Worldshaper-HZBIOX
Overview
Our project this year is to engineer the arginine-probiotics (EcN1917) to boost the immunotherapy of colorectal cancer which inspired by a paper published on Nature. In order to deeply understand the background knowledge of colorectal cancer and immunotherapy, improve the technical route and clarify the proper routs of administration and packaging for our probiotics, we collect information through background survey, public surveys, field trip and expert interviews. Trough the human practices work, we clarified our project goals to cure microsatellite stable bowel cancer and chose the strategy of modulating the tumor microenvironment. Meanwhile, with the help of the results of HP, we adjusted our focus on how to improve intestinal colonization capacity, precise targeting of EcN 1917, how to design and choose the packaging materials for oral administration as well as how to ensure probiotic activity for long storage, etc.
Highlights for Integrated Human Practices
1 Clearer ideas of project goals and strategy
• Through the interview with Dr. SHI, we clarified our project goals to cure microsatellite stable bowel cancer and chose the strategy of modulating the tumor microenvironment.
• According to the suggestion of Dr. ZHU, we explored the lysin gene induced expression by arabinose to break down bacteria to ensure safety.
• According to the suggestion of Dr. WANG, the replacement of E. coli must be taken into account. E. coli iterates every 20 minutes, thus colonization ability in intestine of EcN1917 need to be carefully verified.
2 Clearer understanding of the approval of genetically modified probiotics
From Dr. ZHU, we know that clinical breakthroughs of GM probiotics are still pending in China. However, the modified probiotics drug has been approved as a systematic injection in the United States. It gave us confidence to continue our project.
Determination of drug delivery methods
As for administration mode of probiotics, Dr. Zhu said that there are two ways at present, oral and injection. The effect of injection is better for directly injecting into the tumor tissue, but there is a safety risk. Oral administration is comparatively safer. To ensure safety, we select the oral administration finally after this talk with Dr. Zhu.
Adjustment of experimental design
Due to time restriction and safety consideration, we were not able to do animal experiments, so how to verify the anti-tumor effect remained a problem. According to Dr. YU, we adjusted our design to use cancer cell model to verify the anti-cancer effect. Meanwhile, test the relationship between PD-L1 expression and lactic acid level with arginine to verify its influence to cancer microenvironment.
Inspiration in drug packaging design
• According to Dr. ZHANG, we pay attention to activity of the probiotics in the intestinal environment. And we conducted a market research of drug packaging and further interviewed with Prof. Feng for our specific packaging design of our probiotics.
• According to Prof. FENG, we adjusted the packaging design from powder to microcapsules in order to prevent from damaging by stomach acid. Besides, prof. Feng also suggested to use a single bubble wrap to ensure each capsule of probiotics can have a better activity.
• According to Prof. FENG, we should select coating materials with low oxygen permeability and embed it more deeply to reduce the oxygen transmission rate. We finally selected alginate–chitosan–alginate (ACA) to coat our probiotics.
• It was recommended by Prof. FENG that using enteric capsule to coating arabinose to ensure it being absorbed in the gut or oral liquid for its stable characteristic and water-soluble.
Direction for future project improvement and a deeper understanding of precision therapy.
• From the talk with Prof. Feng, we also further familiarized about drug approval process. Many probiotics are listed as food labels, which is much easier. If it is a drug labels, phase I phase II phase III clinical verification is needed, estimated at least 5 years.
• The dosage of the arginine should be carefully considered
• From the talk with Dr. Fang, we noticed that the balance and competition of intestinal flora should be considered when probiotics are used to treat colorectal cancer.
• Dr. Fang reminded us to pay attention to ethics in medical research. "Patients are people, also have the anger and sadness".
• One of the problems we encounter in colon cancer treatment research is the precise targeting of tumor tissue. According to Prof. Zhang, the molecular markers related genes or proteins maybe a possible solution.
Purposeful and determined science communication
• According to the results of our questionnaire, people are more inclined to traditional treatment methods, and the popularity of immunotherapy and targeted therapy is not high. So, we select this issue as our publicity topic.
• According to Dr. ZHU, a common cause of colon cancer is that people eat too much fat and not enough fiber. In other words, if people make a conscious effort to maintain a healthy diet, their risk of colon cancer can be greatly reduced. So, we popularized colon cancer and healthy diet habits to the general public, hoping to raise everyone's awareness.
