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237 Epitope engineered hematopoietic stem cells to enable multi-specificity CAR-T cells for acute myeloid leukemia
  1. Gabriele Casirati1,
  2. Andrea Cosentino1,
  3. Adele Mucci1,
  4. Mohammed S Mahmoud1,
  5. Iratxe Ugarte Zabala2,
  6. Jing Zeng1,
  7. Denise Klatt1,
  8. Christian Brendel1,
  9. Jerome Ritz3,
  10. Scott Ficarro3,
  11. Jarrod Marto3,
  12. Danilo Pellin1,
  13. Daniel Bauer1,
  14. Scott A Armstrong3 and
  15. Pietro Genovese1
  1. 1Boston Children’s Hospital, Boston, MA, USA
  2. 2DFKZ, Heidelberg, NA, Germany
  3. 3Dana-Farber Cancer Institute, Boston, 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 Acute myeloid leukemia (AML) is associated with an unfavorable outcome for >50% of patients. Whereas novel immunotherapies, such as CD19-CAR-T, demonstrated striking efficacy when targeting dispensable antigens (Ag), the same approach cannot be exploited for AML, due to lack of actionable leukemia-restricted Ags. AML targets are shared with progenitors (HSPCs) or mature myeloid cells, leading to on-target/off-tumor toxicity. We reasoned that precise modification of target epitopes in donor HSPCs used in hematopoietic stem cell transplantation (HSCT) would result in loss of recognition by CAR/mAbs, without affecting protein expression and function. Epitope-editing allows targeting genes essential for leukemia survival regardless of expression in HSPC, minimizing the risk of immune-escape.1

Methods We selected the cytokine receptors FLT3, CD123 and KIT, found in >85% of AML cases. By library screenings, we identified substitutions in their extracellular-domain that avoid detection by therapeutic Abs. We validated the functionality of mutated receptors (ligand affinity, western-blot, proliferation, RNAseq, phospho-proteomics) and their resistance to on-target killing (mAb-affinity, CAR-T co-culture). We optimized a base-editing protocol to introduce these mutations in CD34+HSPCs. We exploited advanced in vivo models with co-engraftment of healthy HSPCs, patient-derived AML xenografts (PDX) and CAR-T to assess selective elimination of leukemia and protection of healthy hematopoiesis.

Results Epitope variants were resistant to in vitro CAR-T killing and did not induce CAR activation. Electroporation of ABE8e mRNA+sgRNAs into CD34+HSPCs achieved 90%, 85% and 75% editing efficiency on FLT3, KIT and CD123. After xenotransplant into NBSGW mice, epitope-edited HSPC sustained long-term multi-lineage hematopoiesis, similar to AAVS1 controls. Upon treatment with FLT3-CAR-T, we observed sparing of HSPCs, granulo-mono progenitors and B-cell subsets derived from FLT3-edited HSPCs, while treatment with CD123-CAR showed protection of epitope-edited myeloid lineages, compared to AAVS1. Concomitantly, co-engrafted PDXs were eradicated by either FLT3- or CD123-CAR-T. Due to AML intra-tumoral heterogeneity and plasticity, targeting several Ags might be required to eradicate leukemia stem cells. To this end, we optimized high-efficiency multiplex-editing to enable targeting of multiple Ags without overlapping toxicities. We confirmed resistance of dual-FLT3/CD123 epitope-edited HSPCs and the superior efficacy of dual-target CAR-T in mice co-engrafted with a PDX partially resistant to FLT3-targeting alone. Additionally, our approach was able to protect HSPCs from the combination of FLT3-CARs with FLT3-tyrosine kinase inhibitor Crenolanib, while controls showed additional toxicity.

Conclusions In conclusion, transplantation of epitope-engineered HSPCs endowed with selective resistance to multi-specifc CAR-T-cells is a novel approach to enable more effective and safer immunotherapies for difficult-to-target tumors such as AML.

Acknowledgements I would like to acknowledge my mentor, Prof. Pietro Genovese, for his kind and thorough supervision.

Additionally, I would like to thank the members of the lab that have collaborated with me to generate the data for this work, among others: Adele Mucci PhD, Andrea Cosentino MD, Mohammed S. Mahmoud PhD, Iraxte Ugarte Zabala, MSc.

Our close collaborators, Dan Bauer’s and Christian Brendel’s lab also deserve a special mention.

Several funding agencies have provided the funding to develop this project, but I would like to specifically thank the American Society for Transplantation and Cellular Therapy and the Pediatric Transplantation and Cellular Therapy Consortium for granting me two New Investigator Awards to advance this work further and start new spin-off projects related to this abstract.


  1. Epitope Editing Enables Targeted Immunotherapies for Acute Myeloid Leukemia, Casirati G, et al. Nature (provisionally accepted)

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