Patent SG-11201907285X-A (SG)
Title: Methods and kits for generating mimetic innate immune cells from pluripotent stem cells
Inventor: ZENG JIEMING
IPC: C12N5/10
Filed: 2018-02-07
Abstract: 1 1-1 O GC N 1-1 00 1-1 O N C (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date 16 August 2018 (16.08.2018) WIPO I PCT olimion °noI0 DIDmoIIVmoIIIII DIIIIII DID (10) International Publication Number WO 2018/147801 Al DO: Seed and culture hPSCs on OP9 cells (51) International Patent Classification: C12N 5/0783 (2010.01) A61K 35/17 (2015.01) C12N 5/10 (2006.01) A61P 35/00 (2006.01) (21) International Application Number: PCT/SG2018/050051 (22) International Filing Date: 07 February 2018 (07.02.2018) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 10201700937P 07 February 2017 (07.02.2017) SG 10201705582S 06 July 2017 (06.07.2017) SG (71) Applicant: AGENCY FOR SCIENCE, TECHNOLO- GY AND RESEARCH [SG/SG]; 1 Fusionopolis Way #20-10 Connexis, Singapore 138632 (SG). (72) Inventors: ZENG, Jieming; c/o Institute of Bioengineer- ing and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669 (SG). WANG, Shu; c/o Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669 (SG). (74) Agent: YUSARN AUDREY; 24 Raffles Place #27-01 Clif- ford Centre, Singapore 048621 (SG). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Declarations under Rule 4.17: as to applicant's entitlement to apply for and be granted a patent (Rule 4.17(ii)) of inventorship (Rule 4.17(iv)) Published: with international search report (Art. 21(3)) with sequence listing part of description (Rule 5.2(a)) (54) Title: METHODS AND KITS FOR GENERATING MIMETIC INNATE IMMUNE CELLS FROM PLURIPOTENT STEM CELLS Figure 1 a Hematepoietic Differentiation OP9 cells e-EVLEM + 20%FBS Lyn', I, it triti Commitment OP9-tLL1 cells s-NIENI + 20 F BS + Cytokntes 1 012: Harvest LH 9: Harvest D20: Harvest D33: Harvest 040 or D47: differentiated cells differentated cells differentiated cells differentiated cells Harvest NK cells and culture on and Culture on and culture on and culture on OP9-OLL1 OP9-0LL1 OP9•01.1.1 OP9-DLL1 (57) : Human pluripotent stem cells (hPSCs), especially induced pluripotent stem cells (iPSCs) provide a promising starting material to produce mimetic innate immune cells such as natural killer (NK) cells and yS T-cells for cancer immunotherapy. To facil- itate consistent mass production, an overall manufacturing scheme to make mimetic innate immune cells from hPSCs was designed and demonstrated. Particularly, a robust protocol to differentiate hPSCs into NK cells or yS T-cells through sequential hematopoietic differentiation on stromal cell line deficient in expressing M-CSF and lymphoid commitment on stromal cell line deficient in expressing M-CSF ectopically expressing DLL1 without employing CD34+ cell enrichment and spin embryoid body formation is established. Using this two-stage protocol, the generation of functional mimetic NK cells and functional mimetic NKT-cells was demonstrated yS from hPSCs, including hESCs, peripheral blood cell-derived iPSCs (PBC-iPSCs), non-T cell-derived iPSCs or yS T cell-derived iPSCs and the use of these mimetic innate immune cells in killing cancer cells.