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155 CD5 knockout enhances the potency of multiplex base-edited allogeneic anti-CD5 CAR T-cell therapy for the treatment of T-cell malignancies
  1. Yinmeng (Amy) Yang,
  2. Ryan Murray,
  3. Adam Camblin,
  4. Faith Musenge,
  5. Lindsey Coholan,
  6. MarkVic Naniong,
  7. David Sweezy,
  8. Scott Haskett,
  9. Lauren Young,
  10. Yingying Zhang,
  11. Amanda Costa,
  12. Hui Wu,
  13. Alden Ladd,
  14. Luis Barrera,
  15. Lisa Schlehuber,
  16. Sarah Smith,
  17. Yeh-Chuin Poh,
  18. Giuseppe Ciaramella and
  19. Jason Gehrke
  1. Beam Therapeutics, Boston, MA, USA


Background T-cell lymphomas and leukemias are a class of diseases lacking durable effective therapies, where median survival for patients suffering from relapsed/refractory disease is often measured in months. Translation of B-cell targeting CAR-T therapeutic success to T-cell malignancies comes with significant challenges. Notably, the shared expression of target antigens on malignant T-cells and in the T-cell product itself results in CAR-T activation and fratricide during manufacturing. To overcome the challenges associated with creating CD5-targeting CAR-Ts, we developed a process to simultaneously base edit five target genes, including CD5 and PD1, to produce potency-enhanced allogeneic anti-CD5 CAR T-cells for use as an off-the-shelf treatment for T-cell malignancies.

Methods Anti-CD5 CAR-Ts were produced in a GMP-compatible process using T-cells isolated from healthy human donors. T-cells were modified using base editing technology to simultaneously knock-out five target genes in a single electroporation step. Edited T-cells were transduced with a lentivirus encoding a second-generation anti-CD5 CAR. Knockout frequencies were evaluated by flow cytometry and next-generation sequencing. Anti-CD5 CAR-Ts were then characterized for their specificity in vitro and potency in in vivo xenograft tumor models.

Results Simultaneous base editing at five genomic loci resulted in anti-CD5 CAR-Ts edited with 94–98% efficiency at each target gene, greatly diminishing the likelihood of GvHD, CAR-T rejection, fratricide, and checkpoint inhibitor activation. In addition, CD5 has an established role as a negative regulator of TCR signaling, and T cells lacking CD5 have enhanced proliferative capacity.1 Anti-CD5 CAR T-cells with or without CD5 KO demonstrated equally potent cytotoxicity and cytokine production in vitro against CD5 expressing tumor lines. However, CD5 KO greatly improved in vivo efficacy of anti-CD5 CAR-Ts in a murine model of T-ALL against established tumor xenografts. Mice previously cleared of tumor underwent a second tumor challenge to assess the persistence of anti-CD5 CAR-T cells and were cleared of tumor a second time, indicating extended persistence of functional anti-CD5 CAR-T cells in vivo.

Conclusions Our approach addresses current technological limitations in developing and applying CAR-Ts that target T-cell malignancies and demonstrates that simultaneous multiplex base editing of up to five targets can create universally compatible, fratricide-resistant, therapeutically active anti-CD5 CAR-Ts. We further demonstrate that CD5 knockout produces CAR-T cells with enhanced potency capable of clearing multiple tumor challenges in vivo. We are progressing this CD5-targeting CAR-T cell product towards potential clinical development for the treatment of T cell malignancies and other CD5+ hematological tumors.


  1. Guillaume V, Peredo G, Romain R. CD5, an Undercover Regulator of TCR Signaling. Frontiers in Immunology 2018;9:2900.

Ethics Approval All animal studies were performed according to the guidelines and approval of the Institutional Animal Care and Use Committee.

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