1 Abstract

Colon cancer is a relatively common digestive tract malignant tumor with a high mortality rate and is one of the three major cancers in the world. Immunotherapy is currently the most promising cancer treatment and can effectively treat colon cancer. Immunotherapy is more effective and less toxic than traditional chemotherapy. Our project consists of two main parts: Escherichia coli Nissle 1917 (EcN1917, or EcN)  engineered bacteria for arginine synthesis and functional validation of the antitumor effect of high concentrations of arginine on colorectal cancer cells. L-arginine has been shown to be an excellent choice for overcoming immunotherapy resistance. The main idea of our project is to use the non-pathogenic bacteria EcN to overcome the arginine inhibition mechanism by inserting the ArgJ gene and deleting the ArgR gene. To ensure biosafety, we inserted the Lysin gene into our designed EcN so that it would be lysed after the bacteria had done their work. It can enter the digestive tract through the mouth and colonize tumor sites in the digestive tract. The engineering bacterium EcN in the project is designed to use the metabolite ammonia of cancer cells as a substrate to continuously produce a large amount of L-arginine in tumors, and at the same time can improve the colonization ability and adhesion ability. Long-term stay in the tissue has great potential for the treatment of digestive system tumors. In addition, our probiotics can also be delivered to the target site by injection and oral administration in experimental animals, so this direction can be developed in future clinical applications.  


2 Cancer immunotherapy and its problem

Immunotherapy is the most promising cancer treatment method so far. It is based on patients’ own immune system, and brings the immune potential into full play to effectively inhibit or even eliminate tumor cells [1]. Recently scientists’ interest in immunotherapy has been intensified due to its established efficacy and less toxicity when compared to traditional chemotherapy [2].


However, cancer cells are constantly evolving and can develop a complex molecular and cellular network within the tumor microenvironment to suppress the immune response. They also change antigens on the cell surface to escape attacks by immune cells such as killer T cells. Therefore, after a period of immunotherapy, some cancer patients have acquired resistance to it due to the tumor-mediated immunosuppression [3].



Figure 1 Superresolution image of a group of killer T cells (green and red) surrounding a cancer cell (blue, center). (The National Institutes of Health, via Wikimedia Commons)


L-arginine supplementation has been shown to inhibit such immunosuppression and is a good candidate to overcome the resistance to immunotherapy.


3 L-arginine supplementation in treating cancer

L-arginine has cytotoxic effects against cancer and can be potentially used to treat cancer. High concentration of L-arginine inhibits the growth and proliferation and induces the apoptosis of various cancer cells such as liver cancer cells and hepatocellular carcinoma cells [4].



Figure 2 Time-lapse microscopy video showing apoptosis of DU145 prostate cancer cells (Egelberg, via Wikimedia Commons)


More importantly, L-arginine can inhibit the tumor-mediated immunosuppression and facilitate anti-tumor immune responses, therefore L-arginine supplementation can be used as a combined treatment option to enhance the efficacy and overcome the resistance of immunotherapy. Low level of L-arginine is shown to suppress T cell proliferation [5] and reduce the interaction of T cells with tumor antigens [6]. L-arginine treatment prolongs the survival of breast cancer-bearing mice treated with anti-PD-1 immunotherapy, partially via reducing the number of immunosuppressive cells (MDSCs) in spleen and tumor tissues [7].





Figure 3: Illustration of a mechanism of immunotherapy


These properties make L-arginine supplementation useful for antitumor therapy in humans, especially as a combined treatment with immunotherapy to improve the clinical outcome of cancer patients.



Figure 4 Structure of arginine (NEUROtiker, Public domain, via Wikimedia Commons)




4 Delivery of high-concentration L-arginine


Several delivery approaches are under consideration for L-arginine supplementation in treating cancer, such as oral administration, or injection into the tumors.


Yet in order to achieve high efficacy of a combined treatment of L-arginine and anti-PD-L1 immunotherapy, a cancer patient with 75 kg body weight will in equivalence need to orally take 150 g L-arginine based on a mice model, which is impractical.  Injection of L-arginine directly into tumors in mice models failed to inhibit the growth of tumor with or without anti-PD-L1 antibodies probably due to the fast diffusion of L-arginine [8]. These methods are unable to maintain high concentration of L-arginine locally within tumors.


