CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility ...of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (
) and TCRβ (
), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1;
), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.
Replication-competent retrovirus/lentivirus (RCR/L) and insertional oncogenesis are potential safety risks with integrating viruses in gene-modified cell therapies. As such, the Food and Drug ...Administration guidances outline RCR/L-monitoring methods throughout the entire gene therapy treatment cycle. We present data for 17 vector lots, 375 manufactured T cell products, and 308 patients post-infusion across both HIV and oncology indications, showing no evidence of RCR/L. Given our data, a Poisson probability model estimates that a single patient, or a group of patients, would need to be followed for at least 52.8 years to observe one positive RCR/L event, highlighting the unlikelihood of RCR/L development. Additionally, we estimate the median time for lentivirus-modified T cell products to fall below the 1% vector sequence threshold in peripheral or whole blood that would trigger vector integration site analysis. These estimated times are 1.4 months in hematologic malignancies, 0.66 month in solid tumors, and 0.92 month in HIV. Based on these considerable safety data in HIV and oncology and recent Biologics License Applications filed for lentiviral-modified T cell products for hematologic malignancies, this may be an opportune time to re-evaluate the current guidelines for T cell gene therapy product testing and long-term patient monitoring.
Replication-competent retrovirus/lentivirus (RCR/L) and insertional oncogenesis are potential safety risks in gene therapy. Marcucci et al. present negative RCR/L data for 17 vector lots, 375 T cell products, and 308 infused patients. Poisson probability estimates at least 52.8 patient follow-up years to observe an RCR/L event, highlighting the unlikelihood of RCR/L.
Medial amygdala projections regulate the metabolic responses to internal and external signals
The brain is a crucial part of the complex system that controls blood glucose in response to internal ...signals and external stressors. However, we do not know whether distinct CNS circuits regulate metabolism in response to internal state (fed vs. fasted) and environmental cues. Here we used detailed mapping of activated neurons, circuit tracing and projection-specific neural manipulation to determine the contribution of medial amygdala (MeA) neurons and circuits to the metabolic response to internal and external signals. Fasting, refeeding and stressors that increase blood glucose significantly activate MeA neurons. MeA neurons are synaptically connected to organs crucial to metabolic control and chemogenetic activation of MeA neurons significantly increased blood glucose, independent of pancreatic hormone release, and suppressed feeding. We identified the bed nucleus of the stria terminalis (BNST) and ventromedial hypothalamus (VMH) as major downstream projections from the MeA. MeA neurons projecting to BNST and to VMH are distinct neural populations activated by metabolic and emotional stress. Targeted activation of MeA neurons projecting to VMH but not to BNST MeA neurons mimicked the glycemic responses to fasting and stress with significantly impaired glucose tolerance, increased hepatic gluconeogenesis and a more rapid recovery from hypoglycemia, without increased corticosterone or epinephrine. Conversely, targeted ablation of MeA neurons projecting to VMH significantly reduced blood glucose and blunted the hyperglycemic response to stress. These data reveal a critical role for the MeA-VMH circuit in glucose regulation in response to internal and external signals. Future work will assess how this pathway is disrupted in diabetes and its contribution to the associations between diabetes and mental health disorders.
Disclosure
K. Devarakonda: None. A. Alvarsson: None. M. Jimenez gonzalez: None. R. Li: None. V. E. Lehmann: None. S. Stanley: None.
Funding
American Diabetes Association/Pathway to Stop Diabetes (1-17-ACE-31 to S.S.); National Institutes of Health (R01NS097184, OT2OD024912); U.S. Department of Defense (W81XWH-20-1-0345, W81XWH-20-1-0156); Charles H. Revson Foundation (18-25); Swedish Society
The use of lentiviral vectors in cell and gene therapy is steadily increasing, both in commercial and investigational therapies. Although existing data increasingly support the usefulness and safety ...of clinical-grade lentiviral vectors used in cell manufacturing, comprehensive studies specifically addressing their long-term stability are currently lacking. This is significant considering the high cost of producing and testing GMP-grade vectors, the limited number of production facilities, and lengthy queue for production slots. Therefore, an extended shelf life is a critical attribute to justify the investment in large vector lots for investigational cell therapies. This study offers a thorough examination of essential stability attributes, including vector titer, transduction efficiency, and potency for a series of clinical-grade vector lots, each assessed at a minimum of 36 months following their date of manufacture. The 13 vector lots included in this study were used for cell product manufacturing in 16 different clinical trials, and at the time of the analysis had a maximum storage time at −80°C of up to 8 years. The results emphasize the long-term durability and efficacy of GMP-grade lentiviral vectors for use in ex vivo cell therapy manufacturing.
