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A next-generation of antivenom could be just around the corner. Besides being safer and more effective, the technology behind it can also be applicable for other diseases, such as cancer and COVID-19.

Christina Adams1, Lise Marie Grav1, Andreas H. Laustsen2

1Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark

1Department of Biotechnology and Biomedicine, Technical University of Denmark

 

Snakebite envenoming is a neglected tropical disease that particularly affects tropical regions of the world. Each year, snakebite envenoming claims around 2-3 million victims, of which around 100,000 die.

The most affected victims are often the (future) bread-winners of poor families living in rural areas, who make their way through fields, trying to earn enough money to feed their wives and children.

Snake venoms are highly complex and diverse; some show local effects, such as tissue damage, while other venoms show systematic effects, such as neuromuscular paralysis. The only thing that helps against the potentially deadly venoms are antivenoms.

Fortunately, we have antivenoms around to deal with the effects that snake venoms cause. But are existing antivenoms really as good as they could be?

The short answer is no. New experiments with mammalian cells suggest that improvements could be made in production of antivenoms.

These experiments also suggest that the technology used to create a new antivenom could be applicable to other diseases ranging from cancer to COVID-19.

But before we get ahead of ourselves, let’s first look at the antivenom we use today.

Antivenom with limitations

 

Today, antivenoms are obtained by milking snakes and immunizing horses or sheep with the venom (figure 1). Afterwards, the blood is drawn from the animal, from which the antibodies are isolated and bottled after formulation.

Scheme demonstrating production of antivenom

Figure 1 Current production of antivenoms. 1. The venom is taken from the snake (‘milking’), 2. The venom is given to a horse (or a sheep) by injection, 3. The horse’s blood is drawn and 4. The antibodies are isolated. 5. The antivenom is concentrated, formulated and bottled. (Modified figure from Kini et. al).

Current antivenoms are effective in neutralizing snake venoms by binding to them and thus, they can be lifesaving.

However, they also impose a serious risk to patients, as the animal-derived antibodies can cause severe allergic reactions and anaphylactic shock.

Besides that, traditional antivenoms often fail to neutralize toxins in limbs, as they cannot reach the limbs fast enough, which leads to disfigurement or the need for amputation.

The venom differs from region to region

One of the largest drawbacks is that these antivenoms cannot neutralize venoms from different geographical regions.

For example, the same cobra species (Naja kaouthia) lives in different parts of India, and the antivenom for a specimen from one region is not effective against the venom from another region’s specimen.

Due to differences in diet and through local adaptation, the venoms may differ greatly (figure 2), which complicates treatment.

A person could die from the snakebite in one region, if this person receives an antivenom for the same snake species from a different region. In the past, inappropriate regional antivenoms have been distributed, which led to cases being more critical in these regions.

Graph showing region A and region B

Figure 2 The figure shows how different the venom composition from the cobra (Naja kaouthia) is in two Indian regions. What the colors stand for is not our point here, the variation is. Region A = Arunachal Pradesh, Region B = West Bengal). (Modified graphic from Laxme et. al)

Next-generation antivenom

As an alternative to animal-derived antivenoms, scientists are developing next-generation antivenoms, which are comprised of human antibody mixtures. These antibodies are produced biosynthetically. They should mimic the antibodies the human body produces after being confronted with a foreign particle; in other words: they replace the human immune reaction. In many cases, antibodies only bind to one specific particle called antigen. For next-generation antivenom, antibodies are mixed as a form of a cocktail. In this cocktail, each of the antibodies targets a different snake venom or toxin. These antibody mixtures can be used in many different ways.

Quick fact:

Scientists discover antibodies against snake toxins using a technique called ‘phage display’. Here, bacteriophages – special viruses – display different antibodies on their surface. The phages are mixed with a target toxin, and the best antibody binder is selected for further development.

The next generation of antivenoms is safer and more effective

In comparison to animal-derived antivenoms, biosynthetic antibody mixtures will have some major advantages.

Their human origin makes them less immunogenic and thus safer in human patients.

Moreover, they can be designed to only include antibodies that have therapeutic value, whereas the animal-derived antivenoms also contain non-therapeutic antibodies.

