(T1330-07-43) Engineered Nanobody-Bearing Exosomes Enable Precision Trop2 Knockdown in Resistant Breast Cancer

Purpose: Trophoblast cell-surface antigen 2 (Trop2) is aberrantly over-expressed in >80 % of triple-negative and endocrine-resistant breast cancers while remaining scarce in normal tissues, making it an ideal “tumor postcode.” Nanobodies, single-domain, camelid-derived antibodies (~15 kDa), retain full antigen affinity yet offer superior solubility and thermal stability; when displayed on nanovesicles they can navigate dense tumor stroma and maintain binding under acidic pH. Leveraging these advantages, we created the first bio-engineered exosome platform that “wears” an anti-Trop2 nanobody (NB60) as a homing device, aiming to knock down surface Trop2, derail its downstream oncogenic circuitry, and re-calibrate the tumor immune milieu in breast cancer.

Methods: Exosomes were bioengineered to display Trop2-specific nanobodies on their surface. A pDisplay plasmid construct encoding the Trop2 nanobody sequence (pNB60) tagged with a Myc epitope was transfected into HEK293T cells. Expression of the nanobody was validated post-transfection via western blotting using an anti-Myc antibody. Large-scale production of NB60 exosomes was performed by culturing NB60-expressing HEK293T cells in a 500 mL PBS vertical wheel bioreactor at 25 rpm for 14 days. Exosomes were isolated through ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) for particle size, ζ potential, and concentration and fluorescent NTA using fluorescent anti-CD9 and CD63 antibodies. The experimental workflow consisted of three phases: (1) evaluating the binding of NB60 exosomes (NB60 E) to Trop2 and their knockdown efficiency in Trop2-expressing cell lines (LTLT-Ca, MDA-MB-468, and Jurkat T cells); (2) investigating the downstream effects of Trop2 knockdown on cancer hallmarks, including proliferation (MTT assay) using LTLT-Ca cells, angiogenesis (In vitro angiogenesis assay) with human umbilical vein endothelial cells (HUVECs), and cytokine signaling (ELISA) in Jurkat T cells; and (3) assessing in vivo biodistribution using Cy7-DSPE-labeled exosomes (Analytik Jena whole body imaging system) and Trop2 knockdown in LTLT-Ca tumor xenografts (western blotting).

Results: Exosome Characterization: Stable transfection of HEK293t cells with the pNB60 plasmid (Figure 1A) was confirmed by immunoblot analysis, which revealed a distinct protein band at ~25 kDa corresponding to NB60, detectable only in transfected cells (NB60-HEK293t) and absent in untransfected (HEK293t) controls. Stable expression was maintained through Geneticin (G418) selection for 30 days. Exosomes isolated from these cells exhibited characteristic sizes within the expected 30–150 nm range, and expression of NB60 was verified alongside established exosomal markers CD9 and CD63, confirming successful isolation of NB60 E (Figure 1B-F). Precision Trop2 Engagement: Three cell lines, letrozole-resistant MCF-7 (LTLT-Ca), MDA-MB-468, and Jurkat, were evaluated for Trop2 expression, with LTLT-Ca cells exhibiting the highest levels. Treatment with NB60 E significantly reduced surface Trop2 expression across all three lines: by 23.5 ± 1.5% in MDA-MB-468, 61.5 ± 1.5% in LTLT-Ca, and 55.8 ± 5.3% in Jurkat cells, relative to the HEK293t-derived control exosomes (HEK293t E) (Figure 1G-H). Oncogenic Shutdown: NB60 E treatment resulted in a marked reduction in LTLT-Ca cell viability by 52.8 ± 0.9% at 48 hours post-treatment (Figure 1I). This was accompanied by downregulation of key oncogenic signaling molecules: phosphorylated ERK (p-ERK) decreased by 30 ± 4%, cyclin D1 by 67 ± 11%, phosphorylated STAT3 (p-STAT3) by 71.8 ± 1.6%, and vimentin by 40.8 ± 1.4% (Figure 2A-D). These molecular alterations collectively support the anti-proliferative effect of NB60 E on Trop2-driven breast cancer cells. Angio-Immune Recalibration: In vitro angiogenesis assay using HUVECs revealed that treatment with NB60 E significantly impaired tubular network formation. Quantitative analysis showed marked reductions in the number of nodes (−45 ± 4.4%), junctions (−55 ± 3.5%), and branch points (−38 ± 1.2%), indicating suppression of angiogenic capacity (Figure 3A). These findings support the role of Trop2 in promoting angiogenesis and demonstrate that its blockade via NB60 E effectively inhibits this function. NB60 E modulated cytokine secretion profiles in Jurkat T cells, promoting a shift from a pro-tumorigenic to an immunostimulatory phenotype. ELISA analysis revealed significant decreases in IL-6 (−57.5 ± 1.5%, 7.4 ± 0.35 pg/mL) and TNF-α (−32.1 ± 0.3%, 6.1 ± 1.2 pg/mL) levels, accompanied by an increase in IL-2 secretion (+22.1 ± 2.7%, 49.2 ± 1.1 pg/mL) (Figure 2E), consistent with enhanced cytotoxic T cell activation and anti-tumor immunity. In Vivo Targeting and Efficacy: Near-infrared Cy7 imaging demonstrated rapid and tumor-selective accumulation of NB60 E within 4 hours post-administration, followed by efficient systemic clearance by 24 hours (Figure 3B), supporting a “hit-and-run” targeting mechanism. Furthermore, quantitative analysis of Trop2 expression in LTLT-Ca tumor xenografts revealed a significant reduction of 41 ± 4% following NB60 E treatment (Figure 3C), confirming efficient targeted delivery.

