Team:ZJU-China/Poster

Poster: ZJU-China

MagHER2some

Presented by ZJU-China 2020 iGEM team.

Qiyu Liang¹, Yutong Liu¹, Weihao Wang², Yihong Cai², Shirui Xu², Mingxiao Wei², Xiner Ying², Yanqing Li², Jiayi Chen², Yurun Jin², Jiajie Su², Hui Zhou², Yujie He², Ming Chen³, Fan Yang³

¹Student Leader, ²Student Menber, ³Team PI, Colleage of Life Science, Zhejiang University, China

Abstract

Breast cancer is a main malignant tumor that threatens women's health. In an effort to overcome the unspecificity and side effect of the current contrast agents used for MRI, we reconstructured the magnetosomes from magnetotactic bacteria Magnetospirillum gryphiswaldense to produce a contrast agent with high biocompatibility that target HER2-positive breast cancer cells specifically. With biological modifications, magnetosomes can link with anti-HER2 antibodies. By antigen-antibody interaction, engineered magnetosomes can conjugate with HER2-positive breast cancer cells, demonstrating a special pattern on the image under magnetic field. Armed with our engineered contrast agent MagHER2some, the efficiency of evaluating the response of HER2-positive breast cancer treatment can be optimized, thus assisting the progress of therapy process. Further application can be made through establishing a platform with biomarkers targeting various diseases as inputs, paving avenue for precise evaluation of other diseases.

Problem: Breast Cancer

● Breast Cancer Worldwide

Breast cancer is a leading cause of death for women worldwide, with incidence and mortality increasing in lower-income countries.
According to the statistics on the Globocan by International Agency for Research on Cancer (IARC), the incidence and mortality rate of breast cancer among women rank the first among all the cancers types worldwide in 2018, which is 24.2% and 15.0% (Figure A, pink region); the number of countries with breast cancer as the top common cancer is 154, ranking the first in the legend (Figure B, pink region).

Figure A. New cases and deaths of breast cancer per year. B. Global maps of the most common type of cancer incidence in 2018. C. Trends in incidence rates. D. Trends in mortality rates.
Data source: https://gco.iarc.fr/today/home

● Breast Cancer in China

In China, breast cancer is one of the most common malignant tumors threatening to female health due to the increasing stress and accelerating pace of life. In the global cancer report released by IARC, the cases number of breast cancer in China accounts for 11.19% of the world's, second to United States. In the past few years, both the incidence rate and mortality rate see a remarkable increase, which speaks of the significance of comprehensive prevention and control strategies (Figure C,D, yellow line).

● Survival Rate Improvement

In the face of these problems, accurate screening is of great significance and can greatly improve survival rate. The current methods mainly include mammography, ultrasonography, MRI and PET.

Inspiration: MRI

● Advantages of MRI

1. The most sensitive modality in the breast cancer imaging.
With higher resolution and multi-dimensional imaging, MRI outperforms mammography in assessing tumor size, examining the soft tissue and detecting additional lesions in 16% to 20% of patients.

2. Free of ionizing radiation.
Compare to computed tomography (CT), which is another routine screening for breast cancer, MRI is free of ionizing radiation and will not trigger gene mutation.

● Contrast Agent

Gadolinium-based contrast agent (GBCA) has been widely used for MRI.
However, gadolinium is not always an ideal contrast agent for breast cancer due to its non-specificity and potential side effects. It has been reported that gadolinium would caused renal fibrosis and would deposits in the brain.

Idea: Magnetosome

In an effort to overcome the shortcomings of the current contrast agent, we reconstructed the magnetosomes from magnetotactic bacteria Magnetospirillum gryphiswaldense to produce a contrast agent with high bio-compatibility that specifically targets HER2-positive breast cancer cells. With biological modifications, magnetosomes can link with anti-HER2 antibodies.

Single-chain variable fragment (scFv) was used to target HER2. As shown in figure below, scFv-Fc fusion protein was expressed in E.coli SHuffle^®, and magnetosomes containing ZZ protein on the membrane surface were generated in magnetotactic bacteria. By interaction between Fc and ZZ in vitro, MagHER2some was built.

Figure. Summary of our biological design.

Armed with our engineered contrast agent MagHER2some, the efficiency of evaluating the response of the treatment of HER2-postive breast cancer treatment can be optimized, thus assisting the progress of therapy process.
Further application can be made through establishment of a platform with biomarkers targeting various diseases as inputs, paving avenue for precise evaluation of other diseases.

Expression of scFv-Fc

Figure A. Western blotting results of scFv-Fc under different inducing concentration of IPTG. B. Western blotting results of scFv-Fc under different induction time.

As the two figures showed, the best concentration for expressing was 2 mM of IPTG, and the longer time for expressing, the higher expression level the cells reached. In the next work, the expression period was carried out under 2 mM of IPTG inducing and 24 h of additional incubating, the gradient concentration expressed protein proved that the fusion protein was expressed successfully.

Expression of mamC-ZZ

Figure A. Western blot results of GST mamC-ZZ under different induction time. B. Western blot results of GST mamC-ZZ under different inducing IPTG concentration.

As Figure A showed above, the best concentration IPTG for expressed mamC-ZZ was 2 mM. In the next work, the expression period was carried out under 2 mM of IPTG inducing and 4 h of additional incubating, the gradient concentration expressed protein proved that the fusion protein was expressed successfully.

Interaction of two fusion protein

Figure A. Western blot results of mamC-ZZ and scFv which introduce different primary antibody. B. Western blot results of input control block.

