Team:iBowu-China/Engineering
Engineering Success
Overview
In our magneto-hyperthermia-based lung cancer therapy, solutions were designed and modified.
• DNA of magnetic protein with a high expression promoter was first designed to be delivered by the liposome to the cancer cells. The magnetic protein can spontaneously form crystals after recombinantly expressed with inka-box (iBox) and PAK4cat.
• Regarding cancer cell targeting, the surface of the liposome was modified with the ligand specific to epidermal growth factor receptor (GE11), which is highly expressed in cancer cell, and PEG for immune escape.
• Moreover, the expressed and iron-loaded magnetic protein was delivered by ADC (Antibody Drug Conjugation), which is usually used as the drug specific delivery.
• Also, the magnetocaloric effect can be control by the area of the localized alternating magnetic field.
Currently, we have successfully expressed the magnetic protein crystal and completed purification. The magnetic properties were also confirmed by magnetic force microscopy. (
See statistics and further explanation in ‘Results’.) Moreover, we uploaded eight parts regarding magnetic protein crystal, GE11, and the lung-tissue-specific promoter.
(See detailed explanation in ‘Parts Overview".)
Research:
Magnetic hyperthermia is advantageous in that the heating source can be located directly at the target region. Local hyperthermia only addresses small areas, such as the tumor per se. Therefore, the utilization of magnetic materials right to tumor areas is expected to improve the therapeutic efficiency. Non-small cell lung carcinoma (NSCLC) is a clinically common type of epithelial cancer, yet is not sensitive to chemotherapy or radiotherapy. Because of this, magnetic hyperthermia has been considered a promising tool.Hypothesis:
We postulate that an engineering protein could be assembled for magnetic sensing and generate substantial magnetocaloric effect. With these features, this protein is an alternative material for magnetic hyperthermia. Moreover, it should be targeted to the tumor cells for the specific therapy.Design:
Our project aims to increase cancer-killing efficacy by inducing magneto-hyperthermia on the magnetic protein crystals (MPCs). The DNA sequence that encodes the magnetic protein is designed to be delivered into the tumor cells through liposomes. DNA is selected as the delivered material instead of mRNA because it is a more stable form of genetic information and can therefore guarantee a successful expression. The designed vector also includes a lung Tissue-Specific Promoter (TSP), SFTBP Promoter, which is a high expression promoter in lung cancer cells (Figure 1). With the SFTBP promoter, the magnetic protein will be highly expressed in lung cancer cells and less-expressed in other healthy cells. Beside ftn-PAK4cat, GFP-PAK4cat is designed to monitor the progress of crystallization.
Figure 1. Schematic of ftn-PAK4cat and GFP-PAK4cat plasmid. GFP and ferritin are linked with inkabox-PAK4cat respectively. Both are promoted by tumor specific promoters, ensuring targeting accuracy.
Build:
Core Component - Magnetic Protein Crystal
Magnetic protein crystal is an engineered protein crystal that contains more than 10 million ferritin subunits. This protein crystalizes spontaneously after expression in mammalian cells and is able to load iron. Crystallization of protein attributes to the conformational changes of the inka-PAK4 complex. PAK4 is a metazoan-specific kinase within the family of p-21 activated kinases. When expressed with its inhibitor Inka1, the catalytic domain of PAK4 binds with Inka1 and furthers the formation of a rod-like crystal. Its structure is illustrated in Figure 2. We engineered ferritin into the hollow channel in inka-PAK4 crystal by expressing them together. With ferritin, the crystal is capable of mineralizing substantial amounts of iron. By applying an external alternating magnetic field, the iron generates heat and causes death of tumor cells through the intense heat. Furthermore, its characteristic of magneto taxis allows another layer of insurance of targeting since the magnetic field can orient the crystal to the tumor tissues.
Figure 2. Structure of Ft-PAK4 Cat protein: showing how ferritin fits into the hollow channel of the crystal.
Delivery System — Phospholipid Capsule
Nucleic acids can be easily decomposed in the biological environment. Thus, liposomal capsule is designed to ensure successful delivery of the nanoparticles. Due to its identical chemical composition with the cellular membrane, liposome guarantees biocompatibility and biosafety. Under the dual targeting of the internal lung-specific promoter and the external alternating magnetic field, the liposomes containing the DNA can fuse with the cancer cells' membranes and deliver the cargo through endocytosis. In the cytoplasm, the phospholipid membrane is disintegrated, and DNAs are released to form proteins through transcription and translation, as illustrated below in Figure 3.
Figure 3. Lipid-mediated transfection in mammalian cells. DNAs are artificially enclosed by the liposome and will be spontaneously released after entering the tumor cell.
Modification — Improved Targeting and Safety
At present, there are still some problems with liposomes. In our design, the liposome is modified with epidermal growth factor (EGF) GE11 and polyethylene glycol (PEG) to achieve active targeting and to prevent detection and destruction by immune cells, respectively.
