Mesothelin/CD3 half-life extended bispecific T-cell engager molecule shows specific tumor uptake and distributes to mesothelin and CD3 expressing tissues

BiTE ® (bispecific T-cell engager) molecules exert antitumor activity by binding one arm to CD3 on cytotoxic T-cells and the other arm to a tumor-associated antigen. We generated a fully mouse cross-reactive mesothelin (MSLN)-targeted BiTE molecule that is genetically fused to a Fc-domain for half-life extension, and evaluated biodistribution and tumor targeting of a zirconium-89 (89Zr)-labeled MSLN HLE BiTE molecule in 4T1 breast cancer bearing syngeneic mice with positron emission tomography (PET). Biodistribution of 50 µg 89Zr-MLSN HLE BiTE was studied over time by PET imaging in BALB/c mice and revealed uptake in tumor and lymphoid tissues with an elimination half-life of 63.4 hours. Compared to a non-targeting 89Zr-control HLE BiTE, the 89Zr-MLSN HLE BiTE showed a 2-fold higher tumor uptake and higher uptake in lymphoid tissues. Uptake in the tumor colocalized with mesothelin expression, while uptake in the spleen colocalized with CD3 expression. Evaluation of the effect of protein doses on the biodistribution and tumor targeting of 89Zr-MSLN HLE BiTE revealed for all dose groups that uptake in the spleen was faster than in the tumor (day 1 vs day 5). The lowest dose of 10 µg 89Zr-MSLN HLE BiTE had higher spleen uptake and faster blood clearance compared to higher doses of 50 µg and 200 µg. 89Zr-MSLN HLE BiTE tumor uptake was similar at all doses. Conclusion: The MSLN HLE BiTE showed specific tumor uptake and both arms contributed to the biodistribution profile. These findings support the potential for clinical translation of HLE BiTE molecules

PET imaging shows dose-dependent pharmacokinetics of a 89Zr-labeled mesothelin/CD3 half-life extended bispecific T-cell engager molecule in a syngeneic mouse model

BiTE® (bispecific T cell engager) immune therapy consists of two connected single-chain variable fragments, one targeting T-cells via CD3, and one targeting the tumor via a tumor-associated antigen. Given their short half-life, these molecules are administered by continuous intravenous (iv) infusion to ensure tumor accumulation. To extend plasma half-life, they are conjugated to an Fc domain, resulting in a molecule with an extended pharmacokinetic profile. To evaluate their biodistribution in a syngeneic mouse model, a molecule targeting murine mesothelin and murine CD3 (muMSLN HLE BiTE®), and a non-targeting control HLE BiTE® molecule were radiolabeled with the positron emission tomography (PET) isotope zirconium-89 (89Zr). METHODS: The control and muMSLN HLE BiTE® were labeled with 89Zr. Immunocompetent BALB/c mice were engrafted in the lower mammary fat pad with mesothelin-positive murine mammary carcinoma cell line 4T1. PET imaging was performed at 1, 3, 5, 7 and 9 days after 50 µg 89Zr-muMSLN HLE BiTE® was administered by iv infusion (n = 8). Next, biodistribution of 10 µg (n = 6), 50 µg (n = 6) and 200 µg 89Zr-muMSLN HLE BiTE® (n = 5) and 50 µg 89Zr-control HLE BiTE® (n = 6) was compared via PET imaging on day 1, 3 and 5. PET-scans were quantified by mean standardized uptake value (SUVmean). Tracer injections were 4 MBq. To compare uptake in multiple groups, an analysis of variance followed by a post-hoc Tukey's multiple comparison test was performed. Values are expressed as mean ± standard deviation. RESULTS: PET imaging following 50 µg 89Zr-muMSLN HLE BiTE® dosed iv revealed a blood elimination half-life of 63.4 hours. Uptake in tumor, spleen, thymus and liver were visible from 3 days post injection (pi). On day 5, tumor uptake was highest (SUVmean = 1.50 ± 0.2) with a tumor to blood ratio of 1.9 ± 0.3 and a spleen to blood of 1.7 ± 0.2. Heart SUVmean 5 days pi revealed that 10 μg 89Zr-muMSLN HLE cleared faster from the blood (0.5 ± 0.1) than the 50 μg (0.8 ± 0.1, P < 0.01) and the 200 μg dose group (0.8 ± 0.1, P < 0.01). Tumor SUVmean was higher in 50 µg (1.5 ± 0.2) than the 10 µg dose group (1.2 ± 0.1, P < 0.01), but similar to the 200 μg dose group (1.3 ± 0.1, P = 0.28). Spleen uptake 5 days pi was dose-dependent (SUVmean: 10 µg = 1.6 ± 0.2; 50 µg = 1.3 ± 0.1; 200 µg = 0.8 ± 0.1, P < 0.01). Spleen and tumor SUVmean 5 days pi of 50 µg 89Zr-muMSLN HLE BiTE® were higher than 50 µg 89Zr-control HLE BiTE® (spleen = 0.5 ± 0.1, P < 0.01; tumor = 0.8 ± 0.1, P < 0.01), while blood SUVmean of 50 µg 89Zr-control HLE BiTE® was similar (0.6 ± 0.1, P = 0.12). CONCLUSION: PET imaging with a 89Zr-labeled muMSLN HLE BiTE® in a tumor-bearing syngeneic mouse model revealed a long blood half-life, specific uptake in tumor and spleen, and dose-dependent pharmacokinetics. Taken together, the HLE BiTE® molecule demonstrates an extended pharmacokinetic profile over previously evaluated small, canonical, BiTE® molecules. Citation Format: Frans V. Suurs, Grit Lorenczewski, Julie M. Bailis, Sabine Stienen, Matthias Friedrich, Elisabeth G.E. de Vries, Derk Jan A. de Groot, Marjolijn N. Lub-de Hooge. PET imaging shows dose-dependent pharmacokinetics of a 89Zr-labeled mesothelin/CD3 half-life extended bispecific T-cell engager molecule in a syngeneic mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2769. ©2020 American Association for Cancer Research

