¹⁸F-labeled Designed Ankyrin Repeat Proteins Targeted To HER2 For PET Imaging

[Background]Tumor targeting is becoming a research heat of molecular imaging recently. The tumor targeting molecular imaging takes advantage of molecular probes that specifically recognize tumor targets and bind specifically to tumor tissue, then uses the corresponding imaging device to image the tumor in vivo, to improve the sensitivity and specificity of tumor detection. At present, the commonly tumor targeting probes are antibody protein molecules and peptides which have their shortcomings, for example, protein molecules are: ① big molecular weight, prone to aggregation; ② low tissue penetration, hard to enter tumor; ③ complex manufacturing process and high production costs; ④ inherent immunogenicity in vivo, injection may elicit an immune response; and peptides are:① half-life short in vivo, easily degradation; ② mainly rely on chemical synthesis, higher cost; ③ chemical modification, the targeting efficiency easily be affected.In recent years, the synthetic libraries and screening technologies provide a non-immunological method to screen the novel specific molecular, for example the high affinity and high specificity binding molecules---esigned ankyrin repeat proteins,(DARPins), AdNectins, Affibodies, and Anticalins. DARPins are small and stable molecules, which can be expressed easily in E. coli cytoplasm with high yield and low production cost. They can get to the target molecule with high affinity and specificity to overcome deficiencies of antibodies and peptides. DARPins repeat protein is a natural derivative, which comprised 4-6 amino acids repeat structure and its molecular weight is about 14-21KDa (simple sequence repeats composed of 33 amino acid residues). By assembling random residues and repeat sequences, a various and complex DARPins library can be produced, which can be used to screen binding molecules that identify any antigen with high specificity and affinity. In 2006, Zahnd C screened and got various DARPins targeting HER2 extracellular domain by using ribosome display technology, and then they got DARPin H10-2-G3 (abbreviated as G3) targeting HER2 with more affinity and specificity in 2007. Introducing the second cysteine in the C-terminal by site-directed mutagenesis, DARPin G3 can be marked fluorescein, nanoparticles, toxins, and radionuclides. The mutation is far from of the active region of DARPin G3, so it can still maintain its original biological functions.Human epidermal growth factor receptor 2 (HER2) is a kind of transmembrane protein with tyrosine kinase activity, which has an intracellular tyrosine kinase domain and an extracellular ligand-binding domain. Normally, HER2 and human epidermal growth factor receptor family (EGFR, HER3 and HER4) form heterodimers playing an important role of signal transduction in cell growth, survival and differentiation. The overexpression or mutation of HER2 could lead directly to malignant transformation and tumor metastatic, and blocking HER2 signaling pathway and inhibition of HER2 activity can inhibit tumor growth and induce apoptosis of tumor cells. Studies have shown that HER2 is overexpression in breast cancer, ovarian cancer, stomach cancer, lung cancer and prostate cancer. Currently, the treatments for HER2 targeted drugs have been widely used in clinical. Clinical studies have shown that HER2 overexpression predicts poor prognosis. The methods of clinical detection of HER2 expression mainly include histological methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) 3 species, which are unable to achieve real-time dynamic monitoring of HER2 expression status in vivo.In recent years, the tumor targeting imaging technologies are developing, which can be used for the real-time visualization, dynamic monitoring of physiological state HER2 expression. To achieve the targeted molecular imaging of HER2, the probe 18F-G3 was synthesized, which is composed by the highly specific targeting ligand HER2 DARPin H10-2-G3 and radionuclide 18F; then PET imaging was operated for achieve real-time dynamic monitoring of HER2 expression status in vivo. In This study, the methods of cytology and pathology in vitro, fluorescence imaging and small animal PET/CT imaging in vivo were used to test the feasibility of this new tumor targeting imaging technology. If true, the new method to target HER2 in vivo will be achieved; it also can be used in treatment options, prognosis of breast cancer, developing targeting drugs, and providing theoretical and practical basis for other DARPins molecular probes.