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272 PD-L1 CAR engineered K-NK cells to target PD-L1+ or PD-L1- tumors
  1. Hardikkumar Jetani1,
  2. Pedro Romero1,
  3. Andre Kunert1,
  4. Katarzyna Franciszkiewicz2,
  5. David Vlerick3,
  6. Aleksandra Nowicka4 and
  7. Robert Y Igarashi1
  1. 1Sanofi Pharma, Amsterdam, Netherlands
  2. 2Sanofi, Paris, Île-de-France, France
  3. 3Sanofi, Ghent, East Flanders, Belgium
  4. 4Sanofi, Cambridge, MA, 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 Chimeric antigen receptor (CAR) engineered immune cells have shown unprecedented efficacy in Relapsed/Refractory hematological malignancies. However, effectiveness against solid tumors remains a challenge. The treatment of solid tumors with checkpoint blockage via PD-1/PD-L1 axis (i.e. Atezolizumab) has shown feasibility and potential benefit of targeting PD-L1 in these patients. However, lack of durable anti-tumor responses remains a major concern. Additionally, patients with low PD-L1 expression at baseline remains ineligible for the treatment.

We propose the development of a PD-L1 targeted CAR-NK cell to target PD-L1 positive or also possibly PD-L1 negative tumors. PD-L1 CAR-NK cells will be made by transducing a αPD-L1 CAR during ex vivo expansion of peripheral blood-derived NK cells, enabled by stimulation with PM21 particles that confers a highly active functional phenotype (referred as K-NK cells). We hypothesize that PD-L1 upregulation by tumor cells after exposure to IFNγ secreted by K-NK cells, upon tumor detection, can further make PD-L1low tumor cells susceptible to PD-L1 CAR K-NK cells. Additionally, these PD-L1 CAR K-NK cells are activatable by CAR and/or additional receptors that bind activating ligands on tumor cells. This allows cytotoxicity towards a range of tumors that are PD-L1+, PD-L1 inducible and also PD-L1-.

Methods PD-L1 CAR K-NK cells were generated by engineering NK cells during expansion with a PD-L1 CAR encoding vector. The expression of PD-L1 CAR and K-NK activating/inhibitory receptors was assessed using flow cytometry. PD-L1 CAR K-NK mediated cytotoxicity and cytokine secretion after co-culture with tumor cells were analyzed in vitro.

Results Stable and high PD-L1 CAR expression on K-NK cells was observed. PD-L1 CAR K-NK cells show enhanced cytotoxicity against panel of solid tumor cells, with varying PD-L1 density, compared to control WT K-NK cells. The enhancement in cytotoxic effect was particularly striking at lower E:T ratios (E:T<1). Significantly higher IFNγ secretion by PD-L1 CAR K-NK cells was observed compared to control K-NK cells, that then further enhanced induction of PD-L1 expression on cancer cell lines that normally have low base-line expression of PD-L1. Confirmatory to this mechanism of action, both enhanced cytotoxicity and enhanced IFNγ secretion by NK cells were not observed with PD-L1 knockout target cells.

Conclusions PD-L1 CAR K-NKs were able to more effectively kill tumors with varying levels of PD-L1 expression and thus have the potential to address unmet medical need for patients with PD-L1+ or also PD-L1- tumors, that currently are not optimal candidates for antibody based PD-1/PD-L1 blockade therapeutics.

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