Ibrutinib May Improve CAR T-Cell Production for Patients With Chronic Lymphocytic Leukemia

Ibrutinib (Imbruvica) during chimeric antigen receptor (CAR) T-cell culture improved the yield and function of CAR T-cell products as treatment of patients with chronic lymphocytic leukemia (CLL) in a study published in the International Journal of Cancer.

The study investigators led by Leapold Sellner, MD wrote, “our study provides evidence that BTK/ITK inhibition with ibrutinib during CAR T-cell generation may improve CLL patient-derived CAR T cell products and may have the potential to enhance CAR T-cell function. Ibrutinib-supplemented CAR T-cell production leads to increased CAR T-cell yields as well as enriches for less-differentiated T cells with lower expression of exhaustion markers and could be an option to further improve the clinical outcome of CLL patients.”

Sellner et al evaluated the absolute cell numbers during CAR T-cell generation by Trypan blue staining. During cell generation, cell proliferation was significantly lower in CLL-derived cells compared with HD-derived cells. The BTK inhibitor allowed for a significant increase in cell expansion of HD-derived cells on day 10 of the CAR T-cell generation (20.67 ± 8.82 × 106 without ibrutinib vs. 25.28 ± 10.29 × 106 with ibrutinib; P =.0248), and ibrutinib appeared to significantly increase the viability of CD3-positive T cells from HDs on day 10 (3% ±8% vs 87% ± 7%; P =.0183) and day 14 (84% ± 8% vs 91% ± 6%, respectively; P =.0202).

On day 10 (60% ± 15% without ibrutinib vs 69% ± 12% with ibrutinib; P =.0086) and 14 (47% ± 14% vs 59% ± 14%, respectively; P =.0142), CLL-derived cells have increased transduction efficiency. The absolute numbers of CLL patient-derived CAR T cells were significantly higher with ibrutinib on days 10 and 14 of the cell generation (1.52 ± 1.74 × 106 without ibrutinib vs. 2.61 ± 4.57 × 106 with ibrutinib; P =.0138 and 4.05 ± 4.37 × 106 vs. 7.20 ± 6.58 × 106; P =.003, respectively). There was only a limited amount of CD20-positive B cells that were potentially malignant on days 10 and 14.

There were higher amounts of CD4-positive T cells and lower amounts of CD8-positive T cells in the CLL patient-derived CAR T cells compared with the HD-derived. Ibrutinib did not appear to have any influence on the distribution of the CD4-positive and CD8-positive T cells during the cultivation of CLL- and HD-derived CAR T cells.

In prior studies, almost all CAR T cells were CD95-positive during the production process, and in the HD-derived CAR T cells, ibrutinib-supplemented production was able to enrich Tn cells on day 10 in all CD3-positive T cells (25% ± 11% without ibrutinib vs 40% ± 11% with ibrutinib; P =.0009), CD3/CD4-positive cells (17% ± 9% vs 32% ± 13%; P =.0016) and CD8-positive cells (without vs with ibrutinib: 34% ± 13% vs53% ± 9%; P =.0007), respectively; this was further increased on day 14 in all CD3-positive cells, without versus with ibrutinib, respectively, (28% ± 12% vs 45% ± 13%, P =.0162), CD3/CD4-positive cells (20% ± 7% vs 33% ± 13%; P =.0139), and CD3/CD8-positive cells (35% ± 15% vs55% ± 8%; P =.0038).

Compared with HD-derived PBMCs, CLL-derived PBMCs had significantly lower proportions of Tn cells (20% ± 16% vs 5% ± 3%; P =.0216) and significantly higher proportions of TEM cells (% ± 12% vs 49% ± 12%; P=.0142), respectively.

Ibrutinib-supplemented CAR T-cell production could significantly enrich for TN cells without vs with ibrutinib on days 10 (CD3-positive: 2% ± 2% vs 5% ± 3%, P = .0213; CD3/CD4-positive: 1% ± 1% vs 4% ± 3%; P =.0122) and 14 (CD3/CD4-positive: 2% ± 2% vs 6% ± 6%; P =.0449), respectively.

