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1 CAR-T .. TCR T .. ICB ICB . PD-1/PD-L1 . CTLA-4 .. HLA . ICB ICB T ICB ICB 1 . T .. A B Proliferating Myeloid cell Myeloid cell ESC or iPSC (MC) (pMC) pMC-DC. pMC Day0 Day 6-7 Day13-14 Day 23-24 Day 26-27 OP9 Feeder GM-CSF. GM-CSF IL-4 + GM-CSF + M-CSF IL-4. C Passage c-Myc transduction Passage GM-CSF pMC Num. of cells GM-CSF / M-CSF 2. GM-CSF 1 M-CSF Medium 100 mm 100 mm 20 mm 20 mm 0. ESC or iPSC Mesoderm Myeloid cell DC-like cell 0 1 2 3 4 day Zhang R. et al. Cancer Immunol. Res. 2015 Proliferation factor GM-CSF or M-CSF .. 1 . GM-CSF 1 IFN . 1 IFN T . pMC A IFN- -pMC B C D Cxcl10 IFN- -pMC IFN- (pg/mL) Relative exp. 4000" 200". IFN- -pMC * 3000" 150". GM-CSF / M-CSF Num. of cells". 2000" 100". 1 GM-CSF M-CSF 1000" 50". Medium 0" 0". 0. 0 1 2 3 4.

2 E F Medium pMC Relative exp.( 10-2) 6 IFN- -pMC Ccl6 Ccl9 Ccl3 4 Ccl2 Il16 2 Ccl22 Cx3cl1 0 Ccl4 Cxcl2 Ccl12 Cxcl9 Ccl5 Cxcl10 1 IFN pMC. 1 IFN .. 1 IFN Distant site Treatment site A B Tumor volume (mm3) Tumor Treatment Treatment 2000" 2000. Mice". 1500" C57Bl/6" 1500. 0 5 6 7 12 13 14 1000". ". Tumor". * 1000 No treatment B16-OVA pMC 500" 500 * IFN- -pMC C day5 0". 5" 10" 15" 20". 0. 5" 10" 15" 20".. Total flux ( 108 photon/sec) (Days) D 20" Mice". C57Bl/6". 20.. day7 15" " 15. Tumor". No treatment 10" B16-OVA 10. Color Scale" 5". * 5 * pMC IFN- -pMC Min = ". day11 Max = 0" 0. 4" 6" 8" 10" 12" 4" 6" 8" 10" 12". (Days) 1 IFN . 500.. Tumor volume (mm3) IFN- (pg / mL) E 400 rIFN- (104 units) F 3000". Mice". 3000. 300 IFN- -pMC C57BL/6". 2000" 2000 No treatment ".

3 1 IFN 200. 1000". Tumor". B16-OVA * 1000 IFN- -pMC rIFN- 100. 0 0" 0. 0 10 20 5" 10" 15" 20" 5" 10" 15" 20". (Days) Treatment tumor Distant tumor Draining lymph node G Day 1 Day 1 Day 1 1 mm 200 m 1 mm 200 m 1 mm 200 m Day 2 Day 2 Day 2 . Day 3 Day 3 Day 3 Day 4 Day 4 Day 4 10 40 10 40 10 40 T . TCR T T CD8+ . A PD-1/PD-L1 B Gzmb C CD8+T . Treatment site Depletion Ab No treatment pMC IFN- -pMC Distant site Control Ab Cd3e Cd8 Gzmb Tumor volume (mm3) ** ** Distant site Treatment site ** Relative exp. Treatment 16" 30" 250" * * ** ** ** 2500" 1000". 25" 200". 12" 2000" 800". 20" 150". 8" 15" 1500" 600". CD8 10" 100" 1000" 400". 4" 50" 500" 200". 5". 0" 0" 0" 0" 0". Distant .. TCR T . D E F T G T .. Treatment site TRB Ranking Top1-50. Peptide Rank TRBV TRBJ CDR3 Reads Frequency Left Rank Spleen Rank Negative" OVA".

