Aptamers, serving as unique targeting ligands, make aptamer-drug conjugates (ApDCs) an attractive strategy for targeted cancer therapy. Su et al. [1] developed a PTK7-targeting ApDC (Sgc8c-M) by conjugating the potent anti-mitotic agent monomethyl auristatin E (MMAE) with the classic PTK7 aptamer Sgc8c. They validated its targeting ability, internalization, and cytotoxicity in vitro, and demonstrated its potent anti-tumor efficacy in triple-negative breast cancer models. Efficacy studies in multiple PTK7-overexpressing cancer types showed that Sgc8c-M effectively induced sustained regression in both cell line-derived and patient-derived xenografts, outperforming unconjugated MMAE, the chemotherapy drug paclitaxel, and a PTK7-targeting antibody-drug conjugate. In certain models, the preclinical efficacy of Sgc8c-M surpassed that of h6M24-vc0101, with a broader therapeutic window. These findings highlight the potential of Sgc8c-M as an effective anti-tumor agent and provide useful insights for the clinical translation of emerging ApDCs.

Synthesis and Characterization of the ApDC (Sgc8c-M)

To construct the ApDC Sgc8c-M, a one-step Michael addition reaction was employed, reacting the 3'-thiol-modified aptamer Sgc8c-SH with the maleimide-modified drug MC-VC-PAB-MMAE (VcMMAE). Successful synthesis and purification of Sgc8c-M were confirmed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) (Figure 1).

Figure 1: Structure and characterization of the ApDC (Sgc8c-M)

Evaluation of Sgc8c-M Targeting, Internalization, and Cytotoxicity In Vitro

The study first assessed the in vitro targeting ability of Sgc8c-M at both the protein and cellular levels. At the protein level, Sgc8c effectively bound to recombinant PTK7 proteins from mouse, rat, cynomolgus monkey, and human, with comparable equilibrium dissociation constants (Kd) across species (Figure 1a). After MMAE conjugation, Sgc8c-M maintained strong binding to PTK7 proteins across species, with Kd values similar to those of the unconjugated aptamer Sgc8c (Figure 1b). In contrast, Ctrl-M showed no binding to human PTK7 recombinant protein. At the cellular level, the aptamer was labeled with Cy5 dye to enable investigation of cell binding by flow cytometry. Using PTK7-positive triple-negative breast cancer cell lines SUM159 and MDA-MB-468 as examples, Cy5-Sgc8c and Cy5-Sgc8c-M were observed to have significantly higher binding affinity to these cells compared to the controls Cy5-Ctrl and Cy5-Ctrl-M (Figure 1c-d). Notably, Cy5-Sgc8c and Cy5-Sgc8c-M showed minimal binding to PTK7-negative Ramos cells. These results indicated that the affinity and specificity of Sgc8c were not compromised by MMAE conjugation.

Figure 2: Validation of Sgc8c-M targeting at the protein and cellular levels in vitro

Subsequently, the study investigated the intracellular internalization process and endocytic pathways of Cy5-Sgc8c-M using flow cytometry. Inhibitors for three classical endocytic pathways were employed: amiloride hydrochloride (AMI) for macropinocytosis, chlorpromazine hydrochloride (CPZ) for clathrin-mediated endocytosis, and methyl-β-cyclodextrin (M-β-CD) for caveolae-mediated endocytosis. These inhibitors were confirmed to be non-cytotoxic to the cells (Figure 3a). In SUM159 cells, internalization of Cy5-Sgc8c-M was significantly inhibited by both AMI and CPZ, with AMI showing a more pronounced inhibitory effect (Figure 3b). These results suggested that Cy5-Sgc8c-M enters SUM159 cells via macropinocytosis and clathrin-mediated endocytosis. Clathrin-mediated endocytosis is a receptor-mediated process, indicating that Cy5-Sgc8c-M specifically recognizes and binds to PTK7 on the cell membrane, subsequently being internalized via this pathway. Regarding macropinocytosis, previous studies have indicated it can be stimulated by the receptor tyrosine kinase EGFR. Notably, PTK7 has been shown to promote metastasis and progression of triple-negative breast cancer through the EGFR/Akt signaling pathway, suggesting a plausible link between PTK7 and macropinocytosis. The endocytic pathways of Sgc8c-M could be further validated in the future via siRNA-mediated knockdown or genetic depletion of key endocytic regulators. In MDA-MB-468 cells, internalization of Cy5-Sgc8c-M was inhibited only by the macropinocytosis inhibitor AMI (Figure 3c). To visualize the internalization process, confocal microscopy was performed, revealing that Cy5-Sgc8c-M rapidly entered the lysosomes of SUM159 cells. At 0.5 hours, Cy5-Sgc8c-M was primarily located on the cell membrane, whereas at 2 hours, the majority of Cy5-Sgc8c-M co-localized with lysosomes (Figure 3d). Finally, the cytotoxicity of Sgc8c-M in SUM159 and MDA-MB-468 cells was evaluated. Results showed that Sgc8c-M exhibited greater toxicity than unconjugated VcMMAE (Figure 3e).

Figure 3: Validation of Cy5-Sgc8c-M internalization process and endocytic pathways in cells

Upon conjugation with the aptamer, it is hypothesized that VcMMAE more readily binds to target cells and is internalized into lysosomes, where the specific linker is cleaved upon arrival, facilitating MMAE release and subsequent cell killing. The study also evaluated the cytotoxicity of Sgc8c-M against human normal ovarian epithelial cells (IOSE80) and PTK7-negative cancer cells (A549). The IC50 values for these cells were approximately six times higher than those for PTK7-positive SUM159 cancer cells (Supplementary Figure 4), indicating that Sgc8c-M can kill PTK7-positive tumor cells with high specificity.

