Impact of Combination Therapies on Graft-versus-Host Disease and Graft-versus-Leukaemia Immunity

Allogeneic haematopoietic stem cell transplantation (alloHSCT) is a curative therapy for haematological malignancies, including leukaemia, that are refractory to conventional chemotherapy and radiotherapy. AlloHSCT aims to generate graft-versus-leukaemia (GVL) immunity, where the alloreactive donor T cells eliminate residual host malignant cells following myeloablation. However, a common and often fatal side effect of this treatment is graft-versus-host disease (GVHD), where these alloreactive donor T cells (graft) recognise the healthy tissues of the transplant recipient (host) as foreign and mount a destructive inflammatory immune response. Despite the use of prophylactic agents, GVHD occurs in 30-60% of patients and results in severe damage to multiple organs including the liver, skin, gastrointestinal tract and lungs. One therapeutic strategy for the prevention of GVHD is the use of post-transplant cyclophosphamide (PTCy). However, while PTCy reduces GVHD occurrence and severity, GVHD still develops in 40-50% of patients and the cellular mechanisms by which PTCy reduces the disease are not fully understood. Additional therapies and a greater understanding of the cellular mechanisms through which PTCy abrogates GVHD are therefore required.

Interleukin (IL)-6 is a pleiotropic cytokine with a central role in T cell differentiation and GVHD development. When IL-6 is present with transforming growth factor β, naive T cells develop into T helper (Th) 17 cells, which have been shown to exacerbate GVHD. However, in the absence of IL-6, the same naive T cells develop into regulatory T cells (Tregs), which have a protective role against GVHD. As such, driving the T cell differentiation pathway away from inflammatory Thl7 cells and toward Treg development is a potential strategy for the prevention of GVHD.

Chapter 3 of this thesis investigated the effect of tocilizumab (TOC), an anti-IL-6 receptor monoclonal antibody (mAb), combined with PTCy on immune cell engraftment and GVHD development in a humanised mouse model. NOD-scid-IL2Rγ^null (NSG) mice were injected intraperitoneally (i.p.) with 2 x 10⁷ human peripheral blood mononuclear cells (hPBMCs) and cyclophosphamide (33 mg/kg) or control diluent on days 3 and 4, then TOC or control antibody (0.5 mg/mouse) twice weekly for 28 days. Combination therapy with TOC and PTCy delayed GVHD onset and increased proportions of human (h) Tregs but did not improve long-term survival outcomes compared to PTCy alone.

T cells are involved in GVHD pathogenesis and GVL responses, thus, it is important to ensure that GVHD preventative strategies do not abrogate GVL immunity. Therefore, Chapter 4 investigated the impact of PTCy alone and combined with TOC on GVL immunity to human THP-1 acute myeloid leukaemia cells in a humanised mouse model of GVL immunity. NSG mice were injected i.p. with 2 x 10⁷ hPBMCs followed by treatment with PTCy and TOC (as above), then injected intravenously with 1 x 10⁶ THP-1 cells on day 14. PTCy did not compromise GVL immunity but reduced both the hepatic and splenic hCD4⁺:hCD8⁺ T cell ratio. Moreover, PTCy increased the proportions of splenic hCD39⁺ Tregs and increased both the hepatic and splenic hThl7:hTreg ratio but had no impact on circulating human cytokines. Further, combination therapy with TOC and PTCy did not impact GVL immunity and reduced histopathology in the liver and lung, with minimal impact on T cell subsets and circulating cytokines.

Although PTCy can reduce GVHD, the precise cellular mechanisms through which it reduces the disease are not fully understood and high doses are associated with toxicities. Therefore, Chapter 5 aimed to determine whether a lower dose of PTCy can reduce GVHD and to examine the effects of low-dose PTCy on human immune cell subsets, T cell exhaustion, and tissue damage at early and late time points in GVHD development in a humanised mouse model. NSG mice were injected i.p. with 2 x 10⁷ hPBMCs and cyclophosphamide (33, 25 or 10 mg/kg) or control diluent on days 3 and 4. Low-dose PTCy at 10 mg/kg abrogated clinical signs of GVHD with comparable efficacy to high-dose PTCy at 33 mg/kg, with improved survival and delayed GVHD onset. Moreover, proportions of PD-l⁺hCD4⁺ and PD-l⁺hCD8⁺ T cells were increased by PTCy at 10 mg/kg but not 25 or 33 mg/kg, while proportions of PD-1⁺ hTregs were increased by PTCy at all doses. Further, proportions of exhausted hCD8⁺ T cells were increased with 33 mg/kg PTCy, but not lower doses of PTCy.

Thl7 cells can exacerbate GVHD, therefore, Chapter 6 investigated the impact of Thl7 cell targeted compounds alone or combined with mafosfamide (MAFOS), an active cyclophosphamide analogue, on the proliferative capacity and proportions of T cell subsets using in vitro proliferation assays. Treatment with 5 μg/mL secukinumab, an anti-IL-17 mAb, had no impact on the proliferation or proportions of the T cell subsets assessed. However, FGIN-1-27 (known to impair murine Thl7 cells) inhibited the proliferation of several T cell subsets involved in GVHD pathogenesis, with the greatest impact on Thl7 cells. Moreover, combining FGIN-1-27 with MAFOS did not have an additive or synergistic effect, with proliferation and subset proportions similar between cells treated with FGIN-1-27 and FGIN-1-27 with MAFOS.

Collectively, this thesis demonstrates that combination therapy with PTCy and TOC can reduce GVHD severity while retaining GVL immunity in humanised NSG mice. Furthermore, this thesis shows that a reduced dose of PTCy abrogates GVHD to a similar extent as a higher dose, and further elucidates the impact of PTCy on human immune cell subsets in humanised NSG mice. Finally, this thesis identifies a novel Thl7-targeted compound, FGIN-1-27, as a potential GVHD therapeutic using in vitro proliferation assays.