Preclinical Evaluation of an Engineered Single-Chain Fragment Variable-Fragment Crystallizable Targeting Human PD-L2

Introduction: Programmed cell death-Ligand 2 (PD-L2), a ligand programmed cell death protein 1 (PD-1), is an immune checkpoint molecule closely related to the efficacy of immune checkpoint inhibitor therapy (ICI). The aim of this study is to design a novel tracer ¹²⁴I-E6scFv-Fc targeting PD-L2 and perform preclinical evaluation to dynamically monitor PD-L2 expression and screen potential beneficiaries of ICI therapy.

Methods: A human single-chain fragment variable (scFv) was generated by phage display, using the extracellular domain of recombinant human PD-L2. The E6scFv was reformatted into a bivalent E6scFv-Fc, based on human IgG₁–fragment crystallizable (Fc). The E6scFv-Fc was radiolabeled with ¹²⁴I by N-Bromosuccinimide (NBS) in PB-buffer at pH 7 (37°C, 60s). In vitro stability in 5% human serum albumin (HSA) and PBS was analyzed using Radio-thin layer chromatography (Radio-TLC). The affinity of the ¹²⁴I-E6scFv-Fc was evaluated using PD-L2 protein by Radio-ELISA. Cellular uptake assays were performed using the transduced PD-L2 expressing lung cancer cell line A549 (A549-PD-L2) and wild-type A549 cells as negative control. The purified reagents were injected into athymic nude mice bearing PD-L2-positive human tumors. Micro-PET/CT imaging was conducted with ¹²⁴I-E6scFv-Fc and static images were recorded with an acquisition time of 15 min. Immunohistochemical and HE staining studies were carried out using the tumor tissue of tumor-bearing mice.

Results: The radiochemical yields of ¹²⁴I-E6scFv-Fc were 84.02±4.89% and the radiochemical purity (RCP) of the tracer was more than 99%. For in vitro stability, the tracer incubated in 0.01 M PBS and 5% HSA maintained relatively high stability (RCP>95%). Radio-ELISA showed that ¹²⁴I-E6scFv-Fc had a high affinity for the PD-L2 protein (Kd = 11.17 nM, R² = 0.92). Cellular uptake experiments confirmed that the uptake of ¹²⁴I-E6scFv-Fc in A549-PDL2 group was higher than that in A549 group and A549-PDL2-block group at each time point. The half‐lives of the distribution phase and elimination phase were 0.18 h and 9.01 h. Micro-PET/CT showed significant uptake in the tumor region of A549-PDL2 tumor-bearing mice (SUVmax = 3.53 ± 0.12 at 40h) compared with other groups. The biodistribution of the at 24h postinjection showed higher tumor uptake in A549-PDL2 mice (20.34±1.09 %ID/g for ¹²⁴I-E6scFv-Fc in A549-PDL2 mice vs 3.25±0.24 %ID/g for ¹²⁴I-E6scFv-Fc in A549 mice vs 3.20±0.51 %ID/g for ¹²⁴I-CKscFv-Fc in A549-PDL2 mice). The dosimetry estimation by using olinda software showed that the effective dose was 7.99E-02 mSv/MBq. HE staining confirmed that the subcutaneous xenograft tumor model A549-PDL2 was successfully constructed, and immunohistochemistry showed that A549-PDL2 was highly expressed in A549-PDL2 mice.

Conclusions: ¹²⁴I-E6scFv-Fc enables easy radiosynthesis and shows excellent in vitro and in vivo PD-L2 targeting characteristics. The high tumor uptake at early imaging time points demonstrate the feasibility of ¹²⁴I-E6scFv-Fc for imaging of PD-L2 expression in tumors and is encouraging for further clinical applications of screening potential beneficiaries of ICI therapy.

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