Project Description

Colorectal Cancer at a Glance

Colorectal cancer is one of the great health challenges of our time. Each year, more than 1.9 million people receive this diagnosis, making it the 3rd most common cancer and the 2nd biggest cause of cancer-related deaths worldwide (World Health Organization, 2023). Often described as a “silent disease”, it typically develops slowly and symptom-free until it has reached an advanced stage[1].

Over the past two decades, a marked increase in CRC incidence has been observed among adults under the age of 50, a condition termed young-onset colorectal cancer (YO-CRC). By 2030, YO-CRC is projected to account for 11% of colon cancers and 23% of rectal cancers[2].

Lifestyle and environmental changes since the mid-20th century, including widespread antibiotic use, reduced physical activity, obesity, and dietary patterns characterized by high intake of red and processed meat and low dietary fiber, are thought to disrupt the gut microbiome and contribute significantly to CRC development[3].

At the molecular level, many colorectal tumors are driven by abnormal activity of the Epidermal Growth Factor Receptor (EGFR). In healthy cells, the EGFR signaling process begins when small molecules, called ligands, attach to the EGFR protein on the cell surface. There are about eleven different ligands that can activate the ErbB family of receptors, including epidermal growth factor (EGF) and transforming growth factor-α (TGF-α)[4].

When a ligand binds, two receptors pair up (dimerization) and switch on their built-in enzyme activity. This activates a chain of signals inside the cell.

Two of the main “switches” turned on by EGFR are the MAPK pathway and the PI3K/AKT pathway. These act like control hubs, switching on transcription factors that regulate how the cell behaves. Through these switches, EGFR can tell a cell when to grow, move, specialize, or even self-destruct if necessary[5].

As part of our investigation, we employed Cetuximab, a chimeric IgG1 monoclonal antibody with established clinical use in multiple epithelial cancers, including metastatic colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Cetuximab works in two main ways: it blocks signals that drive the uncontrolled growth of tumor cells, and it also helps the immune system to recognize and attack those cells[6].

• Slower tumor progression
• Stronger response to chemotherapy and radiotherapy
• In some cases, apoptosis of cancer cells
• Additional anti-tumor activity through immune system involvement

A major challenge is the way Cetuximab is produced. It is typically manufactured in mammalian cell lines, most often Chinese hamster ovary (CHO) cells. This process is not only costly and technically demanding but also resource-intensive, relying on large culture facilities, energy, and animal-derived materials. Such methods raise questions about long-term sustainability and contribute to unequal access, particularly in low- and middle-income countries where the burden of cancer is increasing[7].

A life cycle assessment by Bunnak et al.[8] quantified the environmental impact of conventional mAb production, showing massive resource consumption and emissions:

Amasawa et al.[9] found that most of the environmental impact of CHO-based antibody production comes from energy use (~70%) and waste management (~20%). This makes the process not only environmentally damaging but also expensive, especially under the EU Emissions Trading Scheme[10]. The high production cost directly affects access to Cetuximab: in Germany, a 100 mg vial costs €299.49 and a 500 mg vial €1,454.38[11], adding up to more than €30,000 per patient each year. Despite these barriers, global sales of Cetuximab still reached €1.025 billion in 2023[12].

That’s why we found it exciting to look into synthetic biology as a solution. In our project, we explored using the green alga Chlamydomonas reinhardtii to produce monoclonal antibodies, which offer several important advantages. To make the transformation into Chlamydomonas easier, we used a single-chain fragment – a short but still functioning version of Cetuximab instead of the full antibody.

The Idea of SUSPACT: DNA is first transformed into E. coli for initial expression, and then into Chlamydomonas reinhardtii for sustainable antibody production. The secreted antibodies bind to EGFR on cancer cells, block ligand interaction, inhibit signaling, and ultimately suppress cancer cell growth.

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Key benefits of Chlamydomonas as a production system include:

• Photoautotrophic growth, which allows direct conversion of CO₂ into biomass
• The ability, as a eukaryotic organism, to carry out essential post-translational modifications
• Low cultivation costs, since only simple media are required
• Minimal land and water demands compared to conventional production systems
• A naturally low risk of contamination with human pathogens

[13]

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Our aim is to establish Chlamydomonas reinhardtii as a sustainable and cost-effective production platform for Cetuximab fragments, offering an environmentally friendly alternative to mammalian cell culture systems. Although algae are not yet common in pharmaceutical manufacturing, early studies suggest they can be engineered for antibody expression. This highlights the potential of combining synthetic biology with sustainability goals, making cancer therapies like Cetuximab not only effective but also more accessible. We expect to successfully express and assemble Cetuximab single-chain fragments in Chlamydomonas, confirm their ability to bind EGFR, and demonstrate that algal systems can reduce resource demands compared to CHO-based methods.

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Modular Cloning System (MoClo)

The Modular Cloning (MoClo) system is a synthetic biology method that assembles genes in a standardized, building-block fashion using Golden Gate cloning[14][15]. This approach enables the efficient, one-pot assembly of multiple predefined genetic parts through a hierarchical system:[16] [17]

• Level 0 (L0): Basic genetic elements such as promoters, UTRs, coding sequences, tags and terminators, each stored in standard vectors
• Level 1 (L1): Complete transcription units assembled by combining several L0 parts
• Level 2 (L2): Multigene constructs generated by assembling up to six L1 transcription units in a single reaction

Traditionally, using MoClo in Chlamydomonas required two assembly steps: one to build the gene of interest and another to combine it with a selectable marker. To overcome this, Niemeyer and Schroda (2022)[18] developed new Level 2 destination vectors that already carry an antibiotic resistance marker in position 1.

In our approach, we made use of these vectors: the placeholders in positions 2 onwards were replaced in a single step with our L0 parts encoding the heavy and light chains of Cetuximab.

The advantages are clear:

• Faster workflow: cloning time drops from about 5 days (~100 h) to just 3 days (~50 h)
• Lower error rate: fewer steps mean fewer opportunities for mistakes
• Flexibility: multiple antibiotic markers allow us to choose the best one for our experiment
• Reusability: the gene parts created can be recycled in future assemblies