Comparative Study of the Effect of Radiation Delivered by Lutetium-177 or Actinium-225 on Anti-GD2 Chimeric Antigen Receptor T Cell Viability and Functions

Abstract

Chimeric antigen receptor (CAR) T cells have been relatively ineffective against solid tumors. Low-dose radiation which can be delivered to multiple sites of metastases by targeted radionuclide therapy (TRT) can elicit immunostimulatory effects. However, TRT has never been combined with CAR T cells against solid tumors in a clinical setting. This study investigated the effects of radiation delivered by Lutetium-177 (¹⁷⁷Lu) and Actinium-225 (²²⁵Ac) on the viability and effector function of CAR T cells in vitro to evaluate the feasibility of such therapeutic combinations. After the irradiation of anti-GD2 CAR T cells with various doses of radiation delivered by ¹⁷⁷Lu or ²²⁵Ac, their viability and cytotoxic activity against GD2-expressing human CHLA-20 neuroblastoma and melanoma M21 cells were determined by flow cytometry. The expression of the exhaustion marker PD-1, activation marker CD69 and the activating receptor NKG2D was measured on the irradiated anti-GD2 CAR T cells. Both ¹⁷⁷Lu and ²²⁵Ac displayed a dose-dependent toxicity on anti-GD2 CAR T cells. However, radiation enhanced the cytotoxic activity of these CAR T cells against CHLA-20 and M21 irrespective of the dose tested and the type of radionuclide. No significant changes in the expression of PD-1, CD69 and NKG2D was noted on the CAR T cells following irradiation. Given a lower CAR T cell viability at equal doses and an enhancement of cytotoxic activity irrespective of the radionuclide type, ¹⁷⁷Lu-based TRT may be preferred over ²²⁵Ac-based TRT when evaluating a potential synergism between these therapies in vivo against solid tumors.

Keywords: Actinium-225; Lutetium-177; chimeric antigen receptor; melanoma; neuroblastoma; targeted radionuclide therapy.

Figure 1

Third-generation virus-free CRISPR anti-GD2 CAR T cells. (A) Schematic of the domains of the third generation CAR. The extracellular domain includes the scFV (V_H and V_L chains connected by a linker) and a hinge. The intracellular domain is comprised of 2 costimulatory domains (CD28 and OX40) and a signaling domain (CD3-Zeta). These 2 domains are connected by a transmembrane domain (CD28 transmembrane domain). (B) Schematic of the third generation anti-GD2 CAR construct inserted into the human T cell receptor alpha constant gene (TRAC). (C) Schematic of the anti-GD2 CAR T cell used experimentally. It expresses the anti-GD2 CAR but is devoid of T cell receptor due to the CRISPR knockout of the human TRAC gene. scFv: single-chain variable fragment; V_H: heavy chain; V_L: light chain.

Figure 2

Dose-dependent effect of ²²⁵Ac and ¹⁷⁷Lu on the viability of anti-GD2 CAR T cells. (A) Experimental scheme: anti-GD2 CAR T cells were incubated in cell culture medium containing free ²²⁵Ac or ¹⁷⁷Lu with activities calculated to deliver a radiation dose between 1 and 6 Gy by day 3. The CAR T cells were harvested, and their viability was analyzed by flow cytometry using a live/dead staining. Viable CAR T cells were determined as CD45+ and live/Dead-Ghost red-. (B) Both ²²⁵Ac and ¹⁷⁷Lu led to a dose-dependent CAR T cell death. One-way ANOVA with Tukey’s multiple comparisons correction ** p = 0.0024; *** p = 0.0001; **** p < 0.0001. Each bar represents the mean (triplicate measurements) and error bar represents the standard deviation.

Figure 3

Dose-independent effect of ²²⁵Ac and ¹⁷⁷Lu on anti-GD2 CAR T cell cytotoxicity. (A) Experimental scheme: After irradiation of CAR T cells by ²²⁵Ac or ¹⁷⁷Lu, the CAR T cells were harvested, washed and trypan blue viability assay was performed. The viable CAR T cells after irradiation were co-cultured with the GD2-expressing human neuroblastoma cell line CHLA-20 for 24 h at an E:T ratio of 10:1 (viable anti-GD2 CAR T cells: tumor cells). (B) The viability of the CHLA-20 cells was analyzed by flow cytometry. The exposure of anti-GD2 CAR T cells to radiation delivered by radionuclide enhances their cytotoxicity against CHLA-20 cells to a similar degree for all doses tested for ²²⁵Ac or ¹⁷⁷Lu. One-way ANOVA with Tukey’s multiple comparisons correction *** p < 0.001; **** p < 0.0001; ns: not significant. Each bar represents the mean (triplicate measurements), and the error bar represents the standard deviation. The cytotoxic activity of CAR T cells irradiated with 2 Gy of radiation delivered by ²²⁵Ac was not performed because of very low CAR T cell viability after irradiation.

Figure 4

²²⁵Ac or ¹⁷⁷Lu does not impact the expression of exhaustion and activation markers on anti-GD2 CAR T cells. (A) Experimental scheme: anti-GD2 CAR T cells were incubated in cell culture medium containing ²²⁵Ac or ¹⁷⁷Lu with activities calculated to deliver a radiation dose between 1 and 6 Gy by day 3. CAR T cells were harvested, and the expression of exhaustion and activation markers was analyzed by flow cytometry. (B) The expression of the exhaustion marker PD-1 does not change following irradiation by ²²⁵Ac or ¹⁷⁷Lu. (C) Similarly, no major impact is seen on the expression of the T cell activation marker CD69. (D) Irradiation does not result in a major impact on the activation marker NKG2D. One-way ANOVA with Tukey’s multiple comparisons correction; ns: not significant. Each bar represents the mean (triplicate measurements), and the error bar represents the standard deviation. The effects of 2 Gy irradiation delivered by ²²⁵Ac on the receptors’ expression was not evaluated because of very low CAR T cell viability after irradiation. ns: not significant.