Cytotherapy, Journal Year: 2023, Volume and Issue: 25(6), P. 670 - 682
Published: Feb. 26, 2023
Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they not experienced the same success solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency tumor cells.
Language: Английский
bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown
Published: Feb. 2, 2024
ABSTRACT Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact T-cell phenotypes. CRISPR/Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous receptor alpha constant ( TRAC ) gene resulting in -CAR cells an enriched stem memory population, process that could be further optimized through modifications to composition. In this study we generated anti-GD2 using “metabolic priming” (MP), where were activated glucose/glutamine low and then expanded high media. products evaluated spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays vitro potency against human GD2+ xenograft neuroblastoma models vivo . Compared standard cells, MP showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity reduced IFN-γ, IL-2, IP-10, IL-1β, IL-17, TGFβ production at end manufacturing ex , increased central better persistence observed Metabolic priming during biomanufacturing minimize glycolysis enrich phenotypes which lead responses solid tumors
Language: Английский
Citations
4
bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown
Published: Feb. 23, 2024
Abstract Chimeric antigen receptor (CAR) T cell therapy for solid tumors remains challenging due to the complex manufacturing process and immunosuppressive tumor microenvironment. The condition directly impacts CAR yield, phenotype, metabolism, which correlate with in vivo potency persistence. Optical metabolic imaging (OMI) is a non-invasive, label-free method evaluate single metabolism based on autofluorescent coenzymes NAD(P)H FAD. Using OMI, we identified dominating of media composition over selection antibody stimulation and/or cytokines anti-GD2 activation strength kinetics, phenotype. We demonstrated that OMI parameters were indicative cycle stage optimal gene transfer conditions both viral transduction electroporation-based CRISPR/Cas9. Notably, accurately predicted oxidative phenotype virus-free CRISPR-edited cells correlated higher against neuroblastoma. Our data supports OMI’s potential as robust, sensitive analytical tool enables dynamic increased yield fitness. One sentence summary Autofluorescence informs enhance fitness
Language: Английский
Citations
4
Frontiers in Pharmacology, Journal Year: 2023, Volume and Issue: 14
Published: June 20, 2023
Background: Chimeric antigen receptor T cells treatment targeting B cell maturation (BCMA) is an emerging option for relapsed/refractory multiple myeloma (RRMM) and has demonstrated outstanding outcomes in clinical studies. Objective: The aim of this comprehensive review meta-analysis was to summarize the effectiveness safety anti-BCMA CAR-T patients with (RRMM). Our research identifies variables influencing outcome measures provide additional evidence product updates, trial design, guidance. Methods: Preferred Reporting Items Systematic Reviews Meta-Analyses (PRISMA) standard followed conducting meta-analysis, which submitted PROSPERO (CRD42023390037). From inception study until 10 September 2022, PubMed, Web Science, EMBASE, Cochrane Library, CNKI, WanFang databases were searched eligible Stata software (version 16.0) used assess outcomes. Results: Out 875 papers, we found 21 relevant trials 761 diagnosed as RRMM given treatment. overall response rate (ORR) entire sample 87% (95% CI: 80–93%) complete (CRR) 44% 34–54%). minimal residual disease (MRD) negativity within responders 78% 65–89%). combined incidence cytokine release syndrome 82% 72–91%) neurotoxicity 10% 5%–17%). median progression-free survival (PFS) 8.77 months 7.48–10.06), (OS) 18.87 17.20–20.54) duration (DOR) 10.32 9.34–11.31). Conclusion: According who received have both safety. Subgroup analysis confirmed anticipated inter-study heterogeneity pinpointed potential factors contributing efficacy, may help development studies lead optimized BCMA CAR-T-cell products. Review Registration: Clinicaltrials.gov , PROSPERO, CRD42023390037.
Language: Английский
Citations
10
Nature Reviews Methods Primers, Journal Year: 2024, Volume and Issue: 4(1)
Published: Oct. 10, 2024
Language: Английский
Citations
4
Molecular Therapy — Methods & Clinical Development, Journal Year: 2025, Volume and Issue: 33(1), P. 101437 - 101437
Published: Feb. 19, 2025
CRISPR-Cas9 ribonucleoproteins (RNPs) combined with a nucleic acid template encoding chimeric antigen receptor (CAR) transgene can edit human cells to produce CAR T precise insertion at single locus. However, many have adverse innate immune responses foreign acids, particularly circular double-stranded DNA (dsDNA). Here, we introduce Cleaved, LInearized Protein Template (Cas9-CLIPT), plasmid containing target sequence for the Cas9 RNP, such that during manufacturing, Cas9-RNP binds and cleaves linearize dsDNA in vitro. remains bound linearized is delivered promote knock-in via homology-directed repair Cas9-CLIPT. Cas9-CLIPT Nanoplasmids generate up 1.7-fold higher rates of relative dsDNA, reaching efficiencies 60% non-homologous end joining inhibition. Cas9-CLIPT-manufactured GD2 TRAC-CAR are potent against GD2+ neuroblastoma exhibit an enriched stem cell memory phenotype. On several electroporation instruments approaching clinically relevant yields, successfully manufactured using plasmids large (2-6 kb) transgenes. strategies potential simplify donor production integrate transgenes, allowing more efficient nonviral manufacturing multifunctional, genome-edited therapies.