[Objective]1. To prepare designed ankyrin repeat protein (DARPin) H10-2-G3 targeting to HER2 by prokaryotic expression system, and make preparation of molecular probes and lay the foundation for future clinical applications; to synthesize fluorescent molecular probes G3-5-MF, purifiy and identify it, and through the vitro cytology and pathology experiment and animal fluorescence tomographic imaging to check DARPin G3 targeted to HER2, and explore the feasibility DARPin G3 as probe ligand.2. Based on GE module TRACERlab FXFN nucleophilic fluorination synthesis system to synthesize marker auxiliary group 18F-2-fluoro-propionic acid p-nitrophenyl ester (18F-NFP), then react with designed ankyrin repeat proteins DARPin H10-2-G3 to get radionuclide molecular probe 18F-G3, by Micro-PET/CT imaging of breast cancer-bearing nude mice to examine the targeting tumors in vivo imaging of molecular probe 18F-G3 and viable for in vivo evaluation of HER2 expression status.[Methods]1. Expression and purification of DARPin H10-2-G3In the NCBI database search to a target protein DARPin G3 92% similarity, published gene sequence, which NCBI sequence number AM997265, in which 20 bases different from using the advantages of synonymous codon replacement method, in order to obtain the desired protein highly expressed gene sequences, other site-directed mutagenesis of the first 135 amino acid cysteine. By PCR DARPin G3 cloned gene by restriction enzyme digestion and sequence analysis showed that the sequence of exactly the same sequence and design. This fragment was subcloned into the expression vector pET-32a (+), transformed BL21, induced by IPTG, SDS-PAGE analysis and mass spectrometric detection. The resultant product was purified by Ni2+-NTA His · Bind Resins purification kit was purified to finally obtain a high-purity DARPin G3.2. Synthesis of fluorescent molecular probes G3-5-MFFluorescein maleimide double bond (Fluorescein-5-maleimide,5-MF) maleimide ring may DARPin G3 amino acid sequence with the penultimate C-terminal cysteine mutation mercapto (-SH) residue on the part of the formation of covalent cross-linking, prepared fluorescent probe G3-5-MF. synthesis product was 3K. centrifugal ultrafiltration tubes isolated and purified.3. Evaluate of the biological function of the molecular probes G3-5-MF(1) In vitro binding experiments of fluorescent molecular probes G3-5-MF with human breast cancer tissueHuman breast tissue was gotten from breast surgery, making HE stained paraffin sections; by conventional dewaxing, microwave repair, after BSA closed each slice added rabbit anti-human HER2 antibody 50 microliters,37 ℃ incubated for 2 hours; rejection to PBS, biotin-labeled goat anti-rabbit IGg,50 microliters and incubated 37 ℃ 40 min, avidin working fluid horseradish peroxidase-labeled, incubated 37 ℃ 40 min, PBS washed three times, DAB color, hematoxylin 10 minutes, dehydration were mounted microscope photograph; take with immunohistochemistry used paraffin sections of adjacent level by conventional dewaxing, microwave repair, after BSA closed each added G3-5-MF PBS solution (2x10-4mg/mL) 50 microliters, dark for 30 minutes, PBS washed three times inverted fluorescence microscope photographs; adjacent levels immunohistochemistry and fluorescence micrograph photo integration, verification of DARPin target H10-2-G3 point whether the HER2 receptor.(2) In vitro binding experiments of fluorescent molecular probes G3-5-MF with tumor cellsConventional culture anthropogenic breast cancer cells that were SKBR-3, MDA-MB-231 and MCF-7. With rabbit anti-human HER-2 antibody to detect the three tumor cells expressing HER2 status; tumor cells in combination with fluorescent probes G3-5-MF, placed under an inverted fluorescence microscope to observe the intracellular fluorescence distribution; flow cytometry analysis of the relative fluorescence intensity of the cells. The G3-5-MF and rabbit anti-human HER-2 antibody has incubated SKBR-3 cells were competitive inhibition assay, inhibition of the existence of competition between two different levels and distributions of fluorescence observation.(3) The animal imaging of fluorescent molecular probes G3-5-MF targeting HER2-positive tumors expressThe number of 23 breast cancer SKBR-3 xenograft nude mice model were established by conventional methods. When the tumor diameter of more than 0.5cm, taken randomly 7via tail vein injection of G3-5-MF (2.5mg/ml,150μl), respectively, Oh, 1h,4h,6h,8h, 10h,24h after the mice were sacrificed, tumor isolated organs and tissues, is placed on a clean glass plate, and then developed with a fluorescent imaging device to observe changes over time, the tumor and the organ distribution of fluorescence; take normal naked a mouse via tail vein injection of G3-5-MF,4h after the mice were sacrificed, organ tissues were separated as blank control. Take 12 animal models of breast cancer were randomly divided into two groups, namely,1 hour and 4hour