RPTU-Kaiserslautern

Plant Synthetic Biology

The Problem

Colorectal cancer ranks among the most common cancer diagnoses worldwide, making the search for innovative and targeted therapies more urgent than ever[1]. Monoclonal antibodies have emerged as a promising solution, offering high targeting efficiency and remarkable effectiveness against cancer cells. However, current production methods face significant ecological and economic challenges that limit their accessibility and sustainability [2].

Figure 1: Chlamydomonas reinhardtii cultures growing in Erlenmeyer flasks with minimal media under light conditions, representing a sustainable alternative to mammalian cell culture systems for biologic production.

While antibody therapies set an exciting milestone in the treatment of several different types of cancers, production remains a hurdle to overcome. Several key factors like the use of animal-derived media for mammalian cell culture, the substantial use of water, and the production of large amounts of CO2 make the production of these biologics unsustainable and not feasible for a time when ecological production of modern biologics is a key factor for a healthy future of humanity and the planet[2]. While other organisms have been investigated to produce biopharmaceuticals, they often lack key components which make these systems unsuitable to use. E. coli produces recombinant proteins in high amounts, but due to its prokaryotic nature, it does not possess the capability to properly fold proteins and cannot perform any post-translational modifications [3].

Plant systems show high production of recombinant proteins yet require difficult and expensive downstream purification steps[4]. However, another chassis does not receive much attention in the world of biopharmaceutical production, despite offering a range of beneficial factors for addressing these problems: green algae. This untapped potential led us to explore alternative production systems, with the star of our project: Chlamydomonas reinhardtii.

Meet Chlamydomonas

Why Chlamy?

Chlamydomonas reinhardtii gives us the best of both worlds: the rapid growth and genetic modification flexibility of microorganisms combined with the sophisticated cellular machinery of eukaryotes. Unlike bacteria, Chlamy performs essential post-translational modifications, including glycosylation, ensuring proper protein folding and functionality crucial for therapeutic antibodies[5]. Its photoautotrophic nature eliminates the need for carbon sources like acetate, while its ability to grow mixotrophically allows for flexible cultivation strategies[6]. The established MoClo toolkit makes genetic engineering straightforward, enabling precise and fast control over its modification capability[7]. Most importantly, Chlamy can be nuclear modified to secrete proteins into the surrounding medium, dramatically simplifying downstream purification compared to intracellular expression systems. This unique combination of features positions Chlamydomonas as an ideal chassis for sustainable, cost-effective antibody production.

Figure 2: Fluorescence microscopy image of Chlamydomonas cells expressing the scFv fused with mTFP (monomeric teal fluorescent protein). The cyan fluorescence indicates successful protein expression into the surrounding medium. Scale bar: 10 µm.

What We Build

We successfully engineered Chlamydomonas reinhardtii to produce cetuximab scFv, a single-chain variable fragment targeting the EGFR receptor in colorectal cancer. Our success required using Chlamy specific solutions like codon optimization and integration of Chlamydomonas-specific introns, selection of appropriate secretion signals (cCA, ARS, GLE), and glycosylation tags (SP20) that work specifically in Chlamy[8]. Through Western blot analysis, we detected our scFv in the culture medium, while fluorescence microscopy confirmed successful production and transport through the cells, proving our expression system works effectively. Crucially, we didn't just produce the protein. We validated its biological activity by demonstrating specific binding to its target EGFR receptor by performing a pulldown assay[9], confirming that our Chlamy produced antibody fragment has its intended binding capacity.

To validate real-world applications, we initiated in vitro studies testing our scFv against cancer cell lines that overexpress the EGF Receptor, the same mechanism found in colorectal cancer[10]. Additionally, we've already expanded our therapeutic portfolio by successfully producing trastuzumab scFv targeting HER2 in breast cancer, as confirmed by Western blot analysis. While our cancer cell line experiments are still under optimization, they demonstrate our commitment to translating the results into clinical relevance across multiple cancer types.

Beyond proof-of-concept, we have tackled both scalability and commercial viability. Using various bioreactor systems, we demonstrated scFv production even at larger medium volumes, showing the industrial-scale feasibility of Chlamy. Most importantly, by selecting the non-patented CLiP UVM strain of Chlamydomonas, we have positioned us for potential commercial production without intellectual property barriers[11]. This comprehensive approach establishes a complete pipeline for Chlamy based antibody manufacturing from the lab to the market.

Impact of our Project

Our breakthrough with Chlamydomonas-based scFv production directly addresses the mounting sustainability crisis facing today's biopharmaceutical industry. As environmental regulations tighten globally and carbon footprints become critical business factors, biopharmaceutical companies will need to seek sustainable alternatives.

Our Chlamy platform offers a revolutionary pathway toward sustainable biologics manufacturing. Unlike energy intensive mammalian systems, our photoautotrophic Chlamydomonas harnesses solar energy and thrives on minimal nutrients. The elimination of animal-derived components reduces costs while addressing ethical concerns. Furthermore, algae naturally absorb CO2 during growth, potentially making our system carbon negative. The scalability we have demonstrated through bioreactor cultivation proves this is not just a laboratory approach, it's a possible viable industrial solution that could transform how the industry approaches biologics production.

Our work tackles one of the most significant limitations in plant synthetic biology: the lack of robust platforms for complex therapeutic proteins. By establishing the first functional single chain fragment variable expression system in Chlamydomonas, we have proven that this single-cell photosynthetic organism can keep up with traditional expression systems while maintaining environmental advantages. The successful production and detection of both cetuximab and trastuzumab scFvs demonstrates our platform's versatility across different therapeutic targets, fundamentally changing what's possible with Chlamy based production.

We have accumulated protocols that dramatically lower barriers for future teams exploring Chlamy based therapeutics production:

These resources represent months of optimization work that future teams won't need to repeat, potentially saving years of development time and accelerating the entire field of Chlamydomonas based therapeutic production.

The implications extend far beyond colorectal cancer. Our platform demonstrates proof-of-concept for producing any antibody fragment, opening doors to the production of recombinant proteins for countless other diseases. From an industry perspective, our work offers a solution that reduces production costs while improving sustainability metrics, a rare win-win scenario. We firmly believe that Chlamy based platforms will become the backbone of sustainable therapeutic production, helping create a healthier future for both patients and our planet.

Figure 3: Part of the iGEM RPTU team hard at work in the laboratory, developing Chlamydomonas-based platforms for sustainable antibody production and a healthier future for both patients and our planet.