Arginine supplementation can kill cancer cells directly and more importantly achieve a synergistic effect with immunotherapy against cancer. Our team has developed a modified E. coli Nissle 1917 (EcN1917) that can use the metabolite ammonia of cancer cells as a substrate to persistently produce arginine in tumors and can be used as a therapeutic vehicle against solid tumors. At this stage, our product is designed to be orally administered, and reach the tumor lesions through gastrointestinal (GI) tract, so its target users are colorectal cancer patients. To ensure biosafety, we inserted the Lysin gene with an arabinose inducible promoter into the EcN so that the bacteria is lysed in response to arabinose after the work is done. Our team is currently working on developing alginate–chitosan–alginate (ACA) microcapsules to coat our engineered EcN1917 to achieve safer, longer and stronger therapeutic effect. As a live biotherapeutic product (LBP), our product is still in its early stage of development and will need further improvement and evaluation based on animal models, before it can be further progressed into clinical trials, in order to meet the requirement of LBP criteria. There is still long way to go before possible clinical application in real world.


1 Project goal

This year, our team aimed to tackle the long-standing problem of immunotherapy resistance in treating cancers. Through literature review and HP work including interviewing experts in the field of oncology, we learned that L-arginine has been shown to be an excellent choice for overcoming immunotherapy resistance in cancer patients and itself can also kill cancer cells. From the interview results of Dr. ZHU, we gained valuable advices about how to introduce this chemical into the tumors. We learned that Escherichia coli Nissle 1917 (EcN1917, or EcN) has long been engineered as vehicles for various target therapies.


Therefore,we constructed an EcN strain to be able to continuously synthesize L-arginine in solid tumors. The high concentration of L-arginine produced by our bacteria vehicle in the tumors can kill cancer cells, mainly through activating the anti-cancer immune response. The effect would be most significant when L-arginine supplementation is combined with immunotherapy.  



Figure 1 L-arginine (Photohound, Public domain, via Wikimedia Commons)


2 End User

Our product, the engineered EcN for arginine synthesis, is of great value to solid tumor patients. The engineered EcN is designed to use the metabolite ammonia of cancer cells as a substrate to continuously produce a large amount of L-arginine in the tumors. This process can also improve the colonization and adhesion ability of EcN. Long-term stay in the tissue has great potential for the treatment of tumors. To ensure biosafety, we inserted the Lysin gene with an arabinose inducible promoter into the EcN so that the bacteria is lysed in response to arabinose after the work is done.


In this early stage of development, our product is designed for oral administration. EcN can enter the digestive tract through the mouth and colonize tumor sites in the digestive tract especially the colorectum. Therefore, at the current stage, our product is best suited for colorectal cancer patients.  




Figure 2 Colorectal cancer patients are our major end users (Blausen Medical Communications, Inc., via Wikimedia Commons)


Since L-arginine can achieve a synergistic effect with immunotherapy, our product can greatly benefit cancer patients with immunotherapy. Patients who have the potential of developing immunotherapy resistance or who have already developed it are especially in need for products like ours. Our product is a promising therapy to combine with immunotherapy to improve the short-term efficacy and long-term survival benefit of immunotherapy in treating patients with solid tumors especially colorectal tumors.



Figure 3 Mechanism of anti-PD-1 and anti-PD-L1 drugs in immunotherapy


3 Product operation

Our product is still in its early stage of development and will need further improvement and evaluation based on animal models, before it can be further progressed into clinical trials. There is still long way to go before possible clinical application in real world. And we also talked this issue with Prof. Feng (an expert in probiotic preparation) and Dr. Zhu (an expert in probiotics and synthetic biology), we know that clinical breakthroughs of genetically modified 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.




Figure 4 Illustration of clinical trials required in a typical pharmaceutical development process for drugs (Bibeyjj, via Wikimedia Commons)


To envision the operation of our product, we had interviews with several experts. For safety consideration (according to the advice of Dr. Zhu) and market research results of drug packaging, it will be presented as an oral probiotic for colorectal cancer patients. Since L-arginine has high synergistic effect with immunotherapy, our product is recommended to be applied in combination with immunotherapy. The dosage and timing should be determined by clinical trials. When the treatment is finished or need to be terminated due to patient’s adverse responses, arabinose is orally taken by the patient. Arabinose will induce the lysis of our engineered EcN and thus eliminate the bacteria fully in the system.


Figure 5 The survey result of the share and demand for each packaging in the drug market.


4 Real-world implementations

In order to find out the proper administration mode of probiotics and the specific packaging form of our probiotic drugs, we conducted market research, had interviews with experts in probiotic preparation (Dr. Zhu and Prof. Feng). Since our product is a probiotic engineered for medical purpose, for the implementation in the real world, there are many issues to be considered.

First of all, we have to consider coating our product. Our product is designed to be orally administered, and reach the tumor lesions through gastrointestinal (GI) tract. Gastric acid and bile salts in the gastrointestinal tract often cause the inactivation of probiotics. This can prevent our product from reaching the lesions viably, so protective enteric coating is needed. Besides, because EcN1917 is anaerobic, so we should select coating materials with low oxygen permeability and embed it more deeply to reduce the oxygen transmission rate (Inspired by Prof. Feng). We finally selected alginate–chitosan–alginate (ACA) to coat our probiotics.

ACA microcapsules are expected to achieve the following purpose:

(1) Cheap coating

Chitosan is a wildly found natural polysaccharide, with good mechanical properties and biocompatibility [1]. So, chitosan is a good film-forming material for coating, and it is cheap for mass production.

