Abstract: Immunomonitoring of chimeric antigen receptor (CAR) T cells relies primarily on their quantification in the peripheral blood, which inadequately quantifies their biodistribution and activation status in the tissues. Noninvasive molecular imaging of CAR T cells by PET is a promising approach with the ability to provide spatial, temporal, and functional information. Reported strategies rely on the incorporation of reporter transgenes or ex vivo biolabeling, significantly limiting the application of CAR T-cell molecular imaging. In this study, we assessed the ability of antibody-based PET (immunoPET) to noninvasively visualize CAR T cells. Experimental Design: After analyzing human CAR T cells in vitro and ex vivo from patient samples to identify candidate targets for immunoPET, we employed a syngeneic, orthotopic murine tumor model of lymphoma to assess the feasibility of in vivo tracking of CAR T cells by immunoPET using the 89Zr-DFO-anti-ICOS tracer, which we have previously reported. Results: Analysis of human CD19-CAR T cells during activation identified the Inducible T-cell COStimulator (ICOS) as a potential target for immunoPET. In a preclinical tumor model, 89Zr-DFO-ICOS mAb PET-CT imaging detected significantly higher signal in specific bone marrow–containing skeletal sites of CAR T-cell–treated mice compared with controls. Importantly, administration of ICOS-targeting antibodies at tracer doses did not interfere with CAR T-cell persistence and function. Conclusions: This study highlights the potential of ICOS-immunoPET imaging for monitoring of CAR T-cell therapy, a strategy readily applicable to both commercially available and investigational CAR T cells. See related commentary by Volpe et al., p. 911

Abstract: Allogeneic hematopoietic cell transplantation (HCT) is a well-established and potentially curative treatment for a broad range of hematological diseases, bone marrow failure states, and genetic disorders. Acute graft-versus-host disease (GvHD), mediated by donor T cells attacking host tissues, still represents a major cause of morbidity and mortality following allogeneic HCT. Current approaches to diagnosis of gastrointestinal acute GvHD rely on clinical and pathological criteria that manifest at late stages of disease. New strategies allowing for GvHD prediction and diagnosis, prior to symptom onset, are urgently needed. Noninvasive antibody-based positron emission tomography (PET) (immunoPET) imaging of T-cell activation post–allogeneic HCT is a promising strategy toward this goal. In this work, we identified inducible T-cell costimulator (ICOS) as a potential immunoPET target for imaging activated T cells during GvHD. We demonstrate that the use of the Zirconium-89-deferoxamine-ICOS monoclonal antibody PET tracer allows in vivo visualization of donor T-cell activation in target tissues, namely the intestinal tract, in a murine model of acute GvHD. Importantly, we demonstrate that the Zirconium-89-deferoxamine-ICOS monoclonal antibody PET tracer does not affect GvHD pathogenesis or the graft-versus-tumor (GvT) effect of the transplant procedure. Our data identify ICOS immunoPET as a promising strategy for early GvHD diagnosis prior to the appearance of clinical symptoms.

Abstract: Introduction: Immunomonitoring of chimeric antigen receptor (CAR) T cells relies primarily on their quantification in the peripheral blood, which inadequately quantifies their biodistribution and activation status in the tissues. Non-invasive molecular imaging of CAR T cell therapy by positron emission tomography (PET) is a promising approach providing spatial, temporal and functional information. Reported strategies for PET-based monitoring of CAR T cells rely on additional manipulation of the cell product such as the incorporation of reporter transgenes or ex vivo biolabeling, which significantly limits the wider application of CAR T cell molecular imaging. In the present study, we assessed the ability of antibody-based PET (immunoPET) to non-invasively visualize CAR T cells in vivo. Methods: For analysis of human CAR T cell activation, we analyzed publicly available RNA sequencing data (GSE136891) obtained at serial time points during in vitro culture of CD19.CD28z CAR T cells. We analyzed by mass cytometry (CyTOF) the ex vivo ICOS expression on human CD19-28z CAR T cells obtained from 31 patients receiving axicabtagene ciloleucel (Axi-cel) for relapsed/refractory diffuse large B-cell lymphoma (DLBCL). For in vivo murine experiments, CD19-expressing B-cell lymphoma A20 cells (2.5×10e5 cells) were injected by tail vein intravenously (i.v.) into sub-lethally (4.4 Gy) irradiated Thy1.2+ BALB/c mice. Seven days later, murine CD19.CD28z Luc+ Thy1.1+ CAR T cells (1×10e6) were i.v. injected. ICOS expression was analyzed by flow cytometry on CAR T cells recovered from spleen and bone marrow 5 days after injection. For imaging studies, anti-ICOS monoclonal antibody (mAb) specific for murine ICOS (clone:7E.17G9, BioXcell) was modified with the bifunctional chelator deferoxamine (DFO/p-SCN-Bn-Deferoxamine). The DFO-ICOS mAb conjugate was radiolabeled with 37 MBq (~1 mCi) of 89Zr-oxalate (final specific activity 6 µCi/µg/ml and radiochemical purity of 99%). 89Zr-DFO-ICOSmAb (45 μCi ± 3.6, 7.5 μg± 0.6) was injected i.v. 5 days post-CAR T cell administration and PET-CT imaging performed 48 hours later. Following PET-CT, mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter. Results: Analysis of RNA-sequencing data from human CAR T cells identified ICOS as an activation marker whose transcription was up-regulated and sustained during in vitro culture. ICOS was preferentially expressed on CAR+ T cells recovered at day 7 from axi-cel treated patients compared with CAR- cells (p & lt;0.001; Figure 1A). Phenotypic analysis in a murine model of B cell lymphoma infiltrating the spleen and the bone marrow confirmed preferential ICOS expression on murine CAR T cells compared to resident cells in both spleen (p=0.003) and bone marrow (p=0.008). Figure 1B shows representative volume-rendered technique (VRT) PET/CT images of 89Zr-DFO-ICOS mAb-injected tumor-bearing mice either untreated (left panels) or that received mCD19.28z CAR T cells (right panels). 89Zr-DFO-ICOS mAb similarly accumulated in highly vascularized organs (heart, liver and spleen) of both untreated and CAR T cell treated mice, consistent with the biodistribution and clearance of intact antibodies. We detected pronounced 89Zr-DFO-ICOS mAb-PET signals in the bones of CAR T cell treated mice, particularly prominent in the lumbar spine, iliac bones, femur, tibia and humeral heads (Figure 1B). Region of interest analysis confirmed markedly increased radiotracer uptake in bones rich in bone marrow from CAR T treated mice compared with those of untreated mice (lumbar spine vertebrae p & lt;0.001; iliac bones p=0.001; femur p=0.002; tibia p=0.002). Moreover we observed a slight, but statistically significant increase in radiotracer accumulation in the heart of CAR T cell-treated mice (p=0.004) while no significant differences were detected in spleen and liver. As expected, there was no significant signal difference in the muscle, considered background. Biodistribution analysis using gamma counting of tissues confirmed the PET results. Conclusions: We describe for the first time an immunoPET approach to monitor the in vivo dynamics of CAR T cell migration, expansion, and persistence that does not require the addition of reporter genes or ex vivo labeling, being therefore applicable to the clinical setting for the study of any commercially available and investigational CAR T cell products. Disclosures Miklos: Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Lyell Immunopharma: Consultancy, Current equity holder in private company; NeoImmune Tech: Consultancy; Nektar Therapeutics: Consultancy; Apricity Health: Consultancy, Current equity holder in private company. Gambhir:CellSight Inc: Current equity holder in private company. Negrin:Amgen: Consultancy; BioEclipse Therapeutics: Current equity holder in private company; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; Biosource: Current equity holder in private company; KUUR Therapeutics: Consultancy; UpToDate: Honoraria.

