Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less‐differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long‐term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib‐induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib‐based CART cell generation resulted in an enrichment of less‐differentiated naïve‐like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD‐1 and Tim‐3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib‐treated CART cells was validated in a xenograft mouse model. Intracellular TNF‐α and IFN‐γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib‐based CART cell generation protocols are warranted.
BACKGROUND.Preclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (modified immune cells [MICs]), induced long-term specific immunosuppression against the allogeneic donor. METHODS.In this phase I trial, patients received either 1.5 × 10 6 MICs per kg BW on day -2 (n = 3, group A), or 1.5 × 10 8 MICs per kg BW on day -2 (n = 3, group B) or day -7 (n = 4, group C) before living donor kidney transplantation in addition to posttransplantation immunosuppression. The primary outcome measure was the frequency of adverse events (AEs) until day 30 (study phase) with follow-up out to day 360. RESULTS. MIC infusions were extremely well tolerated. During the study phase, 10 treated patients experienced a total of 69 AEs that were unlikely to be related or not related to MIC infusion. No donor-specific human leukocyte antigen Abs or rejection episodes were noted, even though the patients received up to 1.3 × 10 10 donor mononuclear cells before transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360, whereas reactivity against third-party cells was still preserved. Frequencies of CD19 + CD24 hi CD38 hi transitional B lymphocytes (Bregs) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19-and 68-fold higher, respectively, than in 2 independent cohorts of transplanted controls. The majority of Bregs produced the immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature. CONCLUSION. MIC administration was safe and could be a future tool for the targeted induction of tolerogenic Bregs.
Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in the treatment of chronic lymphocytic leukemia (CLL). However, efficacy seems to be inferior compared to diffuse large B-cell lymphoma or acute lymphoblastic leukemia. Impaired T-cell fitness of CLL patients may be involved in treatment failure. Less-differentiated naïve-like T cells play an important role in CART expansion and long-term persistence in vivo. These cells are sparse in CLL patients. Therefore, optimization of CART cell production protocols enriching less differentiated T cell subsets may overcome treatment resistance. The B-cell receptor inhibitor ibrutinib targeting Bruton's tyrosine kinase (BTK) is approved for the treatment of CLL. Besides BTK, ibrutinib additionally inhibits interleukin-2-inducible T-cell kinase (ITK) which is involved in T-cell differentiation. To evaluate the effect of ibrutinib on CART cell production, peripheral blood mononuclear cells from nine healthy donors and eight CLL patients were used to generate CART cells. T-cell expansion and phenotype, expression of homing and exhaustion makers as well as functionality of CART cells were evaluated. CART cell generation in the presence of ibrutinib resulted in increased cell viability and expansion of CLL patient-derived CART cells. Furthermore, ibrutinib enriched CART cells with less-differentiated naïve-like phenotype and decreased expression of exhaustion markers including PD-1, TIM-3 and LAG-3. In addition, ibrutinib increased the cytokine release capacity of CLL patient-derived CART cells. In summary, BTK/ITK inhibition with ibrutinib during CART cell culture can improve yield and function of CLL patient-derived CART cell products.
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