Project Description

Inspiration

Regarding cancer solutions, remodeling the tumor immune microenvironment (TIME) and enhancing the activation of immune-infiltrating cells through multifaceted strategies is considered one of the most promising avenues to overcome these challenges. Currently, a series of immune checkpoint inhibitors have gained clinical acceptance to improve the immunosuppressive environment within the tumor and enhance the anti-tumor effects of CD8+ T lymphocytes. And synthetic biology serves as a possible approach to enable the coordinated release of defective cytokines in the tumor microenvironment (TME) to be able to produce similar immunomodulatory effects. Targeting the OSCC tumor microenvironment to achieve barrier clearance and immune enhancement through specific components or structures in the TME that induce tumor immunosuppression has aroused our interest. Through studying the literature and communicating with expert predecessors, we focused on choosing the bacterial-encoded hyaluronidase Hy and flagellin FlaB as our key research molecules.

Background knowledge

1. Cancer

• Social Impact

  As the most common non-communicable disease, cancer is one of the leading causes of death in the world. Epidemiological studies have shown that the number of new cancer cases continues to rise globally, and it is expected that by 2020, there will be about 15 million new cases of cancer and more than 12 million deaths. Especially in developing and less developed countries, the number of cancer patients is expected to increase further due to factors such as pollution, tobacco and alcohol.

• Definition

  Cancer, also known as malignant tumor, is a disease caused by uncontrolled cell proliferation. Under normal circumstances, cells divide and die in an orderly manner according to the body's needs. However, in some cases, cells may lose this control and begin to divide without limit, forming tumors. The main difference between malignant and benign tumors is the ability of malignant tumors to invade surrounding tissues and even metastasize to other parts of the body through the blood and lymphatic systems.

  The tumor microenvironment (TME) is a complex system composed of tumor cells, extracellular matrix (ECM), and tumor-associated cells (TACs), which is characterized by a unique hypoxic and acidic environment, excess tumor ECM, and abnormal hemodynamics. This is the culprit for tumor cell metastasis and immune escape.

  Oral squamous cell carcinoma (OSCC) is a malignant tumor that occurs in the oral mucosa and is one of the most common malignancies of the head and neck. The main causative factors include smoking, alcohol consumption, chronic irritation (e.g., dental caries, excessive use of mouthwash, chewing tobacco or betel nut), and so on.

• Common treatments

  Surgery to remove the tumor and surrounding affected tissues; radiation and chemotherapy using drugs to kill cancer cells or stop their growth; targeted therapies that target specific genes, proteins, or other molecules of the cancer cells; and activating or boosting the patient's own immune system to attack the cancer cells are some of the more common treatments, but their maximum effectiveness is often hampered by rapid metastasis, mutation of the target gene, and the presence of a target gene or other molecule that can cause cancer. However, their maximum efficacy is often hindered by factors such as rapid metastasis of cancer cells, mutation of target genes and induction of immunosuppression

2. Introduction of related technologies

Synthetic coding technology: genes encoding specific proteins are inserted into E. coli through genetic engineering techniques to enable the production of these proteins.

Hyaluronidase (Hy) is an enzyme encoded by a microbial gene that has the ability to break down the ECM in order to remove the tumor extracellular barrier. It is a polysaccharide commonly found in the tumor microenvironment and helps form a protective barrier for tumors.

Heterologous flagellin FlaB is a protein commonly associated with bacterial flagella and is used here as an immunostimulant.

PpepT promoter and PsodA promoter are DNA sequences that control gene expression and are designed to be activated only in specific cellular environments, in this case the tumor microenvironment.

Toll-like receptors (TLRs) are part of the immune system and recognize pathogens and damage signals. They are activated upon binding to FlaB and promote an anti-tumor immune response.

Challenges and solutions

In practice, we need to ensure that these proteins are specifically released within the tumor without affecting normal tissues or organs to avoid catastrophic consequences. Our iGEM team designed a dual protection measure:

First, we inserted tumor microenvironment targeting PpepT promoter and lactate-responsive PsodA promoter into the plasmid for integration into E. coli ECN1917, and by doing so ensured that the target proteins, Hy and FlaB, would be expressed only in the high-lactate environment of the TME.

Secondly, we introduced another cleavage system controlled by an arabinose manipulator (araC-PBAD) into the plasmid. Patients can stop this biological reaction by ingesting a certain amount of arabinose after taking the drug for a period of time.

Looking ahead, our team plans to improve the safety and controllability of engineered bacteria in vivo by designing more optimal controlled expression systems. This will provide a more reliable solution for immunotherapy of malignant tumors, aiming to improve therapeutic efficacy while minimizing potential risks.

Program Description

In this study, we propose to construct engineered Escherichia coli with expression of Hy, flagellin FlaB, and targeting of lactic acid in the TME by directed evolution of PpepT and reverse transcription of the PsodA promoter, in order to break down the extracellular barrier of tumors and activate T-cell-mediated cellular immunity.

Our plan is to release Hy into tumor tissue in a controlled manner to specifically break down the extracellular “barrier” formed by excess TECM. Subsequently, FlaB will be expressed in the tumor microenvironment (TME), an immunogenic protein that binds to Toll-like receptors and stimulates antigen-presenting cell (APC) activity. These activated APCs recruited to the TME will enhance the presentation of tumor antigens, thereby promoting and enhancing adaptive immune responses.