Team:ZJU-China/Design
Design
This year, our team is committed to optimizing the examination approaches for breast cancer. Among the existing examination approaches, magnetic resonance imaging (MRI), which is an appealing tumor imaging method for its noninvasive characteristics and high spatial resolution, has attracted our attention. However, the existing MRI techniques have the disadvantages of poor specificity and side effects of contrast agents. To solve the problem, our team create a new kind of contrast agent which have the ability to target HER2-positive breast cancer cells specifically. To be specific, we have developed engineered magnetosomes displaying anti-HER2 scFv for tumor-targeting magnetic resonance imaging of HER2-positive breast cancers.
What is Magnetosomes?
Magnetotactic bacteria, a special kind of bacteria, were discovered by Blakemore in 1975. These bacteria synthesize intracellular magnetic nano-particles, designated as magnetosomes or bacterial magnetic particles, which are enveloped by the cytoplasmic membrane and an iron crystal made of Fe3O4, Fe3S4, Fe2O3, FeS, etc.[1][2] Several strains of magnetotactic bacteria, including Magnetospirillum gryphiswaldense MSR-1, M. magnetotacticum MS-1 and M. magneticum AMB-1, have been isolated and identified so far.
Due to the existence of natural lipid bilayers on the magnetosomes provide ample functional groups for conjugation of other agents, magnetosomes have potential applications in the biological and medicinal fields such as biosensors[1] and carriers of drugs[4][5].
Practical applications of magnetosomes are based on their ferrimagnetism, nanoscale size, narrow size distribution, dispersal ability, and membrane-bound structure[5]. However, the applications of magnetosomes have not yet been developed commercially, mainly because magnetotactic bacteria are difficult to cultivate and consistent, and high yields of magnetosomes have not yet been achieved. Ying Li reported a chemostat culture technique based on pH-stat feeding that yields a high cell density of Magnetospirillum gryphiswaldense MSR-1 in an auto-fermentor. In a large-scale fermentor, magnetosome yield and productivity were 83.23 ± 5.36 mg/L (dry weight) and 55.49 mg/(L·day), respectively[6].
Figure 1. Formation process of magnetosomes in magnetotactic bacteria.
Why Used Magnetosomes?
Researchers have carried out a lot of research on the development of high-performance contrast agents, aiming at providing precise information to determine tumor prognosis and treatment. To improve the sensitivity of MRI, the contrast agents should have extremely high R2 relaxivity[7], which is defined as the slope of the linear regression generated from a plot of the measured relaxation rate 1/T2 versus the concentration of the contrast agent[8]. General contrast agents superparamagnetic iron oxide nanoparticles (SPIOs) with relatively low R2 values are no longer applicable to current developments.
Research shows magnetosomes of M. gryphiswaldense MSR-1, which consist of the nanocrystal of magnetite (Fe3O4) with size ranging from 35 to 120 nm, have extremely high R2 relaxivity[9][10]. Besides, the characteristics of phospholipid bilayer membrane coating make magnetosomes have incomparable bio-compatibility[11]. Those characteristics mentioned above make magnetosomes promising as an excellent MRI contrast agent.
Figure 2. Screen of MRI by using MagHER2some as contrast agent.
Why Used scFv?
In addition to the R2 relaxivity of magnetic nanoparticles, the accumulation of contrast agents in the tumor site is also an important factor to improve the imaging quality[12]. The natural lipid bilayers on the magnetosomes provide good bio-compatibility and ample functional groups for conjugation of tumor-targeting or therapeutic agents. In the past strategies, researchers displayed transgenic-targeted peptides[13] or affibody molecules[14] on the surface of magnetosomes to achieve the accumulation of magnetosomes in tumor sites.
In this work, we plan to display a single-chain variable fragment (scFv) on the surface of magnetosomes. Here, we use scFv instead of the whole antibody because we find that scFv has a better affinity to the target cells[15] and the expression of scFv in prokaryotes has higher efficiency. Variable regions of heavy (VH) and light (VL) chains of the antibody are linked via a flexible linker encoding a short peptide to form scFv[16]. With the high binding ability of scFv against tumor specific antigen, engineered magnetosomes can be enriched in tumor sites.
To display scFv as much as possible on the surface of magnetosome, we chose mamC protein as anchor protein, which is the most abundant magnetosome membrane protein of Magnetospirillum gryphiswaldense strain MSR-1[17].
Figure 3. Expression of scFv in SHuffle®.
How to Display scFv?
The humanized monoclonal antibody against HER2 (trastuzumab, Herceptin®) binds to the extracellular domain of HER2[18]. We plan to develop engineered magnetosomes displaying anti-HER2 scFv for tumor-targeting MRI of HER2-positive breast cancers. In the cytoplasmic environment, the existence of two specialized systems including thioredoxin as well as glutaredoxin pathways results in holding free thiol groups in a reduced state. Due to the presence of two disulfide bond, scFv expression in reducing bacterial cytoplasm may cause the formation of inclusion bodies[19]. As a result, it’s not feasible to fusion express scFv and mamC protein directly in MSR-1.
Here, we use the interaction system of Fc-ZZ as our linker to link the mamC expressed in Magnetospirillum gryphiswaldense and scFv expressed in Escherichia coli SHuffle® T7 Express Cells. SHuffle® is suitable for expression of proteins requiring disulfide bonds for their activity. This strain has △trxB, △gor mutations and also overexpresses cytoplasmic disulfide bond isomerase (DsbC)[19].
To link the scFv onto the magnetosomes, we use the protein interaction system of Fc-ZZ. ZZ is derived from Staphylococcal protein A, which is originally found in the cell wall of bacteria Staphylococcus aureus. Protein A contains five homologous antibody binding regions, each region can bind to the heavy chain of immunoglobulins within the Fc region, most notably IgGs of mamalian species. However, some defects exist in protein A blocking it further application in molecular biology. Therefore, a new synthetic Fc-region binding domain originated from B domain of Staphylococcal protein A designated ZZ is created[20].
Compared to native protein A, ZZ is much smaller in molecular weight so it is more suitable for fusion expression with other protein. What’s more, modification in primary amino acids sequence in ZZ make it more resistance to chemical treatment, especially to hydroxylamine and cyanogen bromide[21].
The magnetosome and scFv were extracted respectively and self-assembled in vitro, then our MagHER2somes come into being!
Figure 4. Combination of scFv-Fc and mamC-ZZ to produce engineered magnetosomes.
References
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