US 12,486,324 B2
Chimeric antigen receptor cells targeting ROBO1, preparation method and use thereof
Huashun Li, Suzhou (CN); Baolei Wang, Suzhou (CN); and Kunkun Han, Suzhou (CN)
Assigned to Asclepius (Suzhou) Technology Company Group Co., Ltd., Suzhou (CN)
Filed by Asclepius (Suzhou) Technology Company Group Co., Ltd., Suzhou (CN)
Filed on May 18, 2023, as Appl. No. 18/319,638.
Application 18/319,638 is a continuation of application No. 16/700,401, filed on Dec. 2, 2019, granted, now 11,697,685.
Application 16/700,401 is a continuation of application No. PCT/CN2017/089517, filed on Jun. 22, 2017.
Claims priority of application No. 201710407619.8 (CN), filed on Jun. 2, 2017.
Prior Publication US 2023/0287116 A1, Sep. 14, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C07K 16/28 (2006.01); A61K 40/11 (2025.01); A61K 40/15 (2025.01); A61K 40/31 (2025.01); A61K 40/42 (2025.01); A61P 35/00 (2006.01); C07K 14/705 (2006.01); C07K 14/725 (2006.01); C12N 5/0783 (2010.01); C12N 15/867 (2006.01); A61K 38/00 (2006.01); A61K 39/00 (2006.01)
CPC C07K 16/2803 (2013.01) [A61K 40/11 (2025.01); A61K 40/15 (2025.01); A61K 40/31 (2025.01); A61K 40/42 (2025.01); A61P 35/00 (2018.01); C07K 14/7051 (2013.01); C07K 14/70517 (2013.01); C07K 14/70521 (2013.01); C07K 14/70578 (2013.01); C12N 5/0636 (2013.01); C12N 5/0646 (2013.01); C12N 15/867 (2013.01); A61K 38/00 (2013.01); A61K 2039/505 (2013.01); A61K 2239/17 (2023.05); A61K 2239/55 (2023.05); C07K 2317/24 (2013.01); C07K 2317/55 (2013.01); C07K 2317/622 (2013.01); C07K 2317/76 (2013.01); C07K 2319/02 (2013.01); C07K 2319/03 (2013.01); C07K 2319/30 (2013.01); C07K 2319/33 (2013.01); C12N 2510/00 (2013.01)] 13 Claims
 
1. A CAR-cell, expressing an extracellular secretory protein and a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain and a costimulatory signaling region, and the antigen-binding domain is capable of specifically binding to FN3 domain of the tumor specific antigen ROBO1, and activating immune cells through the transmembrane domain and the costimulatory signaling region; the extracellular secretory protein is capable of preventing or inhibiting the binding of the wild type PD-1 protein to PD-L1 ligand;
the extracellular secretory protein is the extracellular domain of PD-1 protein or the mutant of the extracellular domain, and the mutant of the extracellular domain is capable of binding PD-L1 with high affinity;
the transmembrane domain is CD8 transmembrane domain, the costimulatory signaling region is 4-1BB and CD3 ζ intracellular domains; the structure of the chimeric antigen receptor is anti-ROBO1-FN3 scFv-CD8 transmembrane-4-1BB-CD3 ζ;
a nucleotide encoding IRES (internal ribosome entry site) is located between a nucleotide encoding chimeric antigen receptor and a nucleotide encoding extracellular secretory protein,
a polynucleotide comprises a nucleotide encoding anti-ROBO1-FN3 scFv-CD8 transmembrane-4-1BB-CD3 ζ-IRES-(the extracellular domain of PD-1 protein or mutant thereof), which has a sequence as shown in SEQ ID NO: 7.