Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia.

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

Potency assessment of IFNγ-producing SARS-CoV-2-specific T cells from COVID-19 convalescent subjects.

The development of new therapies for COVID-19 high-risk patients remains necessary to prevent additional deaths. Here, we studied the phenotypical and functional characteristics of IFN-γ producing-SARS-CoV-2-specific T cells (SC2-STs), obtained from 12 COVID-19 convalescent donors, to determine their potency as an off-the-shelf T cell therapy product. We found that these cells present mainly an effector memory phenotype, characterized by the basal expression of cytotoxicity and activation markers, including granzyme B, perforin, CD38, and PD-1. We demonstrated that SC2-STs could be expanded and isolated in vitro, and they exhibited peptide-specific cytolytic and proliferative responses after antigenic re-challenge. Collectively, these data demonstrate that SC2-STs can be a suitable candidate for the manufacture of a T cell therapy product aimed to treat severe COVID-19.

CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome.

Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

Assessment of Minimal Residual Disease by Next Generation Sequencing in Peripheral Blood as a Complementary Tool for Personalized Transplant Monitoring in Myeloid Neoplasms.

Patients with myeloid neoplasms who relapsed after allogenic hematopoietic stem cell transplant (HSCT) have poor prognosis. Monitoring of chimerism and specific molecular markers as a surrogate measure of relapse is not always helpful; therefore, improved systems to detect early relapse are needed. We hypothesized that the use of next generation sequencing (NGS) could be a suitable approach for personalized follow-up post-HSCT. To validate our hypothesis, we analyzed by NGS, a retrospective set of peripheral blood (PB) DNA samples previously evaluated by high-sensitive quantitative PCR analysis using insertion/deletion polymorphisms (indel-qPCR) chimerism engraftment. Post-HCST allelic burdens assessed by NGS and chimerism status showed a similar time-course pattern. At time of clinical relapse in 8/12 patients, we detected positive NGS-based minimal residual disease (NGS-MRD). Importantly, in 6/8 patients, we were able to detect NGS-MRD at time points collected prior to clinical relapse. We also confirmed the disappearance of post-HCST allelic burden in non-relapsed patients, indicating true clinical specificity. This study highlights the clinical utility of NGS-based post-HCST monitoring in myeloid neoplasia as a complementary specific analysis to high-sensitive engraftment testing. Overall, NGS-MRD testing in PB is widely applicable for the evaluation of patients following HSCT and highly valuable to personalized early treatment intervention when mixed chimerism is detected.

Screening for occult malignancy with FDG-PET/CT in patients with unprovoked venous thromboembolism.

Extensive screening strategies to detect occult cancer in patients with unprovoked venous thromboembolism (VTE) are complex and no benefit in terms of survival has been reported. FDG-PET/CT (2-[F-18] fluoro-2-deoxy-D-glucose positron emission tomography combined with computed tomography), a noninvasive technique for the diagnosis and staging of malignancies, could be useful in this setting. Consecutive patients ≥ 50 years with a first unprovoked VTE episode were prospectively included. Screening with FDG-PET/CT was performed 3-4 weeks after the index event. If positive, appropriate diagnostic work-up was programmed. Clinical follow-up continued for 2 years. Blood samples were collected to assess coagulation biomarkers. FDG-PET/CT was negative in 68/99 patients (68.7%), while suspicious FDG uptake was detected in 31/99 patients (31.3%). Additional diagnostic work-up confirmed a malignancy in 7/31 patients (22.6%), with six of them at early stage. During follow-up, two patients with negative FDG-PET/CT were diagnosed with cancer. Sensitivity (S), positive (PPV) and negative predictive values (NPV) of FDG-PET/CT as single tool for the detection of occult malignancy were 77.8% (95% CI: 0.51-1), 22.6% (95% CI: 0.08-0.37) and 97.1% (95% CI: 0.93-1), respectively. Median tissue factor (TF) activity in patients with occult cancer was 5.38 pM vs. 2.40 pM in those without cancer (p = 0.03). Limitation of FDG-PET/CT screening to patients with TF activity > 2.8 pM would improve the PPV to 37.5% and reduce the costs of a single cancer diagnosis from 20,711€ to 11,670€. FDG-PET/CT is feasible for the screening of occult cancer in patients with unprovoked VTE, showing high S and NPV. The addition of TF activity determination may be useful for patient selection.