Abstract 1134: PET-imaging of 89Zr-labeled bispecific T-cell engagers in syngeneic tumor bearing mice
Authors: Suurs Frans V. , Groot Derk J. de , Domanska Urszula M. , Lorenczewski Grit , Stienen Sabine , Friedrich Matthias , Vries Elisabeth G. de , Hooge Marjolijn N. Lub-de 
Published: August 2019

BACKGROUND Bispecific T-cell engagers (BiTE®) harness the immune system against cancer. BiTE® antibody constructs are small proteins of ~53 kDa existing of two connected single-chain variable fragments. MuS110, a BiTE® with affinity for murine CD3 (KD= 2.9 nM) and murine EpCAM (KD= 21 nM), was radiolabeled with positron emission tomography (PET) isotope zirconium-89 (89Zr) to study its pharmacokinetics and involvement of the immune system in an immunocompetent mouse model bearing a syngeneic tumor.

METHODS MuS110 and two control BiTE® antibody constructs (hyS110 and AMG110) were radiolabeled with 89Zr. HyS110 has affinity for murine CD3 and human EpCAM, AMG110 for human CD3 and human EpCAM. The human and murine variants of EpCAM and CD3 are not cross-reactive. Either immunocompetent BALB/c or immunodeficient nude BALB/c mice were all orthotopically engrafted in the lower mammary fat pad with EpCAM-positive murine breast cancer cell line 4T1. PET-imaging was performed at 0.5, 3, 6, 24, 48 and 72 h after intravenous (iv) administration of 10 µg 89Zr-muS110. Distribution of 10 µg 89Zr-muS110, 89Zr-hyS110 and 89Zr-AMG110 was assessed 24 h after administration in BALB/c and nude BALB/c mice with PET-imaging and ex-vivo biodistribution. In addition, a group of BALB/c mice received 10 µg muS110 iv daily for 5 days followed by 10 µg 89Zr-muS110 or 10 µg 89Zr-AMG110 and PET-imaging 24 h after tracer administration. Values are expressed as median (interquartile range).

RESULTS PET-imaging revealed fast renal clearance of 89Zr-muS110 in the BALB/c mice resulting in a blood half-life of 0.93 h (one-phase decay). Tumor- and spleen-to-blood ratios increased to 3.7 (3.0 to 4.5) and 9.3 (7.5 to 11.1) after 72 h. Ex-vivo biodistribution 24 h after tracer administration showed 89Zr-muS110 and 89Zr-hyS110 accumulation in the spleen with 8.2 % injected dose per gram (ID/g) and 8.7 %ID/g, in comparison to 89Zr-AMG110 (2.3 %ID/g; vs 89Zr-muS110 P&lt;0.05, vs 89Zr-hyS110 P&lt;0.05). In mesenteric lymph nodes (mesLNs), 89Zr-muS110 and 89Zr-hyS110 resulted in a higher uptake (3.5 and 6.4 %ID/g) than 89Zr-AMG110 (2.0 %ID/g; vs 89Zr-muS110 P&lt;0.05, vs 89Zr-hyS110 P&lt;0.05). Tumor uptake did not differ for 89Zr-muS110 and 89Zr-AMG110 (2.6 vs 2.2 %ID/g, P&gt;0.05). In nude BALB/c mice spleen and mesLN uptake of 89Zr-muS110 was lower than in BALB/c mice (spleen: 3.4 vs 8.2 %ID/g, P&lt;0.05; mesLNs: 1.7 vs 3.5, P&lt;0.05). In addition, in nude BALB/c mice, tumor uptake was the same for 89Zr-muS110 and 89Zr-AMG110 (1.5 vs 1.7 %ID/g, P&gt;0.05). 89Zr-muS110 uptake was lower in spleen and mesLNs following 5 days of 10 µg muS110 iv compared to control mice

(spleen: 4.2 vs 8.2 %ID/g, P&lt;0.01; mesLNs: 1.9 vs 3.5, P&lt;0.01), likely representing target saturation.

CONCLUSION Distribution of BiTE® 89Zr-muS110 is predominantly mediated by the affinity for CD3, resulting in uptake in lymphoid tissues.

Citation Format: Frans V. Suurs, Derk J. de Groot, Urszula M. Domanska, Grit Lorenczewski, Sabine Stienen, Matthias Friedrich, Elisabeth G. de Vries, Marjolijn N. Lub-de Hooge. PET-imaging of 89Zr-labeled bispecific T-cell engagers in syngeneic tumor bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1134.