Bruce L. Levine

CD19-directed CAR T-cells (CTL019) can produce durable remissions in chronic lymphocytic leukemia (CLL), but therapeutic success depends on whether autologous T-cells expand, persist, and retain cytotoxic function after manufacturing. Failure of CLL T-cells is often attributed to exhaustion, although many dysfunctional CLL T-cells retain inflammatory cytokine production. We tested whether this paradox reflects immunosenescence, an aging-like program defined by costimulatory loss, DNA damage, inflammatory secretion, repertoire restriction, and proliferative arrest. In response-linked preinfusion CTL019 products, nonresponders and short partial responders had higher senescence and senescence-associated secretory phenotype (SASP) programs than the functional-responder group, comprising complete responders and partial responders with transformed disease. These programs were detectable before infusion and became more prominent during manufacture in susceptible products. They tracked with weak in vivo CAR T-cell expansion and were associated with inferior survival. Serum proteomics revealed a circulating, CLL-associated SASP-like inflammatory milieu in nonresponders. Flow cytometry and T-cell receptor (TCR) profiling showed that poor responders carried CD27⁻CD28⁻ and KLRG1⁺ CD8 T-cells at apheresis, lower product CD27, and reduced product TCR diversity. Research-manufactured CAR T-cells from treatment-naïve CLL samples displayed the same state, most prominently in CD4 CAR T-cells, with SA-β-gal, p16, p53, DNA damage, depletion of less-differentiated states, and senescence-enriched transcriptomes. Under repeated CD19 stimulation, CLL-derived products reached a proliferative ceiling, and p53-high products lost cytotoxic reserve. Ibrutinib improved proliferative fitness, attenuated senescence-associated features and SASP output in paired patient and direct-exposure assays, and enhanced CAR T-cell expansion in an ibrutinib-resistant CLL model. Together, these data identify immunosenescence as a measurable and functionally consequential barrier to CAR T-cell efficacy in CLL and a candidate for therapeutic modulation.

Chimeric antigen receptor (CAR) T cell therapy induces durable remissions in lymphoid malignancies, yet the extent and biology of long-term CAR T cell persistence in B cell lymphoma remain unclear. Here we report the persistence and characteristics of 4-1BB-costimulated anti-CD19 CAR T cells (CART19) up to 10 years after infusion in 38 patients with non-Hodgkin lymphoma. Beyond year five, the CAR19 transgene was detectable in five of eight long-term responders (7.0–10.1 years), with three patients maintaining B cell aplasia, which is consistent with sustained functional activity. In one patient with a progression-free survival of 10.1 years, CART19 cells comprised 1.2% of circulating T cells 9.3 years after infusion. Long-term persisting CART19 cells exhibited a predominant double-negative (CD4 ⁻ CD8 ⁻ ), effector-memory-like phenotype associated with increased aerobic metabolism and T cell activation programs. Longitudinal profiling revealed a progressive transition from CD8 ⁺ to double-negative CAR T cells over time. Persisting CART19 shared transcriptional features with long-term CAR T cells described in acute and chronic leukemias. T cell receptor sequencing demonstrated oligoclonal persistence at 9.3 years, with a dominant clone (70% of CART19 cells) already detectable at low frequency (<0.1%) at day 14. Lentiviral integration-site analysis identified a predominant integration within PACS1 without evidence of known drivers of CAR T cell expansion. These findings demonstrate that CART19 cells can persist for more than 10 years in lymphoma and identify phenotypic, transcriptional and clonal features associated with exceptionally long-term persistence. ClinicalTrials.gov registration: NCT02030834

Chimeric antigen receptor (CAR) T-cell therapy holds great promise for patients with cancer, and the identification of predictive biomarkers is crucial in finding new ways to guide therapy. Major challenges to the application of informatics and machine learning in CAR T-cell therapy include limited sample sizes and non-uniformity in data generation across cancer indications and trials. Here we took a global, pan-haematologic cancer approach, analysing 256 patients across 5 cancer types and 13 clinical trials. We generated data using a framework that included pre-infusion clinical features, over 2 million apheresis T cells analysed by flow cytometry using 17 unique markers, ex vivo T-cell expansion during CAR T-cell manufacture, more than 90,000 measurements of 30 serum markers and serial tracking of circulating CAR T cells using qPCR. From this data resource, we demonstrate the potential of pan-cancer predictive biomarkers that capture generalizable characteristics of treatment response and non-response in CAR T-cell therapy.

