Recent times have witnessed remarkable progress in cancer immunotherapy, drastically changing the cancer treatment landscape. Among the various immunotherapeutic approaches, adoptive cell therapy (ACT), particularly chimeric antigen receptor (CAR) T cell therapy, has emerged as a promising strategy to tackle cancer. CAR-T cells are genetically engineered T cells with synthetic receptors capable of recognising and targeting tumour-specific or tumour-associated antigens. By leveraging the intrinsic cytotoxicity of T cells and enhancing their tumour-targeting specificity, CAR-T cell therapy holds immense potential in achieving long-term remission for cancer patients. However, challenges such as antigen escape and cytokine release syndrome underscore the need for the continued optimisation and refinement of CAR-T cell therapy. Here, we report on the challenges of CAR-T cell therapies and on the efforts focused on innovative CAR design, on diverse therapeutic strategies, and on future directions for this emerging and fast-growing field. The review highlights the significant advances and changes in CAR-T cell therapy, focusing on the design and function of CAR constructs, systematically categorising the different CARs based on their structures and concepts to guide researchers interested in ACT through an ever-changing and complex scenario. UNIVERSAL CARs, engineered to recognise multiple tumour antigens simultaneously, DUAL CARs, and SUPRA CARs are some of the most advanced instances. Non-molecular variant categories including CARs capable of secreting enzymes, such as catalase to reduce oxidative stress in situ, and heparanase to promote infiltration by degrading the extracellular matrix, are also explained. Additionally, we report on CARs influenced or activated by external stimuli like light, heat, oxygen, or nanomaterials. Those strategies and improved CAR constructs in combination with further genetic engineering through CRISPR/Cas9- and TALEN-based approaches for genome editing will pave the way for successful clinical applications that today are just starting to scratch the surface. The frontier lies in bringing those approaches into clinical assessment, aiming for more regulated, safer, and effective CAR-T therapies for cancer patients.

COVID‐19 remains a significant global health problem with uncertain long‐term consequences for convalescents. We investigated the relationships between anti‐N protein antibody levels, severe acute respiratory syndrome (SARS)‐CoV‐2‐associated TCR repertoire parameters, HLA type and epidemiological information from three cohorts of 524 SARS‐CoV‐2‐infected subjects subgrouped in acute phase, seronegative and seropositive convalescents from the Emilia Romagna region. Epidemiological information and anti‐N antibody index were associated with TCR repertoire data. HLA type was inferred from TCR repertoire using the HLA3 tool and its association with clonal breadth (CB) and clonal depth (CD) was assessed. Age above 58 years, male and COVID‐19 hospitalisation were significantly and independently associated with seropositivity (p = 0.004; p = 0.004; p = 0.04), suggesting an association between high antibody titres and symptoms' severity. As for the TCR repertoire analysis, we found no difference in CB among the cohorts, while CD was higher in seronegative than acute (p = 0.04). However, clustering analysis supported that seronegative patients are endowed with broader CB and deeper CD indicating a compensatory mechanism without effective seroconversion. The CD calculated on the TCRs associated with the single SARS‐CoV‐2 ORFs in convalescents is higher when compared to the acute. Lastly, we identified and reported on novel HLAs significantly associated with increased risk of hospitalisation such as HLA‐C*07:02 carriers (OR = 3.9, CI = 1.1–13.4, p = 0.03) and on HLAs that associate significantly with lower or higher TCR repertoire parameters in a population exposed for the first time to SARS‐CoV‐2.

Introduction: Clinical presentation and genetic profile of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are highly variable, hampering their management. Sequencing of circulating tumor DNA from liquid biopsy (LB) has been proposed as a less invasive alternative to solid biopsy (SB). Our aim was to compare the mutational profile (MP) provided by LB with that deriving from SB in GEP-NETs. Methods: SB and LB were derived simultaneously from 6 GEP-NET patients. A comparative targeted next-generation sequencing (NGS) analysis was performed on DNA from SB and LB to evaluate the mutational status of 11 genes (MEN1, DAXX, ATRX, MUTYH, SETD2, DEPDC5, TSC2, ARID1A, CHECK2, MTOR, and PTEN). Results: Patients (M:F = 2:1; median age 64 years) included 3 with pancreatic and 3 with ileal NETs. NGS detected a median number of 55 variants/sample in SB and 66.5 variants/sample in LB specimens (mutational burden: 0.2–1.9 and 0.3–1.8 mut/Mb, respectively). Missense and nonsense mutations were prevalent in both, mainly represented by C>T transitions. ARID1A, MTOR, and ATRX were consistently mutated in SB, and ARID1A, TSC2, MEN1, PTEN, SETD2, and MUTYH were consistently mutated in LB. DAXX mutations were absent in LB. Seventeen recurrent mutations were shared between SB and LB; in particular, MTOR single-nucleotide variants c.G4731A and c.C2997T were shared by 5 out of 6 patients. Hierarchical clustering supported genetic similarity between SB and LB. Conclusions: This pilot study explores the applicability of LB in GEP-NET MP evaluation. Further studies with larger cohorts are needed to validate LB and to define the clinical impact.

Introduction: Hematological patients are at higher risk of severe SARS-CoV-2 infection and exhibit impaired vaccine responses due to disease and therapy. Treatments like Rituximab are known to compromise immune function, reducing the generation of protective antibodies. Methods: In the CORSA trial, we evaluated humoral and cellular responses to messenger RNA vaccines against SARS-CoV-2 in 77 patients with hematological malignancies receiving active treatment and in matched healthy controls. Peripheral blood samples were processed to assess Spike-specific immunoglobulin G titers, T-cell responses by enzyme-linked immunospot assay, and T-cell receptor repertoire sequencing from baseline to six months after vaccination to determine clonal breadth and depth of Spike-specific T-cell receptor clones. Results: Seroconversion occurred in only 8% and 21% of patients after the first and second dose, compared to 93% and 100% of healthy controls. Despite poor antibody responses, 69% of seronegative patients showed measurable T-cell activity, suggesting some level of vaccine-induced protection. Spike-specific TCR repertoire analysis in lymphoma patients (LP) and HC revealed significantly broader clonal breadth (p = 0.0013) and higher clonal depth (p = 0.0265) in HC at day 50 versus baseline, while blunted diversification in LP (p = 0.0407). Conclusions: Lymphoma patients showed significantly weaker Spike-specific clonal responses and reduced diversity compared with healthy controls, supporting the association with increased patient vulnerability.