Use of Anti-P2X7 Biologics to Reduce Graft-Versus-Host Disease

P2X7 is an adenosine 5'-triphosphate (ATP)-gated ion channel involved in various health and disease processes. P2X7 is present on many cell types, most notably leukocytes, and its activation promotes inflammatory and immune responses. Thus, P2X7 is a potential therapeutic target for inflammatory disorders, including graft-versus-host disease (GVHD), a major complication of donor blood stem cell transplantation. A speciesspecific anti-human (h) P2X7 monoclonal antibody (mAb) is available but its use to date has been limited. Moreover, anti-P2X7 nanobodies (Nbs) are available. Neither anti-P2X7 mAbs or Nbs have been examined in GVHD. Therefore, this thesis aims to investigate anti-P2X7 biologics in the reduction of GVHD in a humanised mouse model.

Chapter 3 involved the purification and in vitro testing of the anti-hP2X7 mAb (clone L4). This chapter confirmed the ability of the anti-hP2X7 mAb to bind and block human, but not mouse, P2X7. Moreover, this mAb could prevent the loss of human regulatory T cells (Tregs) and human natural killer (NK) T cells during serum starvation. Consequently, the results in this chapter validated the investigation of the anti-hP2X7 mAb in an in vivo model of GVHD (Chapter 4) and in other chapters of this thesis.

Chapter 4 examined if the anti-hP2X7 mAb could reduce GVHD in a humanised mouse model. This mAb impaired clinical and histological liver and lung GVHD development but had no significant effect on survival. The reduction in GVHD severity corresponded to an increase in proportions of hTregs, hNK T cells, and hNK cells and a decrease in the proportions of human T helper (Th)17 cells and the hTh17:hTreg ratio. Collectively, these results demonstrated for the first time that a biologic targeting P2X7 can reduce GVHD and that donor (human) P2X7 can contribute directly to GVHD progression.

Chapter 5 explored if two different anti-P2X7 Nbs, delivered via an adeno-associated viral vector, could reduce GVHD in a humanised mouse model. The anti-mouse (m)/hP2X7 Nb (1C81) and to a lesser extent the anti-mP2X7 Nb (13A7) reduced clinical and histological GVHD, in the liver, lung and skin. These effects corresponded to, most notably, a decrease in liver hTh17 cells, and to a lesser extent, splenic hTregs and human invariant (i)NK T cells. Further, the presence of circulating anti-P2X7 Nbs in mice was confirmed.

Chapter 6 examined P2X7 expression on human blood leukocyte subsets and whether the anti-hP2X7 mAb could mediate complement-dependent cytotoxicity (CDC) of these cells. This chapter confirmed the expression of cell surface P2X7 on various human blood leukocytes and revealed the presence of P2X7 on human blood MDSCs. Further, it demonstrated that this mAb can mediate CDC in human leukocytes expressing relatively high cell surface P2X7.

Chapter 7 focused on the use of the anti-hP2X7 mAb in mass cytometry while also comparing the expression of P2X7 or CD39 on human blood leukocyte subsets in peripheral blood mononuclear cells (PBMCs) isolated by Ficoll-Paque and SepMate Tube density gradient centrifugation. The suitability of the anti-hP2X7 mAb for mass cytometry was validated by demonstrating P2X7 expression on blood leukocytes. Further, this chapter indicated there are minor but significant differences in P2X7 or CD39 expression between paired hPBMC samples isolated by either Ficoll-Paque or SepMate tube density gradient centrifugation, with the most notable difference with CD39 on hTregs.

In summary, this thesis has identified possible anti-P2X7 biologics to reduce GVHD. Moreover, this thesis further characterised an anti-hP2X7 mAb (clone L4). Collectively, this thesis continues to support evidence that P2X7 blockade is a potential therapeutic for GVHD also providing new biologics that could potentially be used in clinical trials in the future.