Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients.

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.

CAR-T cells targeting IL-1RAP produced in a closed semiautomatic system are ready for the first phase I clinical investigation in humans.

PURPOSE OF THE STUDY: The use of chimeric antigen receptor (CAR)-T cells has demonstrated excellent results in B-lymphoid malignancies. The Advanced Therapy Medicinal Products (ATMP) status and good manufacturing practice (GMP) of CAR-T cells require particular conditions of production performed in a pharmaceutical establishment. Our team developed a new medical drug candidate for acute myeloid leukemia (AML), a CAR targeting interleukin-1 receptor accessory protein (IL-1RAP) expressed by leukemia stem cells, which will need to be evaluated in a phase I-IIa clinical trial. During the preclinical development phase, we produced IL-1RAP CAR-T cells in a semi-automated closed system (CliniMACSࣨ Prodigy) using research grade lentiviral particles. PATIENTS AND THE METHODS: The purpose of this work was to validate our production process and to characterize our preclinical GMP-like medicinal product. IL-1RAP CAR-T cells were produced from healthy donors in 9 days, either in an semi-automated closed system (with GMP-like compliant conditions) or according to another research protocols, which was used as a reference. RESULTS: Based on phenotypic, functional and metabolic analyses, we were able to show that the final product is ready for clinical use. Finally, in a xenograft AML murine model, we demonstrated that the IL-1RAP CAR-T cells generated in a GMP-like environment could eliminate tumor cells and increase overall survival. CONCLUSION: We demonstrated that our IL-1RAP CAR-T cell preclinical GMP-like production process meets standard regulatory requirements in terms of CAR-T cell number, subpopulation phenotype and cytotoxic functionality. Our CAR-T cell production process was validated and can be used to produce medicinal IL-1RAP CAR-T cells for the first phase I clinical trial.

Chimeric antigen receptor T-cells targeting IL-1RAP: a promising new cellular immunotherapy to treat acute myeloid leukemia.

BACKGROUND: Acute 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. METHODS: In 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. RESULTS: We 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. CONCLUSION: In 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.

Efficient and Robust NK-Cell Transduction With Baboon Envelope Pseudotyped Lentivector.

NK-cell resistance to transduction is a major technical hurdle for developing NK-cell immunotherapy. By using Baboon envelope pseudotyped lentiviral vectors (BaEV-LVs) encoding eGFP, we obtained a transduction rate of 23.0 ± 6.6% (mean ± SD) in freshly-isolated human NK-cells (FI-NK) and 83.4 ± 10.1% (mean ± SD) in NK-cells obtained from the NK-cell Activation and Expansion System (NKAES), with a sustained transgene expression for at least 21 days. BaEV-LVs outperformed Vesicular Stomatitis Virus type-G (VSV-G)-, RD114- and Measles Virus (MV)- pseudotyped LVs (p < 0.0001). mRNA expression of both BaEV receptors, ASCT1 and ASCT2, was detected in FI-NK and NKAES, with higher expression in NKAES. Transduction with BaEV-LVs encoding for CAR-CD22 resulted in robust CAR-expression on 38.3 ± 23.8% (mean ± SD) of NKAES cells, leading to specific killing of NK-resistant pre-B-ALL-RS4;11 cell line. Using a larger vector encoding a dual CD19/CD22-CAR, we were able to transduce and re-expand dual-CAR-expressing NKAES, even with lower viral titer. These dual-CAR-NK efficiently killed both CD19KO- and CD22KO-RS4;11 cells. Our results suggest that BaEV-LVs may efficiently enable NK-cell biological studies and translation of NK-cell-based immunotherapy to the clinic.