Memory stem T cells modified with a redesigned CD30-chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma

Abstract

Objectives: Adoptive cell therapy (ACT) with mature T cells modified with a chimeric antigen receptor has demonstrated improved outcome for B-cell malignancies. However, its application for others such as Hodgkin lymphoma remains a clinical challenge. CD30 antigen, expressed in Hodgkin lymphoma cells, is absent in most healthy tissues, representing an ideal target of ACT for this disease. Despite that, efficacy of CD30-chimeric antigen receptor (CAR) T cells for Hodgkin lymphoma remains modest. Here, we have developed and tested a novel CD30-CAR T to improve efficacy of CD30-CAR therapy, using a targeting epitope within the non-cleavable part of CD30 receptor, and memory stem T cells (T_SCM) to improve engraftment, persistence and antitumor activity.

Methods: T_SCM-like cultures were generated and expanded ex vivo and transduced at day 1 or 2 with a lentiviral vector encoding the CD30-CAR. Therapeutic in vivo experiments were performed using NSG mice injected with L540 (sc) or L428 (iv) and treated with CD30-CAR T cells when the tumor was established.

Results: CD30-CAR T_SCM-like cells generated and expanded ex vivo, despite CD30 expression and fratricide killing of CD30⁺ CAR T cells, were not impaired by soluble CD30 and completely eradicated Hodgkin lymphoma in vivo, showing high persistence and long-lasting immunity. In addition, highly enriched CD30-CAR T_SCM-like products confer a survival advantage in vivo, in contrast to more differentiated CAR T cells, with higher tumor infiltration and enhanced antitumor effect.

Conclusion: This study supports the use of a refined CD30-CAR T cells with highly enriched T_SCM-like products to improve clinical efficacy of CAR T for Hodgkin lymphoma.

Keywords: chimeric antigen receptor; immunotherapy; memory stem T cells.

Figure 1

T_SCM‐like are highly enriched, CD30‐CAR‐transduced and expanded in vitro despite CD30 expression. Naïve T cells from healthy donors (n = 7) were cultured with CD3/CD28 costimulation in the presence of IL‐7, IL‐15 and IL‐21 during 10 days. (a) Frequencies of CD4⁺ and CD8⁺ T‐cell subpopulations at the end of culture. T_SCM‐like cells were the most prevalent T‐cell population (mean ± SD). (b) Representative plot of T cells transduced at day 2 with CD30‐CAR‐encoding lentivirus and CD30‐CAR expression and analysed by flow cytometry using an anti‐CD3 and anti‐EGFRt antibodies. (c) Expression of CD30 receptor on CD4⁺ and CD8⁺ T‐cell subpopulations in untransduced cells (white bars) and transduced cells (black bars) during the culture (days 4, 7 and 10; mean ± SD). (d) Percentage of T‐cell viability in untransduced cells (grey line) and transduced cells (black line) at days 4, 7 and 10 of culture (mean ± SD). (e) Fold expansion of CD4⁺ and CD8⁺ T cells during 10‐day culture in untransduced cells (grey line) and transduced cells (black line; mean ± SD). Data are representative of seven independently repeated experiments.

Figure 2

CD30‐CAR T_SCM‐like enriched culture efficiently eradicates HL in vitro. (a) T_SCM‐like enriched cells expressing CD30‐CAR (n = 7) were exposed to CD30⁺ target cells (L540 or L428 tumor cells) and control CD30⁻ cell line (Raji) at different effector:target (E:T) ratios. Specific cytolytic activity was measured at 24 h by bioluminescence assay. Untransduced T_SCM‐like cells were used as negative control (mean ± SD). (b) Cytolytic capacity of untransduced and CD30‐CAR T_SCM‐like cells (n = 3) was measured against CD30⁺ target cells (L540 and L428) at 24 h, in the presence or absence of saturated concentration of soluble CD30 protein (sCD30; 20 μg), at different E:T ratios. Raji cell line (CD30⁻) was used as negative target control (mean ± SD). (c) Cytokine secretion of CD30‐CAR T_SCM‐like enriched culture (n = 3) 24 h after L540, L428 and Raji co‐culture. Untransduced T_SCM‐like cells were used as negative control. Cytokine levels of IL‐2, IL‐6, IL‐10, IFN‐γ and TNF‐α were measured by cytometry‐based multiplex analysis (mean ± SD). Data are representative of seven (a) or three (b, c) independently repeated experiments. For each donor, technical duplicate was used (c).

