leucine-zipper-based-cell-sorting-for-purification-dual-vector

Leucine Zipper-based Cell Sorting for Purification of Dual-vector-transduced Cells

Adoptive T cell therapies, including chimeric antigen receptor (CAR) T cells, have demonstrated impressive clinical activity against hematologic ...

Memorial Sloan Kettering Cancer Center

CD19-targeted chimeric antigen receptor (CAR) T-cells have revolutionized the treatment of lymphoid malignancies, including large B cell lymphoma (LBCL). Following seminal early phase multicenter clinical trials published between 2017 and 2020, three CD19-CAR T-cell products received FDA and EMA approval designations in lymphoma in the third-line setting, paving the way for follow-up studies in the second-line. Meanwhile, investigations into the applications of CAR T-cell therapy have further broadened to treating high-risk patients even prior to completion of first-line conventional chemo-immunotherapy. Furthermore, as early trials excluded patients with central nervous system involvement with lymphoma, several studies have recently shown promising efficacy of CD19-CAR T-cells in primary and secondary CNS lymphoma. Here we provide a detailed overview on clinical data supporting the use of CAR T-cells in patients with LBCL.

CAR T-cell therapy in large B cell lymphoma.

Does BTKi improve CAR T-cell therapy in MCL?

Multiple myeloma is a plasma cell malignancy that is currently incurable with conventional therapies. Following the success of CD19-targeted chimeric antigen receptor (CAR) T cells in leukemia and lymphoma, CAR T cells targeting B-cell maturation antigen (BCMA) more recently demonstrated impressive activity in relapsed and refractory myeloma patients. However, BCMA-directed therapy can fail due to weak expression of BCMA on myeloma cells, suggesting that novel approaches to better address this antigen-low disease may improve patient outcomes. We hypothesized that engineered secretion of the proinflammatory cytokine interleukin-18 (IL-18) and multiantigen targeting could improve CAR T-cell activity against BCMA-low myeloma. In a syngeneic murine model of myeloma, CAR T cells targeting the myeloma-associated antigens BCMA and B-cell activating factor receptor (BAFF-R) failed to eliminate myeloma when these antigens were weakly expressed, whereas IL-18-secreting CAR T cells targeting these antigens promoted myeloma clearance. IL-18-secreting CAR T cells developed an effector-like T-cell phenotype, promoted interferon-gamma production, reprogrammed the myeloma bone marrow microenvironment through type-I/II interferon signaling, and activated macrophages to mediate antimyeloma activity. Simultaneous targeting of weakly-expressed BCMA and BAFF-R with dual-CAR T cells enhanced T-cell:target-cell avidity, increased overall CAR signal strength, and stimulated antimyeloma activity. Dual-antigen targeting augmented CAR T-cell secretion of engineered IL-18 and facilitated elimination of larger myeloma burdens in vivo. Our results demonstrate that combination of engineered IL-18 secretion and multiantigen targeting can eliminate myeloma with weak antigen expression through distinct mechanisms.

IL-18-secreting multiantigen targeting CAR T cells eliminate antigen-low myeloma in an immunocompetent mouse model.

Greater disease burden is a well-established predictor of poorer outcomes following chimeric antigen receptor T-cell (CAR T) therapy. Although bridging therapy (BT) is widely used between leukapheresis and CAR T infusion, limited data have evaluated the impact of BT on CAR T outcomes. In this study, we hypothesized that the quantitative dynamics of radiomic cytoreduction during bridging are prognostic.

Metabolic Tumor Volume Response after Bridging Therapy Determines Chimeric Antigen Receptor T-Cell Outcomes in Large B-Cell Lymphoma.