Introduction
According to the latest data from the National Cancer Center, in China, 370,400 new colorectal cancer cases and 179,600 deaths were estimated in China in 2014. The age-standardized incidence and mortality rate of colorectal cancer has increased by about 1.9% per year for incidence and about 0.9% per year for mortality rate from 2000 to 2014[1]. What is more, in America, there will be an estimated 104,610 new cases of colon cancer and 43,340 cases of rectal cancer diagnosed in 2020. Besides, the mortality rate of colorectal cancer has increased by about 40% from 1995 to 2020[2]. Thus, it could be seen that nowadays people do not put enough attention to this serious disease. A common cause of colon cancer is that people eat too much fat and not enough fiber. In other words, if people make a conscious effort to maintain a healthy diet, their risk of colon cancer can be greatly reduced.
Through background survey, we found that in terms of cancer treatment, traditional chemotherapy not only costs patients a huge amount of money but also causes serious damage to other organs in their bodies. Based on this, our team choose immunotherapy, which has emerged in recent years but has the problem of lack of efficiency now.
Inspiring by the paper published on Nature 2021[3], we want to utilize an engineered probiotic Escherichia coli Nissle 1917 strain (EcN1917) to locally increase intratumoural l-arginine levels to regulate the tumor microenvironment, affecting the expression of PD-L1 in tumor cells, and ultimately improving the viability of T cells.
However, we still feel confused with the following problems which need to be further clarified:
1 Current status of colon cancer treatment and immunotherapy
2 The ability of EcN1917 to colonize the intestine
3 Forms of probiotic pharmaceutical preparations
4 Drug Delivery Methods
5 How to verify the anti-tumor effect when animal experiments are not available
6 Precise Targeting Strategy
1 Public survey
In order to understand the current public opinion on the choice of immunotherapy, the choice of cancer treatment, and traditional therapies, we launched an online questionnaire.A total of 729 questionnaires were collected, with the proportions of age to gender and education
The main results and conclusion are presented as below:
• The incidence of colon cancer is age-dependent, people more than 60 years old is almost two times than that below 60.
• At present, people are more inclined to traditional treatment methods, and the popularity of immunotherapy and targeted therapy is not high.
• There are more reports of side effects to traditional therapies, and people are more concerned about side effects and expect therapies with fewer side effects. It gave us a clearer picture of our goals.
Through analysis and comparison, we found that the knowledge of colon cancer was very similar in both genders and those with a master's degree or lower. Most people had heard about the disease, and the number of highly educated people who were very aware of the disease increased depending on their chosen major.
We found before the age of 60 suffer from in the age structure of colon cancer accounts for 15% of the proportion of the total number of left and right sides, and aged more than 60 years to increase the proportion of people with the disease, almost proportion in the original two times, which proves that the incidence of colon cancer in the older population probability need bigger and more simple and easy treatment plan.
Through investigation, we learned that most of the patients choose through surgery for cancer treatment, most patients without serious use the traditional medical treatment such as chemotherapy, radiotherapy plan, which is the most stable of cancer treatments, and select targeted therapy and immunotherapy of population is relatively small, the lateral proves that such treatment can is very vast development space and prospects.
The disadvantages of traditional treatment are also very obvious, the disadvantages of radiotherapy chemotherapy are most obvious, will have great adverse reactions may occur in patients treated by, loss of appetite, nausea, vomiting, loss of hair fatigue are relatively high and difficult to avoid the symptoms of patients with medical expenses, meanwhile, has become many difficulties, it has bad influence on the part of the treatment of cancer, so we need safer and more effective treatments.
Finally, most people prefer treatment with fewer side effects, so immunotherapy and targeted therapy are very important in the field of cancer treatment.
Expert Interviews
Dr. Zhong Shi
Cancer Hospital Affiliated to University of Chinese Academy of Sciences
We know that the main treatment methods for cancer at present are surgery, chemotherapy and radiotherapy. However, the current traditional treatment methods, such as chemotherapy and radiotherapy, have great disadvantages. In order to further understand the current treatment of colon cancer, we are honored to interview Dr. Shi, Cancer Hospital Affiliated to University of Chinese Academy of Sciences. We asked her about the background of traditional treatment methods and immunotherapy for colon cancer, and shared our ideas on using arginine to boost immunotherapy. Based on her research experience, Dr. Shi shared a lot of knowledge about colon cancer treatment, especially the current status of immunotherapy and the current popular treatment strategies. Through the interview with Dr. SHI, we further clarified our project objectives and strengthened our confidence in achieving relevant research.
