Human chimeric antigen receptor neutrophils, compositions, kits and methods of use

A process for manufacturing a population of chimeric antigen receptor (CAR) neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:

• (a) constructing a PiggyBac transposon plasmid comprising a CAR expression gene;
• (b) delivering the PiggyBac plasmid into a human pluripotent stem cell via nucleofection/electroporation;
• (c) isolating successfully targeted single cell-derived human pluripotent stem cell (hPSC) colonies or hPSC cell mixture for stable CAR-expressing hPSC lines;
• and
• (d) producing CAR-expressing neutrophils according to the process of:
• (i) preparing hPSCs;
• (ii) stimulating said hPSCs with a glycogen synthase kinase 3 β (GSK3β) inhibitor to produce a population of CD34+ hemogenic endothelium cells;
• (iii) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor β (TGFβ) inhibitor to produce a population of CD45+ hematopoietic cells;
• and
• (iv) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony-stimulating factor (GM-CSF) and a retinoic acid receptor agonist to afford a population of CD11b+/CD16+ neutrophils.

A pharmaceutical composition comprising:

• a population of isolated neutrophils obtained by performing a stage-specific process for manufacturing a population of neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:
• (a) preparing hPSCs;
• (b) stimulating said hPSCs with a glycogen synthase kinase 3 β (GSK3β) inhibitor to produce a population of CD34+ hemogenic endothelium cells;
• (c) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor β (TGFβ) inhibitor to produce a population of CD45+ hematopoietic cells;
• and
• (d) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony-stimulating factor (GM-CSF) and a retinoic acid receptor agonist to afford a population of CD11b+/CD16+ neutrophils;
• and
• a pharmaceutically acceptable carrier.

A method of treating cancer in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of a population of the neutrophils obtained by performing a stage-specific process for manufacturing a population of neutrophils from human pluripotent stem cells (hPSCs) comprising the steps of:

• (a) preparing hPSCs;
• (b) stimulating said hPSCs with a glycogen synthase kinase 3 β (GSK3β) inhibitor to produce a population of CD34+ hemogenic endothelium cells;
• (c) stimulating said CD34+ hemogenic endothelium cells with a transforming growth factor β (TGFβ) inhibitor to produce a population of CD45+ hematopoietic cells;
• and
• (d) stimulating said CD45+ hematopoietic cells with granulocyte macrophage colony-stimulating factor (GM-CSF) and a retinoic acid receptor agonist to afford a population of CD11b+/CD16+ neutrophils and a pharmaceutically acceptable carrier, whereupon the subject is treated for cancer.