RT Journal Article SR Electronic T1 Preclinical evaluation of 89Zr-Df-labeled anti-VISTA antibody CI-8993 JF Journal of Nuclear Medicine JO J Nucl Med FD Society of Nuclear Medicine SP 4030 OP 4030 VO 63 IS supplement 2 A1 Burvenich, Ingrid A1 Wichmann, Christian A1 McDonald, Alexander A1 Scott, Fiona A1 Guo, Nancy A1 Rigopoulos, Angela A1 Soikes, Raul A1 Angelides, Steven A1 von Roemeling, Reinhard A1 Scott, Andrew YR 2022 UL http://jnm.snmjournals.org/content/63/supplement_2/4030.abstract AB 4030 Introduction: CI-8993 is a fully human IgG1k monoclonal antibody (mAb) that binds specifically to immune checkpoint molecule VISTA (V-domain Ig suppressor of T-cell activation). Murine anti-VISTA antibodies can overcome anti-PD1 and anti-CTLA4 resistance in orthotopic tumor models. Phase I safety was investigated in NCT02671955 and continues to be explored in Curis’s NTC04475523. To determine the pharmacokinetics and biodistribution of CI-8993 in patients, we aimed to develop 89Zr-labeled CI-8993 and validate PET imaging and quantitation in preclinical models prior to a planned human bioimaging trial.Methods: CI-8993 and isotype IgG1 control were conjugated to the metal ion chelator p-isothiocyanatobenzyl-desferrioxamine (Df). Optimal Df-to-mAb ratio, reaction temperature, reaction time and purification method were established. Quality of conjugates were assessed by SE-HPLC, SDS-PAGE, and FACS. After radiolabeling with zirconium-89 (89Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. 89Zr-Df-CI-8993 alone (1 mg/kg, 4.6 MBq) or in combination with 30 mg/kg unlabeled CI-8993, as well as isotype control 89Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in huVISTA knock-in female (C57BL/6N-Vsirtm1.1(VSIR)Geno) or control C57BL/6 mice bearing syngeneic MB49 bladder cancer tumors; and in BALB/c nu/nu mice bearing pancreatic tumors. Whole-body animal PET/MRI imaging was performed on day of radioconjugate synthesis, day 1 and day 3 (huVISTA KI model) or day 2 and day 7 (Capan-2 model) p.i. Biodistribution was assessed by image analyses and ex vivo biodistribution studies.Results: Conjugation of Df to CI-8993 for 60 minutes at room temperature followed by purification via gel filtration resulted in stable constructs with an average chelator-to-antibody ratio of 1.81. SDS-PAGE showed integrity of CI-8993 was maintained after chelation; and ELISA indicated no impact of conjugation on binding to human VISTA. Radiochemical purity (iTLC) and protein integrity (SE-HPLC) at end of synthesis were > 99% and 93%, respectively. PET imaging and biodistribution in MB49 tumor-bearing huVISTA KI female mice showed specific localisation of 89Zr-Df-CI-8993 to VISTA expressing organs (liver: 14.98 ± 0.50 %ID/g; spleen: 292.00 ± 14.51 %ID/g; n = 3) compared to 89Zr-Df-IgG1 control (liver: 4.615 ± 0.15 %ID/g; spleen: 6.37 ± 0.22 %ID/g; n = 4) or in the presence of competing unlabeled CI-8993 (liver: 8.14 ± 0.50 %ID/g; spleen: 41.14 ± 3.00 %ID/g; n = 5). Tumor-to-blood ratios indicated specific tumor targeting of 89Zr-Df-CI-8993 in the presence of unlabeled CI-8993 (20.47 ± 3.09) compared to trace dose 89Zr-Df-CI-8993 (0.97 ± 0.12; P = 0.0001) or 89Zr-Df-IgG1 control (1.75 ± 0.11; P < 0.0001). Specific tumor uptake was also demonstrated in Capan-2 human pancreatic tumor-bearing BALB/c nu/nu mice. Conclusions: We have validated 89Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that 89Zr-labeled CI-8993 is now suitable for use in the bioimaging clinical trial study planned in solid tumor patients.