Language: Английский
Citations
0
Cancer Medicine, Journal Year: 2025, Volume and Issue: 14(5)
Published: Feb. 27, 2025
ABSTRACT Cell therapy based on chimeric antigen receptor (CAR) T cells has represented a revolutionary new approach for treating tumors, especially hematological diseases. Complete remission rates (CRR) > 80%–97% and 50%–90% overall response (ORR) have been achieved with treatment CAR‐T in patients malignant B‐cell tumors that relapsed or are refractory to previous treatments. Toxicity remains the major problem. Most treated develop high‐grade cytokine release syndrome (CRS) immune effector cell‐associated neurotoxicity (ICANS). However, unprecedentedly high CRR ORR led approval of six cell therapeutics by Food Drug Administration (FDA) European Medicines Agency (EMA), prompting researchers improve existing products ones. By now, around 1000 clinical trials registered at ClinicalTrials.gov : 82% diseases, while remaining 16% solid tumors. As result this increased research, an enormous amount conflicting information accumulated literature, each group follows its manufacturing protocols performs specific vitro testing. This review aimed combine compare preclinical information, highlighting most used provide comprehensive overview world cells, from their characterization. The focus is all steps process, collection patient donor blood enrichment activation anti‐CD3/CD28 beads, interleukin‐2 (IL‐2) IL‐7 IL‐15 (induction more functional memory phenotype), transfection (viral non‐viral methods). Automation crucial ensuring standardized final product.
Language: Английский
Citations
0
Cancer Immunology Immunotherapy, Journal Year: 2025, Volume and Issue: 74(5)
Published: March 21, 2025
Abstract Background This first-in-human phase 1 study (NCT04432597) evaluated the safety and recommended 2 dose (RP2D) of PRGN-2009, a gorilla adenoviral-vector targeting oncoproteins E6, E7 (human papillomavirus (HPV)16/18) E5 (HPV16), as monotherapy (Arm 1A) combined with bifunctional TGF-β “trap”/anti-PD-L1 fusion protein bintrafusp alfa (BA; Arm 1B), in patients recurrent/metastatic HPV-associated cancer. Methods Patients ≥ prior treatment (immunotherapy allowed) received PRGN-2009 (1 × 10 11 particle units or 5 units, subcutaneously) every weeks for 3 doses, then 4 1A), (RP2D, schedule per BA (1200 mg, intravenously) 1B). Primary endpoints were RP2D PRGN-2009; secondary objectives included overall response rate (ORR) survival (OS). Results Seventeen treated. In 1A (n = 6) there no limiting toxicities grade 3/4 treatment-related adverse events (TRAEs), PU was selected RP2D, responses observed, median OS (mOS) 7.4 months (95% CI 2.9–26.8). 1B 11), TRAEs occurred 27% patients, ORR 20% all (22% checkpoint-resistant patients), mOS 24.6 9.6-not reached). Multifunctional HPV-specific T cells increased induced de novo 80% not impacted by anti-vector antibodies. Higher serum IL-8 at baseline associated shorter OS. Conclusions well tolerated, immune observed to PRGN-2009. Encouraging anti-tumor activity noted combination arm, consisting mainly patients. Trial Registration ClinicalTrials.gov Identifier: NCT04432597.
Language: Английский
Citations
0
Science Advances, Journal Year: 2025, Volume and Issue: 11(13)
Published: March 26, 2025
CRISPR-Cas9 systems have revolutionized genome editing, but the off-target effects of Cas9 limit its use in clinical applications. Here, we systematically evaluate FrCas9, a variant from Faecalibaculum rodentium , for cell and gene therapy (CGT) applications compare performance to SpCas9 OpenCRISPR-1. OpenCRISPR-1 is CRISPR system synthesized de novo using large language models (LLMs) has not yet undergone systematic characterization. Using AID-seq, Amplicon sequencing, GUIDE-seq, assessed on-target activity profiles these across multiple genomic loci. FrCas9 demonstrated higher efficiency substantially fewer than Furthermore, TREX2 fusion with reduced deletions translocations, enhancing stability. Through screening 1903 sgRNAs targeting 21 CGT-relevant genes sequential GUIDE-seq analysis, identified optimal each gene. Our high-throughput platform highlights particularly TREX2-fused form, as highly specific efficient tool precise therapeutic editing.
Language: Английский
Citations
0
Frontiers in Immunology, Journal Year: 2025, Volume and Issue: 16
Published: May 8, 2025
This review explores recent advances in the characteristics and manufacturing of CAR T-cell products. Traditional potency assays have been designed based on well-established functionalities. However, advent innovative tools methodologies has revealed a broader spectrum important that correlate with function. Furthermore, as strategies continue to evolve, conventional may no longer fully capture complexity these Therefore, it is essential examine emerging approaches consider development tailored ensure products are characterized.
Language: Английский
Citations
0
Cancers, Journal Year: 2023, Volume and Issue: 16(1), P. 191 - 191
Published: Dec. 30, 2023
Background and purpose. Chimeric antigen receptor (CAR) T cells have been relatively ineffective against solid tumors. Low-dose radiation which can be delivered to multiple sites of metastases by targeted radionuclide therapy (TRT) elicit immunostimulatory effects. However, TRT has never combined with CAR tumors in a clinical setting. This study investigated the effects Lutetium-177 (177Lu) Actinium-225 (225Ac) on viability effector function vitro evaluate feasibility such therapeutic combinations. After irradiation anti-GD2 various doses 177Lu or 225Ac, their cytotoxic activity GD2-expressing human CHLA-20 neuroblastoma melanoma M21 were determined flow cytometry. The expression exhaustion marker PD-1, activation CD69 activating NKG2D was measured irradiated cells. Both 225Ac displayed dose-dependent toxicity enhanced these irrespective dose tested type radionuclide. No significant changes noted following irradiation. Given lower cell at equal an enhancement type, 177Lu-based may preferred over 225Ac-based when evaluating potential synergism between therapies vivo
Language: Английский
Citations
9