Strategy to Improve Functional T-Cell Yield in Peripheral Blood By Co-Inhibition of Beta-2 Adrenergic and CXCR4 Signaling Pathways

Chimeric antigen receptor (CAR-T) therapy has shown notable success in B-cell malignancies. However, lack of treatment efficiency is observed in up to 50% patients due to factors such as low CAR-T activation, reduced in vivo expansion, and heterogeneity in the autologous CAR-T product. Additionally, anti-cancer therapies also lead to decreased peripheral blood (PB) T cell count in up to 20% of apheresis cases. Research shows that positive selection of functional T cell subsets is one approach that can improve success of the CAR-T therapy. Finally, newer gene editing therapies involving in vivo viral transduction of cells can also benefit from T cell mobilization into PB. Here, we discuss a strategy to enhance functional T cell yield in PB utilizing pharmacological co-inhibition of CXCR4 and beta 2-adrenergic receptor (B2AR) signaling.

CXCR4 is widely expressed by hematopoietic cells, including lymphocytes. First, we evaluated total and differential PB white blood cell (WBC) count in mice following a single injection of several CXCR4 inhibitors, including GPC-100 (burixafor), AMD3100 (plerixafor) and BL8040 (motixafortide). Mice were administered these treatments at doses that maximize mobilization in mice. Compared to baseline, GPC-100 (30 mg/kg, IV) and BL8040 (5 mg/kg, SC)-induced mobilization lasted 4-6 hours, whereas in AMD3100 (5 mg/kg, SC) treated mice, increased cell counts returned to baseline by 4 hours. For all treatments, lymphocyte mobilization peaked at 2 hours. GPC-100, but not BL8040, induced increase in lymphocytes was significantly greater than AMD3100.

Previous studies have reported increased lymphocyte retention by CXCR4 in lymph nodes due to the formation of B2AR and CXCR4 heteromers on lymphocytes. Such functional consequences of B2AR and CXCR4 heteromerization have also been demonstrated in cancer cells. Moreover, co-blockade of both receptors in mice enhance mobilization of WBCs and hematopoietic stem cells into PB. Accordingly, we administered propranolol, a B2AR blocker, for 7-days, followed by a single injection of a CXCR4 inhibitor on day 7. A significant increase in PB lymphocyte count was only observed when propranolol was combined with GPC-100, but not with AMD3100 or BL8040. This was also accompanied by increase in PB neutrophils which has been shown to be associated with increased CD8+ T-cells during CAR-T expansion.

Further characterization of lymphocyte subsets revealed that GPC-100 alone increased CD3+T cells, CD19+ B cells, and NKp46+ NK cells in PB, whereas propranolol alone had no effect. Moreover, GPC-100 significantly mobilized both CD4+ and CD8+ T cells and induced a 10% increase in PB CD8+T cells expressing granzyme B, indicating more functional T cells. Propranolol further enhanced T-, B-, and NK-cell mobilization by GPC-100 by 2-3-fold. The most prominent impact was observed on CD8+T cells with a 7-fold increase over vehicle. In vehicle treated mice, 21% of T cells in PB were CD8+, whereas in propranolol plus GPC-100 treated mice, 34% of PB T cells were CD8+. Propranolol and GPC-100 combination also reduced CD4:CD8 cell ratio from 2.7 to 1.5, which suggests that this combination treatment can be used to select for functional T-cell subsets from PB. Further evaluation of the benefits on other mobilized subsets such as NK and B cells is warranted.

Adrenergic stress leads to immunosuppression. Particularly, B2AR plays an exclusive role in suppression of T-cell receptor-mediated human and mouse CD8+ T cell effector function. We tested this via stimulation of human PB mononuclear cells (PBMCs), which leads to release of the functional cytokine interferon gamma (IFNy). Treatment of stimulated PBMC with epinephrine inhibited IFNy release, which was fully reversed when propranolol and GPC-100 were added. Thus, B2AR and CXCR4 co-inhibition can induce enhanced mobilization of functional T-cells to PB during harvest or in vivo viral transduction.

Clinically, GPC-100 shows satisfactory safety profile and effective mobilization of WBC subsets in phase 1 and 2 in healthy volunteers and multiple myeloma patients, respectively. Therefore, we propose that propranolol can be safely combined with GPC-100 for improving CAR-T efficiency. Comprehensive analysis of immune cell subsets mobilized by propranolol and GPC-100 in our clinical study (NCT05561751) is also ongoing to evaluate this treatment for improving current approaches to cell and gene therapy.