Engineering multiple receptors into chimeric antigen receptor (CAR) T cells has emerged as a powerful strategy to prevent antigen-negative relapse and reduce on-target/off-tumor toxicities. However, manufacturing multi-receptor CAR T cells remains challenging, as increasing lentiviral transgene sizes significantly reduces viral titers and T cell transduction rates. Current production workflows often rely on cell sorting to enrich transduced T cells from low-yield productions. Yet, cell sorting techniques do not increase the absolute number of CAR T cells and add further complexity to the already elaborate manufacturing process. Consequently, these limitations impede the clinical translation of multi-receptor CAR T cells and restrict the development of next-generation immunotherapies. Here, we present a detailed, step-by-step production workflow optimized for generating CAR T cells with large lentiviral transgenes. Using this workflow, we demonstrate size-dependent increases in transduction efficiency across a range of transgene sizes, with the most pronounced enhancement of up to 14.8-fold observed for a 10.1 kb lentiviral vector. Importantly, the workflow supports robust T cell expansion and eliminates the need for cell sorting. By overcoming current size limitations in lentiviral gene transfer, this workflow enables the efficient generation of multi-receptor CAR T cells, thereby facilitating the development of advanced immunotherapies.

Engineering multiple receptors into chimeric antigen receptor (CAR) T cells has emerged as a powerful strategy to prevent antigen-negative relapse and reduce on-target/off-tumor toxicities. However, manufacturing multi-receptor CAR T cells remains challenging, as increasing lentiviral transgene sizes significantly reduces viral titers and T cell transduction rates. Current production workflows often rely on cell sorting to enrich transduced T cells from low-yield productions. Yet, cell sorting techniques do not increase the absolute number of CAR T cells and add further complexity to the already elaborate manufacturing process. Consequently, these limitations impede the clinical translation of multi-receptor CAR T cells and restrict the development of next-generation immunotherapies. Here, we present a detailed, step-by-step production workflow optimized for generating CAR T cells with large lentiviral transgenes. Using this workflow, we demonstrate size-dependent increases in transduction efficiency across a range of transgene sizes, with the most pronounced enhancement of up to 14.8-fold observed for a 10.1 kb lentiviral vector. Importantly, the workflow supports robust T cell expansion and eliminates the need for cell sorting. By overcoming current size limitations in lentiviral gene transfer, this workflow enables the efficient generation of multi-receptor CAR T cells, thereby facilitating the development of advanced immunotherapies.

Immune-based cell therapy offers a promising approach to cancer treatment. While autologous chimeric antigen receptor (CAR) T cells have shown success, production is time-consuming, costly and patient specific. Gamma–delta (γδ) T cells are promising for ‘off-the-shelf’ CAR T cell therapy. However, clinical translation of γδ CAR T cells is hampered by low frequency, resistance to genetic manipulation and advanced differentiation after expansion, limiting therapeutic feasibility. Here we demonstrate a method for in vitro activation and expansion of peripheral blood γδ T cells, facilitating high rates of gene editing and efficient CAR integration. Using artificial antigen-presenting cells, we produce minimally differentiated, highly functional γδ CAR T cells. By targeting a US Food and Drug Administration-approved CD19 CAR to the CCR5 locus, we generate CCR5-deficient γδ CD19 CAR T cells (γδ CCR5KI-CAR19), which demonstrated resistance to HIV-mediated depletion and robust antitumour responses against B cell lymphoma and leukaemia. γδ CCR5KI-CAR19 T cells enable the immunotherapy of HIV-associated B cell malignancies. These studies provide preclinical evidence supporting large-scale development of potent allogeneic γδ CAR T cells for diverse immunotherapies.