(2) Long shelf life of EcN--prolonged stability

ACA can prolong the preservation time of our product, as researches have shown that the stabilization period of E. coli encapsulated in ACA was significantly prolonged compared with suspension culture and traditional gel immobilization culture[2]

(3) Prolonged and enhanced bioavailabiliy of EcN in GI tract

ACA has strong pro-mucosal properties and prolonged action to withstand GI fluids, enabling longer residence time of our product in GI tract to carry out its therapeutic effect. As shown in mice model, ACA microcapsules instilled into the stomach remained well preserved in the small intestinal area for 48 h[2]. Recent research also showed that chitosan and sodium alginate had better effect in enhancing the bioavailability of engineered EcN in the GI tract in a mouse model [1].

(4) Good permeability for bacteria activity

The permeability of ACA membrane ensures nutrient supply and the transmembrane transfer of metabolites of the bacteria, so that they can carry out the production mission and deliver arginine to the lesions.

(5) Safe

Chitosan and sodium alginate are FDA-approved materials for food additives. Therefore, ACA is safe for our product.

(6) Dosage control and Stable activity

According to the suggestion of prof. Feng, a single bubble wrap could be used to ensure the better activity of each capsule of probiotics, as well as control of the dosage.


In addition to the above coating concerns for our product, there are other aspects worth reckoning before applying the product into real world:

(1)  Beside gastric acid and bile salts in the tract, environmental factors in the tumor regions, such as pH value, available nutrients and oxygen level can also affect the growth and metabolism of the bacteria. Engineered new pathways may bring additional burden for the bacteria in harsh environments. Therefore, experiments of such influences on the bacteria and more trials based on animals are demanded for our product.

(2)  Since our product is designed for treating disease, it is a live biotherapeutic product (LBP). LBPs should be studied for toxicology diligently and must conform to strict regulations by organizations such as FDA. In America and Europe, LBPs must be safe, reliable, robust and consist in each batch and must go through strictly regulated clinical trials to prove its efficacy and safety for a particular population before it can be used clinically in the real world. To date, no LBP has been approved by FDA yet, though several engineered bacteria have been evaluated in the clinic [3].



Figure 6 Clarification of terminology and corresponding regulatory status [4]


(3) Facilities for the manufacturing, processing and packaging of LBPs are also required to strict regulations under current good manufacturing processes (cGMP). Hence, there will be limited options of manufacturing factories once our product is ready for clinical use.

(4) If the product is approved to be used clinically, throughout the treating process, individualized evaluation of patients’ condition and tailored modification of the treatment by physicians is necessary. Side effects should be monitored and evaluated closely, and patients might demand treatment for such symptoms or termination of the treatment if the side effects are unbearable. The short-term efficacy of the treatment should be analyzed based on standard clinical criteria and guidelines.     


5 Safety  

Due to the genetic modifications and therapeutic characteristics of our product, there are several safety aspects we must be aware of.

Firstly, our product is engineered bacteria with exogenously introduced genes. Such modification needs to be stable over time for safety concerns and hence requires thorough evaluation.

In addition, the cassettes of antibiotic resistance and other genes used for the engineering purpose might transfer to other microbiota both in the body and in feces. And elimination of the bacteria within the body and in feces when its job is done is preferred. Our lysin insertion is one approach to tackle this issue. Our preliminary results showed that EcN1917 transformed with pGLO-Lysin plasmid can be lysed significantly by 10-hour treatment of 30mM arabinose (Figure 7). Oral intake of arabinose and adding arabinose to excrement can lyse the bacteria when needed. And according to Prof. Feng, the arabinose is very stable and water-soluble, so it can be coated with enteric capsule or oral liquid.



Figure 7 Effect of arabinose on EcN1917 transformed with pGLO-Lysin plasmid (Left: bacteria solution without arabinose, right: bacteria solution treated with 30mM arabinose for 10h)



Although the target site is the tumors, the engineered bacteria can also reside in other regions of the body, their biodistribution in those areas and the potential effect on the body might require evaluation.

The bacteria may contain certain components like lipopolysaccharides that could cause damage to the body. Although for the current oral administration method, it is less of a problem, once the product is developed to apply systemically or directly intratumorally, the toxicology of such agent needs to be studied carefully [2].



Figure 8 The general structure of lipopolysaccharides (kupirijo, via Wikimedia Commons)


6 Challenges and prospect 

Animal experiments have shown that it can also be delivered to the target site by injection, and systematical administration might also be an option in the future. The product might benefit patients with other cancer types as the research deepens. However, the actual efficacy and safety of the product in vivo need to be thoroughly studied before application in human, and many safety concerns need to be tackled and regulatory guidelines must be followed as mentioned above. Therefore, more researches and clinical trials are needed before this engineered EcN can truly implement clinically and benefit cancer patients. The guidance for the safety regulations and toxicology criteria for LBPs are currently general and not very specific in published documents. Further research and studies regarding these areas might require more consultation with the authorities in the countries of interest.



[1] Jun Zhou, Maoyi Li, Qiufang Chen, et al. Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery. Nature Communications, (2022), 13:3432.

[2] Fu Yingli, Xiong Ying, Liu Xiudong,et al. Study of Alginate/Chitosan Microcapsules for Immobilization of Escherichia coli DH5a. Chinese Journal of Biotechnology, (2002),18(2): 239.

[3] Mark R. Charbonneau, Vincent M. Isabella, Ning Li, et al. Developing a new class of engineered live bacterial therapeutics to treat human diseases. Nature Communications, (2020), 11:1738.

[4] Cordaillat-Simmons, M., Rouanet, A. & Pot, B. Live biotherapeutic products: the importance of a defined regulatory framework. Exp Mol Med 52, 1397–1406 (2020).


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