Preclinical PET imaging of bispecific antibody ERY974 targeting CD3 and glypican 3 reveals that tumor uptake correlates to T cell infiltrate

BACKGROUND: Bispecific antibodies redirecting T cells to the tumor obtain increasing interest as potential cancer immunotherapy. ERY974, a full-length bispecific antibody targeting CD3ε on T cells and glypican 3 (GPC3) on tumors, has been in clinical development However, information on the influence of T cells on biodistribution of bispecific antibodies, like ERY974, is scarce. Here, we report the biodistribution and tumor targeting of zirconium-89 (89Zr) labeled ERY974 in mouse models using immuno-positron emission tomography (PET) imaging. METHODS: To study both the role of GPC3 and CD3 on the biodistribution of [89Zr]Zr-N-suc-Df-ERY974, 89Zr-labeled control antibodies targeting CD3 and non-mammalian protein keyhole limpet hemocyanin (KLH) or KLH only were used. GPC3 dependent tumor targeting of [89Zr]Zr-N-suc-Df-ERY974 was tested in xenograft models with different levels of GPC3 expression. In addition, CD3 influence on biodistribution of [89Zr]Zr-N-suc-Df-ERY974 was evaluated by comparing biodistribution between tumor-bearing immunodeficient mice and mice reconstituted with human immune cells using microPET imaging and ex vivo biodistribution. Ex vivo autoradiography was used to study deep tissue distribution. RESULTS: In tumor-bearing immunodeficient mice, [89Zr]Zr-N-suc-Df-ERY974 tumor uptake was GPC3 dependent and specific over [89Zr]Zr-N-suc-Df-KLH/CD3 and [89Zr]Zr-N-suc-Df-KLH/KLH. In mice engrafted with human immune cells, [89Zr]Zr-N-suc-Df-ERY974 specific tumor uptake was higher than in immunodeficient mice. Ex vivo autoradiography demonstrated a preferential distribution of [89Zr]Zr-N-suc-Df-ERY974 to T cell rich tumor tissue. Next to tumor, highest specific [89Zr]Zr-N-suc-Df-ERY974 uptake was observed in spleen and lymph nodes. CONCLUSION: [89Zr]Zr-N-suc-Df-ERY974 can potentially be used to study ERY974 biodistribution in patients to support drug development

Interferon-γ Plays Protective Roles in Sodium Arsenite-Induced Renal Injury by Up-Regulating Intrarenal Multidrug Resistance-Associated Protein 1 Expression

Subcutaneous injection of sodium arsenite (NaAs, 12.5 mg/kg) into BALB/c [wild-type (WT)] mice causes acute renal dysfunction characterized by severe hemorrhages, acute tubular necrosis, and cast formation, with increases in serum blood urea nitrogen and creatinine levels. Concomitant enhancement in intrarenal interferon (IFN)-γ expression prompted us to examine its roles in this pathology. IFN-γ-deficient (IFN-γ(−/−)) mice exhibited higher serum blood urea nitrogen and creatinine levels and exaggerated histopathological changes, compared with WT mice. Eventually, IFN-γ(−/−) mice exhibited a high mortality (87.5%) within 24 hours after NaAs challenge, whereas most WT mice survived. The intrarenal arsenic concentration was significantly higher in IFN-γ(−/−) mice later than 10 hours after NaAs treatment, with attenuated intrarenal expression of multidrug resistance-associated protein (MRP) 1, a main transporter for NaAs efflux, compared with WT mice. NF-E2-related factor (Nrf) 2 protein, a transcription factor crucial for MRP1 gene expression, was similarly increased in the kidneys of both strains of mice after NaAs treatment. In contrast, the absence of IFN-γ augmented transforming growth factor-β-Smad3 signal pathway and eventually enhanced the expression of activating transcription factor 3, which is presumed to repress Nrf2-mediated MRP1 gene expression. Thus, IFN-γ can protect against NaAs-induced acute renal injury, probably by maintaining Nrf2-mediated intrarenal MRP1 gene expression

The Absence of Interleukin-6 Enhanced Arsenite-Induced Renal Injury by Promoting Autophagy of Tubular Epithelial Cells with Aberrant Extracellular Signal-Regulated Kinase Activation

Sodium arsenite (NaAs)-induced autophagic cell death (ACD) of a mouse renal tubular epithelial cell line (mProx24), which expresses enhanced levels of interleukin-6 (IL-6), was reduced by the suppression of autophagy by 3-methyladenine or Atg7 knockdown. The inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) signal pathway by anti-IL-6 antibody or a Jak2 inhibitor (AG490) exaggerated ACD of mProx24 cells after NaAs challenge, attenuating STAT3 activation and reciprocally enhancing extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, an ERK inhibitor, PD98059, reduced NaAs-induced ACD in mProx24 cells. Subcutaneous injection of NaAs (12.5 mg/kg) into BALB/c (wild-type) mice enhanced intrarenal expression of IL-6, mainly produced by tubular cells, and caused severe renal injury characterized by hemorrhages, acute tubular necrosis, cast formation, and brush border disappearance, with increases in serum urea nitrogen (blood urea nitrogen) and creatinine levels. In addition, IL-6-deficient (IL-6−/−) mice exhibited exaggerated histopathological changes with higher blood urea nitrogen and creatinine levels. Moreover, in IL-6−/− mice treated with NaAs, ACD in renal tubular cells was significantly augmented, along with diminished STAT3 activation and reciprocal enhancement of ERK signaling, compared with wild-type mice. Finally, the administration of exogenous IL-6 into wild-type mice significantly reduced NaAs-induced ACD along with diminished ERK activation and eventually alleviated acute renal dysfunction. Thus, IL-6/STAT3 signal pathway could inhibit ERK activation, a crucial step for ACD, eventually attenuating NaAs-induced renal dysfunction