To sum up, from the interview with her, we mainly learned the following suggestions.
• Microsatellite stable bowel cancer is about 95% of stage 4 bowel cancer. It's going to be a little bit more meaningful to choose to cure it.
• The price of PD-1 antibody drugs was very high when it is just launched, and has declined in recent years.
• Combination therapy is more effective than either treatment alone.
• The PD1 monoclonal antibody has an effective rate of zero percent. Improving efficacy of immunotherapy is highly in-need.
• Modulating the tumor microenvironment is reliable strategy to improve the efficacy of immunotherapy.
Dr. Haiying Wang
Institute of Microbiology
To understand the potential of EcN1917 as a probiotic drug and its colonization in the gut, we were honored to have an online interview with Dr Wang from the Institute of Microbiology.
from the interview with him, we mainly learned the following suggestions.
1. Dr. Wang pointed out that although E. coli that produces arginine in the intestine can be implanted, the replacement of E. coli must be taken into account. E. coli iterates every 20 minutes, so it is necessary to achieve a permanent fixation of this bacterium in the intestine.
2. The effective rate of the drug is also worth studying, and efforts should be made to ensure that 30% of the drug effect is the best. But it is possible to do this with ECN.
3. The aim of this experiment is to produce a bacterium that can highly express arginine in the human gut and perform immune activation. For bowel cancer, Dr. Wang said there are many types of cancer, and the body produces about three to four million cancer cells every day, which cause different cancers in different places.
4. Finally, Dr. Wang pointed out that our project is relatively large. At the initial stage of research, we need to break down the objectives, gradually implement them, and focus on the training of thinking ability and practical ability.
Dr. Yongliang Zhu
Suzhou Precision Biotechnology Co.
Dr. Zhu is the founder of Suzhou Preisen Biotechnology Co., LTD. The company has accumulated many years of technology in intestinal microbiota detection, live bacteria drugs, bacterial gene editing, synthetic biology and other aspects.
In order to find out the proper administration mode of probiotics and the problem of tumor location colonization, we talked with Dr. Zhu, and in general, he gave us a lot of helpful advices, as follows:
1. At present, the clinical response rate of PD1 is generally about one third, that is, about two thirds of people do not respond or is called drug resistance. To solve this problem, methods need to be developed. For example, targeting small or large molecules, including PD1 inhibitors, this immune system activation is multifaceted, so we have to start with some other novel mechanisms.
2. Regarding the mainstream of cancer treatment, Zhu said that from the perspective of developing new drugs, colon cancer for example, most of the genetic factors are not much, mainly related to the environment and diet caused by cancer. This is also the reason we publicized this issue to public.
3. China has not yet from the regulatory level of approval, clinical breakthroughs are still pending. However, the modified probiotics drug has been approved as a systematic injection in the United States, so there is no problem in clinical practice.
4. Besides, Dr. Zhu pointed out that for modified EcN1917 drug development, we need to concern not only the effective of the anti-cancer but also the safety for human body. If a sudden influx of bacteria in the blood, there is a risk of sepsis. Thus, the strategies for expression controlled and kill the bacteria is in need. This suggestion inspired us to explore the lysin gene induced expression by arabinose to break down bacteria when needed.
5. Currently, it is indicated that the bacteria accumulate and enter the tumor when the tumor is relatively large. So, Dr. Zhu suggested us to find a way to let the bacteria to recognize the tumor and increase its viability in the tumor environment.
6. As for administration mode of probiotics, Dr. Zhu said that there are two ways at present, oral and injection. The effect of injection is better for directly injecting into the tumor tissue, but there is a safety risk. Oral administration is comparatively safer. To ensure safety, we select the oral administration finally after this talk with Dr. Zhu.
Field Trip to Transcenta ltd.
Due to time restriction and safety consideration, we were not able to do animal experiments, so how to verify the anti-tumor effect remained a problem. To find the answer, we had a field trip to Transcenta ltd. We were welcomed by the manager and was shown around in the building. After the visit, we were honored to have a face-to-face talk with Dr. Zhang and Dr. Yu.