4 10" 1 TRBV16 TRBJ2-7 CASSLELGGREQYF 2172 1 - tetramer tetramer (-) 2 TRBV5 TRBJ2-3 CASSQGLGGAETLYF 1740 - - 3 TRBV5 TRBJ1-6 CASSQDPHSYNSPLYF 1473 - - Tetramer 8 4 TRBV5 TRBJ2-7 CASSPPGGKEQYF 1347 - - 0" 1 TRBJ 5 TRBV13-1 TRBJ1-6 CASRGDNSPLYF 1061 58 - wtAdpgk ASMTNRELM". 11. 13. 19. 21. 23. 1. 3. 9. 15. 17. 5. 7. 6 TRBV16 TRBJ2-5 CASSVTVGQDTQYF 1019 - - TRBV 7 TRBV20 TRBJ2-4 CGARGAENTLYF 1017 - - 8 TRBV13-3 TRBJ2-7 CASSLGGYEQYF 905 32 - mAdpgk ASMTNMELM". Distant site 9 TRBV5 TRBJ2-3 CASSPPGGSETLYF 854 - - 10 TRBV13-2 TRBJ2-1 CASGDGGNYAEQFF 758 8 4. 11 TRBV20 TRBJ1-2 CGATRGANSDYTF 585 7 3. CD8 9" 12 TRBV1 TRBJ1-5 CTCSRDNQAPLF 570 - EphA2 - 6" 13 TRBV2 TRBJ2-5 CASSQVGPDTQYF 565 - - 3" 14 TRBV13-1 TRBJ2-1 CASRDSLAEQFF 562 46 - 8. 15 TRBV26 TRBJ2-1 CASSLPAIDNYAEQFF 416 - - 0" 1 TRBJ 16 TRBV16 TRBJ2-3 CASSLDNRGAETLYF 408 61 - Number of spots 11.

5 13. 19. 21. 23. 1. 3. 9. 15. 17. 5. 7. Lysis (%) TRBV 17 TRBV4 TRBJ2-3 CASSLGAAETLYF 368 70 - 100" 1500". 18 TRBV15 TRBJ1-2 CASSLESSDYTF 361 36 1 80". Spleen 19 TRBV14 TRBJ1-6 CASSFHRDYNSPLYF 356 - - Ag peptide 1000". 60". 20. 21. TRBV5. TRBV13-3. TRBJ2-5. TRBJ2-3. CASSPPGGEDTQYF. CASGQGSAETLYF. 354. 354. 14. - - - 40". Medium 500". ** 3" 22 TRBV20 TRBJ1-3 CGARDNSGNTLYF 301 83 59. 23 TRBV13-2 TRBJ2-2 CASGDVGNTGQLYF 269 - 20". 2" - 0". (-)". wtAd". mAd". wtRe". mRe". MC38". EphA2 Db". 24 TRBV13-1 TRBJ1-4 CASQGADERLFF 267 - - 0". 1" 8 25 TRBV5 TRBJ2-4 CASSQTQGGSQNTLYF 255 - - TRBJ 0" 1 26 TRBV2 TRBJ2-7 CASSQEGDNYEQYF 255 57 - 11. 13. 19. 21. 23. 27 TRBV2 TRBJ2-1 CASSQDTGFYAEQFF 247 1. 3. 9. - - 15. 17. 5. 7. TRBV 28 TRBV5 TRBJ2-3 CASSQDYRLGSSAETLYF 241 - - 29 TRBV13-3 TRBJ2-1 CASSGQGNYAEQFF 238 97 - E: T ratio 30 TRBV19 TRBJ2-4 CASSRTGGSQNTLYF 236 - - RMA-S DC.

