Targeting chronic lymphocytic leukemia with B-cell activating factor receptor CAR T cells

Abstract

The challenge of disease relapsed/refractory (R/R) remains a therapeutic hurdle in chimeric antigen receptor (CAR) T-cell therapy, especially for hematological diseases, with chronic lymphocytic leukemia (CLL) being particularly resistant to CD19 CAR T cells. Currently, there is no approved CAR T-cell therapy for CLL patients. In this study, we aimed to address this unmet medical need by choosing the B-cell activating factor receptor (BAFF-R) as a promising target for CAR design against CLL. BAFF-R is essential for B-cell survival and is consistently expressed on CLL tumors. Our research discovered that BAFF-R CAR T-cell therapy exerted the cytotoxic effects on both CLL cell lines and primary B cells derived from CLL patients. In addition, the CAR T cells exhibited cytotoxicity against CD19-knockout CLL cells that are resistant to CD19 CAR T therapy. Furthermore, we were able to generate BAFF-R CAR T cells from small blood samples collected from CLL patients and then demonstrated the cytotoxic effects of these patient-derived CAR T cells against autologous tumor cells. Given these promising results, BAFF-R CAR T-cell therapy has the potential to meet the long-standing need for an effective treatment on CLL patients.

Keywords: BAFF‐R; B‐cell malignancies; CAR T cells; chronic lymphocytic leukemia; immunotherapy.

FIGURE 1

Verification of antigen‑specific functionality of B‐cell activating factor receptor (BAFF‐R) chimeric antigen receptor (CAR) T cells. (A) This schematic diagram depicts BAFF‐R scFv and additional domains of the CAR engineered into the lentiviral expression vector. (B and C) Antigen‐specific cytotoxicity of BAFF‐R CAR T cells were evaluated by a CD107a assay with representative data shown here. CAR T cells were co‐incubated with target cells (WT or BAFF‐R‐knockout [KO] Nalm‐6 cells) at an E:T ratio of 2:1. Cytotoxic analysis was focused on CD8⁺ CAR T cells (B) or CD4⁺ CAR T cells (C); non‐CAR T served as control samples. The BAFF‐R CAR T cells that were generated from three donors and when incubated with target cells showed similar cytotoxicities (Table S2). (D) Interferon‐gamma (IFN‐γ) was measured from the harvested supernatant of BAFF‐R CAR T cells that were co‐cultured with target cells (WT or BAFF‐R‐KO Nalm‐6 cells) at an E:T ratio of 4:1 for 72 h. The results were presented as mean ± SEM from four replicates and representative from three independent experiments (^∗∗∗ p < 0.001; ns, no significant differences).

FIGURE 2

Cytotoxicity of B‐cell activating factor receptor (BAFF‐R) chimeric antigen receptor (CAR) T cells on acute lymphocytic leukemia (ALL) cells directly confirms cytolysis of target cells. (A) Using a direct killing assay, BAFF‐R CAR T cells or non‐CAR T cells were co‐cultured with either green fluorescent protein (GFP)‐labeled tumor cells (Nalm‐6 WT or BAFF‐R‐knockout [KO] cells) at 20:1 (E:T ratio) for 24 h. The live GFP‐expressing tumor cells were quantified with a gating strategy that gated on the live cells first and then the GFP‐positive target cells. These dot plots are representative of data obtained from three independent experiments. (B) By setting the non‐CAR T cells at 100% for each of three experiments, the non‐CAR T group served as a baseline to normalize the percentile of live tumor cells in BAFF‐R CAR T group. The results were showed as mean ± SEM from four replicates and representative from three independent experiments (^∗∗∗ p < 0.001).

FIGURE 3

Cytotoxicity of B‐cell activating factor receptor (BAFF‐R) chimeric antigen receptor (CAR) T cells against three chronic lymphocytic leukemia (CLL) cell lines in vitro. BAFF‐R (A) or CD19 (B) expression in MEC‐1 WT, HG‐3, or CII cell lines were characterized by BAFF‐R‐AF647 or CD19 APC antibodies staining and assessed using flow cytometry. (C and D) BAFF‐R CAR T cells were incubated with MEC‐1 WT, HG‐3, or CII cells at an E:T ratio of 2:1. CD107a‐positive cell analysis, which was utilized to indicate antigen‐specific cytotoxicity, was focused on CD8⁺ (C) or CD4⁺ CAR T cells (D); non‐CAR T cells were used as negative controls. (E) Additionally, cytotoxicity of BAFF‐R CAR T cells was observed with the co‐incubated with either MEC‐1 WT, HG‐3, or CII cell lines at an E:T ratio of 4:1. Interferon‐gamma (IFN‐γ) was measured from the harvested supernatant after 72 h. Non‐CAR T cells were used as controls. The results were showed as mean ± SEM from four replicates and representative from three independent experiments (^∗∗∗ p < 0.001).

