Human epidermal growth factor receptor 2 (HER2) protein overexpression is found in ~30% of invasive breast carcinomas and in a high proportion of noninvasive ductal carcinomas in situ. Targeted cancer therapy is based on monoclonal antibodies and kinase inhibitors and reflects a new era of cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. Furthermore, there are many disadvantages with the current anti-HER2 drug, including drug resistance and adverse effects. Nanobodies (15 kDa), single-domain antibody (sdAb) fragments, can overcome these limitations. This study produced the recombinant sdAb against the HER2-tyrosine kinase (HER2-TK) domain using phage display technology. Three specific anti-HER2-TK sdAbs were selected for further characterization. Hallmark V_HH residue identification and amino acid sequence analysis revealed that clone numbers 4 and 22 were VH antibodies, whereas clone number 17 was a V_H H antibody (nanobody). The half-maximal inhibitory concentration of V_HH17 exhibited significantly greater HER2 kinase-inhibition activity than the other clones. Consistent with these results, several charges and polar residues of the HER2-TK activation loop that were predicted based on mimotope analysis also appeared in the docking result and interacted via the CDR1, CDR2 and CDR3 loops of V_HH17. Furthermore, the cell-penetrable V_HH17 (R9 V_HH17) showed cell-penetrability and significantly decreased HER2-positive cancer cell viability. Thus, the V_H H17 could be developed as an effective therapeutic agent to treat HER2-positive breast cancer.

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of proteins and are involved in downstream signal transduction, plays prominent roles in cell growth regulation, proliferation, and the differentiation of many cell types. They are correlated with the stage and severity of cancer. Therefore, EGFRs are targeted proteins for the design of new drugs to treat cancers that overexpress these proteins. Currently, several bioactive natural extracts are being studied for therapeutic purposes. Cannabis has been reported in many studies to have beneficial medicinal effects, such as anti-inflammatory, analgesic, antibacterial, and anti-inflammatory effects, and antitumor activity. However, it is unclear whether cannabinoids reduce intracellular signaling by inhibiting tyrosine kinase phosphorylation. In this study, cannabinoids (CBD, CBG, and CBN) were simulated for binding to the EGFR-intracellular domain to evaluate the binding energy and binding mode based on molecular docking simulation. The results showed that the binding site was almost always located at the kinase active site. In addition, the compounds were tested for binding affinity and demonstrated their ability to inhibit kinase enzymes. Furthermore, the compounds potently inhibited cellular survival and apoptosis induction in either of the EGFR-overexpressing cell lines.

Background: The epidermal growth factor receptor (EGFR) overexpression is found in metastatic colorectal cancer (mCRC). Targeted molecular therapies such as monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKI) are becoming more precise, targeting specifically for cancer therapeutics. However, there are adverse effects of currently available anti-EGFR drugs, including drug-resistant and side effects. Nanobodies can overcome these limitations. Our previous study has found that cell-penetrable nanobodies targeted at EGFR-tyrosine kinase were significantly reduced EGFR-positive lung cancer cells viability and proliferation. The aim of the present study was to determine the effect of cell-penetrable nanobody (R9VH36) on cell viability and proteomic profile in EGFR-positive human colorectal cancer cell lines. Methods: The human colorectal carcinoma cell line (SW480) was treated with R9VH36, compared with gefitinib. Cell viability was monitored using the MTT cell viability assay. The proteomic profiling was analyzed by LC–MS/MS . Results: The half-maximal inhibitory concentration (IC₅₀) values determined for R9VH36 and gefitinib against SW480 were 527 ± 0.03 nM and 13.31 ± 0.02 μM, respectively. Moreover, both the gefitinib-treated group and nanobody-treated group had completely different proteome profiles. A total 6626 differentially expressed proteins were identified. PCA analysis revealed different proteome profiling in R9VH36 experiment. There were 8 proteins in R9VH36 that significantly exhibited opposite expression directions when compared to gefitinib. These proteins are involved in DNA-damage checkpoint processes. Conclusion: The proteomics explored those 6,626 proteins had different expressions between R9VH36 and gefitinib. There were 8 proteins in R9VH36 exhibited opposite expression direction when comparing to gefitinib. Our findings suggest that R9VH36 has the potential to be an alternative remedy for treating EGFR-positive colon cancer.

Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor within the ErbB family that plays a pivotal role in the progression of various aggressive cancers. HER2-positive tumors often develop resistance to standard therapies, necessitating the exploration of innovative treatment options. Cannabinoids, bioactive compounds from Cannabis sativa such as cannabidiol (CBD), cannabigerol (CBG), and cannabinol (CBN), have gained attention for their potential anticancer properties. This study evaluates the efficacy of CBD, CBG, and CBN in targeting HER2-positive ovarian cancer through kinase inhibition assays, surface plasmon resonance (SPR), molecular docking, and cell viability assessments. SPR analysis revealed that cannabinoids bind strongly to HER2-tyrosine kinase (HER2-TK), with CBD showing the highest affinity (K _D = 6.16 μM), significantly better than afatinib (K _D = 26.30 μM), and CBG demonstrating moderate affinity (K _D = 17.07 μM). In kinase inhibition assays, CBG was the most potent inhibitor (IC₅₀ = 24.7 nM), followed by CBD (IC₅₀ = 38 nM), suggesting their ability to disrupt HER2-mediated signaling pathways. Molecular docking studies highlighted critical interactions between cannabinoids and essential HER2 residues (Leu796, Thr862, Asp863). In cell viability assays, CBD and CBG effectively inhibited the growth of HER2-positive SKOV3 cells (IC₅₀ = 13.8 μM and 16.6 μM, respectively), comparable to traditional tyrosine kinase inhibitors. These findings underscore the therapeutic potential of cannabinoids, particularly CBD and CBG, as alternative or adjunct therapies for HER2-positive cancers, with the promise of mitigating resistance and adverse effects associated with existing treatments.

Rattanabunyong S, et al. J Vet Sci. 2023 Aug;24:e67. https://doi.org/10.4142/jvs.22326