Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia

Nadia El Khawanky,Amy Hughes,Michael P Brown,Angel F Lopez,Markus G Manz,Agnes S M Yong,Lutz Hein,Justus Duyster,Jade Clarson,Khalid Shoumariyeh,Sanaz Taromi,Tony Matschulla,Janaki Manoja Vinnakota,Konrad Aumann,Renier Myburgh,Timothy P Hughes,Cornelius Miething,Deborah L White,Wenbo Yu,Robert Zeiser

doi:10.1038/s41467-021-26683-0

Abstract

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5′-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.

Highlights

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low chimeric antigen receptors (CARs) T cell persistence
In order to develop anti-CD123 CAR T cell therapy for AML, we first confirmed the expression of CD123 on primary human AML bone marrow (BM) versus healthy donor (HD) BM specimens (Supplementary Figs. 1 and 2a)
While its success has demonstrated improved survival outcomes for patients with certain B-lymphoid malignancies[5,6], no comparable success has been achieved with CAR T cells directed against myeloid malignancies

Summary

Introduction

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. We develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. AZA has been shown to upregulate the expression of leukemia-associated antigens on AML and other cancer cells This activates a type I interferon (IFN) signaling response thereby increasing T cell efficacy[15]. Here, that AZA treatment led to the upregulation of the target antigen, CD123 This resulted in reduced numbers of cytotoxic T lymphocyte antigen-4 (CTLA-4) expressing CAR T cells in vivo following infusion. CLTA-4 is a regulator of T cell activation and function, in which T cells with a low expression of this molecule showed increased longevity and activity[22]

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