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As the use of GMP-grade lentiviral vectors for clinical cell therapy manufacturing increases, the high cost, limited production facilities, and long queues necessitate that vector lots remain stable in long-term storage for extended use. This comprehensive analysis demonstrates long-term stability by titer, product transduction efficiency, and potency.
Nicotinic acid, N-methylpyridinium ion, and trigonelline are well studied nutritional biomarkers present in coffee, and they are indicators of thermal decomposition during roasting. However, no ...method is yet available for their simultaneous determination. This paper describes a rapid and validated HPLC-diode array detector method for the simultaneous quantitation of caffeine, trigonelline, nicotinic acid, N-methylpyridinium ion, 5-caffeoylquinic acid, and 5-hydroxymethyl furfural that is applicable to three coffee matrixes: green, roasted, and instant. Baseline separation among all compounds was achieved in 30 min using a phenyl-hexyl RP column (250×4.6 mm, 5 μm particle size), 0.3% aqueous formic buffer (pH 2.4)-methanol mobile phase at a flow rate of 1 mL/min, and a column temperature at 30°C. The method showed good linear correlation (r2>0.9985), precision (less than 3.9%), sensitivity (LOD=0.023-0.237 μg/mL; LOQ=0.069-0.711 μg/mL), and recovery (84-102%) for all compounds. This simplified method is amenable for a more complete routine evaluation of coffee in industry.
We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to ...third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)-negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment.
CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy. This article is highlighted in the In This Issue feature, p. 101.
•Reinfusion extended CAR T-cell persistence in 52% of patients reinfused for relapse prevention, thereby potentially reducing relapse risk.•Reinfusion induced remissions in 50% of patients with CD19+ ...relapses after initial CART, but durability was limited without further therapy.
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Relapse after CD19-directed chimeric antigen receptor (CAR)–modified T cells remains a substantial challenge. Short CAR T-cell persistence contributes to relapse risk, necessitating novel approaches to prolong durability. CAR T-cell reinfusion (CARTr) represents a potential strategy to reduce the risk of or treat relapsed disease after initial CAR T-cell infusion (CARTi). We conducted a retrospective review of reinfusion of murine (CTL019) or humanized (huCART19) anti–CD19/4-1BB CAR T cells across 3 clinical trials or commercial tisagenlecleucel for relapse prevention (peripheral B-cell recovery BCR or marrow hematogones ≤6 months after CARTi), minimal residual disease (MRD) or relapse, or nonresponse to CARTi. The primary endpoint was complete response (CR) at day 28 after CARTr, defined as complete remission with B-cell aplasia. Of 262 primary treatments, 81 were followed by ≥1 reinfusion (investigational CTL019, n = 44; huCART19, n = 26; tisagenlecleucel, n = 11), representing 79 patients. Of 63 reinfusions for relapse prevention, 52% achieved CR (BCR, 15/40 38%; hematogones, 18/23 78%). Lymphodepletion was associated with response to CARTr for BCR (odds ratio OR, 33.57; P = .015) but not hematogones (OR, 0.30; P = .291). The cumulative incidence of relapse was 29% at 24 months for CR vs 61% for nonresponse to CARTr (P = .259). For MRD/relapse, CR rate to CARTr was 50% (5/10), but 0/8 for nonresponse to CARTi. Toxicity was generally mild, with the only grade ≥3 cytokine release syndrome (n = 6) or neurotoxicity (n = 1) observed in MRD/relapse treatment. Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may reduce relapse risk in a subset of patients, and can reinduce remission in CD19+ relapse.
Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive ...tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-β. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-β receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion, >98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-β-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.
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