6 DTx DC . DC . A B E Distant site Treatment site Tumor volume (mm3) Tumor Treatment Treatment 4000" Mice" 4000". CD11c-DTR WT 3000" " 3000" No treatment 0 4 5 6 7 11 12 13 14 B16-OVA" DTx 2000" WT 2000". IFN- -pMC 1000" IFN- -pMC / DTx DTx DTx DTx DTx 1000". 0" 0". 5" 10" 15" 20" 25" 5" 10" 15" 20" 25". (Days) DC DC . C D T . T . CD11c+ XCR1+ DC CD11c+ XCR1- DC CD169+ F4/80- M CD11c+ XCR1+ DC CD11c+ XCR1- DC CD169+ F4/80- M 1 IFN .. T . ( H2-Kb/OVA) ( CD86) 1500" 1500" 1500". 16000" 16000" 16000". MFI". MFI". 1000" 1000" 1000" 12000" 12000" 12000". 8000" 8000" 8000". 500" 500" 500". 4000" 4000" 4000". 0" 0" 0". 0" 0" 0".. GFP NUGC-4(GFP) iPS-ML(PKH-26) Cancer". iPS-ML".. Spleen SCID mouse 200 m . PKH26 iPS-ML MKN45 Cancer". Liver". Meta". Portal region Spleen".

7 IPS-ML". Tumor" Tumor". 200 m". Metastases 1 IFN . Day 2 9" 16 23 30 80 Tumor growth". 70 Control 60 50 40 control Control 30 IFNb- ML 20 10 ES-ML/IFN ". 0 day2 day9 day16 day22 day30 Days after cancer cell implantation survival (%) c o ntro l 100 IF N b - ML. ES-ML/IFN ". ML/IFN 80. s u r v iv a l ( %). 60. 40. P< 20. Control 0. 0 30 60 90 120 150. D a y s e la p s e d Days after cancer cell implantation Colon26 > BALB/c iPSC 1/2 IFN . 1 IFN 2 IFN 90". Day 10 17 24 31 38 45 80". Tumor growth". 70" Control 60". 50". Control 40". 30". 20". 10" iPS-ML/IFN + ". 0". 10 17 24 31 38 45 100". 90" Day 10 survival (%) 80" 2 4 70" ML/IFN + ". 60". 50" iPS-ML/IFN + ". 40". 30". 20". 10" Control 0". 0" 20" 40" 60" 80" 100" 120". SK-Hep-1 > SCID Days after cancer cell implantation iPSC 1/2 IFN.

8 Cell Processing Center . F Sealer WAVE . PRO Regulatory readiness Cellbag bioreactors The ReadyToProcess WAVE 25 system is suitable for Cellbag bioreactors are single-use bags for the noninvasive biomanufacturing of regulated products under various mixing of culture medium and cells during cultivation (Fig 8). Quality Management Systems. The use of UNICORN software Cellbag bioreactors require no sterilization or cleaning steps. in a 21 CFR Part 11 and GAMP 5 compliant manner enables The disposable bioreactors provide a suitable environment use of the system in a regulated environment. Individual for cell growth while minimizing the cross-contamination risk. user access permissions can be set and individual users The bags are manufactured from multilayer, laminated, clear are password-protected.

9 Active processes can be locked USP Class VI plastics and are easily connected to the full suite to enable unattended operations without the risk of of ReadyToProcess cell culture, purification, and fluid handling unauthorized interference. All records are stored in a single, products. The selection of Cellbag bioreactors is listed in unalterable database, including results and extended run Table 2. For more information about Cellbag bioreactors, see documentation. Additional validation support includes data file 28-9511-36. comprehensive documentation on control system validation as well as IQ/OQ services. Available validation support Optical documentation includes: DO sensor embedded in bag Air outlet Detailed description of the development model used for Optional filter UNICORN software tubing Air inlet filter 21 CFR Part 11 system assessment in checklist format Inoculation/.

10 Harvest lines Optical pH sensor Audit report and 21 CFR Part 11 conclusion on embedded in bag functionality by an external, independent expert Cellbag rod Optional Networking capabilities tubing UNICORN software operates in Windows XP and Windows 7. environments and the network ability allows real-time Needleless Optional tubing control from a remote or local computer. Communication sampling port is Ethernet-based and each instrument is controlled by a Fig 8. Presentation of typical Cellbag bioreactor fittings. dedicated instrument server. One database can be connected to 32 systems and up to three instruments can be controlled Table 2. Selection of Cellbag bioreactor sizes simultaneously from one UNICORN are saved Cellbag bioreactor* Working volume WAVE-25 locally, in the rocker, during the run and then stored on the Cellbag 2 L.