Background | iGEM Hamburg 2025

Amanita Phalloides

Amanita phalloides, commonly known as the “death cap”, harbors the highly potent toxin α-amanitin. This compound consists of eight highly modified amino acids and is remarkably stable, resisting both heat and desiccation. The death cap is a common α-amanitin-containing mushroom in Germany, but there are many other mushrooms species containing the toxin in Germany and worldwide. It exerts its lethal effect primarily by damaging the liver through inhibition of vital cellular processes.[1]

Alpha-amanitin — *Amanita Phalloides* Licensed under CC SA 2.0, via Wikimedia Commons

The main danger of the death cap (Amanita phalloides) lies in its potential for confusion with edible mushrooms. Inexperienced foragers often mistake it for the field mushroom or the horse mushroom (Agaricus arvensis), as young specimen also have white caps and pale gills. It can also be confused with the green-cracking russula (Russula virescens), a popular edible mushroom, due to its greenish cap. Such misidentifications are particularly hazardous, as even small amounts of Amanita phalloides can cause life-threatening poisoning.

Diagnosing an α-amanitin poisoning is particularly challenging, since the first symptoms such as nausea, vomiting, and diarrhea usually appear only 6–12 hours after ingestion. This delayed onset often leads to misinterpretation as a harmless gastrointestinal infection. After this acute phase, patients frequently experience a deceptive period of apparent recovery, during which clinical symptoms improve. However, severe liver and kidney damage is already progressing silently, often resulting in sudden organ failure within a few days. This course makes timely diagnosis and effective treatment extremely difficult.[2]

α-Amanitin enters hepatocytes via specific transporters. Uptake occurs through the basolateral bile acid transport systems, which normally carry bile acids from the portal blood into the liver cells. Two transport proteins of approximately 54 kDa and 48 kDa are involved in binding the toxin. The active transport includes both a sodium-dependent and a sodium-independent component. Through these targeted transport mechanisms, α-amanitin is efficiently taken up into hepatocytes, where it exerts its toxic effects. [5]

The primary mechanism of α-amanitin is the inhibition of RNA polymerase II in the cell nucleus, leading to a complete arrest of mRNA transcription. As a result, hepatocytes are no longer able to synthesize essential proteins, which ultimately causes cell death. The cumulative damage to hepatocytes is so severe that it progresses to acute liver failure. The initial gastrointestinal symptoms of poisoning are mainly caused by other compounds in Amanita phalloides, such as phallotoxins. In contrast, some other α-amanitin-containing mushrooms, for example species of Lepiota, lack phallotoxins and therefore present primarily with liver failure as the first clinical symptom. [2]

The estimated lethal dose for humans is as low as 0.1 mg per kg body weight. This means 5 to 8 mg of α-amanitin can cause fatal damage, an amount that may already be present in a single mushroom. According to the World Health Organisation, tens of thousands of mushroom poisonings occurr worldwide each year and the mortality ranges from 10 to 30 %. More than 90 % of all lethal mushroom poisoning worldwide can be linked to α-amanitin. The mortality rate strongly depends on the patient’s condition, the timing of medical intervention and the available clinical resources. With immediate and intensive treatment the fatality rate can be reduced to around 5 %. The actual number of cases is probably much higher if the required treatment isn’t available, because diagnosing α-amanitin poisoning is difficult and its unspecific symptoms are often mistaken for other diseases. [1] [3]

Currently, there is no specific antidote available for poisoning with α-amanitin. However, several agents are used to reduce its toxic effects. One example is Silibinin, which inhibits the OATP transporters in hepatocytes and thereby prevents further uptake of the toxin into liver cells. While Silibinin shows promising protective effects, treatment is extremely costly, often amounting to several thousands of dollars. A more affordable alternative is N-acetylcysteine, which acts by replenishing intracellular cysteine levels, stimulating glutathione synthesis and reducing oxidative stress. Increased glutathione levels can bind and neutralize toxic metabolites, thereby alleviating hepatic injury. In addition, high-dose intravenous penicillin is historically used and is believed to compete with the hepatic uptake of amatoxins, although clinical evidence remains limited. Activated charcoal can reduce toxin absorption from the intestinal tract when administered early after ingestion and may also limit reabsorption during enterohepatic recirculation. Cyclosporine has been shown in in vitro studies to inhibit OATP transporters; however, its clinical application in amatoxin poisoning remains limited to case reports. Several other therapies, including intravenous cimetidine and thioctic acid, have also been investigated, but supporting evidence derives only from animal studies. In severe cases of liver injury, liver transplantation may represent the only life-saving intervention. Dialysis can be initiated in renal failure to support kidney function, but it does not effectively eliminate amatoxins from the bloodstream, even when started early. [2] [6]