The absolute yield of Tn CAR T cells is of importance, besides during the distribution of different T cell subsets. The BTK inhibitor was able to significantly increase the absolute number of CD3-positive Tn CAR T cells on days 10 without versus with ibrutinib (1.69 ± 1.16 × 106 vs 4.39 ± 2.23 × 106; P =.0478) and 14 (4.55 ± 1.2 × 106 vs 22.45 ± 10.34 × 106; P =.0488) of CART cell production, respectively. This significant increase was observed in the CD4-positive Th cell on days 10 without versus with ibrutinib (0.64 ± 0.25 × 106 vs 2.2 ± 1.02 × 106; P =.0447) and 14 (1.72 ± 0.59 × 106 vs 3.98 ± 1.71 × 106; P =.0412), as well as CD8-positive T cell on days 10 (0.99 ± 0.94 × 106 vs 2.22 ± 1.51 × 106; P =.0393) and 14 (2.83 ± 1.49 × 106 vs 14.61 ± 7.95 × 106; P =.0357), respectively.

Investigators noted in the paper that the increased production of CD3-positive Tn CAR T cells was also observed in production with CLL-derived PBMCs on days 10, without versus with ibrutinib, (2.32 ± 1.87 × 104vs 9.76 ± 0.06 × 104; P =.0017) and 14 (6.81 ± 9.18 × 104 vs 25.21 ± 30.32 × 104; P =.0439), respectively, and the increased yield of TN CAR T cells was observed on days 10 and 14 in the CD4-positive (0.70 ± 0.60 × 104vs 5.76 ± 0.03 × 104; P =.0049; 2.52 ± 2.58 × 104vs 16.21 ± 18.90 × 104; P =.0500) and CD8-positive (0.99 ± 1.03 × 104vs 2.39 ± 0.05 × 104; P =.0102 and 3.14 ± 5.87 × 104vs 6.66 ± 9.37 × 10; P =.0403) T-cell fractions.

There were no differences observed in CXCR3 expression between both production strategies in HD- and CLL-derived CAR T cells, but a significantly higher expression of CD62L was seen after cultivation with ibrutinib in CLL-derived CAR T cells on day 14 (P =.0455).

PD-1 expression was also reduced in CLL-derived CAR T cells at days 10 (59% ± 24% vs 52% ± 20%; P=.0063) and 14 (44% ± 22% vs 32% ± 16%; P =.0149) without versus with ibrutinib, respectively, while TIM-3 expression was significantly lower with ibrutinib-based production in HD-derived CAR T cells on days 10 (90% ± 10% vs 74% ± 20%; P =.0224) and 14 (73% ± 20% vs 56% ± 28%; P =.0425), as well as CLL-derived CAR T cells (96% ± 3% vs 82% ± 17%; P =.0305 and 86% ± 11% vs 72% ± 20%; P =.0066). LAG-3 expression was also decreased with ibrutinib in HD-derived and CLL-derived CAR T cells on days 10 (37% ± 16% vs 18% ± 10%; P =.0178 and 69% ± 21% vs49% ± 19%; P =.0002) and 14 (34% ± 17% vs 22% ± 14%; P =.0242) without versus with ibrutinib, respectively.

Significantly higher intracellular production of TNF-α were generated with the presence of ibrutinib in CAR T cells from CLL patients (without vs with ibrutinib: 47% ± 11% vs 60% ± 18%; P =.0392), particularly in the CD8-positive CAR T cell subpopulation (35% ± 12% vs 51% ± 18%; P =.0322). IFN-γ was also higher for CD8-positive CART cells generated with ibrutinib (without vs with ibrutinib: 52% ± 12% vs 63% ± 8%; P =.0428). No significant differences, however, were observed for TNF-α and IFN-γ-producing multifunctional CAR T cells between both production strategies.

Ibrutinib did not appear to have significant effects on in vitro cytotoxic capacities of HD- and CLL-derived CAR T cells against CD19-positive Daudi cells in a functional analysis for CAR T cell-mediated cytotoxicity. There was also no relevant unspecific cytotoxicity against CD19-negative K-562 cells.

“CD19-CAR T cell therapy is a promising treatment approach for patients with CD19-positive B-cell malignancies,” the investigators wrote. “However, efficacy is clearly lower for CLL patients compared to acute lymphocytic leukemia and diffuse large B-cell lymphoma. Therefore, novel strategies are needed to improve the clinical benefit using CAR T cells for CLL patients.”

In conclusion, the investigators noted that further analyses, including in vivo validation of the findings from this study, are needed to prove this concept for CAR T-cell therapy in patients with CLL.

_Reference

_{Fan F, Yoo HJ, Stock S, et al. Ibrutinib for improved chimeric antigen receptor T-cell production for chronic lymphocytic leukemia patients. Int. J. Cancer. 2020; 1-10. doi: 10.1002/ijc.33212}