Tracking T Cell Activation By OX40 ImmunoPET: A Novel Strategy for Imaging of Graft Versus Host Disease (2019)

Graft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) mediated by donor immune cells reacting against host tissues. GvHD diagnosis is often challenging and superior noninvasive imaging strategies are critically needed. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F FDG) has largely been confounding, mainly due to its low specificity. Monitoring T cell activation and expansion using Tcell targeted PET tracers seems a more promising approach. We recently reported a novel immunoPET tracer (64CuDOTAmAbOX40) that enables noninvasive imaging of activated T cells expressing the cell surface activation marker OX40. In the present work, we evaluated the utility of this immunoPET strategy to image activated T cells in a major MHCmismatch mouse model of acute GvHD. Lethally irradiated Balb/C (H2Kd) recipients received 5 × 10e6 Tcell depleted bone marrow (BM) cells intravenously (i.v.) with or without 10e6 CD4 and CD8 T cells from C57BL/6 (H2Kb) mice. AntiOX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (average ratio of 1.4 DOTAs per mAb) and radiolabeled with 64CuCl2 (final specific activity 10 15μCi/μg, radiochemical purity >99%). Mice were tail vein injected with 64CuDOTAmAbOX40 (100 μCi, i.v.) at day 7 after HCT and PETCT performed 24h after injection. In vivo OX40ImmunoPET revealed increased radiotracer uptake in spleen, mesenteric LN and abdominal region of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64CuDOTAmAbOX40 uptake in spleen, mesenteric LN and abdominal region positively correlated with the GvHD score [spleen, r=0.6, p=0.0018; mesenteric LN, r=0.42, p=0.042; abdomen, r=0.77, p < 0.0001]. Immediately following PETCT radioactivity was measured in dissected weighed tissues using a gammacounter. Biodistribution analysis confirmed the PET results showing the same trends; significantly increased uptake in GvHD mice compared with BM controls in spleen (p < 0.01), cervical LN (p < 0.01), mesenteric LN (p < 0.01), small intestine (p < 0.05), colon (p < 0.05) and skin (p < 0.01). In conclusion, the OX40 immunoPET tracer enabled specific imaging of OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64CuDOTAmAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting. Graft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) mediated by donor immune cells reacting against host tissues. GvHD diagnosis is often challenging and superior noninvasive imaging strategies are critically needed. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F FDG) has largely been confounding, mainly due to its low specificity. Monitoring T cell activation and expansion using Tcell targeted PET tracers seems a more promising approach. We recently reported a novel immunoPET tracer (64CuDOTAmAbOX40) that enables noninvasive imaging of activated T cells expressing the cell surface activation marker OX40. In the present work, we evaluated the utility of this immunoPET strategy to image activated T cells in a major MHCmismatch mouse model of acute GvHD. Lethally irradiated Balb/C (H2Kd) recipients received 5 × 10e6 Tcell depleted bone marrow (BM) cells intravenously (i.v.) with or without 10e6 CD4 and CD8 T cells from C57BL/6 (H2Kb) mice. AntiOX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (average ratio of 1.4 DOTAs per mAb) and radiolabeled with 64CuCl2 (final specific activity 10 15μCi/μg, radiochemical purity >99%). Mice were tail vein injected with 64CuDOTAmAbOX40 (100 μCi, i.v.) at day 7 after HCT and PETCT performed 24h after injection. In vivo OX40ImmunoPET revealed increased radiotracer uptake in spleen, mesenteric LN and abdominal region of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64CuDOTAmAbOX40 uptake in spleen, mesenteric LN and abdominal region positively correlated with the GvHD score [spleen, r=0.6, p=0.0018; mesenteric LN, r=0.42, p=0.042; abdomen, r=0.77, p < 0.0001]. Immediately following PETCT radioactivity was measured in dissected weighed tissues using a gammacounter. Biodistribution analysis confirmed the PET results showing the same trends; significantly increased uptake in GvHD mice compared with BM controls in spleen (p < 0.01), cervical LN (p < 0.01), mesenteric LN (p < 0.01), small intestine (p < 0.05), colon (p < 0.05) and skin (p < 0.01). In conclusion, the OX40 immunoPET tracer enabled specific imaging of OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64CuDOTAmAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting. Figure 1.