When the antibodies are isolated from the blood of an animal, all antibodies from the animal are isolated – even those not related to snake venom, which imposes an unnecessary health risk to the snakebite victim, who, therefore, will receive more antibodies than necessary. This may increase the risk of adverse reactions.

Another major advantage of the described next-generation antivenom approach is that different antibodies can be mixed in many ways. Examples would be to mix antibodies that neutralize venoms from different snake species or local variations of one snake species. The first step in this process would be to decide, which different snake species (or specimens of the same species) to target. Scientists can then mix certain antibodies that neutralize these certain toxins. This means that each antibody has a known specific therapeutic value for neutralizing one or more toxins.

The antibody cocktail against different snake venoms especially comes in handy, if the victims are unable to identify the snake they were bitten by. As we realized, next-generation antivenoms have a big potential.

The remaining question is now – how do we manufacture these antivenoms at a low cost?

Manufacturing the next generation of antivenoms

One thing is designing and developing a biopharmaceutical product, another thing is manufacturing it. How much does it cost to produce a mixture of biosynthetic antibodies?

Scientists calculated the costs of the traditional and next-generation methods and they are actually fairly comparable, making the next generation method cost-competitive.

The price of both the traditional and next-generation method varies widely, depending on the venoms of snake species they can neutralize and how effective they are.

Animal plasma-derived antivenoms cost anywhere from 13 to 1,120 USD per treatment, while a mixture of recombinant antibodies can be manufactured at a cost of 48 to 1,354 USD per treatment. This was an important calculation. Even if a next-generation antivenom was better, it would likely not find its way to the market if it was much more expensive than the current treatment. Given the fact, that these antibody cocktails are cost-competitive and exhibit major advantages, there is a large possibility that such next generation antivenoms will prevail in the future.

Antibody production pipelines are often time-consuming

One way to produce recombinant antibodies is using mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

An advantage of these cells over other production hosts (bacteria, yeast, plants) is that they modify antibodies in a way that resembles human antibodies, so that the human body does not react negatively to them.

For years, the common way to produce antibodies in CHO cells was to introduce the DNA of the wanted antibody to a cell population. The DNA was then randomly inserted into any part of the genome of individual cells in the population.

This made the resulting cells very different from each other, in the sense that they produced different amounts of the antibody, depending on where the DNA was inserted in the cell’s genome (see figure below).

Consequently, it was very time-consuming to find the best antibody producer among all cells, if there was any.

Random integration

Figure 3 The ‘old fashioned’ method is called random integration, as we cannot control where in the CHO cell genome the antibody gene is inserted (figure by Christina Adams).

New production pipeline can save time and money

Quick fact:

The CRISPR approach mimics a bacterial defense mechanism against viruses.

The bacterium inserts viral DNA into its genome and then produces short matching DNA fragments (CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats), which can recognize the viral DNA.

If a virus infects the bacterium afterwards, the short matching repeats recognize the viral DNA, which subsequently is cut by an enzyme to prevent any damage to the cell.

In bacteria, this is a defense mechanism. Scientists have exploited this system to cut DNA in numerous species to insert a new gene at the exact place where they cut the DNA.

They introduce matching DNA fragments of the DNA they want to cut together with the cutting enzyme. This makes it possible to insert DNA into one specific site, which yields a consistently high amount of antibody being generated.

Scientists can circumvent these drawbacks by using special ‘molecular scissors’, such as the CRISPR approach (see fact box). With this approach, we can choose a site in the genome, where we know that proteins are produced well to ensure stable and high production of, for example, antibodies. This can save a lot of time and money in comparison to the random integration method (figure 3), as for the latter, good antibody producers first have to be found among all the cells. When each antibody gene is integrated in the exact same site, antibody production will be comparable, resulting in cell lines producing one antibody each in similar amounts. This leads to an important standardization of production. Like this, it could be possible to grow several cell lines in one vessel, as they behave the same and will thus have the same needs. Before, that was hard to achieve. Using only one vessel is very favorable, since it saves time and money.

As good as this new approach may sound, researchers first need to find a good integration site in the CHO cell genome, which ensures good production of the antibodies.