FIGURE 4

Cytotoxicity of B‐cell activating factor receptor (BAFF‐R) chimeric antigen receptor (CAR) T cells against CD19‐knockout (KO) MEC‐1 cell line. (A) BAFF‐R or CD19 expression in MEC‐1 WT or MEC‐1 CD19‐KO were characterized by BAFF‐R‐AF647 or CD19 APC antibodies staining and analyzed with flow cytometry. (B and C) BAFF‐R or CD19 CAR T cells were incubated with MEC‐1 WT or MEC‐1 CD19‐KO at an E:T ratio of 2:1, respectively. Cell surface expression of CD107a was analyzed on gated CD8⁺ CAR T cells (B) or CD4⁺ CAR T cells (C); non‐CAR T cells were used as controls. (D) BAFF‐R or CD19 CAR T cells were co‐cultured with either MEC‐1 WT or MEC‐1 CD19‐KO cell lines at an E:T ratio of 4:1. IFN‐γ was assessed from the harvested supernatant after 72 h. Non‐CAR T cells were used as control. The results were showed as mean ± SEM from four replicates and representative from three independent experiments (^∗∗∗ p  < 0.001; ns, no significant differences).

FIGURE 5

Immunophenotyping of isolated primary B tumor cells from chronic lymphocytic leukemia (CLL) patients. (A) Primary B tumor cells from CLL patients were isolated from peripheral blood mononuclear cells (PBMCs) to serve as target cells in chimeric antigen receptor (CAR) T‐cell function assays. Enriching B cells significantly reduces the proportion of T cells in the target cell population. (B) B‐cell activating factor receptor (BAFF‐R) expression in the enriched B cells from the three CLL patients was characterized using BAFF‐R‐AF647 antibodies, followed by flow cytometry analysis. Isotype antibody served as staining controls.

FIGURE 6

Cytotoxicity of B‐cell activating factor receptor (BAFF‐R) chimeric antigen receptor (CAR) T cells from healthy donors against primary B tumor cells from chronic lymphocytic leukemia (CLL) patients. The BAFF‐R CAR T cells from healthy donors were produced and then incubated with primary B tumor cells from three CLL patients at 2:1 ratio, followed by a CD107a assay. Used MEC‐1 WT cells as a positive control. The analysis focused on CD8⁺ or CD4⁺ CAR T cells, with non‐CAR T cells as negative controls. Cytotoxicity of BAFF‐R CAR T cells derived from healthy donor A against primary B tumor cells from three CLL patients was observed: CLL patient 1 (CD8⁺ in A; CD4⁺ in B); CLL patient 2 (CD8⁺ in C; CD4⁺ in D); and CLL patient 3 (CD8⁺ in E; CD4⁺ in F).

FIGURE 7

Generation of B‐cell activating factor receptor (BAFF‐R) chimeric antigen receptor (CAR) T cells derived from chronic lymphocytic leukemia (CLL) patients. (A) The growth curves of BAFF‐R CAR T and their matched non‐CAR T cells, obtained from T cells of three CLL patients, were displayed side by side for comparison. (B) The identity (CD3‐positive staining) greater than 80% and potency (tEGFR staining) greater than 10% were detected in the patient‐derived CAR T cells.

FIGURE 8

Chronic lymphocytic leukemia (CLL) patient‐derived B‐cell activating factor receptor (BAFF‐R) chimeric antigen receptor (CAR) T elicited ex vivo cytotoxicity against autologous B cells. Using a CD107a degranulation assay, the cytotoxic efficacy of CLL patient‐derived BAFF‐R CAR T cells was assessed against a collection of previously established target cells: Nalm‐6 WT, BAFF‐R‐KO Nalm‐6, and MEC‐1 WT cells. Most significantly, the cytotoxic efficacy of CLL patient‐derived BAFF‐R CAR T cells showed cytotoxicity against autologous B tumor cells. Non‐CAR T cells from the same patient were used as a control. Cytotoxicity analysis of was performed on the CD8⁺ CAR T‐cell populations from CLL patient 1 (A), CLL patient 2 (B), and CLL patient 3 (C). Note that the cytotoxicity was also gated on the CD4⁺ CAR T‐cell population and included in Figure S2A‒C.