G-2882

Development of a Novel Anti-PDGFRα Single-Chain Variable Fragment for Enhanced Molecular Diagnosis of Thyroid Cancer

 Atiyeh Dabouian ¹ © ℗, Mohammad Ebrahim Khamseh ²   

 
Abstract

Abstract Introduction: Thyroid cancer, particularly papillary thyroid carcinoma (PTC), has emerged as a growing global health concern, with incidence rates increasing by approximately 3% annually over the last ten years. Current diagnostic methods, such as ultrasound and fine-needle aspiration biopsy, often lack the molecular precision required for early-stage detection and accurate risk stratification. Platelet-derived growth factor receptor alpha (PDGFRα), a transmembrane tyrosine kinase receptor, is overexpressed in 60–80% of thyroid malignancies and plays a critical role in tumor angiogenesis and metastatic spread. Targeting PDGFRα with high-specificity probes could revolutionize both diagnosis and therapy. Single-chain variable fragments (scFvs), owing to their compact structure and target-binding efficiency, are ideal candidates for molecular imaging. This study focuses on the development and in vitro evaluation of a novel anti-PDGFRα scFv designed to enhance diagnostic accuracy in thyroid cancer. Materials and Methods: A codon-optimized DNA sequence encoding the anti-PDGFRα scFv was cloned into pColdII vector and expressed in E. coli SHuffle® T7 cells. Protein purification was performed using Ni-NTA affinity chromatography. Binding specificity and cytotoxicity were evaluated in PDGFRα-positive thyroid cancer lines (BCPAP, 8305C) and negative CHO-K1 cells. Methods included flow cytometry, cell-based ELISA, MTT proliferation assays (scFv concentrations: 1–100 μg/mL, 24–72 h), and Annexin V/PI staining for apoptosis (1–10 μg/mL, 48 h). PDGFRα expression was quantified using qRT-PCR. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (p 0.05 considered significant). Results: The scFv was successfully expressed with a molecular weight of approximately 28 kDa. Flow cytometry and ELISA confirmed its dose-dependent binding to PDGFRα in BCPAP and 8305C cells, with negligible binding to CHO-K1. Dissociation constants (Kd) were ≈148.5 μg/mL (BCPAP) and ≈138.8 μg/mL (8305C). Cytotoxicity assays showed no significant viability reduction at ≤100 μg/mL (P 0.05). qRT-PCR verified higher PDGFRα expression in BCPAP compared to 8305C and CHO-K1. Discussion and Conclusion: The anti-PDGFRα scFv demonstrates high specificity and moderate affinity for PDGFRα-expressing thyroid cancer cells, supporting its potential as a molecular imaging probe. The absence of cytotoxicity at tested therapeutic doses combined with apoptosis induction at higher concentrations highlights its diagnostic and potential therapeutic versatility. Future studies should optimize in vivo targeting and assess clinical applicability. This work advances scFv-based strategies for precision oncology in thyroid cancer.

Keywords: PDGFRα, single-chain variable fragment (scFv), thyroid cancer, molecular imaging