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418 The development of ‘off-the-shelf’ manufacturing strategies of iPSC-based gamma-delta T cells
  1. Yanjie Li1,
  2. Lei Ding1,
  3. Jixue Li1,
  4. Mariska Ter Haak2,
  5. Kate Rochlin4 and
  6. Lawrence Lamb1
  1. 1IN8bio Inc., Birmingham, AL, USA
  2. 2IN8Bio Inc., New York, NY, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Gamma-delta (γδ) T cells are depleted during cancer progression resulting in the progressive loss of anti-cancer activity. Elevated numbers of γδ T cells are associated with greater survival outcomes in both hematopoietic and solid malignancies. Induced pluripotent stem cell (iPSC) derived γδ T cells could address the therapeutic challenges of multiple allogeneic γδ T cell infusions as iPSCs possess nearly unlimited self-renewal and multi-lineage differentiation potential. These can be genetically modified, selected, and propagated to provide a source of potentially ‘off-the-shelf’ immune cells.

Methods Precursor cells obtained from healthy volunteer donors were reprogrammed into iPSCs using non-integrating Yamanaka factors. A feeder-free multi-step strategy was used to differentiate iPSCs, leading to the generation of Vδ1+ γδ T cells. Characterization of the Vδ1+ T cell product included multiplex genomic PCR assays and Sanger sequencing to examine the rearrangement of the TCRγ and TCRδ gene loci, and G-band karyotype analysis. Pluripotent markers (Tra-1–60, OCT3/4 & SSEA4), HPC markers (CD34, CD43), γδ T cell surface markers (CD3, γδ TCR, CD4, CD8, CD16, CD56), effector memory markers (CD45RA, CD27), natural cytotoxicity receptors (NKG2D) were identified using multiparameter flow cytometry. T cell function was determined by flow cytometric cytotoxicity assays against K562, OLM13, U87MG, OVSAHO, OVCAR-3, KURAMUCHI targets at increasing Effector to Target (E:T) ratios. Th1/2/17 cytokine release was determined following PMA/ionomycin stimulation and LEGENDplex™ bead-based immunoassays.

Results We generated Vδ1T-iPSC lines (iVδ1T) identified as Vγ5-to-Jγ1/2 and Vδ1-to-Jδ1 recombination. One iPSC line showed normal karyotype with 99% cells expressing OCT3/4 & SSEA4. The differentiation process generated 70+ million iVδ1T cells from 3 million iPSCs expressing γδ T cell markers CD45, CD3, Vδ1-TCR, CD16, CD56, NKG2D, CD45RA, and CD27. Cytokine release following PMA/ionomycin stimulation showed increases of at least 50x for Granzyme A, 300x for IFN-γ, 1400x for TNF-α, ~10 to 20x for Granzyme B, ~5 to 10x for Perforin, ~6x for Granulysin. IL-6 was not detected either before or after stimulation, and IL17A was at low concentration. At a 16:1 E:T ratio, preliminary data shows that Vδ1+ γδ T cells killed K562 (CML) 95.7%; MOLM13 (AML) 60.3%; U87MG (glioblastoma) 70.3%; and ovarian cancer lines OVSAHO 57.1%, OVCAR-3 69.6%, and KURAMUCHI 55.1%.

Conclusions We generated Vδ1+ iPSC derived γδ T cells with effector cytokine phenotype and low risk for cytokine release syndrome. Robust cytotoxic activity was seen across a variety of cancer cell lines, potentially providing an off-the-shelf platform for allogeneic cell therapy.

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