Axl and EGFR Dual-Specific Binding Affibody for Targeted Therapy in Nasopharyngeal Carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is a tumor of the head and neck, with a higher incidence in southern China and Southeast Asia. Radiotherapy and chemotherapy are the main treatments; however, metastasis and recurrence remain the main causes of treatment failure. Further, the majority of patients are diagnosed in the late stage due to lack of tumor-specific biomarker for early diagnosis. Therefore, an effective treatment and early detection can improve the outcome of patient with NPC. Axl and EGFR are co-expressed in NPC tissues and play key roles in tumor proliferation, migration, and invasion, which are often correlated with poor prognosis and therapy resistance. In this study, we generated a novel bispecific affibody (Z_239-1907) for the dual targeting and inhibition of Axl and EGFR expression in NPC-positive cells both in vitro and in vivo. The in vitro experiments demonstrated that Z_239-1907 had more pronounced antitumor effects than either modality alone (Z_AXL239 or Z_EGFR1907) in NPC-positive cells. Further, mice bearing NPC-positive tumors showed significant inhibition in tumor growth after treatment with Z_239-1907 compared to Z_AXL239 and Z_EGFR1907. The in vivo tumor targeting ability and imaging also showed that Z_239-1907 specifically and selectively targeted NPC xenograft mice models and accumulate at tumor site as early as 30 min and disappeared within 24 h post-injection. Collectively, these results suggest that Z_239-1907 dual-target affibody is a promising therapeutic agent and a molecular imaging probe for early diagnosis in NPC.

Keywords: Axl; EGFR; affibody molecules; nasopharyngeal carcinoma; targeted therapy.

Figure 1

Protein expression and purification. (A) Schematic representation of pET21a (+)/Z_239-1907 plasmid construct. (B) Three-dimensional structure prediction by Swiss model. (C) SDS-PAGE and Coomassie brilliant blue staining of the recombinant proteins. M, protein marker; 1, empty E. coli BL21; 2, transformed pET21a (+) empty vector of E. coli BL21; 3–6, E. coli BL21 transformed with pET21a (+)/Z_239-1907, pET21a (+)/Z_AXL239, pET21a (+)/Z_EGFR1907, and pET21a (+)/Z_WT induced with IPTG, respectively. (D) Purified proteins analyzed by SDS-PAGE. (E) Protein identification was further confirmed by Western blot.

Figure 2

Binding analysis. (A,B) Sensograms measuring the binding interaction of Z_239-1907, Z_AXL239, Z_EGFR1907, and Z_WT to Axl and EGFR, respectively. (C) Sensograms obtained after injection of the highest concentration 10 μM of affibodies to flow over the sensor chips. Z_WT was used as a negative control. Three independent experiments were performed in triplicate.

Figure 3

Immunofluorescence assay. (A) Western blot protein expression of NPC-positive cell lines and NPC-negative cell line. (B) C666-1, NPC/HK-1, MKN-45, and TC-1 cell lines were treated with Z_239-1907, Z_AXL239, Z_EGFR1907, or Z_WT. Corresponding antibodies are labeled with FITC (green), and cell nuclei are stained with Hoechst 3342 (400×). (C) Mean fluorescence intensity of Z_239-1907, Z_AXL239, Z_EGFR1907, and Z_WT-treated cell lines. Data are given as mean ± SD (n = 3). ** p < 0.01; *** p < 0.001 compared to SPA-Z scaffold (ZWT).

Figure 4

NIR fluorescence tumor imaging. (A) Images were recorded at different time points after injection with Dylight-755-labeled Z_239-1907, Dylight-755-labeled Z_AXL239, or Dylight-755-labeled Z_EGFR1907. Dylight-755-labeled Z_WT was set as negative control. (B,C) Calculated tumor/skin fluorescence intensity ratio post-injection of affibody molecules. Data are given as the mean ± SD (n = 3). ** p < 0.01; *** p < 0.001 compared to SPA-Z scaffold (Z_WT).

Figure 5

Affibody molecules inhibited NPC-positive cell proliferation. (A–C) CCK-8, wound-healing, and EdU were used to determine the effect of Z_239-1907, Z_AXL239, and Z_EGFR1907 on the proliferation of NPC-positive cells. Z_WT set as negative control. Data are given as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 compared to SPA-Z scaffold (Z_WT).

Figure 6

Signal transduction pathway. (A) Downregulation of p-MEK after Z_239-1907 treatment in a concentration- and time-dependent manner. (B) Treatment with either Z_239-1907, Z_AXL239, or Z_EGFR1907 downregulated signaling proteins and transcription genes. (C) Image of the schematic representation of Z_239-1907 affibody molecule blocking MAPK-ERK signaling pathway. Experiments were performed in triplicate.

Figure 7

Assessment of therapeutic efficacy of affibody molecules. (A) Schematic illustration of affibody molecules treatment in mouse model. (B,C) Tumor volume of mice with different treatments (PBS, Z_WT, Z_239-1907, Z_AXL239, Z_EGFR1907, and cisplatin, n= 6/group). (D,E) Tumor separated from mice under different treatment regimens. (F,G) Tumor weights in different groups of mice. Data are given as mean ± SD (n = 5). * p < 0.05; ** p < 0.01 compared to SPA-Z scaffold (Z_WT).