Abstract: BACKGROUND 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 non-invasive imaging strategies specifically detecting early GvHD are critically needed to improve clinical care of HCT recipients. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F-FDG) have largely been confounding, mainly due to their low specificity. Monitoring T cell activation and expansion using T-cell targeted PET tracers seems a more promising approach (Ronald et al., Cancer Res 77(11) 2893, 2017). We recently reported a novel immuno-PET tracer (64Cu-DOTA-mAbOX40) that enables non-invasive imaging of activated murine T cells expressing the cell surface activation marker OX40 (Alam et al., JCI 128(6) 2569, 2018). In the present work, we evaluated the utility of this immuno-PET strategy to image activated T cells in a major MHC-mismatch mouse model of acute GvHD. METHODS Balb/C (H-2Kd) recipients were irradiated with 8.8 Gy and on the same day received intravenously (i.v.) 5 x 10e6 T-cell depleted bone marrow (BM) cells with or without 1 x 10e6 CD4 and CD8 T cells positively selected from C57BL/6 (H-2Kb) mice. Severity of GvHD was assessed by clinical GvHD scoring. Flow cytometry of lymphoid organs from BM control and GvHD mice was performed at day 7 after HCT to determine OX40 protein expression on immune cells. For imaging studies, anti-OX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86, BioXcell) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (an average ratio of 1.4 DOTAs per mAb was obtained) and subsequently radiolabeled with 64CuCl2 (final specific activity 10-15μCi/μg and radiochemical purity 〉 99%). Mice were tail-vein injected with 64Cu-DOTA-mAbOX40 (100 µCi, i.v.) at day 7 after HCT and PET-CT imaging performed 24 hours after injection. Immediately following PET-CT mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter. RESULTS Flow cytometry analysis of OX40 expression in lymphoid organs isolated at day 7 after HCT revealed significantly higher proportions and absolute numbers of OX40 expressing cells in the spleen and cervical lymph nodes (LN) isolated from mice that received BM + T cells (GvHD group) compared with mice having received BM cells alone (p 〈 0.05). In vivo OX40-ImmunoPET performed at day 8 after HCT revealed increased radiotracer uptake in spleen (p 〈 0.0001), mesenteric LN (p 〈 0.01) and the abdominal region (p 〈 0.001) of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64Cu-DOTA-mAbOX40 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]. Biodistribution analysis using gamma counting of tissues 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) and GvHD target organs e.g. small intestine (p 〈 0.05), colon (p 〈 0.05) and skin (p 〈 0.01). Importantly, outcome analysis of GvHD mice receiving tracer doses of OX40 mAb at day 7 after HCT did not reveal any significant worsening of GvHD in terms of survival, body weight loss or GvHD score, compared with mice receiving the appropriate isotype control, supporting the safety of this OX40-targeted imaging approach. CONCLUSION The OX40 immuno-PET tracer enabled specific imaging of alloreactive OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64Cu-DOTA-mAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting. FIGURE 1. 64Cu-DOTA-AbOX40 PET-CT imaging in a mouse model of acute GvHD. (A) Representative day 8 64Cu-DOTA-AbOX40 PET-CT images in BM controls or GvHD group. H, heart (including cardiac muscle and blood); Li, liver; Sp, spleen; Bl, blood vessels and venous sinuses; BM, bone marrow; Ab, abdomen. (B) Quantitative region of interest PET image analysis of indicated organs in BM controls (n=12, blue filled boxes) or GvHD mice (n=12, red filled boxes). Outliers are represented as dots. [Mann-Whitney test , ****p 〈 0.0001, ***p 〈 0.001, **p 〈 0.01, *p 〈 0.05]. Disclosures Gambhir: CellSight Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.