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@#Objective To prepare platelet-derived growth factor receptor β (PDGFRβ)-targeted near-infrared molecular probe and evaluate its potential in optical molecular imaging of lung cancer. Methods PDGFRβ-specific affibody Z-tri was recombinantly expressed in Escherichia coli (E. coli) and purified using affinity chromatography. In vitro cell-binding of Z-tri was analyzed by flow cytometry. Cellular distribution of Z-tri in tumor grafts was determined by protein-tracing. The molecular probe CF750-Z-tri was prepared by conjugating near-infrared fluorescent dye CF750 to Z-tri. The optical images of xenografts of lung cancer were obtained by using CF750-Z-tri combined with optical imaging system. Results PDGFRβ-specific affibody Z-tri was highly expressed in E. coli and purified to homogeneity. Z-tri could bind PDGFRβ-positive cells but not PDGFRβ-negative cells cultured in vitro. In the tumor xenografts of human lung cancer, intravenously injected Z-tri was predominantly distributed on cells overexpressing PDGFRβ. The near infrared fluorescent dye CF750 was efficiently conjugated to Z-tri. Optical images with high contrast of lung cancer xenografts were produced by using the near-infrared fluorescent probe CF750-Z-tri combined with optical imaging system. Conclusion The near-infrared fluorescent probe CF750-Z-tri can be used for optical imaging of human lung cancer, which takes great potential in optical imaging-guided surgery of lung cancer.
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Objective To prepare the 99Tcm-labeled human epidermal growth factor receptor type 2 (HER2) affibody molecule ZHER2:342 and evaluate its receptor binding specificity in vitro.Methods The molecular ZHERa:342 was labeled with 99Tcm using the ligand exchange method.The labeling efficiency and radiochemical purity were measured by HPLC.The major factors,such as the mass of SnC12 and NaOH and reaction time were analyzed,and the optimal method was summarized.Cell binding kinetics and cellular retention of the probe were investigated in HER2-expressing SKOV-3 cells and MDA-MB-231 cells with low HER2 expression respectively.HER2 binding specificity of 99Tcm-ZHER2:342 was analyzed by a pre-injection of excess unlabeled ZHER2:342 to saturate HER2 receptors.One-way analysis of variance and two-sample t test were used.Results The optimal labeling procedure was as follows:5 μg (1 g/L) of ZHER2:342 was mixed with 5 μg of NaOH (1 g/L),then 8.8 μg SnC12(1 g/L,solution) was added,followed by 150 μl (37 MBq) 99TcmO4-solution,and finally the mixture was slightly vortexed and incubated for 1 h at room temperature.99TcmZHER2:342 was stable in vitro with a high labeling efficiency of (98.10± 1.73)％.The radiochemical purity was ＞ 98％,and was more than 85％ after the incubation for 24 h in saline and fresh human serum.The cell binding of 99Tcm-ZHER2:342 with HER2-expressing SKOV-3 cells gradually increased over time with a peak of (9.95± 1.02)％ at 6 h.The binding of 99Tcm-ZHER2:342 in SKOV-3 cells was significantly higher than that in MDA-MB-231 cells at every time point (5.68-9.88 vs 0.56-2.11 ; t:from-34.50 to-13.14,all P＜0.01).The labeled molecular probe retained the capacity to bind specifically to HER2-expressing SKOV-3 cells since the cell binding decreased from (9.95 ± 1.02) ％ to (2.11 ±0.27) ％ after receptor saturation (t =-13.14,P＜0.01).Conclusions 99Tcm-ZHER2:342 has a high labeling efficiency,good stability and optimal binding specificity.These characteristics enable it to be a promising molecular probe for HER2-targeting imaging.
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Objective To label the modified human epidermal growth factor receptor 2 (HER2) affibody with 68Ga at cysteine position of carboxyl-terminal Gly-Gly-Gly-Cys (GGGC) sequence,and to image the mice model bearing HER2-expressed breast cancer with microPET.Methods The HER2 affibody with carboxyl-terminal cysteine was produced by gene engineering.The 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid (DOTA) was conjugated to the thiol (SH)-group of cysteine by DOTA-10-maleimidoethylacetamide (MMA-DOTA).The newly eluted 68Ga was labeled to DOTA-HER2 affibody at 90 ℃.The labeled mixture was purified by C18 cartridge and eluted with anhydrous alcohol.The purified 68Ga-labeled affibody was prepared for cell binding analysis and microPET imaging.The HER2-expressed breast cancer cells MBA-MD-361 were used to analyze cell binding and establish cancer model with BALB/c nude mice.68Ga-labeled affibody (3.7 MBq) was administered to 4 nude mice with breast cancer via tail vein for microPET imaging at 20 and 60 min post injection.The mice were sacrificed immediately after imaging in unconscious state and their tissue/organs (tumor,muscle,bone and heart) were removed for weighing and radioactivity counting by γ counter.The tumor to normal organ ratios of radioactivity (tumor to liver,muscle,bone and heart)were calculated.Results The purity of HER2 affibody was more than 95％.The radiochemical purity of 68Ga-labeled HER2 affibody was 97％.The KD value (affinity) of 68Ga-labeled HER2 affibody was 1.5 nmol/L in HER2positive cells.MicroPET imaging showed that 68Ga-labeled HER2 affibody could bind HER2-positive cancer rapidly and excreted mainly through urinary system.The radioactivity ratios of tumor to liver,muscle,bone and heart were 17.4±1.0,35.1±10.9,20.7±6.2 and 20.9±4.0,respectively.Conclusions 68Ga could be labeled to HER2 affibody.The 68Ga-labeled HER2 affibody may be useful for PET imaging of HER2-positive tumor.
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Objective: To construct a phage-displayed random combinatorial library of IgA affibodies and to analyze its directed in vitro evolution, so as to study the relationship of IgA affibody structure with its function. Methods: The coding sequences of two affibodies, ZA1 and ZA2, were generated by overlapping PCR. The affibodies with a random linking peptide coding sequence in the 3′ terminal were randomly ligated and cloned into the K pn I site of the phagemid pCANTAB5S to construct a combinatorial phage library. Totally four rounds of in vitro human IgA directed evolution were conducted, and selected phage clones were prepared individually to test the IgA binding activity by ELISA technology. Results: The combinatorial phage library was successfully constructed; it contained about 3.4 × 107 clones with a titer of 1.6 × 1012 TU/L, and the positive clones accounted for more than 79%. Sequence analysis showed that the two single affibodies were randomly linked by random linking peptides. The composition of the phage clones displaying three or four affibodies in tandem increased remarkably along with the rounds of selection, which indicated the successful IgA directed evolution. Three new arrangements of two, three or four affibodies in tandem were obtained. ELISA results demonstrated a significantly enhanced IgA binding activity of three or four affibodies in tandem. Conclusion: A series of new IgA binding recombinants have been obtained by directed in vitro evolution of combinatorial phage library displaying randomly linked IgA affibodies. The three or four affibodies in tandem have much higher IgA binding activity than others, indicating that the in vitro molecular evolution is an effective way to produce IgA binding proteins with high activity.