Figure 5 Equivalent amount of L-arginine orally taken for a human being based on mice models in treating cancer


Bacteria are able to survive and proliferate in tumors. In this project, we developed a practical way to produce high local concentrations of L-arginine in tumors by using engineered non-pathogenic bacteria.



Figure 6 Treating tumors using engineered bacteria (image made based on Jlabanimation and Sarbasst Braian, via Wikimedia Commons)


5 E. coli Nissle 1917 (EcN) 

EcN is a non-pathogenic Escherichia coli (E. coli) strain. Because it is a probiotic with proven human safety record and genetic tractability, EcN is a common and favorable microbe for delivering therapeutic modalities to the sites of diseases [9]. EcN is highly targeted to solid tumors, can specifically colonize the hypoxic area of the tumor, and can be used as a delivery vehicle to deliver anti-tumor active proteins to the tumor area [10].  


The metabolism of tumor cells generates ammonia gas. Ammonia can be channeled into arginine synthesis by EcN, because EcN has the full set of enzymes required for this biosynthesis route. The partial pathway of arginine biosynthesis by EcN is shown in the figure below [11].



Figure 7: L-Arginine synthesis pathway in EcN


Because of its well-established record as a delivering vehicle in treating various diseases, its growing tool boxes for engineering, its innate arginine synthesis route and its ability to survive and thrive in tumors and gastrointestinal tract, EcN is an appropriate bacteria strain to be engineered to produce and maintain high level of L-arginine in tumors.


6 Our project

Our project aimed to provide a safe, effective and practical way to bring and maintain high level of L-arginine in solid tumors, especially in colorectal tumors, to facilitate cancer immunotherapy. The main idea of the project is to utilize the non-pathogenic bacterium, EcN, which can enter the digestive tract orally and colonize the tumor site in the digestive tract. EcN is engineered in this project to be able to keep producing large amounts of L-arginine in tumors by using the metabolic product of the cancer cells, ammonia, as substrates. After the treatment, a regulatory mechanism is introduced to eliminate the EcN in the system to ensure security.

Our project consists of two major components: the engineering of EcN for arginine synthesis and the functional validation of the antitumor effect of high-concentration arginine on colorectal cancer cells.



Figure 8 Project scheme


6.1 Arginine synthesis engineering in EcN

To construct an engineered EcN to continuously produce arginine in high concentration environment and be therapeutically used in tumors for patients, two aspects need to be considered. Firstly, we have to break the negative feedback route in the natural arginine synthesis route of the bacteria, so that EcN will not stop the production of arginine as arginine start to accumulate in tumors. Secondly, we need to ensure that EcN can be safely erased from the system and will not linger in the body once it finishes its therapeutic role.

Problem #1: Arginine inhibition of arginine synthesis in EcN (ArgA and ArgR genes)

In native E. coli bacteria, the negative inhibition of the arginine biosynthesis is achieved by an arginine repressor called ArgR protein encoded by argR gene and the arginine inhibition of N-acetylglutamate synthetase (NAGS) encoded by Arg A gene.

The arginine repressor, ArgR, is allosterically activated by internal L-arginine. Activated ArgR represses the transcription of all arginine biosynthetic genes in the pathway through binding to the 18bp conserved palindromic operator sequences in close proximity of or overlapping the promoter regions of these genes. In this way, the expression of enzymes needed for arginine biosynthesis from glutamate to arginine is negatively regulated by L-arginine levels in the bacteria cells. In this project, we deleted argR gene to exclude ArgR protein in EcN.


NAGS catalyzes the synthesis of N-acetylglutamate (NAG) from glutamate and CoA and is inhibited by internal arginine. By studying the arginine synthesis pathway of Corynebacterium glutamicum, we found ornithine acetyl transferase encoded by argJ gene also converts L-glutamate into N-acetyl-L-glutamate and can function well at high concentration of arginine. Therefore, ornithine acetyl transferase is a good substitute for NAGS to avoid the negative feedback of arginine in the biosynthetic pathway. In this project, we transferred argJ gene into our engineered bacteria to bring ornithine acetyl transferase into the arginine synthesis pathway.

Problem #2: Biosafety

The engineered bacteria may have biological safety problems in vivo over a long period of time. Therefore, we transferred a lysin gene with an arabinose-induced promoter into the engineered bacteria. After the antitumor effect of the engineered bacteria is completed, the patient can consume arabinose via oral administration or inject, etc, and the promoter of the lysin gene will be activated by arabinose and express lysin protein, causing the bacteria in tumors to commit suicidal lysis.


