Project Description

GI tract cancers are an enormous public health issue, and together are responsible for more deaths than any other form of cancer. One of the major difficulties with diagnosing and treating these cancers, especially when localized to the intestine, is access to the tumour - patients often do not exhibit obvious symptoms until the later stages, when treatment options are limited.

We looked into a few different ways of tackling this issue, from improving existing diagnostic methods to developing new avenues for treatment. One of the consistent themes that came up in our brainstorming process was the potential for us to use the host's cell-mediated immune response to fight cancer. Cancer immunotherapy is a rapidly growing field, and clinical trials for certain forms of leukemia are already underway.

Thus, this year, McMaster iGEM sought to augment the power of the host immune system to fight against GI tract cancers, using a specially engineered strain of commensal lactobacillus bacteria. When completed, our bacteria will be able to sense the presence of tumours in the gut, bind to specific receptors on tumour cells, and begin secreting pro-inflammatory cytokines in this tumour microenvironment. This will recruit T cells to the site of the tumour and elicit an anti-cancer response, effectively stopping the cancerous growth using the body's own toolkits. This summer, we aim to develop a proof-of-concept of our idea, and create a bacterial strain that can secrete IL-2 under tightly controlled, tumour-specific conditions

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In the past May of 2016, mGEM collaborated with Hamilton Health Sciences to present Discovery Day at McMaster. This constitutes a day filled with workshops where secondary school students are able to explore a wide variety of careers in medicine and health sciences. One of the workshops lead by mGEM allowed students to apply their basic knowledge of cellular structures to a common laboratory practice where they were able to stain and visualize the cells. The second workshop reviewed the basic structure of DNA approached in the science curriculum, and further applied this to characteristically separate DNA in PCR, a common laboratory practice. This collaboration between mGEM and Hamilton Health Sciences further strengthened our efforts to educate other students on synthetic biology and its scientific relevance in the working world today. The reputability of Hamilton Health Sciences was effective in attracting secondary school students for the workshops prepared by mGEM.

Gastrointestinal (GI) cancers have one of the highest cancer mortality rates in Canada due to difficulties in tumour diagnosis and treatment1. Common anti-cancer therapies used today can lack specificity and have off-target effects, damaging the body in the process2. Our goal is to develop the foundation for a novel form of GI cancer immunotherapy that exploits commensal gut bacteria to specifically target GI tumour cells for destruction by the immune system. Our bacteria of choice is Lactobacillus brevis, which has been designed to bind and aggregate towards Her2+ GI tumours through the use of a cell-wall anchored, anti-Her2 DARPin. Like antibodies, DARPins constitute high-specificity interactions that have the potential to mitigate many off-target effects3. With sufficient bacterial density, a quorum sensing mechanism (Lux and Las) triggers the production of a vital T cell activating cytokine, interleukin-2 (IL-2), to recruit tumour-specific T cells for an anti-cancer response. Future steps will include simulations of the bacteria in a possible tumour environment, which will help drive quorum-sensing design (e.g. promoter sensitivity).

Ethical concerns were addressed through research into the feasibility of such a treatment, particularly into methods to minimize health risks, make the treatment financially accessible, and abide relevant legislation.

References:

1. Canadian Cancer Society’s Advisory Committee on Cancer Statistics. Canadian Cancer Statistics 2015. Toronto, ON: Canadian Cancer Society; 2015.
2. Dy, G. K.; Adjei, A. A. CA. Cancer J. Clin. 2013, 63 (4), 249–279.
3. Stumpp, M. T.; Binz, H. K.; Amstutz, P. Drug Discov. Today 2008, 13 (15), 695–701.