group developing imaging group. Each group were randomly selected by three tail vein injection of fluorescent probe G3-5-MF (2.5mg/ml,150 μ1), the other three injections PBS (150μ1). Upon injection of 1 and 4 hours mice were sacrificed and placed in-80 ℃ refrigerator rapid refrigeration 2h, using a cutting machine cut coronal sectional layer, the layer was cut slices of animal flat on a clean glass plate with fluorescence imaging instrument for imaging fluorescent tumor was observed and the organ uptake, tumor uptake values measured fluorescence, fluorescence difference between the two groups in tumor uptake values. After separation of tumor imaging, placed in-80 ℃ refrigerator rapid refrigeration 30min, Frozen sections after DAPI-stained, fluorescence microscopy to observe green fluorescence distribution.4. Synthesis probes 18F-G3 and PET/CT imaging study (1) Synthesis PET molecular probe 18F-G3Starting from 2-bromo-propionate by 18F-nucleophilic substitution, potassium hydroxide hydrolysis, and bis (p-nitrophenyl) carbonate (NPC), reaction of the activated radiolabeled auxiliary group 18F-NFP; 18F-NFP and DARPin G3 in anhydrous dimethyl sulfoxide (DMSO) solvent with N, N- diisopropyl ethyl amine (N, N-diisopropylethylamine, DIPEA) as base,40 ℃ reaction 20min, multi-step anti It should synthesized 18F-G3. Product obtained after the measured radiochemical purity by TLC, live meter measuring radioactivity, calculated radiochemical yield.(2) The study of micro PET/CT imaging of PET molecular probe 18F-G3 to Nude mice bearing breast cancerConventional methods to establish SKBR-3 breast cancer in nude mice bearing subcutaneous 3, when the tumor diameter of more than 0.5cm, as experimental animals. Nude mice were injected via the tail vein 3.70～5.55MBq (100～150 μCi) 18F-G3, in 30min,60min,120min,200min performed after Micro-PET/CT static imaging using Inveon Research workplace (IRW2.2) workstation for data processing, PET CT attenuation correction data after the use of filtered back projection reconstruction of the coronal plane, cross-sectional, sagittal tomographic image is analyzed and compared with CT image fusion, manual outlining the tumor region of interest, the workstation is automatically calculated SUVmax.[Results]1. Expression and purification of DARPin H10-2-G3DARPin G3 expressed by prokaryotic expression system, separated and purified is a colorless transparent liquid, after vacuum drying apparatus and dried as a white powder. By SDS-PAGE analysis, and there is a clear 15KD bands, mass spectral analyzes peak molecular weight of 14634, a molecular weight of 14.5 KD consistent with the literature, and detects two peptide sequences DARPin G3 match, prompting synthesis is correct.2. Synthesis of fluorescent molecular probes G3-5-MFSynthetic G3-5-MF is a fluorescent yellow liquid, fluorescent probe G3-5-MF can be used to verify the in vivo experiments.3. Evaluate of the biological function of the molecular probes G3-5-MF(1) In vitro binding experiments of fluorescent molecular probes G3-5-MF with human breast cancer tissueImmunohistochemistry confirmed HER2 (+++), HER2 (+) and HER2-negative specimens. Fluorescent probe G3-5-MF with human breast cancer tissue binding experiments:HER2 (+++) specimens, a significant green fluorescence microscope with diffuse distribution in tissues; HER2 (+) samples, microscopic only there are a few green fluorescence distribution; HER2-negative specimens, no green fluorescence microscope distribution. Thereby confirming pre-designed ankyrin repeat proteins DARPin H10-2-G3 can specifically bind to HER2-positive human breast cancer tissue, and the distribution is consistent with HER2.(2) In vitro binding experiments of fluorescent molecular probes G3-5-MF with tumor cellsHER2 antibody immunofluorescence experiments:Appears on SKBR3 cell membrane obvious red fluorescence in the MCF-7 cell membranes have a faint red fluorescence, validated SKBR-3 cells overexpressing HER2, MCF-7 weakly expressed HER2; whereas in MDA-MB no red fluorescence on the cell membrane-231, suggesting that the cells do not express HER2. After the fluorescent probe G3-5-MF tumor cells were incubated inverted fluorescence microscope:significant green fluorescence on the cell membrane SKBR-3, suggesting that the probe can be combined with SKBR-3 cells; while in MCF-7 and MDA-MB-231 does not appear green fluorescence on the cell membrane, suggesting that the probe is not available with the above-described cell binding. Flow cytometry analysis:by adding a fluorescent probe were incubated G3-5-MF culture, SKBR-3 cells significantly fluorescence uptake than the control while in MCF-7 and fluorescence profiles MDA-MB-231 cells MCF-7 MDA-MB-231 cells, and the curve, only a slight positional distribution skewed to the right (MCF-7 cells than the right degree of partial MDA-MB-231 cells were somewhat obvious), G3-5-MF does not prompt the two kinds of cell binding. Competitive inhibition experiments SKBR-3:The green fluorescent probes degree G3-5-MF and red fluorescence level HER2 antibody incubation compared with each individually, have weakened, two fluorescent fusion diagram shows red fluorescence and green fluorescence complementary relationship on the cytomembrane.