Figure A indicated the FLAG scFv-Fc and the interacted mamC-ZZ was then immobilized on the resin, whereas the unbound proteins were washed away with TBS. Subsequently, the protein–protein complex was eluted. The right block was only incubated with the secondary antibody, improving mamC-ZZ was pulled down by the interaction of scFv-Fc. Furthermore, according to lane 1 and lane 2 (Figure A), purified proteins showed stronger interaction than unpurified proteins. However, it suggested an inspiring result, which meant scFv-Fc could bind mamC-ZZ in a complicated environment. These results implied us an easier way to purify and enrich mamC-ZZ from cell lysate directly.

Target HER2-positive cell

Figure A-D. Flow cytometry results of MDA-MB-453 and MDA-MB-231 after incubated with scFv-Fc.

As results shown above, the fluorescence intensity of MDA-MB-453 cells which incubated with scFv-Fc was significantly higher than the other MDA-MB-453 cells from the negative control group, which indicated that scFv-Fc could specifically bind with the certain target on MDA-MB-453 membrane (Figure A).

At the same time, we can also see that MDA-MB-231 has an obvious fluorescence peak after incubated by scFv-Fc (Figure B).

Then, the following analysis shows that although scFv-Fc can target both MDA-MB-453 and MDA-MB-231 cells, the fluorescence peaks were significantly different.

Obviously, the high HER2 expression cell line (MDA-MB-453) showed a higher fluorescence than that of the low HER2 expression cell line (MDA-MB-231), indicating that scFv-Fc is more targeted to HER2, and can distinguish breast cancer cells with high and low expression of HER2 (Figure C). And the fluorescence of MDA-MB-453 was about 10 times higher than MDA-MB-231's, which was corresponding with the difference of HER2 expression level between HER2-positive and negative cells mentioned in existing studies.

To put it more bluntly, compared with MDA-MB-231 which is HER2-negative, MDA-MB-453 had significantly higher fluorescence intensity when both cells were alive. This results indicates clearly that scFv can specifically bind to HER2 on tumor cell membrane (Figure D).

Model

To know more about the process of target products production, combination and functioning in vivo, we have established some models including three parts as follows.
1) Two models were used to describe the reactions in E.coli and magnetotactic bacteria separately.
2) A deterministic model was used to determine the combination and disaggregation of scFv-Fc and modified magnetosomes in vitro.
3) Two models were used to describe the movements of modified magnetosomes and its combination with HER2 in vivo.

Figure A. Combination of scFv-Fc and modified magnetosomes. B. Concentration field of magnetosome in tissue fluid. C. Metabolism of magnetosomes in the body for a long time. D. The combination of different concentrations of magnetosomes in a short time after injection.

After production, scFv-Fc and modified magnetosomes are combined in vitro (Figure A). It can be seen from the figure that the combination process is quick and thorough, which could be finished in 0.004 min.

The diffusion of modified magnetosomes is described by a partial differential equation (PDE). We can see the concentration change of magnetosome in different areas during 100 s (Figure B).

The concentration of combined magnetosomes is described by some ordinary differential equations (ODEs). We can see the concentration change of combined magnetosomes during 2 hours (Figure C) and during 1×10^-3min of different initial concentration of unbounded magnetosome (Figure D).

Future

For the epidemic situation of the COVID-19, a lot of experimental work had been delayed, so we prepared the plans for future work detailly, including the construction of functionalized magnetosomes and the specificity validation.

To obtain the modified magnetosomes, the MSR-1 strain which is able to encode mamC-ZZ protein is required, and this will be realized by the bi-parental conjugation of the E.coli donor harboring recombinant plasmid and the recipient, MSR-1.

To obtain higher quanlity and quatity of MagHER2some, several genes control the quantity of magnetosomes and the size of magnetosomes will be modified genetically. Yet small size of magnetosome is always related with low production, thus, we may try to find the optimum balance between size and production.

Taking advantage of the efficient combination of ZZ and Fc, anti-HER2 antibodies were displayed on the surface of magnetosome successfully. In the same way, diverse scFv modified magnetosomes can be designed according to different tumor targets. Therefore, we aim to develop a versatile targeted imaging platform apply to the clinical diagnosis of more types of tumors.

Except for being used in MRI, magnetosomes are also excellent agents for magnetic thermotherapy. So in the distant future, on the basis of targeting cancer cells, thermotherapy will be operated at the same time to make it reliable for integration of diagnosis and treatment.

Human Practices

Our product, MagHER2some is tightly related to the clinical use of MRI method. On one hand, what we are working on is derived from what we learn from the reality through dialogues with patients, medical specialists in breast cancer, radiologist, researchers in pharmocochemistry, etc. On the other hand, we interviewed contrast agent company and referred to relevant regulations to learn how our product will influence the world in consideration of all aspects, including effects, manufacturing, safety, ethics, etc. We also did some preliminary work by consulting medicine company for our versatile imaging target system.

Entrepreneurship

● The Business Idea

There is no targeted contrast agent available for breast cancer MRI clinical use. We improve the magnetosomes to produce a functional contrast agent which can specifically targets HER2-positive cells.

● Market

From the hospital's point of view, women over 45 need to do screening annually. The market for targeted MRI in the future will be very broad.

● Risk analysis

Risks: production risks, competitive risks, financial risks, manage risks.

Countermeasures: We would continue to carry out research, development follow-up and seize market opportunities. We will adopt a variety of financing methods and establish good credit relations with public institutions.

● Implementation Schedule

Acknowledgements

This work was supported by Undergraduate School of Zhejiang University and Zhejiang University Education Foundation in the financial. Laboratory was provided by Colleage of Life Sciences Zhejiang University and Zhejiang University Biology Lab Center. Thanks Snapgene for provided free sofeware useage for us.

Poster Authors
Poster design: Hui Zhou
Wiki code: Qiyu Liang
Content: Yanqing Li, Jiayi Chen, Yutong Liu, Mingxiao Wei, Yurun Jin, Shirui Xu, Qiyu Liang