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259 iPSC-derived NK cells engineered with a novel TGFβ signal redirector receptor exhibit enhanced performance against solid tumors
  1. Eigen Peralta,
  2. Dan Lu,
  3. Hui-Yi Chu,
  4. Justin Rahman,
  5. Diana Galvan,
  6. Amit Mehta,
  7. Eric Sung,
  8. Jeffrey Chen,
  9. Masanao Tsuda,
  10. Elena Demeester,
  11. Earl Avramis,
  12. Alec Witty,
  13. Tom Lee and
  14. Bob Valamehr
  1. Fate Therapeutics Inc., San Diego, CA, USA


Background Transforming growth factor beta (TGFβ) is an immuno-suppressive cytokine commonly present in the tumor microenvironment (TME) that creates considerable challenges for the treatment of solid tumors. Here we describe a unique strategy where induced pluripotent stem cell (iPSC)-derived NK (iNK) and T (iT) cells engineered to express a chimeric TGFβ signal redirector receptor (TGFβ-SRR) block the TGFβ-mediated repressive signaling and redirect the signal to potentiate effector cell function and improve cell fitness.

Methods To identify iNK cell-specific pathways for TGFβ signal redirection, candidate cytokines were tested for their ability to mitigate suppression of iNK cell anti-tumor activity in the presence of recombinant TGFβ. Next, we developed TGFβ-SRR constructs where selected cytokine endodomains were fused to TGFBR2 ectodomain. TGFβ-SRR constructs were then engineered into iPSCs and differentiated into iNK cells. Phospho-flow for pSMAD2/3 was used to test for blockade of TGFβ signaling in recombinant TGFβ-treated cells. Antibody-dependent cellular cytotoxicity (ADCC) from TGFβ-SRR iNK cells was tested in co-cultures with SKOV-3, PC3, and MDA-MB-231 targets, then measured using xCELLigence readout. Innate killing mechanism was tested via serial restimulation assay with Raji targets and measured by flow cytometry. Co-cultures were performed in the presence of recombinant TGFβ.

Results Engineered iPSCs expressing candidate TGFβ-SRR constructs were successfully differentiated into iNK cells (>95% CD56+), uniformly expressing TGFβ-SRR transgene (TGFβ-SRR; >95% positive). Analysis for pSMAD2/3 in recombinant TGFβ-treated cells showed 95% reduction of SMAD2/3 phosphorylation in top performing TGFβ-SRR motif, indicating successful blockade of TGFβ signaling. Evaluation of ADCC toward multiple solid tumor lines and using various monoclonal antibodies (Herceptin, Cetuximab, and Avelumab) showed superiority of TGFβ-SRR iNK cells (>80% cytolysis) over parental iNK cell control (<40% cytolysis) in the presence of recombinant TGFβ. Innate killing mechanism was tested in the serial restimulation assay, where TGFβ-SRR iNK cells expanded 3.5-fold over parental iNK cells after the first round of co-culture. Notably, the TGFβ-SRR iNK cells exhibited enhanced functional persistence, completely controlling tumor growth through three rounds of co-culture despite the addition of suppressive quantities of recombinant TGFβ, unlike parental iNK cells which failed to control tumor growth after the first round.

Conclusions Collectively, the data illustrate that a customized TGFβ-SRR construct can redirect TGFβ-mediated suppression and potentiate effector cell function to enhance the anti-tumor activity of iNK cells. This novel synthetic receptor represents an innovative strategy to enable adoptively-transferred cell therapy to overcome the immunosuppressive TME for the successful treatment of bulky tumors.

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