Biodistribution of a CD3/EpCAM bispecific T-cell engager is driven by the CD3 arm

Bispecific T-cell engager (BiTE) molecules are designed to engage and activate cytotoxic T cells to kill tumor cells. Little is known about their biodistribution in immunocompetent settings. Methods: To explore their pharmacokinetics and the role of the immune cells, BiTE molecules were radiolabeled with the PET isotope Zr-89 and studied in immunocompetent and immunodeficient mouse models. Results: PET images and ex vivo biodistribution in immunocompetent mice with [Zr-89]Zr-DFO-N-suc-muS110, targeting mouse CD3 (dissociation constant [K-D], 2.9 nM) and mouse epithelial cell adhesion molecule (EpCAM; K-D, 21 nM), and with [Zr-89]Zr-DFO-N-suc-hyS110, targeting only mouse CD3 (K-D, 2.9 nM), showed uptake in the tumor, spleen, and other lymphoid organs, whereas the human-specific control BiTE [Zr-89]Zr-DFO-N-suc-AMG 110 showed similar tumor uptake but lacked spleen uptake. [Zr-89]Zr-DFO-N-suc-muS110 spleen uptake was lower in immunodeficient than in immunocompetent mice. After repeated administration of nonradiolabeled muS110 to immunocompetent mice, Zr-89-muS110 uptake in the spleen and other lymphoid tissues decreased and was comparable to uptake in immunodeficient mice, indicating saturation of CD3 binding sites. Autoradiography and immunohistochemistry demonstrated colocalization of [Zr-89]Zr-DFO-N-suc-muS110 and [Zr-89]Zr-DFO-N-suc-hyS110 with CD3-positive T cells in the tumor and spleen but not with EpCAM expression. Also, uptake in the duodenum correlated with a high incidence of T cells. Conclusion: [Zr-89]Zr-DFO-N-suc-muS110 biodistribution is dependent mainly on the T-cell-targeting arm, with a limited contribution from its second arm, targeting EpCAM. These findings highlight the need for extensive biodistribution studies of novel bispecific constructs, as the results might have implications for their respective drug development and clinical translation