citations

Cellular immunotherapy targeting CLL-1 for juvenile myelomonocytic leukemia

Authors:

Werner J, Lee AG, Zhang C, Abelson S, Xirenayi S, Rivera J, Yousuf K, Shin H, Patiño-Escobar B, Bachl S, Mandal K, Barpanda A, Ramos E, Izgutdina A, Chaudhuri S, Temple WC, Bhatnagar S, Dardis JK, Meyer J, Morales C, Meshinchi S, Loh ML, Braun B, Tasian SK, Wiita AP, Stieglitz E

In:

Source: Nat Commun.
Publication Date: (2025)
Issue: :

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Gene Expression

Basic Research

Molecular Biology

Regenerative medicine

Drug Discovery

Cells used in publication:

T cell, human stim.: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® 96-well Systems

Experiment

TRAC-KO
Cas9 protein (40 µM;Macrolab, University of California, Berkeley) and sgRNA (80 µM; Synthego Corporation) were mixed in 1:2 molar ratio and incubated at 37 °C for 15min. 2e6 of primary T cells were spun down, resuspended in 20 µL of P3 nucleofection solution (P3 Primary Cell 96-well Nucleofector Kit from Lonza, V4SP-3096) plus ribonucleoprotein (RNP) complex, and nucleofected using EH-115 nucleofection program in a Lonza 4D-Nucleofector. Eighty microliters of warm CTS OpTmizer T Cell Expansion SFM was plated into each well and incubated at 37 °C for 15min, then transferred to an appropriately sized plate or flask supplementedwith IL-7 and IL-15 to recover for 72 h.

Abstract

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder that predominantly affects infants and young children. Hematopoietic stem cell transplantation (HSCT) is standard of care, but post-HSCT relapse is common, highlighting the need for innovative therapies. While adoptive immunotherapy with chimeric antigen receptor (CAR) T cells has improved outcomes for patients with advanced lymphoid malignancies, it has not been comprehensively evaluated in JMML. In the present study, we use bulk and single-cell RNA sequencing, mass spectrometry, and flow cytometry to identify overexpression of CLL-1 (encoded by CLEC12A) on the cell surface of cells from patients with JMML. We develop immunotherapy with CLL-1 CAR T cells (CLL1CART) for preclinical testing and report in vitro and in vivo anti-leukemia activity. Notably, CLL1CART reduce the number of leukemic stem cells and serial transplantability in vivo. These preclinical data support the development and clinical investigation ofCLL- 1-targeting immunotherapy in children with relapsed/refractory JMML.

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