Figure 4: Validation of cell killing activity

Potent Anti-tumor Efficacy of Sgc8c-M in Triple-Negative Breast Cancer Models

PTK7 is highly expressed in triple-negative breast cancer (TNBC) and correlates with poor prognosis. Several studies have investigated PTK7 ADCs to treat TNBC and improve this poor outcome. To further advance these efforts, the study first tested the therapeutic potential of Sgc8c-M in TNBC. To validate the in vivo targeting capability of Sgc8c-M, in vivo fluorescence imaging was performed on mice bearing TNBC MDA-MB-468 tumors using Cy5-Sgc8c-M (Figure 5). Results showed that at 90 and 120 minutes post-injection, the fluorescence intensity of Cy5-Sgc8c-M at the tumor site was significantly stronger than that of Cy5-Ctrl-M (Figure 5a). After euthanizing the mice at 2 hours, ex vivo imaging revealed significantly greater accumulation of Cy5-Sgc8c-M in tumors compared to Cy5-Ctrl-M (Figure 5c). Quantitative fluorescence analysis indicated that the concentration of Cy5-Sgc8c-M in tumors was more than twice that of Cy5-Ctrl-M (P < 0.05), while no significant differences were observed in normal organs between the two treatment groups (Figure 5d). These findings highlight the excellent in vivo targeting ability of Sgc8c-M, demonstrating its specific enrichment in PTK7-overexpressing tumors.

Figure 5: Validation of Sgc8c-M in vivo targeting capability

To elucidate the targeting mechanism of Sgc8c-M, the study further investigated co-localization of Sgc8c-M with PTK7 protein in another TNBC model, SUM159, using immunofluorescence imaging. Cy5-Sgc8c-M showed clear co-localization with PTK7 protein in both cells and xenograft tumor sections (Figure 6). These findings provide direct visual evidence that Sgc8c-M specifically targets PTK7-overexpressing cancer cells both in vitro and in vivo.

Figure 6: Co-localization study of Sgc8c-M and PTK7 in SUM159 cells and tumor samples

For ovarian cancer, the study utilized the OVCAR3 cell line-derived xenograft (CDX) model, characterized by PTK7 overexpression and commonly used to evaluate the therapeutic efficacy of PTK7-targeting ADCs, including h6M24-vc0101 and MTX-13 (Figure 7a). Cytotoxicity assay results at 72 hours were consistent with observations in HT-29 and NCI-H1975 cells, with an IC50 value of 27.94 nM for Sgc8c-M compared to 105.3 nM for VcMMAE (Figure 7b-c). Treatment with Sgc8c-M at doses of 7 mg/kg administered every 4 days (q4d × 5) and every 7 days (q7d × 5) resulted in significantly more sustained regression of OVCAR3 tumors in vivo compared to paclitaxel (Figure 7d). These findings suggest that Sgc8c-M demonstrated superior therapeutic efficacy in treating OVCAR3 tumors, which literature indicates exhibit rapid recurrence, compared to h6M24-vc0101 (3 mg/kg, q4d × 4). This result prompted the construction of an ADC, h6M24-VcMMAE, combining the antibody from h6M24-vc0101 with the same MMAE payload used in Sgc8c-M for further comparison. The Kd value of h6M24-VcMMAE for human PTK7 protein was determined to be 3.18 nM (Figure 7e), comparable to the binding affinity reported for h6M24-vc0101 in the literature. Impressively, as shown in Figure 7f, in the OVCAR3 model, Sgc8c-M (3.5 mg/kg, q4d × 4) demonstrated significantly stronger therapeutic efficacy than h6M24-VcMMAE (5 mg/kg, q4d × 4).

Figure 7: Preclinical efficacy of Sgc8c-M in the OVCAR3 CDX model

This study successfully developed a potent PTK7-targeting aptamer-drug conjugate (ApDC) with broad, robust anti-tumor activity and a favorable safety profile. Clinical efficacy of h6M24-vc0101 indicates that PTK7 is a promising therapeutic target. In certain models, the preclinical efficacy of Sgc8c-M surpassed that of h6M24-vc0101, with a broader therapeutic window. These preclinical results suggest that Sgc8c-M may offer a new treatment option for PTK7-expressing solid tumors in the clinical setting and hold promise for advancing the clinical translation of aptamer-based targeted cancer therapies to improve patient outcomes.

TekBiotech has established a comprehensive SELEX platform, capable of providing targeted aptamer discovery services using various screening methods, including MB-SELEX, Cell-SELEX, and CE-SELEX, to meet the requirements of different target types. Additionally, we offer a full range of high-quality downstream services, including aptamer modification, bispecific aptamer development, aptamer-drug conjugate (ApDC) development, and various downstream development and validation services such as in vitro affinity validation (including EC50 validation, BLI/SPR affinity validation), cytotoxicity and targeting validation, in vivo animal imaging validation, and animal model efficacy validation. These services provide novel approaches and technical support for our clients' research projects and small molecule drug development.

References

[1] Su, M., Liu, Y., Lin, H. et al. An aptamer-drug conjugate for promising cancer therapy with comprehensive evaluation from rodents to non-human primates. Sig Transduct Target Ther 10, 316 (2025).