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A new promising CAR.CD30 T-cell therapy for CD30+ lymphoma.
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Figure 2. 28.OX40.ζ CAR T cells exert more efficient lymphoma control compared to 28.4-1BB.ζ CAR T cells at low effector:target ratios. (A-D) In vitro 51Cr cytotoxic assay to evaluate cytolytic activity of non-transduced (NT) T cells (empty circle), 28.4-1BB.ζ T cells (gray circle) and 28.OX40.ζ T cells (black circle), on CD30+ lym- phoma cell lines, namely Karpas-299 (A), HDML-2 (B) and L428 (C) cells, as well as on the CD30– leukemia cell line BV173 (D). (E-G) The long-term 7-day co-culture assay was performed at decreasing effector:target ratios, from 1:1 to 1:32. In particular, 28.4-1BB.ζ (gray bars), 28.OX40.ζ (black bars) and NT (white bar) T cells were co-cultured with CD30+ Karpas-299 (E), HDML-2 (F) and L428 (G) cell lines. (H-J) Interferon-gamma (IFN-γ) production after 24 h of co-culture was measured. Data from seven healthy donors are expressed as average ± standard deviation. *P≤0.05; **P≤0.01; ***P≤0.001 and ****P≤0.0001.
Similarly, the higher CAR MFI observed since day 0 in CAR.CD30.CD28.OX40 T cells compared to CAR.CD30.CD28.4-1BB T cells was retained stably over time during “stressed” co-culture experiments (Figure 3D).
Furthermore, CAR.CD30.CD28.OX40 T cells showed a significantly higher activation profile over time after tumor exposure compared to CAR.CD30.CD28.4-1BB T cells, in terms of cytokine production (Figure 3E-G), with a lower level of IL10 (produced by Karpas-299 cells, as shown by the red bar in Figure 3H.
Dynamic evolution of memory and exhaustion profiles in response to cytokine or tumor stimulation in CAR.CD30 T cells
At day +15 after transduction, most expanded CAR T cells cultured with IL2 had an effector memory (EM) phenotype (Figure 4A), with no substantial differences observed when compared with NT T cells. IL7/IL15 sig- nificantly reduced the central memory (CM) compart- ment, in favor of EM and effector terminal (EMRA) cells in both CAR.CD30. Interestingly, the “stressed” co-cul- ture significantly changed the profile of CAR T cells.
After 5 days of co-culture the naïve compartment decreased rapidly from 26.1% ± 13.0% to 2.6% ± 1.3% for 28.OX40.ζ (P=0.029) and from 30.1% ± 16.5% to 4.0% ± 3.0% for 28.4-1BB.ζ T cells (P=0.034). Tumor-cell encounter significantly modulated CAR T-cell subsets, while the profile of NT T cells compared to day 0 remained stable. In particular, the naïve subset was sig- nificantly reduced in both CAR T-cell types, whereas the CM subset increased from day 0 to day +20 (see Figure 4B).
We sought to evaluate the exhaustion profile modula- tion of CAR T cells after tumor challenge. Although no difference was recorded at day 0 (Figure 4C), we observed a significant upregulation of LAG3, TIM3, and PD-1 receptors after the first tumor-cell exposure, with CAR.CD30.CD28.OX40 T cells showing higher expres- sion of TIM3 over time compared to CAR.CD30.CD28.4-1BB cells. Nevertheless, TIM3 was also significantly upregulated in NT T cells upon tumor exposure (Figure 4D). Interestingly, no differences were observed between III-CAR-T-cell types in terms of per- centage of exhausted cells with triple positivity for LAG3/TIM3/PD-1 (Figure 4D).
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