Dr. Yu suggested that we might try to construct a colon tumor model, use drug with arginine on it and see if the tumor cells are eliminated. Then we could make further efforts and determine the range of arginine that could be used. Another piece of advice he gave was to design control groups more carefully. As we ultimately use T-cells to kill the tumor tissues, we should test out which factor causes the rise in the number of T-cells, or to say if it is caused by the probiotics. Finally, he advised us to look for references to determine the period of medicine intaking, such as medicines sold online.
We also received valuable opinions from Dr. Zhang. He concluded the ways of evaluating the security of the pill we were about to design, like the concentration of the effective ingredient, biomarkers, etc. About the drug conservation, Dr. Zhang suggested us to refer to drugs on the market. The most possible way is keeping in the state of dry powder. However, except for the conservation time, we should also pay attention to the activeness of the probiotics in the intestinal environment, so some simulation might also be needed before we finally finish the pill. According to his advices, we conducted a market research of drug packaging and further interviewed with Prof. Feng for our specific packaging design of our probiotics.
Prof. Hai Feng
Zhejiang University of Technology
Research interests in probiotic preparation
In order to find out the specific packaging form of our probiotic drugs, we conducted market research. It was found that the market share and user demand of powder, tablet and capsule decreased successively (See the figure below). Thus, our initial selection of packaging is powder. Besides, we felt confused about how to packaging the inducer drug arabinose which is responsible for lysis of the bacteria. On these issues, we consulted Professor Feng, a probiotic preparation expert. The suggestions we gained from Prof. Feng are as follows.
1 To maximize probiotic activity, Prof. Feng suggested to use microcapsules or hard capsule in order to prevent from damaging by stomach acid. Besides, it is better to use a single bubble wrap to ensure each capsule of probiotics can have a better activity.
2 Because our probiotic is anaerobic bacteria, Prof. Feng recommended that when making preparations, we should select coating materials with low oxygen permeability and embed it more deeply to reduce the oxygen transmission rate, so that the probiotics could have better survival.
3 As for preparation of arabinose, it was recommended that using enteric capsule to coating arabinose to ensure it being absorbed in the gut or oral liquid for its stable characteristic and water-soluble.
4. From the talk with Prof. Feng, we also further familiarized about drug approval process. Many probiotics are listed as food labels, which is much easier. If it is a drug labels, phase I phase II phase III clinical verification is needed, estimated at least 5 years.
Dr. Fang Zhengyu
Director of intestinal Department
Provincial Hospital of traditional Chinese Medicine
After we basically clarified the design of probiotic drugs, we interviewed Dr. Fang about the choice of tumor treatment drugs. Through this face-to-face talk with Dr. Fang, we realized that doing science, especially medical research, is a serious thing that needs to be rigorous and caring. It also makes us aware of that there is still a long way to go from lab research to clinical use.
1 The dosage of the arginine should be carefully considered
2 The balance and competition of intestinal flora should be considered when probiotics are used to treat colorectal cancer.
3 Dr. Fang reminded us to pay attention to ethics in medical research. "Patients are people, also have the anger and sadness".
Dr. Zhang Hong
Zhejiang university
Research interests in molecular imaging
To evaluate the possible strategy to achieve precise targeting, we spoke to experts in molecular imaging of cancer. Through the interview with Professor Zhang, we have a clearer direction for future project improvement and a deeper understanding of precision therapy.
To sum up：
1. Compared with traditional medical imaging, molecular imaging observes structural changes, while molecular imaging observes biochemical changes at the molecular level earlier than structural changes, which provides great convenience for disease detection.
2. There are three main groups of diseases that benefit from this: first, the structure of the tumor system; The second category is neurological disorders; The third category is cardiovascular disease. One of the problems we encounter in colon cancer treatment research is the precise targeting of tumor tissue.
3. Changes in colon cancer at different stages of development, especially at the molecular level.
4. Some suggestions for tumor tissue targeted therapy are put forward. One is a protein that is highly expressed on the surface of tumor cells. In addition, there are labeled radioisotope ratios.
5. In the study of different stages of colon cancer development, many molecular markers can be detected, such as specific genes, or proteins expressed by specific genes, etc.
References:
1 Rongshou Zheng, Hongmei Zeng, Siwei Zhang, et.al. The epidemiology of colorectal cancer in China. Global Health Journal, 2 (3), 8-20 (2018).
3 Canale, F.P., Basso, C., Antonini, G. et al. Metabolic modulation of tumours with engineered bacteria for immunotherapy. Nature 598, 662–666 (2021).