Comparative Preclinical Analysis of Anti-B7-H3 CAR-T Cells Targeting Neuroblastoma

Abstract

Background: Neuroblastoma is a childhood tumor that is usually fatal after relapse in high-risk patients. Most clinical trials of CAR-T therapy for neuroblastoma are based on targeting the disialoganglioside GD2. B7-H3, a protein from the immunoglobulin superfamily, is a specific marker for neuroblastoma and a number of other solid tumors. We conducted a preclinical study of three variants of anti-B7-H3 CAR-T cells in order to justify the selection of the best candidate for subsequent clinical trials. Methods: The expression level of B7-H3 was measured in a number of cell lines and neuroblastoma tissue samples via flow cytometry. The functional activity of CAR-T cells was compared using an NFAT-inducible reporter assay, a cytotoxicity test, cytokine production, and a repeated stimulation assay. Results: The obtained CAR-T cells carrying all resulting CAR variants specifically recognized and killed B7-H3-positive tumor cells in vitro. Nevertheless, TE9-28z and 8H9-28BBz demonstrated superior activation and cytokine production compared to the second-generation 8H9-BBz construct. TE9-28z and 8H9-28BBz exhibited functional differences in expansion, exhaustion markers, and cytokine secretion in co-cultures with target cells in vitro. In particular, TE9-28z induced higher IFNγ production, while 8H9-28BBz showed increased TNFα release. Despite comparable cytotoxicity, TE9-28z and 8H9-28BBz CAR-T cells exhibited varying persistence depending on the tumor type, and showed signs of functional exhaustion upon prolonged exposure to the target antigen. Conclusion: TE9-28z and 8H9-28BBz were selected for further preclinical development as promising candidates for the effective targeting of B7-H3-expressing malignancies.

Keywords: B7-H3; CAR-T cell functional activity; cancer target; chimeric antigen receptor (CAR); neuroblastoma; preclinical study; solid tumor.

Figure 1

Schematic representation of anti-B7-H3 CAR constructs.

Figure 2

Analysis of B7-H3 expression on different tumor cells. (A)—A schematic representation of the gating strategy for the analysis of neuroblastoma patient samples. (B)—The histogram plots of B7-H3 expression in different tumor cell lines. Live cells were gated prior to the analysis. The lines show the percentage of B7-H3-positive cells.

Figure 3

Generation of functional active anti-B7-H3 CAR Jurkat cells. (A)—A comparative activation of the three Jurkat-NFAT-GFP CAR variants. The data were adjusted against the level of GFP expression in Mock T cells (tonic signaling) (data = experimental data − tonic signaling). (B)—A schematic representation of Jurkat-NFAT-GFP reporter cell workflow. (C)—Fluorescence assay on Jurkat-NFAT-GFP cells transduced with 8H9-BBz, TE9-BBz, and 8H9-28BBz backbones and activated in the presence of different B7-H3+ target cells with increasing B7-H3 expression. Baseline (tonic) activation and B7-H3-negative Daudi and Jurkat cells were used as a negative control. All data represent the mean ± SD. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, ns indicates ‘not significant’. The p values were determined via multiple unpaired t-tests.

Figure 4

Analysis of the functional activity of anti-B7-H3 CAR T cells. (A)—CAR-T cell transduction level. (B)—Expression of CAR in CD4+ and CD8+ T cells. (C)—The differentiation status of CD4+ and CD8+ of the 8H9-BBz, TE9-BBz, and 8H9-28BBz anti-B7-H3 CAR-T cells. Tem—effector memory T cell, Tcm—T cell central memory, T naive—T cell-naive, T eff RA—terminally differentiated T cells, Tscm—T memory stem cell. (D)—Cytokine secretion (IFNγ and TNFα) by anti-B7-H3 CAR-T cells upon stimulation with LAN1 and 143B target cells. (E)—Cytokine secretion (IFNγ and TNFα) by anti-B7-H3 CAR-T cells upon stimulation with wild-type (WT) and B7-H3+ Jurkat cells. Mock T cells were used as a negative control. PMA/Ionomycin stimulation was used as a positive control. (F)—The comparison of cytotoxic activity of anti-B7-H3 CAR-T cells against two B7-H3-expressing cell lines—LAN-1 (neuroblastoma) and 143B (osteosarcoma)—at varying effector-to-target ratios (E:T—2:1; 1:1; 1:2). The data were adjusted against a negative control (Mock T cells). All data represent the mean ± SD. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001. The p-values were determined by multiple unpaired t-tests.

Figure 5

Sequential killing assay and exhaustion analysis of anti-B7-H3 CAR T cells. (A)—Experimental timeline. CAR-T cells were co-cultured with B7-H3-positive target cells (LAN-1 or 143B) in a sequential killing assay. Fresh target cells were added every 2 days to maintain continuous antigenic stimulation. On days 0, 2, and 9 of the experiment, CAR-T cells were analyzed via flow cytometry for the expression of exhaustion markers (LAG-3, TIGIT, PD-1). (B)—Sequential killing assay on CAR-T cells against two B7-H3-expressing cell lines—LAN-1 (neuroblastoma) and 143B (osteosarcoma). The absolute number of live CAR-T cells (left panel) and live target cells (right panel) was monitored over time during repeated stimulation. (C)—Heatmap of exhaustion marker expression (LAG-3, TIGIT, PD-1) on anti-B7-H3 CAR-T cells on days 0, 2, and 9 of the sequential killing assay with LAN1 and 143B target cells. The color represents the number of marker-positive cells (%).