Our research group identified sites that ensure high product formation (see here and here), where we can directly insert our antivenom antibody DNA.

With that knowledge, we developed an even faster and smarter way of generating cells that produce the wanted antibodies. First, we use the CRISPR approach to insert a so-called ‘landing pad’ in the good site. This first step of integrating the landing pad only has to be done once.

The landing pad contains a ‘dummy gene’, which can easily be exchanged by any antibody gene we want (figure 4) so we can make antibody 1, antibody 2 etc. in parallel. This is much faster than making use of the CRISPR approach every time we want to make a new antibody.

Scheme of improved way

Figure 4: Improved way of inserting an antibody gene into the CHO genome. First, a landing pad with a dummy gene is inserted into the genome through CRISPR engineering. Later, the dummy gene is swapped with the desired antibody gene. This ensures insertion in the same site for every cell and thus a standardized product formation. (Illustration: Christina Adams)

Using this technique, we can generate numerous cell lines, of which each contains one antibody gene in the exact same site, producing comparable amounts of antibodies. Because these cell lines behave in a similar way and exhibit a standardized production, we can mix them in one vessel, as mentioned above. By mixing the kindred cell lines, we can finally produce several antibodies in one vessel at a time (figure 5B). This way, less equipment is needed, and costs are reduced compared to producing each antibody in one vessel (figure 5A).

Example of ways to produce antibody mixtures.

Figure 5: Example of ways to produce antibody mixtures. A: Each cell line produces one antibody in one vessel; antibodies are purified and mixed to formulate and bottle the drug. B: All cell lines are cultivated in one vessel and thus all antibodies are produced in one vessel. The antibody mixture is purified and bottled. (Figure: Christina Adams)

One antivenom to rule a region

With our improved technique, we can mix a potent recombinant antivenom against complex snake venoms. Several antibodies against several components of one complex venom will mimic the therapeutic content of an animal-derived antivenom. Again, the advantage compared to existing animal-derived products is that the antibodies in recombinant antivenom are only of therapeutic purpose and compatible with the human immune system.

Our research group, for example, found antibodies against black mamba venom toxins and we were able to show the neutralization capacity of these antibodies in mice. This means that mice received the venom, the antibodies bound to the key venom toxins, and the mice survived.

However, it would even be possible to mix various antibodies that can neutralize all venoms of the same snake species in different geographical regions. Products like these could prevent the maldistribution of antivenom in the future, as they would neutralize the venoms in more than one region – leading to less fatal cases.

As mentioned above, this is a big problem, which scientists could solve with next-generation antivenoms.

What’s next?

The following, bigger step is mixing antibodies that can neutralize different venoms from different snake species: For example, all mambas or cobras.

Such antivenoms are particularly useful, if it is unknown what snake species bit the victim. We call these antibody mixtures broadly-neutralizing antivenoms. Doctors can administer such an antivenom to patients that e.g. did not see the snake, providing a faster and safer treatment.

Applicability from cancer to COVID-19

The here described production pipeline does not stop with the most complex antivenom. Scientists can use this technique to produce antibody mixtures against all kinds of diseases.

An example is cancer, for which scientists already mix several antibodies as a therapy to help stopping tumor growth in the body.

There are also examples of antibody mixtures targeting infectious diseases, such as tetanus, rabies or even COVID-19.

The pharmaceutical company Regeneron, for example, has shown that their cocktail of two antibodies targeting COVID-19 reduces the risk of death in hospitalized patients, and the US government purchased 1.5 million doses of this cocktail. It was recently authorized under an Emergency Use Authorization by the FDA. This means that doctors can treat patients with mild to moderate symptoms, who are at risk for progressing to severe symptoms, with that medicine.

These advances in therapy of diseases are promising, and there are possibilities that the production of such mixtures could be improved even further by using CRISPR, targeted integration, and production of all antibodies in one vessel.

The broad applicability and the big potential of mixing different antibodies makes one thing clear: The future holds many possibilities for biosynthetic antibody mixtures.

 

Declaration of interest

No conflict of interest exists.

We wish to confirm that there are no known conflicts of interest associated with this article and there has been no significant financial support for writing this article.

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