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260 Novel CLL-1-directed CAR-T cells mediate potent antigen-specific cytolytic activity in mouse models of acute myeloid leukemia
  1. Brikena Gjeci,
  2. Reid Williams,
  3. Hillary Hoyt,
  4. Amanda Halfond,
  5. Giacomo Canesin,
  6. Julia Etchin,
  7. Guy Mundelboim,
  8. Mariana Silva,
  9. John Lydeard,
  10. Julian Scherer and
  11. Tirtha Chakraborty
  1. Vor Biopharma, 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 Acute myeloid leukemia (AML) is the most common form of leukemia in adults. However, the clinical outcome for high-risk patients remains poor, highlighting the urgent need for the development of new therapeutic strategies [1]. Chimeric antigen receptor (CAR) T cell therapy holds promise as an immunotherapeutic strategy and targeting C-type lectin-like molecule-1 (CLL-1, CD371) represents an attractive approach, as CLL-1 is highly expressed on AML blasts and leukemic stem cells [1]. Here, we present preclinical data detailing the development and functional characterization of novel CLL-1-directed CAR-T cells to identify top candidates from a panel of 24 CLL-1 binders.

Methods CLL-1-directed binders were identified by phage display technology and evaluated by flow cytometric, ELISA and Octet analyses. Selected binders were used to generate second generation CLL-1-directed CAR constructs with a 4–1BB co-stimulatory domain. CAR constructs were transduced into primary T cells using lentiviral vectors and investigated for antigen-specific cytolysis by flow cytometry-based assays. Co-culture experiments were conducted for 24 and 48 hours with CLL-1-expressing WT and CLL-1 knockout (KO) HL60 target cells. CAR candidates showing robust antigen-dependent activity were further evaluated for potency by bioluminescent-based assays at low effector to target (E:T) ratios, for long-term persistence in repeated stimulation assays, and for avidity measurements in acoustic force microscopy assays. The top CLL-1 CAR candidates were further studied in an in vivo murine xenograft model using HL60 AML cells in NSG mice.

Results We completed an in-depth in vitro characterization of 24 CLL-1-directed CAR-T cells and identified top candidates that exhibited 1) potent and specific cytotoxicity of CLL1-expressing targets with minimal nonspecific killing of CLL1 KO targets, 2) high levels of antigen-dependent activation, and 3) significant antigen-dependent cytokine secretion. Importantly, top candidates effectively killed CLL1-expressing targets at low E:T ratios, demonstrated superior persistence after repeated stimulation with target cells, and displayed similar high binding avidity. Lead CLL-1 CAR-T cell candidates significantly reduced in vivo tumor growth as assessed by IVIS imaging and flow cytometric analyses, which also indicated tumor cell clearance and CAR T cell expansion.

Conclusions Altogether, our preclinical data demonstrate highly efficacious and antigen-specific CLL-1-directed CAR-T cells, with potent in vitro and in vivo cytolytic activity. These results support further clinical development of the lead CLL-1 CAR candidate either as a stand-alone treatment or in combination with the VOR eHSC platform to eliminate on-target off-tumor toxicity to fully benefit high-risk AML patients in need.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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