Mutated GM‐CSF‐based CAR‐T cells targeting CD116/CD131 complexes exhibit enhanced anti‐tumor effects against acute myeloid leukaemia

Abstract

Objectives As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)‐T cells for AML are more challenging than those targeting CD19 in B‐cell malignancies. We recently developed piggyBac ‐modified ligand‐based CAR‐T cells that target CD116/CD131 complexes, also known as the GM‐CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR‐T cells. Methods To further improve the efficacy of the original GMR CAR‐T cells, we have developed novel GMR CAR vectors incorporating a mutated GM‐CSF for the antigen‐binding domain and G4S spacer. All GMR CAR‐T cells were generated using a piggyBac ‐based gene transfer system. The anti‐tumor effect of GMR CAR‐T cells was tested in mouse AML xenograft models. Results Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR‐T cells exhibited potent cytotoxic activities against CD116 + AML cells in vitro . Furthermore, GMR CAR‐T cells incorporating a G4S spacer significantly improved long‐term in vitro and in vivo anti‐tumor effects. By employing a mutated GM‐CSF at residue 21 (E21K), the anti‐tumor effects of GMR CAR‐T cells were also improved especially in long‐term in vitro settings. Although GMR CAR‐T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. Conclusions GMR CAR‐T cell therapy represents a promising strategy for CD116 + R/R AML.

Current Limitations and Perspectives of Chimeric Antigen Receptor-T-Cells in Acute Myeloid Leukemia

“…Multiple additional antigens have been tested preclinically or in a clinical setting in the quest for better CAR-T cell target proteins. Apart from those described above, the following antigens have either been examined for AML or are being investigated: CD7, CD13, CD19, CD34, CD38, CD56, CD117, B7-H3, mesothelin, NKG2D ligands, IL-10 receptor, GM-CSF receptor, ILT3, TIM-3, MUC-1, Lewis Y, and folate receptor β [ 46 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 ] ( Table 1 ).…”

Natural Receptor- and Ligand-Based Chimeric Antigen Receptors: Strategies Using Natural Ligands and Receptors for Targeted Cell Killing

“…While the relative affinities of natural receptors and ligands can also be tuned to optimize CAR functionality in a similar manner to scFvs, the natural affinity that receptors and ligands have for their binding partners may prove to be a better starting point for preclinical CAR design. Indeed, tuning the affinity of one natural ligand-based CAR improved in vivo functionality [ 62 ]. However, it is important to note that the level of affinity for the target receptor is highly contextually specific and needs to be optimized for each individual CAR construct.…”

“…One solution is to design the ligand so that it can bind to the target receptor but is unable to transduce a signal. Indeed, as described above, the GMR-targeted ligand-based CAR T cells utilize a mutant GM-CSF ligand in this context and observe enhanced anti-tumor functionality [ 62 ]. GMR CAR T cells incorporating this mutant GM-CSF design are currently being evaluated in first-in-human clinical trials in Japan (jRCT2033210029).…”

“…The α subunit, CD116, is highly expressed in AML, normal myeloid cells, and a rare subtype of AML known as juvenile myelomonocytic leukemia (JMML) [ 173 ]. These observations have led to the production of a ligand-based CAR in which the GM-CSF protein is utilized as a binding domain to target CD116+ cells [ 62 , 114 ]. Indeed, in vitro co-culture of CAR T cells with CD116+ cell lines and primary JMML patient samples induced significant cytotoxicity, while co-culture with cord blood and bone marrow from a healthy donor did not.…”

“…Therefore, to decrease the chance of unwanted receptor signaling and enhance anti-tumor efficacy, GMR CAR T cells were optimized by replacing the native GM-CSF protein with an E21K GM-CSF mutant to improve long-term in vitro anti-tumor activities. Indeed it was shown that optimized GMR CAR T cells do elicit enhanced anti-tumor responses both in vitro and in murine models with disseminated AML [ 62 ]. The GMR CAR T cell therapeutic is currently being investigated in an investigator-initiated, first-in-human trial currently recruiting in Japan (jRCT2033210029).…”