Why nanobodies?

We came across nanobodies first in lectures at university, where they were introduced as powerful new tools in diagnostics and therapeutics. We instantly fell in love. So when we started to collect ideas for our iGEM project, nanobodies were always in the back of our heads. As the idea of an antidote against alpha-amanitin emerged, we needed a way to bind and neutralize the toxin in cells and the bloodstream. A perfect fit for our favorite tool! But what exactly are nanobodies, and why are they so powerful?

A quick guide to nanobodies

In 1989, Professor Raymond Hamers of the Vrije Universiteit Brussel came across a curious result in his research: he was purifying antibodies from the serum of a camel, when he noticed unusually small fractions in the gel. Further investigation showed that those small antibodies only consisted of two heavy chains, unlike conventional antibodies that are built from two heavy and two light chains. Nevertheless, those heavy-chain only antibodies were still able to bind a broad range of epitopes. They are naturally produced in Camelids but subsequent research also enabled recombinant production of only the variable antigen-binding domain of the heavy chain (VHH). Antibodies just consisting of this VHH are called single domain antibodies or nanobodies [1] [2].

Image of a nanobody and a conventional antibody — *Figure 1:* Conventional antibodies (left) are built from two heavy chains (blue) and two light chains (green). Heavy-chain only antibodies are exactly what their name suggests: built only from two heavy chains (blue). Single-domain antibodies or nanobodies only consist of one of the upper domains of heavy-chain only antibodies. Published by David Goodsell and licensed under CC BY 3.0.

Below are several advantages and challenges:

Conventional antibodies bind to epitopes using the variable antigen-binding domain of both heavy and light chains. Each of those carries three hypervariable complementarity-determining regions (CDR) for antigen specificity. As they have a binding domain on each site, they can hold onto antigens almost like tweezers. Nanobodies only consist of one variable antigen-binding domain. They also have three CDRs and their CDR3 is extended. This way, a nanobody can bind to certain epitopes that would be inaccessible to bigger antibodies.Although they only have one binding domain, nanobodies can still hold onto antigens with very high affinities (equilibrium dissociation constant, KD, can reach nano- to picomolar range) [2].

As conventional antibodies consist of four chains in total, they have a molecular weight of around 150 kDa. Nanobodies are ten times smaller than that, with only 15 kDa. This way, they can penetrate tissues much better and even cross the blood-brain barrier. [2].

Nanobodies are very stable regarding high temperatures and pH changes. They are able to refold after being heated to up to 95 °C. Additionally, they are highly soluble (expert interview with Alejandro).

Interestingly, nanobodies do not lead to immune responses when tested in vivo [2].

While antibodies are complex in structure and often carry post-translational modifications, nanobodies are simpler, unmodified and can be produced in procaryotic and eukaryotic expression systems, such as Escherichia coli or Saccharomyces cerevisiae [2].

Due to their small size, nanobodies are usually filtered out of the bloodstream very quickly by the kidneys. Conjugation to proteins like albumin or to polyethylene glycol (PEG) can help to keep them in the system for longer (expert interview with Waldemar, [2]).

Usually, nanobodies are produced in Camelids by immunizing them with the respective antigen. However, those nanobodies still need to be humanized so they will not pose any risk of immunological reactions when used as therapeutics (expert interview with Waldemar, [2] ). Additionally, although the immunization and harvesting of nanobody-producing cells from Camelids are minimally invasive, the ethical question of their usage in research remains.