(3) The animal imaging of fluorescent molecular probes G3-5-MF targeting HER2-positive tumors expressSKBR-3 breast cancer in nude mice bearing tumors and organs fluorescence imaging revealed:Oh significant fluorescence imaging in tumor uptake; 1h,4h,6h,8h and 10h fluorescence imaging in tumor tissues were uptake was in 4h strongest fluorescence uptake, after the fluorescence intensity over time gradually decreased, 24h almost no fluorescence imaging in tumor uptake; organ imaging aspect, kidney, gallbladder significant fluorescence uptake, gastrointestinal tract, muscle, brain uptake moderate fluorescence intensity, liver, spleen, heart, lung uptake slight fluorescence; fluorescence distribution in various organs of normal mice Ibid. SKBR-3 breast cancer in nude mice bearing tumor fluorescence imaging coronal tomographic slice display:1h and 4h, injecting fluorescent probes G3-5-MF nude mice, which are fluorescent tumor tissue uptake in nude mice injected with PBS, the tumor tissue no distribution of fluorescence; fluorescence distribution and 4h higher than 1h. Were compared tumor fluorescence quantitative uptake values:1 hour fluorescent tumor uptake value (3273.50 ± 901.68),4 hour group tumor fluorescence uptake value (6634.83 ± 662.08), two fluorescent tumor uptake values are significantly different (t=-7.360; P<0.001). Frozen sections of fluorescence microscope observation and coronal slice tomography imaging with similar results.4. Synthesis probes 18F-G3 and PET/CT imaging study(1) Synthesis PET molecular probe 18F-G3Prepared by GE module Tracerlab FXFN commonly used auxiliary group 18F-NFP. 2-bromo-propionate after nucleophilic fluorination, KOH hydrolysis and NPC three-step activation "one-pot" reaction of the initial 18F-NFP, then the semi-preparative liquid column isolated radiochemical purity> 98% 18F-NFP. Its decay corrected yield of 26%, took 100 min. Dry 18F-NFP and then after protein conjugation DARPin G3 Plus C18 column solid phase separation of purity, radiochemical purity successfully obtain> 98% 18F-G3, its total decay corrected yield (5±2)%(n=3, counted from 18F).(2) The study of micro PET/CT imaging of PET molecular probe 18F-G3 to Nude mice bearing breast cancerMicro-PET bearing SKBR-3 breast cancer xenograft model in nude mice/CT imaging:30min imaging, tumor tissue radioactivity uptake see, heart, liver, kidneys and intestine see uptake in the blood than the background high, see significant uptake in the gallbladder, intestine and kidney uptake value higher, indicating that 18F-G3 mainly through the hepatobiliary and urinary system metabolism; no brain distribution of radioactivity showed 18F-G3 may not pass the blood brain barrier; 60min imaging, tumor tissue radioactivity uptake increased, heart, liver, kidneys and intestinal radioactive concentration also increased; 120min and 200min imaging, further increasing the distribution of radioactivity within tumor tissue, and heart, liver, kidneys and intestine distribution of radioactivity gradually reduced.[Conclusion]1. In this study, the preparation of designed ankyrin repeat protein DARPin H10-2-G3 targeting HER2 was done by prokaryotic expression system, and confirmed the expression of the right through SDS-PAGE analysis and mass spectrometric detection.2. Fluorescent probe G3-5-MF was synthesized with fluorescein -5-maleimide to mark DARPin H10-2-G3. Through confirmed in vitro cytology experiments, the fluorescent probe G3-5-MF can bind with HER2-positive-cells, and HER2 expression status and the degree of integration was a positive correlation. The fluorescent bio-distribution experiments of HER2 overexpressing-breast cancer -bearing nude mouse models confirmed fluorescent probe G3-5-MF could specifically bind to tumor tissue in vivo, having targeting to HER2.3. Synthesis of hydroxyl-containing activation markers cofactor 18F-NFP, side-chain amino-reactive DARPin H10-2-G3 with synthetic PET molecular probe 18F-G3, radiochemical purity greater than 98%, lay the foundation for further clinical application the foundation.4. SKBR-3 breast cancer bearing nude mice bearing subcutaneous imaging of experiments show that the expression of 18F-G3 probe molecule in vivo specifically target HER2 expression in breast tumor tissue, it can be used for PET imaging in the diagnosis of HER2 status.