ETH_Zurich

CATE - Cancer-Targeting E. coli

Abstract

Many cancer therapies exist today. They can be divided into systemic and local treatments. Systemic approaches are based on administering therapeutic substances to the whole organism. These substances have different levels of specificity for tumor cells and typically significant off-target effects. Local treatments involve either manual removal of malignant tissue by surgical interventions or irradiation. While systemically administered toxins can damage off-target sites in the body, local treatments oftentimes lack precision in terms of excision of the whole tumor tissue or are themselves carcinogenic, such as radiotherapy. .

In our project, we aim to develop a system that combines the advantages of both strategies, while minimizing their disadvantages. A non-pathogenic strain of E. coli with the ability to populate solid tumors upon intravenous (systemic) administration is used in our experiments. The strain will be engineered to have an Imaging module and a Payload module. The Imaging module includes a cell density sensor, which triggers expression of a magnetic resonance imaging (MRI) contrast agent and accumulation of a toxic agent only if a certain bacterial cell density has been reached. The Payload module consists of a thermo-inducible toxin release system, so that a spatially controlled focused ultrasound pulse can activate cell lysis and toxin release. In a cancer patient treated with our novel strain, the bacterial growth is restricted to tumor tissue and the contrast agent will mark all tumors present. The medical doctor will investigate the MRI and confirm that the therapeutic bacteria have colonized the correct location in a sufficient amount to avoid sub-dosaging. The doctor then uses focused ultrasound to increase the temperature locally, which activates cell lysis and subsequently toxin release. This way, a highly potent anti-cancer therapeutic is delivered intravenously, but activated only in the malignant tissue, minimizing damage to healthy cells.

This high level of interplay between therapeutic agent and treatment supervisor has not been achieved yet and paves the way towards a new generation of cancer therapeutics.

Detailed Description

In the last couple of years, the understanding and ability to manipulate living organisms has increased greatly. It is now possible to program organisms to execute defined functions and interactions with their environment. However, these astounding advancements have not found applications in cancer therapy yet. Classical strategies such as chemotherapy, surgery and radiation therapy remain most successful and are still the backbone of cancer therapy.

These classical approaches, however, share several drawbacks: first, they are prone to incomplete killing of cancer cells and thus often result in a relapse. One reason for this is the potential development of resistance to the therapeutic molecules by evolution. Second, they exert considerable stress on the patient by damaging off-target tissue. This is common in chemo- and radiotherapy, where the whole body is exposed to toxic agents with only a limited preference for cancer cells, or the healthy tissue between tumor and skin gets exposed to carcinogenic radiation. Normal tissue toxicities therefore typically limit the dose for both chemo- and radiotherapy. A subpopulation of tumor cells might survive and develop resistance to the therapy. Surgical procedures fundamentally prevent development of evolutionary acquired resistance. But especially in difficult areas, they are not always feasible, and pose the risk of bleeding, pain, infections or anesthesia related reactions. Furthermore, it is challenging for the surgeon to differentiate tumor tissue from healthy tissue, resulting in incomplete excision of the tumor. The residing cancer cells after surgery can easily lead to regrowth of the tumor.

The aim of our project is to design a tumor-targeting, cytotoxic agent-delivering microorganism to overcome limitations of current treatment modalities. Inherent tumor targeting capabilities (primarily due to ability to survive only in neoplastic microenvironment) and lack of pathogenicity make the probiotic E. coli Nissle 1917 an ideal chassis for development of precisely controllable features. The presence of bacterial cells close to tumor sites further enhances the recruitment of additional immune cells, thereby directing the immune response to immunoevasive cancer cells. We plan on harvesting this inherent therapeutic potential and extending it by developing the bacteria to act as anti-cancer agents in a tissue-specific, safe and controlled manner. The engineered bacteria will have two modules to ensure safety and control. These modules will consist of a set of tightly controlled and fine-tuned functions that will be engineered and tested individually.