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BBa_K1929300

Interleukin-2 (IL-2) with designed RBS

Interleukin-2 (IL-2) protein coding sequence. A designed RBS with spacer DNA was added preceding the IL-2 for protein expression. This biobrick can be inserted after promoter of choice. IL-2 will be ultimately incorporated into the Las and Lux quorum sensing systems. With sufficient stimulation of the quorum sensing mechanism by an aggregation of bacteria, IL-2 production will occur and be released by the bacteria into the extracellular environment. Il-2 is a signalling cytokine and stimulates the production and differentiation of T cells (adaptive immunity). There is a silent point mutation at base pair insert 272 (A>T) to make the gene BioBrick compatible (XbaI site was removed).

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This project was worked on by many undergraduate students in the McMasterU iGEM 2016 Team, listed here by sub-team and alphabetically:

Wet Lab Project Design, DNA Constructs Design, and Benchwork: Chirayu Bhatt, Karanbir Brar, Tony Chen, Yosef Ellenbogen, Melodie (Na-yoon) Kim, Vivian Lau, Dhanyasari Maddiboina, Maxwell Ng, Yu Fei Xia

Website, Graphics Design, and Public Relations: Tony Chen, Farhaan Kanji, Nicolas Lehman, Maxwell Ng, Candy Niu, Yu Fei Xia, Kaijie Zhang

Computational and Software Lab: Ian Fare, Nicolas Lehman, Lauren Liu, Maxwell Ng, Christine Wang, Kaijie Zhang

Human Practices: Mohammed Ahmed, Karanbir brar, Yosef Ellenbogen, Melodie (Na-yoon) Kim, Jinny lee, Clara Long, Hwa Young (Haley) Yun

Community Outreach: Mohammed Ahmed, Chirayu Bhatt, Tony Chen, Yosef Ellenbogen, Vivian Lau, Clara Long, Ramneet Mann, Maxwell Ng, Rui Sun, Hwa Young (Haley) Yun

Administration and External Liaisons: Mohammed Ahmed, Beenish Ajmal, Karanbir Brar, Farhaan Kanji, Samaher Ramzan, Michael Xie

We would like to thank all our sponsors and mentors for guiding us and giving us the platform to investigate our research questions and conduct our project. It took an extraordinary amount of collaboration and support and we could not have done it without you.

Dr. Rosa da Silva, Ms. Alison Cowie, Mr. Alastair Tracey, and the McMaster University Biology Department, for their support, guidance, provision of reagents, and lab space.

Dr. Michael Surette and the Farncombe Institute for providing us with lab space, L. brevis strain and reagents to work with.

Dr. Dawn Bowdish and the McMaster Immunology Research Centre, for providing us with lab space and tissue culture reagents.

Dr. Indranil Biswas from the University of Kansas for providing the pIB184-Km plasmid.

Hamilton Health Sciences
Integrated DNA Technologies
McMaster Student Union USIF
Faculty of Science
Faculty of Health Sciences, BHSc Program
Department of Biochemistry and Biomedical Sciences
Biotechnology Program

As we develop our IL-2 delivery mechanism, we have considered the ethical, practical and legal implications of implementing such treatment in the current Canadian oncology institutions.

How reasonable is it to mass produce for regular usage? What would the cost be? Who will be paying? How will it be used? Who will regulate it? How secure is our product?

First, we introduce our envisioned final product: the lactobacilli in a pill form, liken to a probiotic pill.

Product Description:

The pill will contain our engineered bacteria condensed within a microcapsule to protect them from the upper GI tract and help localize to the colon. Specifically, we are aiming to use resistant starch which cannot be metabolized by pancreatic enzymes but is fermented by gut microflora in the colon, releasing our bacteria (1). In addition, starch is non-toxic and is a normal component of human diet, making it less likely to cause adverse reactions. Additionally, we know from past work that lactic acid bacteria that is protected by the potato starch granules can survive for 6 months in room temperature, and 18 months when frozen. This indicates relatively moderate shelf life if our product is to be mass produced and distributed all over the country, in a variety of environmental conditions (1).

Cost in Mass Production?

