CA (US)
Filed on Nov. 3, 2020, as Appl. No. 17/088,237.
Application 17/088,237 is a division of application No. 15/565,063, granted, now 10,849,992, previously published as PCT/US2016/026485, filed on Apr. 7, 2016.
Claims priority of provisional application 62/144,277, filed on Apr. 7, 2015.
Prior Publication US 2021/0121583 A1, Apr. 29, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/543 (2006.01); A61K 49/00 (2006.01); C07K 16/28 (2006.01); G01N 21/64 (2006.01); G01N 33/533 (2006.01); G01N 33/542 (2006.01)
CPC A61K 49/0056 (2013.01) [C07K 16/2863 (2013.01); G01N 21/6428 (2013.01); G01N 33/533 (2013.01); G01N 33/542 (2013.01); G01N 2333/705 (2013.01); G01N 2500/02 (2013.01); G01N 2500/10 (2013.01)] 16 Claims
 
1. A method of screening for a sortilin binding antagonist, comprising:
(a) contacting a cell expressing a sortilin protein on its cell surface with an agent and a sortilin ligand under conditions in which the sortilin protein is capable of binding to the sortilin ligand,
wherein the sortilin ligand is attached to a fluorescent detection donor moiety and wherein the fluorescent detection donor moiety is Terbium cryptate;
wherein the sortilin protein is attached to a fluorescent detection acceptor moiety and wherein the fluorescent detection acceptor moiety is d2;
(b) exciting the sortilin ligand attached to Terbium cryptate with light of a first wavelength sufficient for fluorescence resonance energy transfer (FRET) to occur between the Terbium cryptate and the d2; and p1 (c) detecting fluorescence emitted by the Terbium cryptate at a second wavelength and fluorescence emitted by the d2 at a third wavelength; wherein
(i) d2 is directly coupled to the sortilin protein, and Terbium cryptate is coupled to Streptavidin, which is bound to biotin coupled to the sortilin ligand; or
(ii) d2 is coupled to an antibody that specifically binds to the sortilin protein, and Terbium cryptate is coupled to streptavidin, which is bound to biotin coupled to the sortilin ligand; and
wherein a decrease in the ratio of the fluorescence emitted by the d2 at the third wavelength to the fluorescence emitted by the Terbium cryptate at the second wavelength, as compared to the ratio in the absence of the agent, indicates that the agent is a sortilin binding antagonist.