Manufacturing and preclinical validation of CAR T cells targeting ICAM-1 for advanced thyroid cancer therapy.
While the bulk of thyroid cancer sufferers are simply treatable, these with anaplastic or poorly differentiated recurrent thyroid carcinomas have a really poor prognosis with a median survival of lower than a 12 months. Previously, we’ve proven a big correlation between ICAM-1 overexpression and malignancy in thyroid cancer, and have pioneered the use of ICAM-1 focused CAR T cells as a novel therapy modality.
For medical translation of this novel modality, we designed CAR T cells possessing micromolar moderately than nanomolar affinity to ICAM-1 to keep away from cytotoxicity in regular cells with basal ranges of ICAM-1 expression. Herein, we report the automated course of of CAR T cell manufacturing with CliniMACS Prodigy (Miltenyi Biotec) utilizing cryopreserved peripheral blood leukocytes from apheresis collections.
Using Prodigy, thawed leukopak cells had been enriched for CD4+ and CD8+ T cells, subjected to double transduction utilizing lentiviral vector, and expanded in tradition for a complete of 10 days with a remaining yield of 2-4 × 109 cells. The ensuing CAR T cells had been formulated for cryopreservation for use straight for infusion into sufferers after thawing with no additional processing.
We examined cross-reactivity of CAR T cells towards each human and murine ICAM-1 and ICAM-1 expression in human and mouse tissues to exhibit that each efficacy and on-target, off-tumor toxicity could be studied in our preclinical mannequin.
Selective anti-tumor exercise within the absence of toxicity offers proof-of-concept that micromolar affinity tuned CAR T cells can be utilized to focus on tumors expressing excessive ranges of antigen whereas avoiding regular tissues expressing basal ranges of the identical antigen.
These research help the initiation of a section I research to guage the security and potential efficacy of micromolar affinity tuned CAR T cells in opposition to newly identified anaplastic and refractory or recurrent thyroid cancers.
OMIP-060-30-Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells.
We developed this complete 28-color circulation cytometry panel with the purpose to measure a spread of T cell effector capabilities together with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4+ and CD8+ αβ T cells (Table 1).
The effector capabilities measured on this panel embrace activation and co-stimulatory molecules (CD69, CD137, and CD154), cytokines (IL-2, IL-13, IL-17A, IL-21, IL-22, TNF, and IFNγ), the chemokine IL-8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a.
In addition, Ki67 permits the identification and evaluation of lately activated T cells. To characterize regulatory T cells (Tregs ), we included CD25, CD39, and the canonical Tregs transcription issue FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin.
However, we efficiently examined different sorts of stimulation akin to staphylococcus enterotoxin B (SEB) or a mixture of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza. Published 2019. This article is a U.S. Government work and is within the public area within the USA.