Chimeric antigen receptor (CAR) T cells targeting CD19 induce durable remissions in patients with relapsed or refractory non-Hodgkin lymphoma (NHL), but many patients experience treatment-related toxicity. Cytokine release syndrome and immune effector cell-associated neurologic syndrome are extensively characterized. However, limited data exist on the burden, predictors, and implications of acute kidney injury (AKI) after CAR T-cell therapy. On initial screening of the Food and Drug Administration adverse event reporting system, we identified a disproportionately high rate of renal adverse events among nearly 6,000 CAR T adverse event reports, suggesting it is clinically important in this patient population. In a subsequent single-center analysis of 399 NHL patients treated with CD19 CAR T cells, we found a substantial burden of AKI after CAR T infusion (10% and 5% of any grade and grade ≥2 AKI) with pre-renal causes being predominant (72%). Evolution to chronic kidney disease was rare, however, three patients required hemodialysis. Importantly, patients experiencing cytokine release syndrome and/or neurotoxicity as well as those with low serum albumin and high inflammatory cytokines, including IL-6 and TNF-α, were more likely to develop AKI. While pre-CAR T renal dysfunction was not associated with adverse outcomes, patients developing post-CAR T AKI had lower overall survival compared to their counterparts. Our findings indicate that renal dysfunction is a common toxicity of CAR T-cell therapy with meaningful prognostic impact. Notably, the link between systemic inflammation and renal dysfunction, suggests that readily available biomarkers may inform on renal injury risk after CAR T-cell therapy.

Predictors and implications of renal injury after CD19 chimeric antigen receptor T-cell therapy.

Resistance to chimaeric antigen receptor (CAR) T cell therapy develops through multiple mechanisms, most notably antigen loss and tumour-induced immune suppression. It has been suggested that T cells expressing multiple CARs may overcome the resistance of tumours and that T cells expressing receptors that switch inhibitory immune-checkpoint signals into costimulatory signals may enhance the activity of the T cells in the tumour microenvironment. However, engineering multiple features into a single T cell product is difficult because of the transgene-packaging constraints of current gene-delivery vectors. Here we describe a cell-sorting method that leverages leucine zippers for the selective single-step immunomagnetic purification of cells co-transduced with two vectors. Such 'Zip sorting' facilitated the generation of T cells simultaneously expressing up to four CARs and coexpressing up to three 'switch' receptors. In syngeneic mouse models, T cells with multiple CARs and multiple switch receptors eliminated antigenically heterogeneous populations of leukaemia cells coexpressing multiple inhibitory ligands. By combining diverse therapeutic strategies, Zip-sorted multi-CAR multi-switch-receptor T cells can overcome multiple mechanisms of CAR T cell resistance.

Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors.

Barriers to Chimeric Antigen Receptor T-Cell Therapy.

Concomitant group A Streptococcus and disseminated mpox infection mimicking relapsed diffuse large B-cell lymphoma.

Although 3 commercial CD19-targeted CAR T-cell therapies are available for large B-cell lymphoma (LBCL), no randomized clinical trials have compared their efficacy and safety. In this retrospective multicenter cohort study, we evaluated real-world clinical outcomes of patients with relapsed/refractory LBCL treated with axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel), or lisocabtagene maraleucel (liso-cel). Between April 2016 and July 2024, 624 patients received CD19-targeted CAR T-cell therapies (344 axi-cel, 142 tisa-cel, and 138 liso-cel). At a median follow-up of 20.9 months, estimated 2-year PFS and OS rates were 46%/63% for axi-cel, 30%/45% for tisa-cel, and 45%/58% for liso-cel. After adjusting for potential confounders in multivariable analyses, tisa-cel was associated with inferior progression-free survival (PFS) (hazard ratio [HR] = 2.25; 95% confidence interval [CI]: 1.65-3.06; p < 0.001) and overall survival (OS) (HR = 1.68; 95% CI: 1.19-2.36; p = 0.003) compared to axi-cel. No significant survival differences were found between liso-cel and axi-cel. Propensity score and subanalyses of patients treated in the second-line vs. third-line or later settings yielded similar outcomes. Compared to axi-cel, the objective response rate at 100 days was higher for liso-cel (odds ratio [OR] = 2.31; 95% CI: 1.21-4.80; p = 0.016) and lower for tisa-cel (OR = 0.36; 95% CI: 0.23-0.57; p < 0.001). Rates of CRS, ICANS, ICAHT, and febrile neutropenia were significantly higher with axi-cel. However, no significant differences in the cumulative incidence of infections or non-relapse mortality were found. Axi-cel was associated with faster vein-to-vein time (axi-cel: 35 days, tisa-cel: 43 days, liso-cel: 41 days; p < 0.001) and fewer out-of-specification products (axi-cel: 2%, tisa-cel: 4%, liso-cel: 11%; p = 0.004). These results provide insights into potential differential outcomes depending on product selection.