To offset the high cost of growing a large amount of bacteria for medical applications, it would be beneficial to purchase effluent waste products from agricultural processing of grains and vegetables as cultivation media for our bacteria. These waste products contain high levels of proteins, carbohydrates, sugars, and vitamins, and thus can be used as low-cost growth media for lactic acid bacteria, including the species of lactobacillus that will act as our treatment (2). This reduction in production cost will assist in keeping the treatment burden low for the patients.

Dosage deliberations:

Determining an appropriate dosage of the bacteria that provides an optimal level of IL-2 production in GI tract cancer patients is an important consideration, one that will require several rounds of animal trials and phase 1 clinical trials in order to perfect. Prior work using lactobacilli in the treatment of IBD has used a dosage of two tablets a day containing 1010 colony-forming units (CFU), for a period of six to nine months (3, 4). As our treatment would not require substantial colonization of the gut as in IBD trials, we predict a lower dosage, ideally at one tablet a day. However, as bacteria have never before been used in cancer treatment, dosing experiments on animal models are required before any reliable approximations of human dose can be made.

Concerns on Safety:

Lactobacilli, including Lactobacillus brevis, are “generally regarded as safe” worldwide. Support for this classification stems from its long history of use in food products and from clinical studies examining the safety of many lactobacilli strains (5). In fact, this safety status is considered one of the major benefits of using lactobacilli as a vector for the therapeutic targeting of GI pathologies (6).

IL-2 is a regulatory cytokine, and too much of it in the body can have severe adverse effects. The most common manifestation of IL-2 toxicity is capillary leak syndrome, when fluids within the vascular system leaks into the tissue outside the bloodstream (7). This results in low blood pressure and poor blood flow to the internal organs. Capillary leak syndrome is characterized by the presence of 2 or more of the following 3 symptoms; low blood pressure, swelling, and low levels of protein in the blood (7). During the treatment phase, it is vital that patients are monitored and regularly screened for symptoms that is common to IL-2 toxicity.

Who should be excluded?

The therapy requires the body to able to respond to IL-2. Therefore, patients with IL-2 receptor deficiency would be unaffected by this treatment. Additionally, patients undergoing total body irradiation as part of a bone marrow transplant would be unable to respond to this treatment. This treatment is limited to tumours present in the intestinal lumen, and not within the intestinal tissues. Furthermore, consideration must be made to individuals with any type of immunodeficiency disorder as they might not respond optimally. Deliberations must be made case-by-case.

Frequency of treatment:

After entering the gut, lactobacillus brevis leaves 24 hours later (8). Therefore this pill will have to be administered daily to maintain constant level of therapy.

Monitoring therapy:

This pill is designed so that the patients would be able to self-administer, without the need of entering the hospital. However, we highly recommend strict regular check-ups for early detection of adverse effects and progression of disease.

To check for the presence of our bacteria in the gut, we will collect and culture patient stools during weekly follow-up visits after commencing the treatment (3). In addition, blood assays testing for the level of colon cancer tumor markers will be carried out both prior to and at monthly intervals during treatment to assess its effectiveness (9). Magnetic resonance imaging (MRI) will also be used at regular intervals to assess tumor size and remission. Patient will be asked series of questions that screen for symptoms of IL-2 toxicity, and blood assays will test for the localization of IL-2 release, to be isolated from the rest of the body.

Ethical Considerations:

Our product must follow the Canadian Medical Association’s code of ethics (last reviewed 2015) as radical therapeutic to a vulnerable population group (10). This therapeutic approach will be artificially modifying the levels of cytokine IL-2 in patients, many with affected immune system. Although safety mechanisms are designed in the bacteria to limit the release of IL-2 to specific cancer regions (see wet-lab), multiple follow-up trials must be conducted before this drug can be put into large-scale practice. These protocols must be clearly defined and standardized. Doctor - patient communication is an important factor to consider when introducing any novel approach to cancer therapy. Before the treatment takes place, the patient must be fully informed of the possible side-effects before obtaining consent.

