Chimeric antigen receptor (CAR) modified T Cells in acute myeloid leukemia: limitations and expectations.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis despite the advent of novel therapies. Consequently, a major need exists for new therapeutic options, particularly for patients with relapsed/refractory (R/R) AML. In recent years, it has been possible to individualize the treatment of a subgroup of patients, particularly with the emergence of multiple targeted therapies. Nonetheless, a considerable number of patients remain without therapeutic options, and overall prognosis remains poor because of a high rate of disease relapse. In this sense, cellular therapies, especially chimeric antigen receptor (CAR)-T cell therapy, have dramatically shifted the therapeutic options for other hematologic malignancies, such as diffuse large B cell lymphoma and acute lymphoblastic leukemia. In contrast, effectively treating AML with CAR-based immunotherapy poses major biological and clinical challenges, most of them derived from the unmet need to identify target antigens with expression restricted to the AML blast without compromising the viability of the normal hematopoietic stem cell counterpart. Although those limitations have hampered CAR-T cell therapy translation to the clinic, there are several clinical trials where target antigens, such as CD123, CLL-1 or CD33 are being used to treat AML patients showing promising results. Moreover, there are continuing efforts to enhance the specificity and efficacy of CAR-T cell therapy in AML. These endeavors encompass the exploration of novel avenues, including the development of dual CAR-T cells and next-generation CAR-T cells, as well as the utilization of gene editing tools to mitigate off-tumor toxicities. In this review, we will summarize the ongoing clinical studies and the early clinical results reported with CAR-T cells in AML, as well as highlight CAR-T cell limitations and the most recent approaches to overcome these barriers. We will also discuss how and when CAR-T cells should be used in the context of AML.

Next-generation BCMA-targeted chimeric antigen receptor CARTemis-1: the impact of manufacturing procedure on CAR T-cell features.

PURPOSE: CAR therapy targeting BCMA is under investigation as treatment for multiple myeloma. However, given the lack of plateau in most studies, pursuing more effective alternatives is imperative. We present the preclinical and clinical validation of a new optimized anti-BCMA CAR (CARTemis-1). In addition, we explored how the manufacturing process could impact CAR-T cell product quality and fitness. METHODS: CARTemis-1 optimizations were evaluated at the preclinical level both, in vitro and in vivo. CARTemis-1 generation was validated under GMP conditions, studying the dynamics of the immunophenotype from leukapheresis to final product. Here, we studied the impact of the manufacturing process on CAR-T cells to define optimal cell culture protocol and expansion time to increase product fitness. RESULTS: Two different versions of CARTemis-1 with different spacers were compared. The longer version showed increased cytotoxicity. The incorporation of the safety-gene EGFRt into the CARTemis-1 structure can be used as a monitoring marker. CARTemis-1 showed no inhibition by soluble BCMA and presents potent antitumor effects both in vitro and in vivo. Expansion with IL-2 or IL-7/IL-15 was compared, revealing greater proliferation, less differentiation, and less exhaustion with IL-7/IL-15. Three consecutive batches of CARTemis-1 were produced under GMP guidelines meeting all the required specifications. CARTemis-1 cells manufactured under GMP conditions showed increased memory subpopulations, reduced exhaustion markers and selective antitumor efficacy against MM cell lines and primary myeloma cells. The optimal release time points for obtaining the best fit product were > 6 and < 10 days (days 8-10). CONCLUSIONS: CARTemis-1 has been rationally designed to increase antitumor efficacy, overcome sBCMA inhibition, and incorporate the expression of a safety-gene. The generation of CARTemis-1 was successfully validated under GMP standards. A phase I/II clinical trial for patients with multiple myeloma will be conducted (EuCT number 2022-503063-15-00).

Monitoring of kinetics and exhaustion markers of circulating CAR-T cells as early predictive factors in patients with B-cell malignancies.

Purpose: CAR-T cell therapy has proven to be a disruptive treatment in the hematology field, however, less than 50% of patients maintain long-term response and early predictors of outcome are still inconsistently defined. Here, we aimed to optimize the detection of CD19 CAR-T cells in blood and to identify phenotypic features as early biomarkers associated with toxicity and outcomes. Experimental design: In this study, monitoring by flow cytometry and digital PCR (dPCR), and immunophenotypic characterization of circulating CAR-T cells from 48 patients treated with Tisa-cel or Axi-cel was performed. Results: Validation of the flow cytometry reagent for the detection of CAR-T cells in blood revealed CD19 protein conjugated with streptavidin as the optimal detection method. Kinetics of CAR-T cell expansion in blood confirmed median day of peak expansion at seven days post-infusion by both flow cytometry and digital PCR. Circulating CAR-T cells showed an activated, proliferative, and exhausted phenotype at the time of peak expansion. Patients with increased expansion showed more severe CRS and ICANs. Immunophenotypic characterization of CAR-T cells at the peak expansion identified the increased expression of co-inhibitory molecules PD1 and LAG3 and reduced levels of the cytotoxicity marker CD107a as predictors of a better long-term disease control. Conclusions: These data show the importance of CAR-T cells in vivo monitoring and identify the expression of PD1LAG3 and CD107a as early biomarkers of long-term disease control after CAR-T cell therapy.

Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System.

In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.

Analysis of p.Tyr307Asn variant in the LRP10 gene in Parkinson's disease in southern Spain.

Lipoprotein receptor-related protein 10 (LRP10) has been proposed as a novel causative gene for autosomal dominant Parkinson's disease (PD), and the c.919T>A (p.Tyr307Asn) variant has been identified as possibly involved in the development of familial PD and PD with dementia. We screened for the p.Tyr307Asn variant in a southern Spain population of 679 PD patients, of who 129 were familial cases, and 1217 unrelated healthy controls. A total of 3 carriers of the LRP10 p.Tyr307Asn variant were identified: 1 PD patient and 2 healthy controls. Together with the absence of a family history of PD, this finding might suggest a low penetrance variant as well as a limited role for p.Tyr307Asn in PD in our cohort. Nevertheless, a family history of Alzheimer's disease in the LRP10 p.Tyr307Asn carriers provides evidence for a possible association with dementia.