-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

4811 Preclinical Study of HLA Class I-Edited Allogeneic iPSC-Derived Virus-Specific T Cells Targeting Epstein-Barr Virus-Associated Lymphomas

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies other than CAR-T Cells: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research
Monday, December 9, 2024, 6:00 PM-8:00 PM

Kosuke Matsuzaki1*, Midori Ishii1*, Shintaro Kinoshita1*, Ayaka Goto1*, Yoko Azusawa2*, Yoshiki Furukawa1*, Jun Ando1,2*, Hiromitsu Nakauchi3,4* and Miki Ando1

1Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan
2Division of Cell Therapy & Blood Transfusion Medicine, Juntendo University School of Medicine, Tokyo, Japan
3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford
4Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

Extranodal NK / T-cell lymphoma, nasal type (ENKL) is a highly aggressive lymphoma invariably infected by Epstein-Barr virus (EBV) with type II latency expressing latent membrane protein (LMP1/LMP2). Therefore, antigen-specific cytotoxic T lymphocyte (CTL) therapy targeting these antigens is a promising strategy to selectively kill these lymphoma cells. However, continuous exposure to their target antigens often exhausts CTLs. To overcome this problem, we have successfully rejuvenated exhausted virus-specific CTLs (rejTs) by iPSC technology, demonstrating that rejTs can more robustly proliferate, show younger memory T cell phenotype, and have stronger antitumor effect than original CTLs (Cell Stem Cell 2013, Stem Cell Reports 2015, Haematologica 2020). Unfortunately, generating rejTs from each patient is time-consuming, which has been an obstacle in clinical application. Therefore, in this study, we generated HLA-class I-edited allogeneic LMP2-specific rejTs from a healthy donor using CRISPR/Cas9 gene editing to evade rejection from patients' immune cells.

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.

Previous Abstract | Next Abstract >>
*signifies non-member of ASH