The treatment starts with intravenous administration of our engineered bacteria (bacterial therapeutic) to the cancer patient. The intrinsic properties of the used strain (E. coli Nissle 1917) lead to selective colonization of primary tumors and metastatic nodules. Colonization of the neoplastic tissue leads to the activation of the Imaging module via Quorum Sensing (QS) and subsequently production of both the toxin and the contrast agent is triggered. Now the patient is examined by MRI and the doctor can assess the location of the contrast agent. If the signal corresponds to the tumor tissue, the procedure can be continued. The doctor applies focused ultrasound (FUS) to the location of interest, which increases the temperature to around 42 °C. This activates the Payload module via a Thermal Switch (TS), bacterial cells get lysed and the previously accumulated toxin is released to the tumor cells.

The Imaging module is designed to locate a single or multiple tumors and to assure sufficient colonization by the therapeutic bacteria. Only if the number of bacteria in the tumor reaches a threshold, a contrast agent will be produced and the doctor will be able to visualize the colonization of the tumor via MRI. We decided to use MRI since almost all currently used FUS treatment protocols are MRI-guided. Additionally, the sensitivity, spatial resolution, lack of ionizing radiation and availability of contrast agents make MRI the most attractive imaging system to use in this application. The Imaging module constitutively produces a small amount of N acyl homoserine lactone (AHL). AHL can diffuse easily through tissues and its concentration can be sensed via QS. Once the population density and therefore AHL concentration exceeds a threshold, the Imaging module will trigger expression of bacterioferritin, a type of ferritin-like proteins produced in bacteria. Upon accumulation of bacterioferritin, there is a change in magnetic properties of the tissue and thereby areas populated by bacteria become visible in MRI as local reductions in signal intensity. The doctor can assess the localization of the bacteria in 3D, which should correlate well with the tumor and decide for the procedure to be continued. In addition to bacterioferritin, expression of a cytotoxic agent is triggered at the same time. It will accumulate in the therapeutic cells and be ready for the next step of the treatment.

Once accumulation of a sufficient amount of bacteria in the tumor site is confirmed via MRI, the doctor will trigger temperature increase via FUS. FUS is a novel treatment technology, recently introduced into the clinics. It non-invasively raises temperature in a small volume of tissue with high precision. However, when used alone, high temperatures are required to ablate the tumor, thus inevitably leading to damage in the surrounding tissue. In our approach, only a slight temperature increase is needed to site-specifically activate a thermo-inducible pathway which leads to delivery of cytotoxic molecules by the engineered bacteria, thus minimize side effects related to high temperatures. To couple the release of the cytotoxic protein to the temperature increase, we propose to use a thermal bioswitch (TlpA) that activates transcription of protein E at a temperature of 42 ºC.7 Protein E causes bacterial cell lysis by forming a tunnel through the cell membrane. Once the cells lyse, the previously accumulated toxin is delivered in a controllable manner. For the cytotoxic agent, we propose to use the effector domain of azurin. It has already been proven as a potent anticancer peptide and is currently being tested in phase 1 clinical trials.

We plan to build new BioBricks for the TS and optimize the QS to work in the appropriate AHL concentration range. Furthermore, we will engineer a system of synchronized cell lysis to ensure delivery of an ample amount of azurin. Electronic hardware developments will accompany these building blocks to demonstrate that their practical use is possible. A self-made ultrasound device will enable us to stimulate the bacteria in conditions as close as possible to in-vivo ones. To emulate the thermal activation process of our strain, we plan to focus ultrasound waves on a Petri-dish inoculated with our thermo-inducible bacteria and locally trigger the expression of a reporter gene. After engineering the parts individually, we aim to combine and test them in a working proof-of-concept strain.

To conclude, we propose a novel treatment modality for local killing of both solid tumors and solitary metastatic nodules, previously never achievable in one simple, non-invasive step. In our opinion, this way of safe local drug delivery to multiple neoplastic sites is a potentially valuable tool in the fight against cancer and offers significant advantages over existing local treatments. Additionally, achieving a good local control allows for a decrease in doses of systemic therapy, therefore offering advantages not only for confined disease, but also for late-stage, disseminated cancer.