A novel RXR agonist, IRX4204, reduces GvHD while maintaining GvL effect

Allogeneic hematopoietic stem cell transplantation (HSCT) may be able to cure hematological malignancies through a phenomenon referred to as the graft-versus-leukemia (GvL) effect, where alloreactive donor T cells deplete tumor cells. However, its efficacy is compromised by graft-versus-host disease (GvHD)-related mortality.¹ There is an unmet need to identify immunomodulatory therapies that reduce GvHD without impacting GvL.

The nuclear receptors, retinoid X receptors (RXRs), exert immunomodulatory functions to control inflammation and metabolism, and studies in mice have reported that RXRα activation can impact T cell effector functions.^2,3 Targeting RXRs may, therefore, have therapeutic potential in the treatment of acute GvHD, as suggested by a recent study by Thangavelu et al. reporting on the RXR selective agonist, IRX4204, which can ameliorate acute GvHD while maintaining GvL responses in mice. This study was recently published in Blood and is summarized below.¹

Methods

• GvHD study: An in vivo model of GvHD was performed in lethally irradiated mice transplanted with allogeneic non–T cell-depleted bone marrow (BM) with or without donor splenocytes or purified splenic T cells. Mice were treated with either tacrolimus (FK506) or IRX4204, administered daily either from Days -3 to 11 or Days 3 to 17 post transplantation.
• GvL study: For GvL studies lethally irradiated B6 recipient mice were given T cell-depleted BM ± tumor cells ± T cells on day of transplantation to assess GvL responses. As tumor cell lines, the investigators used either a myeloid leukemia cell line or a B-cell lymphoma cell line, both expressing the luciferase gene for tumor burden measurement by imaging.
• In vitro and ex vivo studies consisted of T cells suppression assay, T cells proliferation study, mixed lymphocyte reactions (humans), inducible regulatory T cells (Tregs) generation, RNA sequencing of T cells, and serum cytokine analysis.

Results

In mice transplanted with allogeneic BM and splenocytes/T cells, administration of IRX4204 lead to significantly prolonged survival compared to vehicle control or FK506. Furthermore, IRX4204 led to lower GvHD scores in target organs (liver, small and large intestine) and reduced intestinal injury as measured by blood levels of non-absorbable fluorescein isothiocyanate-labeled dextran, when compared to vehicle control.

• Furthermore, IRX4204 inhibited allogeneic donor T-cell proliferation, as shown by significantly reduced donor T-cell infiltration in target organs without impact on donor T-cell homing in the thymus and peripheral or mesenteric lymph nodes (MLNs). IRX4204 had no impact on donor T-cell apoptosis rates.
• In addition, IRX4204 significantly reduced percentages of IFN-γ+ or TNF-α+ producing donor T cells in the spleen on Days 7 and 14, and the lamina propria lymphocytes of the large intestine on Days 13 and 21, compared to vehicle control treated mice, indicating reduced T helper 1 cell differentiation.
• To better understand the mechanism by which IRX4204 reduced gut injury, nanostring transcriptional analysis of donor T cells isolated from intestines of GvHD mice treated with IRX4204 was performed, revealing significant decreases in transcripts regulating pro-inflammatory pathways, such as Sema7a, Stat1, Irf1, and Jun, while anti-inflammatory IL-4 expression was increased.
• Treatment of mice with IRX4204 resulted in a significantly higher percentage of Tregs in the spleen, MLNs, large intestine lamina propria lymphocytes, and blood compared to vehicle-treated controls, indicating that part of the anti-inflammatory activity of IRX4204 was mediated through Treg cells. No effect was seen in effector/memory cells. This finding was confirmed in human mixed-lymphocyte reaction experiments showing increase Treg differentiation from naïve CD4+ T cells by IRX4202. In an in vivo experiment, fluorescence-activated cell sorting (FACS)-purified Foxp3-GFP-negative donor T cells from Foxp3-GFP knock-in mice were injected in IRX4204-treated recipient mice, leading to a more than two-fold increase of CD4 GFP+ (Foxp3+) donor T cells in the spleen, MLNs, and small intestine.
• To assess the impact on GvL effect, two luciferase-positive tumor cell lines (C1498ff, an acute myeloid leukemia and TBL12, a B-cell lymphoma) were transplanted together with BM into mice leading to rapid tumor-induced death by Day 25. The addition of donor T cells inhibited tumor growth while leading to GvHD-induced death. However, addition of IRX4202 in these mice significantly increased survival without detectable tumor, indicating that IRX4204 maintained GvL responses while reducing GvHD.

Conclusions

IRX4204 has the ability of suppressing acute GvHD while preserving alloreactive responses against tumors, indicating that targeting RXRs with IRX4204 could be used as a novel approach to prevent and treat acute GvHD in the clinic.

Data from previous studies suggest that there is a threshold in donor T-cell mediating GvHD versus GvL effect.^5,6 It is possible that treatment with IRX4204 does not impair T cell responses to levels lower than the GvL threshold. Currently, IRX4204 is being tested in clinical trials (NCT02991651) as an anti-cancer agent, however, given these recently discovered immunomodulatory effects, it may be a candidate for patients with GvHD.