Lilrb2 and notch-mediated expansion of hematopoietic precursor cells

Abstract

The current disclosure describes methods of expanding precursor cells for hematopoietic transplantation in subjects. The methods culture precursor cells in media containing an immobilized high molecular weight LILRB2 agonist or an LILRB2 agonist in combination with a Notch agonist. The expanded cells can be used to treat a variety of hematopoietic disorders.

1 . A method of expanding hematopoietic stem cells and/or hematopoietic progenitor cells ex vivo, comprising culturing the cells with: (i) a Notch agonist; and (ii) a leukocyte immunoglobulin-like receptor B2 (LILRB2) agonist immobilized on a first solid phase, wherein the immobilized LILRB2 agonist is an antibody to the LILRB2 receptor, or an antigen-binding fragment of said antibody. 
     
     
         2 . The method of  claim 1 , wherein the culturing comprises adding fresh media every 3 or 4 days for a period of 10 to 20 days. 
     
     
         3 . The method of  claim 1 , wherein the culturing is within a serum-free culture media. 
     
     
         4 . The method of  claim 1 , wherein the culture media comprises 10-100 ng/mL stem cell factor (SCF), 5-100 ng/mL thrombopoietin (TPO), 10-100 ng/mL Flt3 ligand (Flt3-L), and 1-100 μg/mL of retronectin. 
     
     
         5 . The method of  claim 1 , wherein the hematopoietic stem cells and/or hematopoietic progenitor cells are human cells obtained from bone marrow, umbilical cord blood, placental blood, or Wharton's jelly. 
     
     
         6 . The method of  claim 1 , wherein the cells are hematopoietic stem cells or hematopoietic progenitor cells. 
     
     
         7 . The method of  claim 1 , wherein the cells are hematopoietic stem cells and hematopoietic progenitor cells. 
     
     
         8 . The method of  claim 1 , wherein the LILRB2 agonist is an Fv, Fab, Fab′, F(ab′) 2 , or single chain Fv fragment (scFv). 
     
     
         9 . The method of  claim 1 , wherein the LILRB2 agonist is immobilized on the first solid phase at a concentration of 0.08 to 25 μg/mL. 
     
     
         10 . The method of  claim 1 , wherein the Notch agonist is immobilized on a second solid phase at a concentration of 0.025 to 5 μg/mL. 
     
     
         11 . The method of  claim 1 , wherein the Notch agonist is Delta ext-IgG . 
     
     
         12 . The method of  claim 1 , wherein the Notch agonist is an antibody that specifically binds to Notch-1 or an antibody that specifically binds to Notch-2. 
     
     
         13 . A method of expanding hematopoietic stem cells and/or hematopoietic progenitor cells ex vivo, comprising culturing the cells with: (i) a Notch agonist; and (ii) a leukocyte immunoglobulin-like receptor B2 (LILRB2) agonist immobilized on a first solid phase, wherein the immobilized LILRB2 agonist is Angiopoietin-like 5 (Angptl 5). 
     
     
         14 . The method of  claim 13 , wherein the culturing is in a serum-free media comprising 1-100 ng/mL stem cell factor (SCF), 1-100 ng/mL thrombopoietin (TPO), 1-100 ng/mL FLt-3 ligand (Flt3-L), 1-100 ng/mL interleukin-6 (IL-6), 1-100 ng/mL interleukin-3 (IL-3), 1-100 ng/mL FGF1, and 1-100 μg/mL heparin. 
     
     
         15 . The method of  claim 13 , wherein Angptl 5 is immobilized on the first solid phase at a concentration of 0.08 to 25 μg/mL. 
     
     
         16 . A chimeric reporter system for screening agonists and antagonists of leukocyte immunoglobulin-like receptor B2 (LILRB2) comprising:
 cells expressing:
 a fusion protein comprising at least one extracellular domain of LILRB2 and transmembrane and cytoplasmic domains of paired immunoglobulin-like receptor β (PILRβ); and 
 an adapter protein that associates with the cytoplasmic domain of the fusion protein and activates a transcription factor, 
   and   a reporter gene within the cells responsive to the transcription factor.   
     
     
         17 . The system of  claim 16 , wherein the fusion protein is encoded by a gene on a retroviral vector. 
     
     
         18 . The system of  claim 16 , wherein the cells are mouse T cell hybridoma cells. 
     
     
         19 . The system of  claim 16 , wherein the transcription factor is nuclear factor of activated T cells (NFAT). 
     
     
         20 . The system of  claim 16 , wherein the adapter protein is DAP12.