Figure 9 Main problems tackled in our project


Detailed design for engineering EcN

Step 1: Deletion of argR

We first knocked out argR in EcN by the λ Red homologous recombination system and developed a 1917argR strain incapable of expressing the arginine repressor, argR protein. The λ Red homologous recombination system works by replacing the gene of interest, which is argR gene in our project, with a donor gene using a set of enzymes. We used the kanamycin resistance (kanR) gene from the pKD4 plasmid as the donor gene, which gives bacteria immunity to kanamycin. Therefore, we could determine whether the argR gene was knocked out through kanamycin resistance.


To achieve the above goals, we constructed parts H1-argR-H2 (BBa_K4410005) and H1-KanR-H2 (BBa_K4410006). Click here for more details. A detailed schematic illustration of the roles of those parts in the λ Red homologous recombination system used in this project is shown in Figure 10.




Figure 10: Schematic illustration of λ Red homologous recombination system used in our project


Step 2: Insertion of argJ gene

We inserted the argJ gene from the chromosome of Corynebacterium glutamicum into a conventional vector plasmid, pGLO-J23100, by In-Fusion cloning technique and constructed a pGLO-J23100-argJ plasmid. This recombinant plasmid was introduced into bacterial DH5Alpha to be amplified in large quantities. Purified pGLO-J23100-argJ plasmids were transferred into the 1917argR strain to form a 1917argR(pGLO-J23100-argJ)strain that can produce bifunctional glutamate N-acetyltransferase/amino-acid acetyltransferase and can’t produce the arginine repressor.


To achieve this, we constructed a part J23100+RBS+argJ+terminator) (BBa_K4410007). Click here for more details. It is the key part that is responsible for the expression of bifunctional glutamate N-acetyltransferase/amino-acid acetyltransferase.




Figure 11: Schematic illustration of In-Fusion cloning technique  



Step 3: Insertion of lysin gene

We isolated plasmid DNA from the MG1655 bacteria by plasmid purification. In this plasmid DNA, araC gene and araBAD promoter are upstream of the Lysin gene, which together control the transcription and expression of the Lysin gene by arabinose concentration. This plasmid was transfected into our engineered EcN. In this way, the EcN can produce lysin proteins to kill the bacteria by cell lysis, when arabinose concentration is above the threshold.


Step 4: Function Verification

To test the ability of the engineered bacteria to produce arginine, our engineered EcN was cultured and native EcN was used as a control. The rate of arginine synthesis was detected by an amino acid analyzer,after 48 hours of incubation.


To test the function of the lysin gene in our engineered EcN, we added arabinose to the colonies and analyze the bacterial growth curve to detect the lysis.


6.2 Determine Arginine anti-tumor effect

We co-cultured a colorectal cancer cell line CT26 with our engineered EcN (EcN1917-ARG) and compared the cell viability of the CT26 cells with that of the CT26 cells co-cultured with native EcN1917. In this way, we validated the antitumor effect of our engineered EcN against colorectal cancer.


7 Overall design and prospect

All in all, in order to use high concentration arginine locally to synergize the antitumor effect of immunotherapy against solid tumors such as colorectal cancer, our project used the probiotic EcN as a therapeutic vehicle to produce large amount of L-arginine in tumors. We engineered the bacteria to overcome the arginine inhibition of the arginine synthesis pathway by adding the argJ gene and deleting the argR gene. To ensure biosafety, we inserted the Lysin gene in our engineered EcN to lyse the bacteria once their jobs are done. Experiments are designed to validate the efficacy and safety of the engineered bacteria and the treatment effect of arginine on colorectal cancer cells.


Figure 12: Overall design of our project


Probiotic EcN has a tumor-targeting effect, which can specifically accumulate in tumor tissue and act as a targeting transporter [12].

Our engineered probiotic can be rapidly colonized in the intestinal tract by oral administration, has a good adhesion rate on porcine intestinal epithelial cells [13], can stay in the tumor tissue in the intestinal tract for a long time, and has potential for the treatment of digestive system tumors with positive effects. In addition, our probiotics can also be delivered to target sites by injection in experimental animals, so this direction can be developed in future clinical applications. Our genetically engineered bacteria will not be limited to oral intake into the digestive system, but will be extended to various solid tumors.



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