Figure 3

CD30‐CAR T_SCM‐like function persists after CD30⁺ antigen re‐exposition. (a) Representative graphic of untransduced and CD30‐CAR T_SCM‐like enriched cells exposed to CD30⁺ target cells (L540) at different effector:target (E:T) ratios every 72 h after each antigen re‐exposition (total stimulations: 3). Specific cytolytic activity was measured after 24 h of each antigen stimulation by bioluminescence. (b) Mean fluorescence intensity (MFI) of CD30‐CAR expression and (c) percentage of PD‐1‐TIM‐3⁺ T cells during antigen re‐exposures (mean ± SD). *P < 0.05; **P < 0.01. Data are representative of three independently repeated experiments.

Figure 4

Therapeutic treatment with CD30‐CAR T_SCM‐like enriched cell products induces a potent antitumor response against HL in vivo. NSG mice (n = 4 for both experimental and control groups) were injected with (a) 2.5 × 10⁶ L540 tumor cells (sc) on day 0 and treated 3 days later with 5 or 10 × 10⁶ CD30‐CAR T_SCM‐like enriched cells (iv), or (b) 2 × 10⁶ L428 tumor cells (iv) on day 0 and treated with 5 or 10 × 10⁶ CD30‐CAR T_SCM‐like enriched cells (iv) 22 days after tumor challenge. In both models, control mice received 10 × 10⁶ untransduced (UN) T_SCM‐like enriched cells. Mice were monitored every other day for survival, and tumor growth was measured by in vivo bioluminescence. (c) Expression of exhaustion markers (PD1 and TIM3) in CD4⁺ and CD8⁺ tumor‐infiltrating T cells found in L540‐bearing mice treated with 5 × 10⁶ CD30‐CAR T_SCM‐like enriched cells, 48 days after tumor challenge, compared with CD4⁺ and CD8⁺ pre‐infused T cells (mean ± SD). *P < 0.05; **P < 0.01. Data are representative of two independent experiments.

Figure 5

T_SCM‐like enriched cells expressing CD30‐CAR have high persistence in vivo and confer long‐lasting immunity against HL. (a) NSG mice surviving from L428 tumor administration (n = 4) were rechallenged again with the same tumor dose (2 × 10⁶ cells per mouse, iv), 79 days (arrow) after the first tumor challenge (grey discontinued line). An age‐matched mouse group (n = 4) was injected with 2 × 10⁶ L428 tumor cells (iv) as tumor control (pointed line). Black line represents control mice receiving 10 × 10⁶ untransduced (UN) T_SCM‐like enriched cells. Mice were followed every other day for survival, and tumor growth control was done by in vivo bioluminescence. (b) Bone marrow and lymph nodes from surviving CD30‐CAR‐treated mice were analysed for CAR T cell by flow cytometry (mean ± SD). (c) T‐cell subpopulations were analysed in bone marrow and lymph nodes by flow cytometry (mean ± SD). (d) Expression of exhaustion markers (PD1, TIM3 and LAG‐3) was analysed by flow cytometry in T cells found in lymph nodes and bone marrow (mean ± SD). Data are representative of two independent experiments.

Figure 6

CD30‐CAR T_SCM‐like ^H products have higher antitumor efficacy and T‐cell persistence than CD30‐CAR T_SCM‐like ^L products. (a) Composition of T_SCM‐like ^H _and T_SCM‐like ^L cultures at the day of treatment (mean ± SEM). (b, c) NSG mice (N = 4/group) were challenged with 2.5 × 10⁶ L540 tumor cells (sc) on day 0 and treated when the tumor was well established (day 5) with 5 × 10⁶ CD30‐CAR T_SCM‐like ^H (pointed line and ▲) or T_SCM‐like ^L (discontinued line and ■) cells (iv). A group of mice were injected with 2.5 × 10⁶ L540 tumor cells (sc) as tumor control (black line and ●). Mice were monitored every other day for (B) tumor growth and (c) survival, measured by in vivo bioluminescence (mean ± SD). (d) Percentage of CD30‐CAR expression in tumor‐infiltrating T cells was analysed in T_SCM‐like ^H and T_SCM‐like ^L treated mice by flow cytometry (e) CD30‐CAR T‐cell detection in blood from mice treated with T_SCM‐like ^H or T_SCM‐like ^L cultures. (mean ± SD). *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of two independent experiments.