Since nanobodies only have one binding domain and no tweezer effect, they can have problems binding to flat or linear epitopes [2].

First of all, our nanobody should effectively neutralize alpha-amanitin. Since the toxin binds to the RNA polymerase with very high affinity (KD = 3-4 nM [3] ), our nanobody needs to have a high affinity to the toxin as well to be able to compete with the polymerase. This is generally possible to achieve, although it can be much harder to design nanobodies with high affinities against small targets such as peptides (expert interview de Marco and Schöder).

We also aim for as little side effects as possible, as patients that would be treated with our antidote are already weakened by the toxin and often in critical condition. Fortunately, nanobodies already exist as therapeutics and show no immunological effects [4] , making them an ideal candidate for our application.

Since the RNA polymerase is located in the cell nucleus, we need to deliver our nanobody into the nucleus of affected cells. To achieve this, we added a nuclear localization sequence (NLS) originally derived from the simian virus 40 [5] . This NLS is recognized by transporters that help import respective proteins into the nucleus [6].

Nanobodies are also promising regarding transport and storage. As they are highly stable and do not need to be cooled down to extreme temperatures, they provide a treatment alternative for remote locations or hospitals that lack elaborate cooling options.

To make our antidote affordable, we were looking for an inexpensive way of production that can be upscaled easily. Since nanobodies can be produced recombinantly in procaryotic expression systems, we chose the commonly available E. coli strain BL21(DE3) for production. This way, our antidote production is also animal-free.

We want to be able to produce a pure nanobody that is free of any procaryotic traces. To achieve this, we added two things to our design: first, we want our nanobody to be extracellularly secreted by our E. coli. We added a signal peptide called pelB to the nanobody, which directs the protein into the periplasm of the bacteria. This helps form the disulfide bond our nanobody contains and leads to improved folding. Once in the periplasm, proteins can be further secreted into the medium around the bacteria using an aspartate linker [7] . The extracellular secretion can lead to higher protein production and easier downstream isolation and purification [8] . Secondly, we added tags for affinity purification to our nanobody. As our nanobody is a rather small protein, we chose two small tags, a strep-tag II and a 6x Histidine tag, and created constructs with each of them, respectively.

Finally, we anticipated optimization and engineering steps necessary to further develop our nanobody. We added protease sites for the TEV protease and the HRV 3C protease. This way we are able to cut off the aspartate linker required for secretion and the tag required for purification to minimize our nanobody’s immunogenicity for therapeutic applications. We also included several restriction enzyme recognition sites into our design. Thus, we can cut out the nuclear localization sequence to create a nanobody capturing the toxin in the bloodstream or in the cytosol of the cells. We are also able to switch out the coding sequence for the nanobody, so we can use our constructs to produce nanobodies with different targets, for example against other intracellular toxins.

Important note

Unfortunately, we did not check our first design with iGEM’s parts requirements. We included restriction enzyme recognition sites that are not allowed in the BioBrick RFC[10] system. Additionally, our nanobody construct also includes restriction sites incompatible with the Type IIS RFC[1000] system.

Why lipid nanoparticles?

During expert interviews and our research, we were facing the fact that nanobodies get cleared rapidly from the body which causes a risk for efficient therapy. To ensure transport into cells affected by the poison, we wanted to use some sort of transport vehicle. With their well known use as vectors for mRNA-vaccines, lipid nanoparticles quickly came into our mind while brainstorming for an appropriate transport system. A very versatile and well-studied type of lipid nanoparticles are liposomes. They show a good loading capacity and are adapted to encapsulate a wide range of compounds. They seemed to be the perfect choice for the nanobody delivery. A quick guide to liposomes:

Amphiphilic molecules such as lipids show the ability of self-assembly into lipid bilayers in aqueous solutions. If the lipid concentration is high enough, spherical vesicles are formed. Those vesicles can be uni- or multilamellar and are called liposomes. In the 1960s, investigations on liposomes as drug carriers started. The typical size-range of liposomes for therapeutic use is 50-150 nm. Liposomes can entrap hydrophilic as well as hydrophobic active substances. Hydrophilic drugs can be encapsulated in the liposomes core whereas hydrophobic compounds are integrated in the membrane. Furthermore, main characteristics such as the stability, the pharmacokinetic profile or the interactions with the cargo can be determined by the lipid composition used to fabricate liposomes (Fig.2). [7] [8] [9]