Conclusion: We present a first-in-field, nanobody-grafted exosome therapy that precisely homes to Trop2-enriched breast cancers, silences multiple growth-and-invasion pathways, blocks angiogenesis, and rewires cytokine crosstalk, achieving potent antitumor effects with self-clearing, biomimetic carriers. This modular platform is readily adaptable to other tumor antigens and sets the stage for combination with checkpoint inhibitors or payload loading for next-generation, “smart” nano-immunotherapies in refractory solid tumors.


Figure 1. Establishment of NB60-expressing HEK293T cell line and Characterization of exosomes. A. Plasmid construct design of Trop2 nanobody sequence using pDisplay vector. B. Expression of NB60 in HEK293T cells after transfection. C. Expression of NB60 in HEK293t cells after geneticin (200 mg/mL) selection for 30 days. D. Expression of NB60 in the NB60 E exosomes was confirmed by western blotting. E. Fluorescent NTA of the exosomes tagged with fluorescent CD9/CD63 antibodies. F. NTA results characterizing HEK293t and NB60 E. G. Cell lines expressing Trop2, namely, MDA-MB-468, letrozole-resistant MCF-7 (LTLT-Ca), and Jurkat cells were checked for Trop2 expression by western blot. H. Respective cell lines were treated with either HEK293t E or NB60 E for 48 h. After exosome treatment, protein lysates were collected and ran for western blot to check Trop2 knockdown. I. LTLT-Ca cells were seeded onto a 96-well plate and were treated for 48 h with varying concentration of the respective exosomes. After treatment of cells with the indicated concentrations of exosomes, MTT assay was performed to determine the viability of the cells. (*, p< 0.05; **, p<0.01; ***, p<0.001).


Figure 2. Effect of NB60 E on downstream Trop2 signaling pathways and cytokine regulation. LTLT-Ca cells were seeded onto a 10 cm plate and were treated for 48 h with 5 × 10 10 particles/ ml of respective exosomes (HEK293t E or NB60 E). After treatment of cells with exosomes, the cells were collected, and protein lysates were prepared for western blot. Different proteins related to cell signaling were probed, specifically, A. Cell proliferation/Cell cycle, B. Apoptosis/AKT-PARP pathway, C. IL-6/Stat pathway, and D. EMT-related signaling pathway. E. Jurkat cells were seeded onto a 12-well suspension plate and activated with 50 ng/ml of PMA and 1 µM of ionomycin overnight. The activated cells were then treated with respective exosomes (HEK293t E or NB60 E) and supernatant was collected at two time points (24 and 48 h) post exosome treatment. The levels of A. IL-2, B. IL-6, and TNF-α were quantified using ELISA. (+/-) are cells that were activated but no exosome treatment. (*, p< 0.05; **, p<0.01; ***, p<0.001).


Figure 3. In vitro angiogenesis and In vivo tracking of NB60 E in the tumor. A. HUVEC cells resuspended in respective exosome-enriched supernatant (HEK293t E or NB60 E supernatant) were plated into 96-well plate, previously coated with 100 μl of Matrigel. Negative control: untreated HUVEC cells. Positive control: treated with 50 ng/ml of EGF. Images were taken after 8-h incubation using NI OLYMPUS IX73 microscope, and tubular formation was analyzed using NIH ImageJ software (version 1.54). B. BALB/c athymic nude mice were subcutaneously injected with LTLT-Ca cells on their right flank region. When tumor growth becomes visible (150 mm3) (encircled), Cy7-DSPE-labeled exosomes (100 μg in 100 μl PBS, NB60 E -1×1011 particles/animal) were intraperitoneally injected into the mice. Imaging of the live mice was done at different time points (0, 2, 4, 6, 24 h). Fluorescence intensity was measured using the built-in software of the instrument, Analytik Jena UVP iBox. C. BALB/c athymic nude mice were subcutaneously injected with LTLT-Ca cells on their right flank region. When tumor growth becomes visible (150 mm3) (encircled), NB60 E (1×1011 particles/animal) were injected directly into the tumor. After 24 h of exosome treatment, the tumor was excised, and protein lysates were collected for western blotting. Tumors collected from mice not treated with NB60 E serve as the control. (*, p< 0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001).