Targeting
Active Targeting: EGFR ligand: Epidermal growth factor receptor (EGFR) displays tyrosine kinase activity which is essential for DNA synthesis and mitosis. This transmembrane protein is overexpressed in a variety of cancer cell types including lung cancer. GE11 is used to target EGFR. It is a polypeptide chain which has an affinity toward EGFR and is able to form a steady conjugation with lysosome (Figure 4). The integration of GE11 ligand and EGFR induces structural change of the receptor and further promotes endocytosis. Polypeptide demonstrate a low immunogenic potential and can be easily synthesized. Therefore, GE11 shows great characteristics of an active targeting.
Figure 4. Chemical structure of GE11
Lung Tissue-Specific Promoter:
SFTBP promoter is incorporated in the vector to avoid the expression of engineered DNA outside lung tissue. SFTBP gene codes for pulmonary surfactant protein B, which is an important protein for preventing the collapse of alveoli after exhalation. Due to the property of only promoting expression in lung cells, the use of SFTBP promoter ensures the nanoparticles act solely on lung tissues.
Safety
PEG-laytion: When the nanoparticles enter the biological environment, it will be rapidly covered by a large number of biomolecules including proteins. This NP-protein interaction provides the nanoparticles bio-identity which can be detected by the macrophages. PEG-laytion prevents uptake by macrophages and elongates circulation time, enabling the generation of enough heat to kill the tumor cells. The chemical structure of polyethylene glycol shown in Figure 5.
Figure 5. Chemical structure of a unit of polyethylene glycol. Polyethylene glycol is a macromolecule with a number of identical monomers. The figure shows only one unit of it.
In general, our magnetic protein crystal encapsulated in liposome delivery system was modified with targeting ligand GE11 and immune escaping molecule PEG (Figure 6).
Figure 6. Schematic of the plasmid containing liposome, modified with PEG and GE11 peptide.
Test:
Despite the challenges and restrictions of COVID-19, we have completed several stages of our experiments. We have successfully expressed and isolated magnetic protein in vitro. But results have indicated that loading iron into the magnetic protein is difficult to achieve in vivo. Further research is required to validate this approach.
Learn:
To solve the problem of iron loading and improve targeting in late practical application, we sought a better delivery systems. We studied relevant research papers and consulted numerous experts in the fields related our project, the main topics of discussion being active targeting and delivery strategies. They have recommended a few alternatives to GE11: HER2 (Human Epidermal Growth Factor Receptor 2), CD19, and CD20. Other delivery methods were also mentioned: ADC (Antibody Drug Conjugation), Soy Protein, and Albumin.
Improve:
Based on the suggestions mentioned above, we have decided to express magnetic protein crystals in vitro, load iron into the expressed crystals, then deliver the crystals to the tumor cells (Figure 7). This approach would address the issue of not being able to load iron in vivo.
Figure 7. Schematic of the magnetic protein crystal containing liposome, modified with PEG and GE11 peptide.
In addition, we developed another system of delivering the magnetic protein crystals to the tumor cells--through antibody drug conjugate (ADC). ADC is biopharmaceutical drug composed of humanized monoclonal antibody conjugated with drug particles by chemical linkers. Through ADC conjugation, we are able to link the humanized anti-TROP2 monoclonal antibody to the magnetic protein crystal domain (Figure 8). We use the domain because it is easier to transport than the crystals due to its smaller size. The protein domain is smaller yet still fully functional. Therefore, we have successfully designed an ADC nanoparticle. However, due to time constraints, we will use this as part of our experimental plan for the future.
Figure 8. ADC-based magnetic protein delivery: the magnetic protein is conjugated with a specific antibody, which is capable of targeting tumor-associated antigen, by a chemical linker.
References
[1] Margana, R. K. , & Boggaram, V. . (1997). Functional analysis of surfactant protein b (sp-b) promoter. sp1, sp3, ttf-1, and hnf-3alpha transcription factors are necessary for lung cell-specific activation of sp-b gene transcription. Journal of Biological Chemistry, 272(5), 3083-3090.
[2] Li, T. L. , Wang, Z. , You, H. , Ong, Q. , & Cui, B. . (2019). Engineering a genetically encoded magnetic protein crystal. Nano Letters, 19(10).
[3] Baskaran, Y. , Ang, K. C. , Anekal, P. V. , Chan, W. L. , & Robinson, R. C. . (2015). An in cellulo-derived structure of pak4 in complex with its inhibitor inka1. Nature Communications, 6, 8681.
[4] Genta, I. , Chiesa, E. , Colzani, B. , Modena, T. , Conti, B. , & Dorati, R. . (2017). Ge11 peptide as an active targeting agent in antitumor therapy: a minireview. Pharmaceutics, 10(1).
[5] Bardia, A. , Mayer, I. A. , Diamond, J. R. , Moroose, R. L. , Isakoff, S. J. , & Starodub, A. N. , et al. (2017). Efficacy and safety of anti-trop-2 antibody drug conjugate Sacituzumab govitecan (immu-132) in heavily pretreated patients with metastatic triple-negative breast cancer. Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology, 35(19), JCO2016708297.