Theranostic vNAR-Based Immunoconjugates Achieve Selective Intracellular Cisplatin Delivery in Embedded 3D HER2-Positive Breast Cancer In Vitro Model

Background/Objectives: Precise intracellular delivery of chemotherapeutics remains a major challenge in HER2-positive breast cancer, where intratumoral heterogeneity and limited tissue penetration constrain efficacy. A key contributor is the tumor-restricted epidermal growth factor receptor variant III (EGFRvIII), a constitutively active, ligand-independent mutant generated by deletion of exons 2–7. Although classically associated with glioblastoma, lung (NSCLC), head/neck, and prostate cancers, EGFRvIII is also present in subsets of HER2-positive breast cancers, where low-abundance subclones drive aggressive phenotypes and attenuate therapeutic responses. HER2–EGFRvIII co-expression amplifies oncogenic signaling, supported by frequent co-expression in ErbB2-positive primary tumors and metastases, and by sustained receptor phosphorylation in the absence of EGFR gene amplification, depicting EGFRvIII as a compelling therapeutic target. Methods: We evaluated the shark-derived single-domain antibody vNAR R426 as a modular theranostic platform for receptor-mediated cisplatin delivery. Conjugation to cisplatin and fluorescein enabled simultaneous intracellular drug transport and immunofluorescence-based detection in EGFRvIII-positive SKBR3 cells and 3D spheroids. The compact vNAR-based immunoconjugates support efficient receptor recognition, internalization, and intracellular trafficking, features rarely achieved by conventional IgG antibodies. Results: vNARCDDP elicited robust, receptor-mediated cytotoxicity, achieving an IC50 of 2.68 µM—approximately 50-fold lower than that of free cisplatin—while unconjugated vNAR maintained scaffold biocompatibility. In three-dimensional spheroid models, the theranostic vNAR (vNARCDDP+FITC) exhibited deep and uniform penetration throughout tumor-like architectures, with immunofluorescence intensity closely correlating with regions of intracellular drug delivery and the initiation of cytotoxic responses. Notably, cisplatin conjugation did not impair tissue diffusion or receptor engagement, facilitating effective payload delivery to both peripheral and central cell populations. Conclusions: By integrating tumor-restricted targeting and efficient intracellular drug delivery within a modular single-domain scaffold, vNAR R426 represents a next-generation theranostic platform capable of addressing intratumoral heterogeneity. This approach combines potent cytotoxic activity with immunofluorescence-based detection, thereby advancing the rational design of precision therapeutics for HER2-positive breast cancer.