As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating cryopreservation steps becomes a logistical ...necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells PBMNCs) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservation-related subtle micro-cellular damage needs further study.
Panch et al. demonstrate that CART cryopreservation during manufacture minimally affects final product characteristics, in vivo levels, persistence, and clinical response. However, increases in early apoptotic markers and cell damage pathways in cryo-thawed CARTs require careful consideration to better formulate dosing strategies.
Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our ...experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on our phase 1 dose-escalation study of a novel murine stem cell virus (MSCV)-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBζ) for children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives included laboratory investigations, CAR T-cell expansion and cytokine profiling. Twenty patients, ages 5.4 to 34.6 years, with B-ALL received CD19.22.BBζ. The complete response (CR) rate was 60% (12 of 20) in the full cohort and 71.4% (10 of 14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having ≥ grade 3 CRS and only 1 experiencing neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% confidence interval CI: 42.4%-94.9%) and 57.7% (95% CI: 22.1%-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBζ compared with EF1α-CD22.BBζ prompted laboratory investigations comparing EF1α vs MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBζ, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28ζ/CD22.BBζ construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBζ in a heavily pretreated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations (www.clinicaltrials.gov #NCT03448393).
•CD19.22.BBζ CAR T cells were well tolerated and effective in pediatric B-ALL, but persistence and CD22 targeting were limited.•A novel bicistronic CD19.28ζ/CD22.BBζ CAR T-cell enhanced dual-targeting efficacy and cytokine production in preclinical models.
Display omitted
The Centre for Ecology & Hydrology – Gridded Estimates of Areal Rainfall (CEH-GEAR) data set was developed to provide reliable 1 km gridded estimates of daily and monthly rainfall for Great Britain ...(GB) and Northern Ireland (NI) (together with approximately 3500 km2 of catchment in the Republic of Ireland) from 1890 onwards. The data set was primarily required to support hydrological modelling. The rainfall estimates are derived from the Met Office collated historical weather observations for the UK which include a national database of rain gauge observations. The natural neighbour interpolation methodology, including a normalisation step based on average annual rainfall (AAR), was used to generate the daily and monthly rainfall grids. To derive the monthly estimates, rainfall totals from monthly and daily (when complete month available) rain gauges were used in order to obtain maximum information from the rain gauge network. The daily grids were adjusted so that the monthly grids are fully consistent with the daily grids. The CEH-GEAR data set was developed according to the guidance provided by the British Standards Institution. The CEH-GEAR data set contains 1 km grids of daily and monthly rainfall estimates for GB and NI for the period 1890–2012. For each day and month, CEH-GEAR includes a secondary grid of distance to the nearest operational rain gauge. This may be used as an indicator of the quality of the estimates. When this distance is greater than 100 km, the estimates are not calculated due to high uncertainty. CEH-GEAR is available from doi:10.5285/5dc179dc-f692-49ba-9326-a6893a503f6e and is free of charge for commercial and non-commercial use subject to licensing terms and conditions.
Purpose of Review
Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable remission induction rates for relapsed/refractory B cell malignancies. However, loss of the CAR-targeted ...antigen, known as antigen escape, accounts for a substantial percentage of relapses following CAR therapy and is a major barrier to durable remission. Here, we discuss mechanisms for antigen escape and strategies to prevent this pattern of relapse, including the use of multi-specific CARs, which recognize and target multiple tumor-associated antigens simultaneously.
Recent Findings
Preclinical and early clinical trial data indicates that multi-specific CAR therapy for B cell malignancies is both safe and effective. Optimal combinations of target antigens, as well as different multi-specific CAR formats, are currently being evaluated.
Summary
Although still in early stages of development, multi-specific CAR therapy represents a promising approach to mitigate antigen loss–related relapses and improve durability of remission in patients with refractory B cell malignancies, and may be applicable to other types of cancer.
Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell–associated protein have demonstrated potent activity against ...relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.
•Adoptive transfer of T cells genetically modified to express anti-TSLPR chimeric antigen receptors can cure B-ALL in xenograft models.•Anti-TSLPR CAR constructs containing a CH2CH3 spacer domain were inactive against TSLPR-overexpressing B-ALL.
In this issue of
Blood
,
Nagel et al
provide evidence supporting the presence of premalignant leukemic clones in nonlymphoid compartments of patients with
BCR-ABL1
–positive B-cell precursor acute ...lymphoblastic leukemia (ALL) as a cause of lineage switch after CD19 immunotherapy, extending the lineage switch mechanism beyond mixed-lineage leukemia (MLL)-rearranged ALL.
1
This observation suggests that CD19-targeted therapies alone may not suffice in the eradication of underlying disease in
BCR-ABL1
p210- and p190-positive patients, and that strategies to target the stem cell compartment may be needed for durable remissions in this high-risk population.
PDF
