Session: 703. Cellular Immunotherapies other than CAR-T Cells: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research
iPSCs were first established from a healthy donor's HLA-A2402 restricted LMP2-CTL clone. Next, we knocked out B2M gene to eliminate HLA class I antigen expression and introduced B2M-HLA-A24-fusion and trimer peptide-B2M-HLA-E fusion genes to avoid NK-cell attack via bw4 epitope of HLA-A24/KIR3DL1 and HLA-E/NKG2A bindings (Cell Reports Medicine 2023).
These HLA-edited iPSCs efficiently differentiated into rejTs and suppressed alloreactivity when cocultured CD8+ T cells in a mixed lymphocyte reaction assay. CD107a expression levels were significantly lower on the allogeneic CD8+ T cells upon culture with HLA-edited rejTs than unedited-rejTs (p=0.0421). HLA-A24 and HLA-E dual knock-in (KI) rejTs significantly suppressed NK cell cytotoxicity compared to either single KI-A24-rejTs (p<0.0001) or KI-E-rejTs (p<0.0001) in a cytotoxicity assay at an effector to target (E:T) ratio of 9:1 (KI-A24&E-rejTs 19.2%, KI-A24-rejTs 31.4%, KI-E-rejTs 45.4%).
To analyze the antitumor effect of HLA-edited LMP2-rejTs against ENKL, we performed in vitro cytotoxic assay. The cytotoxicity of HLA-edited-LMP2-rejTs was higher against ENKL cells (NK-YS and ENKL-J1), than that of original LMP2-CTLs (NK-YS; 74.3% vs 59.8%, ENKL-J1; 78.8% vs 34.7%) at an E:T ratio of 2.5 : 1. Next, to observe the tumor suppressive effect of LMP2-rejTs in vivo, firefly luciferase-labeled NK-YS cells were injected into NOG mice. Mice were divided into an untreated group and two treatment groups (original LMP2-CTLs or HLA-edited LMP2-rejTs). Although there was no significant difference in the bioluminescent signal treated with original CTLs compared to untreated mice (p=0.3919), the signal was significantly lower in mice treated with HLA-edited LMP2-rejTs compared to untreated mice on Day 28 (p=0.0219). To compare gene expression in original CTLs and EBV-rejTs, we performed single-cell RNA sequencing. EBV-rejTs expressed higher level of genes associated with cytotoxicity (IFNG, PRF1) and lower level of genes associated with exhaustion (TIGIT, LAG-3) compared to original CTLs.
In conclusion, we have successfully generated minimally immunogenic HLA-edited LMP2-rejTs from a healthy donor that can evade allogeneic immune responses while enhancing T cell function. To bring this platform to the clinic, we have established a master cell bank of HLA class I-edited iPSCs derived from a healthy donor-derived LMP2-CTL clone at our cell processing center. We are currently preparing to start investigator-initiated Phase I clinical trials using these cells for HLA-A2402+ patients. Once established, such antigen-specific CTL derived, HLA-edited iPSCs could provide an unlimited source of “off-the-shelf” T cell therapy targeting EBV-associated lymphomas.
Disclosures: Ando: AbbVie Inc.: Honoraria; Novartis: Honoraria. Nakauchi: ReproCELL: Consultancy, Current equity holder in private company; Megakaryon: Consultancy, Current equity holder in private company; Century Therapeutics: Consultancy, Current equity holder in publicly-traded company. Ando: AbbVie: Honoraria; Novartis Pharma: Honoraria; Chugai Pharmaceutical: Research Funding; Century Therapeutics: Research Funding; Sumitomo Pharma: Research Funding; Kyowa Kirin: Research Funding; AstraZeneca: Honoraria; Astellas Pharma: Honoraria.
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