Lifang Zhang

Human papillomavirus (HPV) 18 E6 oncoprotein drives cervical carcinogenesis by degrading p53, enabling uncontrolled cell proliferation. In this study, we developed novel trans-activator of transcription (TAT)-conjugated Affibody molecules (TAT-Z_HPV18E6) targeting HPV18 E6 for therapeutic applications. High-affinity Affibody variants were screened by phage display using recombinant HPV18 E6 expressed in Escherichia coli. Three candidates (TAT-Z_HPV18E6: 4, 59, 352) exhibited high binding affinity, with equilibrium dissociation constant (K_D) ranging from 10^-6 to 10^-4 M, with TAT-Z_HPV18E6: 59 showing superior specificity for native HPV18 E6 in HeLa229 cells, as validated by surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and immuno-fluorescence. In vivo near-infrared fluorescence imaging of DyLight 755-labeled TAT-Z_HPV18E6: 59 in tumor-bearing mice demonstrated rapid tumor accumulation (peak at 2 h) and prolonged retention (> 12 h). Mechanistically, TAT-Z_HPV18E6: 59 restored p53 stability and upregulated pro-apoptotic factors (Bax, PUMA) and cell cycle regulator p21. Notably, combined treatment with TAT-Z_HPV18E6: 59 and E7-targeting TAT-Z_HPV18E7: 228 combinatorial enhanced apoptosis and suppressed HeLa229 proliferation, as confirmed by CCK-8 and clonogenic assays. These results demonstrate the utility of TAT-Z_HPV18E6 Affibody molecules as targeted agents, highlighting combinatorial E6/E7 targeting as a potent strategy for HPV18-driven cervical cancer therapy. KEY POINTS: • TAT-Z_HPV18E6: 59 achieved high affinity for HPV18 E6, enabling precise molecular targeting. • Demonstrates dual utility for in vivo imaging and p53-dependent tumor suppression. • Dual targeting of E6/E7 yields synergistic therapeutic efficacy in cervical cancer models.

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Gastric cancer (GC) has a poor prognosis and high mortality because it is often diagnosed at an advanced stage. Targeted therapeutics are considered an important class for advanced GC treatment. However, the fewer effective therapeutic targets and the poor coverage of the GC population limit the use of GC targeted therapies. Recent research suggests that the AXL receptor tyrosine kinase (AXL) plays an vital role in the survival and proliferation of GC cells, and blocking AXL pathway may be an effective strategy for targeted therapies. On the other hand, the affibody molecule, with its small size and faster penetration of tissue, has great potential in tumor imaging and targeted therapy. In this study, we report the novel AXL-binding affibody molecules (Z_AXL:239) screened by a phage-displayed peptide library. The Z_AXL:239 could specifically bind and interact with AXL proteins in vitro and in vivo, as demonstrated by surface plasmon resonance, co-immunoprecipitation, immuno-fluorescence co-localization, and near infrared fluorescent imaging. In addition, Z_AXL:239 affibody molecules could significantly inhibit the proliferative activity and induce apoptosis of AXL-positive GC cells by decreasing the phosphorylation levels of the PI3K/AKT1 and MEK/ERK pathway, leading to the suppression of the downstream nuclear protein c-myc. Moreover, Z_AXL:239 was found to have significant anti-tumor effects in AXL-positive GC transplantation tumor nude mouse models. In brief, we provide strong evidence that the novel Z_AXL:239 affibody molecules have great potential as a potent tumor-specific molecular imaging and targeted therapeutic agents for GC.

Gastrointestinal cancer is a common malignant tumor with a high incidence worldwide. Despite continuous improvements in diagnosis and treatment strategies, the overall prognosis of gastrointestinal tumors remains poor. Carcinoembryonic antigen (CEA) is highly expressed in various types of cancers, especially in gastrointestinal cancers, making it a potential target for therapeutic intervention. Therefore, the expression of CEA can be used as an indication of the existence of tumors, chosen as a target for molecular imaging diagnosis, and effectively utilized in the targeted therapy of gastrointestinal cancers. In this study, we report the selection and characterization of affibody molecules (Z _CEA 539, Z _CEA 546, and Z _CEA 919) specific to the CEA protein. Their ability to bind to recombinant and native CEA protein has been confirmed by surface plasmon resonance (SPR), immunofluorescence, and immunohistochemistry assays. Furthermore, Dylight755-labeled Z _CEA affibody showed accumulation within the tumor site 1 h post injection and was continuously enhanced for 4 h. The Dylight755-labeled Z _CEA affibody exhibited high tumor-targeting specificity in CEA+ xenograft-bearing mice and possesses promising characteristics for tumor-targeting imaging. Overall, our results suggest the potential use of Z _CEA affibodies as fluorescent molecular imaging probes for detecting CEA expression in gastrointestinal cancer.

SARS-CoV-2 and its variants are threatening the whole world. Although a full dose of vaccine injection showed great preventive effects and monoclonal antibody reagents have also been used for a specific treatment, the global pandemic persists.

Nasopharyngeal carcinoma (NPC), is an Epstein-Barr virus (EBV) associated malignancy most common in Southern China and Southeast Asia. In southern China, it is one of the major causes of cancer-related death. Despite improvement in radiotherapy and chemotherapy techniques, locoregional recurrence and distant metastasis remains the major causes for failure of treatment in NPC patients. Therefore, finding new specific drug targets for treatment interventions are urgently needed. Here, we report three potential Z_LMP1-C affibody molecules (Z_LMP1-C15, Z_LMP1-C114 and Z_LMP1-C277) that showed specific binding interactions for recombinant and native EBV LMP1 as determined by epitope mapping, co-localization and co-immunoprecipitation assays. The Z_LMP1-C affibody molecules exhibited high antitumor effects on EBV-positive NPC cell lines and displayed minimal cytotoxicity towards EBV-negative NPC cell line. Moreover, Z_LMP1-C277 showed higher antitumor efficacy than Z_LMP1-C15 and Z_LMP1-C114 affibody molecules. The ability of Z_LMP1-C277 decrease the phosphorylation levels of up-stream activator phospho-Raf-1^(Ser338), phospho-MEK1/2^{(Ser217/Ser221)}, phospho-ERK1/2^{(Thr202/Thr204)}, thereby leading to downstream suppression of phospho-p90RSK^(Ser380) and transcription factor c-Fos. Importantly, tumor growth was reduced in tumor-bearing mice treated with Z_LMP1-C277 and caused no apparent toxicity. Taken together, our findings provide evidence that Z_LMP1-C277 as a promising therapeutic agent in EBV-associated NPC.