This schematic overview highlights the most important properties of liposomes. They are able to encapsulate
hydrophilic as well as hydrophobic drugs — *Figure 2:* This schematic overview highlights the most important properties of liposomes. They are able to encapsulate hydrophilic as well as hydrophobic drugs, referred here to as active pharmaceutical ingredients (APIs), for delivery. Crucial characteristics such as stability or pharmacokinetics are influenced by the lipid composition. Typically liposomes consist of phospholipids and additional helper lipids as cholesterol which improve the lipid bilayer integrity. Surface modifications can be easily introduced and ligands can be used to achieve delivery to specific organs or cell types. The figure “liposomes overview” by Remo Eugster and Paola Luciano is licensed under CC BY 4.0.

Liposomes can be easily prepared by thin-layer-hydration followed by extrusion. While the thin-layer-hydration method leads to a very heterogeneous distribution of liposome sizes and lamellarity, the extrusion is used as a means for size reduction and narrowing the size distribution. The extrusion device typically is easy to use and no big set-up is needed for liposome fabrication. [10] [11]

Liposomes enable the delivery of hydrophobic as well as hydrophilic active substances due to their amphiphilic building blocks. The interaction between cargo and liposome can be tuned through the lipids chosen to form the liposome. [9]

As hepatocytes are predominantly affected by α-amanitin, it is urgent to target those cells specifically. When administered to the blood, Apolipoprotein E is adsorbed to the liposome’s surface and forms a protein corona. This corona triggers endocytosis via low density lipoprotein receptors which are found in extensive numbers on hepatocytes. This already existing mechanism for targeted delivery to hepatocytes can be further enhanced by tuning the lipid composition. [12] [13]

Liposomes show no to minimal adverse effects upon administration which makes them overall a safe option as a delivery system for the nanobody. Nevertheless, it is possible that the complement system is activated and an immune response triggered.8 [14]

In terms of stability, one must distinguish between in vivo stability of liposomes and their shelf-life. In the body, liposomes are circulating for several hours before they get cleared. This time is determined by how easily a protein corona can be formed on the liposomes surface. If the formation of the protein corona is hindered, the half-life is extended, as it is when polyethylene glycol (PEG) lipids are added to the lipid mix. The shelf-life is determined as well by the lipid composition. Charged lipids lead to repulsive forces between vesicles and prevent aggregation as well as PEG-lipids do by steric hindrance. Tests have shown that liposomes can easily be stable for up to six months even with the cargo already encapsulated. [9] [14] [15]

Important properties of liposomes depend on their lipid composition which can be easily changed. Additionally, the modification of the surface with ligands can help to extend the half-life or to direct it to a specific target. [9]

For a homogeneous size distribution of liposomes, some sort of filtering step must be included. In a laboratory, the typical method is extrusion which unfortunately is restricted to relatively small batch sizes. Adaptation of an alternative size reduction method is needed to meet the conditions for large scale production. [10] [11]

Once the liposome arrives in the cell, it needs to release its cargo into the cytosol. This process must happen efficiently as we need as much of the nanobody as possible to bind the toxin in affected cells. Oftentimes liposomes are more quickly re-excreted than they can release their load into the cell. [16]

Background | iGEM Hamburg 2025

Amanita Phalloides

Why nanobodies?

A quick guide to nanobodies

Below are several advantages and challenges:

Mode of binding

Size

Stability

Immunogenicity

Recombinant production

Serum persistence

Production in Camelids

Mode of binding

Neutralization

Side effects

Nuclear localization

Transport and storage

Affordable and animal-free production

Easy downstream purification

Engineering

Important note

Why lipid nanoparticles?

Fabrication

Cargo Loading

Targeted cell-uptake

Immunogenicity

Stability

Versatility

Fabrication

Cargo Release

Sources