Comparative real-world outcomes of CD19-directed CAR T-cell therapies in large B-cell lymphoma.

While CD19-directed CAR T-cell therapy represents a transformative immunotherapy for relapsed/refractory large B-cell lymphoma (r/r LBCL), more than 50% of patients ultimately progress or relapse. Recently, the International Metabolic Prognostic Index (IMPI) - incorporating age, stage, and metabolic tumor volume (MTV) - was shown to improve prognostication for LBCL frontline treatment. Here, we examine its utility to predict toxicity and survival in CAR-T recipients. This multicenter observational study spanning six international sites included 504 patients with available FDG-PET/CT imaging at last response assessment prior to lymphodepletion. Optimal CAR-adapted MTV thresholds were identified in a development cohort (n = 256) and incorporated into a CAR-T-specific IMPI ("CAR-IMPI"). The prognostic performance of CAR-IMPI was validated in an independent cohort (n = 248). CAR-IMPI risk categories, defined by the median (1.35) and terciles (1.07, 1.58), demonstrated significant discrimination for progression-free survival (PFS; p < 0.0001) and overall survival (OS; p < 0.0001) in both cohorts. Multivariate Cox regression confirmed CAR-IMPI as an independent predictor of survival, accounting for pre-lymphodepletion LDH and CRP, performance status, treatment center, and CAR-T product. Patients in the CAR-IMPI high-risk category experienced increased severity of CRS and ICANS, and higher rates of intensive care unit (ICU) admissions. In an exploratory analysis, combining CAR-IMPI with established indices of high-risk systemic inflammation (CAR-HEMATOTOX, InflaMix) further enhanced survival stratification. The CAR-IMPI may provide a potent and validated PET-based tool for risk stratification of clinical outcomes in patients with r/r LBCL receiving CD19 CAR-T therapy. Our data highlight the utility of combining clinical and radiological modalities, with implications for patient selection and the anticipated level-of-care for toxicity management.

Article	Year
CAR T-cell therapy in large B cell lymphoma. Hematological oncology\| PubMed ID: 37294963	2023
Does BTKi improve CAR T-cell therapy in MCL? Blood\| PubMed ID: 38386430	2024
IL-18-secreting multiantigen targeting CAR T cells eliminate antigen-low myeloma in an immunocompetent mouse model. Blood\| PubMed ID: 38579288	2024
Metabolic Tumor Volume Response after Bridging Therapy Determines Chimeric Antigen Receptor T-Cell Outcomes in Large B-Cell Lymphoma. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 39259292	2024
Predictors and implications of renal injury after CD19 chimeric antigen receptor T-cell therapy. Haematologica\| PubMed ID: 39568416	2025
Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors. Nature biomedical engineering\| PubMed ID: 39715901	2024
Barriers to Chimeric Antigen Receptor T-Cell Therapy. JAMA oncology\| PubMed ID: 40372728	2025
Concomitant group A Streptococcus and disseminated mpox infection mimicking relapsed diffuse large B-cell lymphoma. JAAD case reports\| PubMed ID: 40530178	2025
Comparative real-world outcomes of CD19-directed CAR T-cell therapies in large B-cell lymphoma. Blood advances\| PubMed ID: 40815804	2025
Optimization and validation of the international metabolic prognostic index for CD19 CAR-T in large B-cell lymphoma. Blood cancer journal\| PubMed ID: 40858552	2025

Alexander P. Boardman

Biography

JoVE Journal Publications

Other Publications