Page 74 - 2021_04-Haematologica-web

M. Guercio et al.
Results
Characterization of CAR.CD30 T-cell in vitro activity CAR.CD30 molecules, either second-generation or third-generation (II-CAR or III-CAR), did not induce any proliferative changes in genetically modified T cells as compared to control non-transduced (NT) T cells (Online Supplementary Figure S1B). Moreover, regardless of the high level of CAR expression, detected with either anti- CD34 (Figure 1B) or protein L (Figure 1C, Online Supplementary Figure S1C), in vitro co-culture experiments showed that II-CAR.CD30.4-1BB was characterized by the worst anti-tumor activity compared to all the other configurations (Online Supplementary Figure S3A and B). The II-CAR.CD30.CD28 exerts a similar anti-lymphoma activity as the III-CAR also including the OX40 co-stimu- latory domain; however, at a very low E:T ratio (1:8 effec- tor T cells vs. L428 target cells), we observed that only the III-CAR showed significant anti-lymphoma activity com- pared to NT T cells (Online Supplementary Figure S3B). Moreover, we observed a superior trend, although not statistically significant, of a higher percentage of CAR+ T cells (Online Supplementary Figure S1D) and interferon-γ production upon CD30+ tumor encounter (Online Supplementary Figure S3C and D) in CAR.CD30.CD28.OX40 T cells with respect to II-CAR. Considering these data, together with the clinical evi- dence of the unsatisfactory outcome of patients receiving II-CAR.CD30 therapy,21,22 we continued our study focus-
ing on III-CAR.CD30.
CAR.CD30 T cells did not exert fratricidal activity
The percentage of CD30+ T cells is high after in vitro stimulation, but then declines over time, with no signifi- cant difference between NT and CAR.CD30 T cells (Online Supplementary Figure S4A), indicating that CAR.CD30 T cells do not exert fratricidal activity. Moreover, CAR.CD30 T cells did not proliferate when stimulated by irradiated autologous activated T/CAR cells (Online Supplementary Figure S4B). Notably, mean flu- orescence intensity (MFI) of CD30 in lymphoma cells was significantly higher with respect to activated T cells (Online Supplementary Figure S4C).
Characterization of third-generation CAR.CD30 T cells in extended ex vivo culture
We sought to verify whether different culture conditions could influence the percentage of total CAR+ T cells during standard and prolonged in vitro ex vivo expansion (day 30) to stress the system. CAR.CD30 T cells expanded in IL2 showed a significant reduction of CAR during extended in vitro culture (Figure 1D). The percentage of CAR.CD30.CD28.4-1BB decreased from 76.09% ± 9.80% (day +5) to 49.20% ± 13.35 (day +15, P=0.002), and to 55.93% ± 18.09% after long-term culture (day +30, P=0.027 with respect to day +5). Similar results were also observed for CAR.CD30.CD28.OX40 T cells: 87.27% ± 5.07% (day +5), 65.15% ± 10.92% (day +15, P=0.003); and 73.05% ± 6.41% (day +30, P=0.009 compared to day +5). Moreover, IL7/IL15, compared to the IL2 condition, signif- icantly improved the total percentage of CAR+ T cells at day +15 (a standard time for CAR T-cell manufacturing); 76.84% ± 5.45% CAR.CD30.CD28.OX40 and 66.44% ± 10.95% CAR.CD30.CD28.4-1BB; P=0.004 and P=0.02, respectively).
While CAR.CD30 T cells did not show a significant proliferative advantage over NT T cells (Figure 1E and F), IL7/IL15 improved the expansion rate compared to IL2 (Online Supplementary Figure S5A-C), without any pre- dominant T-cell receptor Vβ family selection (Online Supplementary Figure S6A and B). Independently of the co- stimulatory combination used, we did not observe a sig- nificant difference in the suicide gene inducible caspase 9 (iCasp9) activity, either in vitro (Online Supplementary Figure S7A) or in vivo (Online Supplementary Figure S7B-E). Details are provided in the Online Supplementary Results.
CAR.CD30.CD28.OX40 T cells exert a superior in vitro anti-lymphoma activity compared to CAR.CD30.CD28.4-1BB T cells
Both types of III-CAR.CD30 T cells significantly lysed the Karpas-299 NHL cell line (Figure 2A), as well as two HL cell lines (namely, HDML-2 and L428) (Figure 2B and C, respectively). Cytotoxicity was specific, since negligi- ble lysis was observed against CD30-negative BV173 cells (Figure 2D). The anti-lymphoma activity was also con- firmed in a 7-day co-culture assay (Figure 2E-G).
Interestingly, at a very low E:T ratio, the anti-tumor activity of CAR.CD30.CD28.OX40 was superior to that exerted by CAR.CD30.CD28.4-1BB T cells: E:T ratio 1:8 T cells:Karpas-299 cells; P=0.03 (Figure 2E) and E:T ratios 1:16 and 1:32 T cells:HDML2 cells; P=0.03 and P=0.01, respectively (Figure 2F). The enhanced activity of CAR.CD30.CD28.OX40 T cells compared to that of CAR.CD30.CD28.4-1BB T cells was also demonstrated considering IFNγ production upon stimulation by Karpas- 299 cells (Figure 2H) and HDML2 cells (Figure 2I). However, IFNγ production was comparable when we used the cell line L428 as the target (Figure 2J).
Long-term tumor control and CAR.CD30 T-cell selection in a “stressed” co-culture model
To evaluate the lytic potential of CAR.CD30 T cells, we “stressed” the co-culture conditions by re-challenging Karpas-299 cells every 5 days (Figure 3A). At each time- point, we evaluated the percentage of residual tumor, CAR expression and its relative MFI, the relative produc- tion of IFNγ, TNFα, IL2 and IL10 (24 h after each tumor re-challenge), the CD4+/CD8+ distribution and the mem- ory/exhaustion profile.
Both types of III-CAR.CD30 T cells exhibited high tumor control even after multiple exposures to Karpas- 299. Although we did not observe significant differences in the in vitro anti-tumor activity between constructs, CAR.CD30.CD28.4-1BB T cells showed high intra-donor variability in terms of tumor elimination. By contrast, CAR.CD30.CD28.OX40 T cells were endowed with more stable and predictable lymphoma recognition and killing (residual tumor cells at day +20: 8.6% ± 5.3% for 28.OX40.ζ and 27.9% ± 29.5% for 28.4-1BB.ζ T cells) (Figure 3B). Interestingly, while the percentage of CAR+ cells increased after the first tumor encounter, subsequent tumor re-challenging negatively affected the percentage of residual CAR.CD30.CD28.4-1BB T cells in the in vitro culture (93.8% ± 2.7% at day +5 and 63.0% ± 32.30% at day +20; P=0.041) (Figure 3C); by contrast, the percentage of CAR.CD30.CD28.OX40 T cells remained stable over time (Figure 3C). This phenomenon led to a significant difference of CAR+ cells at day +20 between the two types of CAR T cells (P=0.043) (Figure 3C).
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haematologica | 2021; 106(4)