Outside of practice, this product as a commercialized object is a point of discussion. This therapeutic approach is devised as a pill form that may be taken outside of a hospital. Therefore, the cost falls on the patients, not on the government’s health coverage, OHIP (11). As a result, this potential therapy may be inaccessible to patients with little finances. Regulations or subsidies on behalf of the government could elevate this issue.

Regulations:

Health Canada, the governing body that regulates probiotic use in Canada, has permitted the use of lactobacilli strains, including Lactobacillus brevis, in many products (12).

Probiotics used for therapeutic purpose are classified as either natural health products (NHP) sold over the counter under the Natural Health Products Regulations, or as prescription drug under the Food and Drugs Act (13,14). Depending on the intended use, delivery and composition of the product, different regulations apply (15).

Health Canada, specifically the Health Products and Food Branch, is also responsible for the regulation of products derived from living sources including cytokines through the Biologics Program. Depending on the type of the biopharmaceutical, different centres carry part of the responsibilities of the Biologics Program. Specifically for cytokines, the Centre for Evaluation of Radiopharmaceuticals and Biotherapeutics reviews and evaluates the products and documents to assure safety and quality, and performs further testing for research. (16) The Centre establishes regulations and standard operating procedures of such products, and ensures that the clinical trials conducted were properly designed and had no serious adverse reactions. They also conducts pre-market review of the products and their labels, authorizing for sale in Canada. Even after the products have been released into the market, Health Canada assesses complaints, adverse events, and perform Health Risk Assessments, as part of the Lot Release Program (17). They provide correct information about the product to health care practitioners, consumers, and the general public, as well as visit product manufacturing facilities to conduct evaluations. Lastly, they conduct further research on the methods and the science of the products through academic collaboration nationally and internationally (18).

Security:

Lactobacilli is considered to be non-pathogenic. Furthermore, because of the acidity and enzymes present, it is likely that multiple dose is required for any effect to be seen. Therefore, the potential for this product to be an effective weapon is extremely low. However, in the sight of biosecurity, measures must be taken so that the probiotic is kept in a designated, safe place during research settings. Precautions should be taken to ensure it cannot be used for malicious purposes.

References:

1. http://www.sciencedirect.com.libaccess.lib.mcmaster.ca/science/article/pii/S0924224407000350
2. http://onlinelibrary.wiley.com.libaccess.lib.mcmaster.ca/doi/10.1002/jsfa.4367/full
3. http://journals.lww.com/jpgn/Fulltext/2000/10000/Is_Lactobacillus_GG_Helpful_in_Children_With.24.aspx
4. http://www.gastrojournal.org/article/S0016-5085(00)14499-3/fulltext
5. http://cid.oxfordjournals.org/content/46/Supplement_2/S104.full
6. http://www.sciencedirect.com/science/article/pii/S1369527413000763
7. http://chemocare.com/chemotherapy/drug-info/il-2.aspx
8. Not yet
9. http://www.oncolink.org/treatment/article.cfm?id=296
10. https://www.cma.ca/Assets/assets-library/document/en/advocacy/policy-research/CMA_Policy_Code_of_ethics_of_the_Canadian_Medical_Association_Update_2004_PD04-06-e.pdf
11. Insert
12. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023597/
13. http://www.hc-sc.gc.ca/fn-an/label-etiquet/claims-reclam/probiotics_qa-qr_probiotiques-eng.php
14. http://www.hc-sc.gc.ca/dhp-mps/prodnatur/index-eng.php
15. http://www.hc-sc.gc.ca/dhp-mps/prodnatur/index-eng.php
16. http://www.hc-sc.gc.ca/ahc-asc/performance/eval/biologics-biologiques-eng.php
17. http://www.hc-sc.gc.ca/dhp-mps/brgtherap/applic-demande/guides/lot/gui_sponsors-dir_promoteurs_lot_program-eng.php#a1_0
18. http://www.hc-sc.gc.ca/dhp-mps/prodpharma/applic-demande/guide-ld/ich/securit/s6(r1)-step4etape-eng.php

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