Chronic myeloid leukemia (CML) is a chronic disease resulting in myeloid cell expansion through expression of the BCR-ABL1 fusion transcript. Tyrosine kinase inhibitors (TKI) have significantly increased survival of patients with CML, and deep responders may consider stopping the treatment. However, more than 50% of patients relapse and restart TKI, subsequently suffering unknown toxicity. Because CML is a model immune system-sensitive disease, we hypothesize that chimeric antigen receptor (CAR) T cells targeting IL1 receptor-associated protein (IL1RAP) in quiescent CML stem cells may offer an opportunity for a permanent cure. In this study, we produced and molecularly characterized a specific monoclonal anti-IL1RAP antibody from which fragment antigen-binding nucleotide coding sequences were cloned as a single chain into a lentiviral backbone and secured with the suicide gene iCASP9/rimiducid system. Our CAR T-cell therapy exhibited cytotoxicity against both leukemic stem cells and, to a lesser extent, monocytes expressing IL1RAP, with no apparent effect on the hematopoietic system, including CD34 þ stem cells. This suggests IL1RAP as a tumor-associated antigen for immunotherapy cell targeting. IL1RAP CAR T cells were activated in the presence of IL1RAP þ cell lines or primary CML cells, resulting in secretion of proinflammatory cytokines and specifically killing in vitro and in a xenograft murine model. Overall, we demonstrate the proof of concept of a CAR T-cell immunotherapy approach in the context of CML that is applicable for young patients and primary TKI-resistant, intolerant, or allograft candidate patients. Significance: These findings present the first characterization and proof of concept of a chimeric antigen receptor directed against IL1RAP expressed by leukemic stem cells in the context of CML.

Background Genetically engineered chimeric antigen receptor (CAR) T lymphocytes are promising therapeutic tools for cancer. Four CAR T cell drugs, including tisagenlecleucel (tisa-cel) and axicabtagene-ciloleucel (axi-cel), all targeting CD19, are currently approved for treating B cell malignancies. Flow cytometry (FC) remains the standard for monitoring CAR T cells using a recombinant biotinylated target protein. Nevertheless, there is a need for additional tools, and the challenge is to develop an easy, relevant, highly sensitive, reproducible, and inexpensive detection method. Molecular tools can meet this need to specifically monitor long-term persistent CAR T cells. Methods Based on 2 experimental CAR T cell constructs, IL-1RAP and CS1, we designed 2 quantitative digital droplet (ddPCR) PCR assays. By targeting the 4.1BB/CD3z (28BBz) or 28/CD3z (28z) junction area, we demonstrated that PCR assays can be applied to approved CD19 CAR T drugs. Both 28z and 28BBz ddPCR assays allow determination of the average vector copy number (VCN) per cell. We confirmed that the VCN is dependent on the multiplicity of infection and verified that the VCN of our experimental or GMP-like IL-1RAP CAR T cells met the requirement (< 5 VCN/cell) for delivery to the clinical department, similar to approved axi-cel or tisa-cel drugs. Results 28BBz and 28z ddPCR assays applied to 2 tumoral (acute myeloid leukemia (AML) or multiple myeloma (MM) xenograft humanized NSG mouse models allowed us to quantify the early expansion (up to day 30) of CAR T cells after injection. Interestingly, following initial expansion, when circulating CAR T cells were challenged with the tumor, we noted a second expansion phase. Investigation of the bone marrow, spleen and lung showed that CAR T cells disseminated more within these tissues in mice previously injected with leukemic cell lines. Finally, circulating CAR T cell ddPCR monitoring of R/R acute lymphoid leukemia or diffuse large B cell lymphoma (n = 10 for tisa-cel and n = 7 for axi-cel) patients treated with both approved CAR T cells allowed detection of early expansion, which was highly correlated with FC, as well as long-term persistence (up to 450 days), while FC failed to detect these events. Conclusion Overall, we designed and validated 2 ddPCR assays allowing routine or preclinical monitoring of early- and long-term circulating approved or experimental CAR T cells, including our own IL-1RAP CAR T cells, which will be evaluated in an upcoming phase I clinical trial.

Although chimeric antigen receptor CAR) T cell immunotherapies are an undeniable and unequivocal success, knowledge obtained from the monitoring of the first clinical trials targeting the CD19 antigen in B malignancies, either refractory/relapsed acute lymphoid leukemia (ALL) or lymphomas, contributed to the identification of tumor cell escape in about 30–50% of B-ALL. Resistance occurred due to loss of surface expression of the antigen (rCD19−) or to the early disappearance or inactivation of CAR T cells (rCD19+). In a recently reported clinical case, rCD19− relapse resulted from masking of the antigen by the CAR at the surface of B-ALL leukemia cells following the unexpected viral transduction of a leukemic cell present in the cytapheresis sample. The objective of this work was to reproduce this epitope-masking resistance model, in the context of acute myeloid leukemia (AML), based on our immunotherapeutic CAR T cell model targeting the accessory protein of the interleukin-1 receptor (IL-1RAP) expressed by leukemic stem cells. As AML primary blasts express different levels of IL-1RAP, we modeled transduction of different AML tumor cell lines screened for density of antigenic sites with our lentiviral vectors carrying a third-generation IL-1RAP CAR, an iCASP9 suicide gene, and a truncated CD19 surface gene. We demonstrated that primary AML blasts can be easily transduced (74.55 ± 21.29%, n = 4) and that CAR T cytotoxicity to IL-1RAP is inversely correlated with epitope masking in relation to the number of antigenic sites expressed on the surface of IL-1RAP+ lines. Importantly, we showed that, in vitro, a 24-h exposure of IL-1RAP+/CAR+ leukemia lines to Rimiducid eliminated >85% of the cells. We confirmed that the expression of IL-1RAP CAR by an IL-1RAP+ leukemic cell, by decreasing the membrane availability of the targeted antigen, can induce resistance while a high epitope density maintains sensitivity to CAR T cells. Moreover, the presence of the iCASP9/Rimiducid suicide system safety switch makes this immunotherapy approach safe for application in a future phase 1 clinical trial.

BackgroundAcute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are the basis of refractory/relapsed (R/R) disease in 80% of patients with AML not receiving allogeneic transplantation.MethodsIn this study, we showed that the interleukin-1 receptor accessory protein (IL-1RAP) protein is overexpressed on the cell surface of LSCs in all subtypes of AML and confirmed it as an interesting and promising target in AML compared with the most common potential AML targets, since it is not expressed by the normal hematopoietic stem cell. After establishing the proof of concept for the efficacy of chimeric antigen receptor (CAR) T-cells targeting IL-1RAP in chronic myeloid leukemia, we hypothesized that third-generation IL-1RAP CAR T-cells could eliminate AML LSCs, where the medical need is not covered.ResultsWe first demonstrated that IL-1RAP CAR T-cells can be produced from AML T-cells at the time of diagnosis and at relapse. In vitro and in vivo, we showed the effectiveness of IL-1RAP CAR T-cells against AML cell lines expressing different levels of IL-1RAP and the cytotoxicity of autologous IL-1RAP CAR T-cells against primary cells from patients with AML at diagnosis or at relapse. In patient-derived relapsed AML xenograft models, we confirmed that IL-1RAP CAR T-cells are able to circulate in peripheral blood and to migrate in the bone marrow and spleen, are cytotoxic against primary AML cells and increased overall survival.ConclusionIn conclusion, our preclinical results suggest that IL-1RAP CAR T-based adoptive therapy could be a promising strategy in AML treatment and it warrants the clinical investigation of this CAR T-cell therapy.