By Nurşen Öğütveren, Yale SOM '26

 Can you tell me a little bit about your background? How did you become interested in drug delivery and brain diseases?

I'm a developmental biologist by training; I trained with a famous French developmental biologist, Nicole Le Douarin. At the time I started my career, I was working on chicken embryos! So my entry into science was not at all motivated by building pharmacological tools or starting companies, but by understanding mechanisms of development.

When I started my lab, we discovered that molecules known to guide axons towards their targets also are expressed in blood vessels. Well, that immediately raised the interesting idea that maybe you could use these molecules to guide blood vessels away from a growing tumor, or towards an infected area. We spent 15 to 20 years trying to understand which of these factors were the most powerful ones, and how they affected the vascular system. In the course of these studies we discovered the guardian receptor that we're talking about today.

The guardian receptor has a small role in modulating vascular development, but it turns out to have a much more impressive role in maintaining the blood brain barrier; that was our first exciting discovery.

And how was it that you made that discovery?

We discovered this with a mouse mutant, where we knocked out this receptor in the blood vessels of the mouse. We observed that when we do this in newborn mice, they would keel over and develop seizures. Well, because we had only encountered this receptor on blood vessels, this brain defect suggested to us that perhaps the blood brain barrier was affected. Indeed, when we injected dyes into this baby mouse, we could see that there was a leak in the brain of the mutant, and the controls had no leak and an intact barrier. So that was cool.

So then we wanted to find out what activates this receptor. We found a guardian ligand that when you knock it out in mice, leads to the mouse developing a blood brain barrier leak. So we then had a system with an activator and receptor that maintains the blood brain barrier.

That's when we decided we wanted to make an antibody tool where we could specifically inhibit the binding of this ligand to this receptor. Because, as often happens in biology, molecules are not monogamous. They're not only talking to one partner, they have various interactions. And so it was important to have a tool that would specifically block this one interaction. When we made that tool and injected it into a wild type mouse -- a normal mouse-- we could open the blood-brain-barrier in a reversible manner. A short time after we delivered the antibody, there was a leak. That was the most important scientific finding -- proving that this ligand and this receptor need to "talk" to each other to keep the blood brain barrier intact.

So D2B3 is an extension of this finding?
 

Once we had this antibody tool, I started thinking, well, this could be a very useful tool to deliver drugs into the diseased nervous system. As much as you need the blood brain barrier to protect your brain when you're healthy, if you get sick, this barrier becomes a big obstacle because all the drugs that you could deliver into the brain that would cure the disease cannot pass the blood brain barrier. We're now trying to find out what kind of drug classes we can deliver across the barrier with this antibody tool, and how it can be beneficial for treating brain tumors, neurodegenerative diseases such as Alzheimer's disease, brain infection, or even intellectual disability. There's a whole class of diseases that cause intellectual disability in children, for example, some are caused by mutations in genes that encode for lysosomal storage. Tools are available that could be used to prevent these diseases, but currently you cannot deliver them across the barrier. So we're thinking of developing D2B3 as a platform technology for various brain diseases.

What are the risks associated with opening the blood-brain-barrier with D2B3?

That is a great question. There are several parts of this answer. Firstly, based on clinical experiments, we know it does not necessarily immediately cause brain damage if you were to open the blood brain barrier. There are already nonselective approaches to open the blood brain barrier. For example, you could use focused ultrasound devices with micro bubbles. That creates a mechanical stress on the endothelium and creates openings through which drugs can be delivered into the brain. It has reversibility such that the blood brain barrier stays open around 12 hours. That is already clinically approved for certain indications, and is fairly safe.

However, if you think about the case where you have a diffuse brain tumor, then all of a sudden, this focused ultrasound approach is not quite so attractive. Whereas our antibody-based technology targets all the 400 miles of capillaries that you have in your brain. That should be a lot more effective. We are now comparing the two technologies side-by-side in ongoing experiments with a collaborator that specializes in this focused ultrasound.

Also, with D2B3, our antibody is actually unstable. We know that it opens the blood brain barrier for only 6-8 hours. The majority of the drugs that we deliver get delivered in the first three hours. So that is a fairly favorable opening characteristic that should be amenable to drug delivery.

Finally, there are already antibodies against the ligand in question that have been tested in patients with peripheral cancers. These patients have been dosed with an anti-ligand antibody for up to 17 times every two weeks; no adverse side effects were reported in the patients. This shows the pathway itself is already clinically de-risked, in a way. Since our reagent is so selective for only this ligand and this receptor, we think it should have a favorable safety profile as well.
 

What are your next steps with this discovery, and with your winnings from this prize?

From the moment that you have a tool that works in your laboratory -- in an animal, a mouse -- and the moment where you inject this tool into a person, there is a huge gap that you have to bridge. The wonderful help from Yale Ventures is in facilitating this. They help us with necessary business advice, planning a clinical trial, navigating discussions with the FDA, figuring out what we need to show them to bring this into the clinic, etc. All of these things have been so facilitated by Yale Ventures, and that's why I'm so appreciative of being able to work with them.

How else has Yale supported your journey as an innovator?
 

Yale Ventures has this wonderful pitch program. You send in a very short application, so it doesn't cost you a lot of time to apply, then they provide feedback on your application, so that you can improve it. You have several rounds of this with innovators, and people that work in the industry provide feedback on your goals and help you determine what your next immediate goal should be to bring it forward. I was lucky enough to win this award. So now, with the money I won, we are able to have this experiment repeated by a contract research organization. This is an important part -- to ensure reproducibility of the technology outside your laboratory. We are using the first part of the money to do exactly that. Other experiments ongoing with the money are optimizing the antibody and doing studies in non-human primates so that we can be certain that it doesn't only work in a mouse, it also works in a larger animal.

Yale Ventures helped with filing the patent, and following that, they also helped in actually transforming the idea and the patent into a company. That's how we created a real company, D2B3, and it has advisors, an executive business director, etc. Yale Ventures helped with all of this as well. You get multiple rounds of advice and feedback from people working in the industry. That is really so valuable in understanding what you should and shouldn't do to bring your technology into the market. I'm very excited about this process. And I think it's one of the most fun things I've done in my career. We're doing research because we want to improve human health. This technology really has the potential to do that, and that's why I decided I was going to embark on this business journey in addition to the stuff that you usually do as a professor -- getting grants, writing papers and such.
 

You really highlighted some of these really beautiful moments of discovery. You almost made it sound like it was serendipitous. What were some of the biggest challenges that you faced during the development of D2B3?
 

I love this question. So, it's true that I make it sound serendipitous, because actually, the discovery process started with a control experiment in a dish. So we treated endothelial cells with a siRNA that would knock down our receptor. And then we were looking at what the signaling consequences were. My postdoc, Kevin Boyé, who is the inventor of all this, put an additional loading control into his Western blot, just to make sure that there was the same amount of endothelial protein in all the samples. We had an antibody, and he just felt that it was his due diligence to add an extra loading control. He used CLDN5, which is a major tight junction protein in the brain. But then we saw that there was so much less CLDN5 in the mutant! We were so surprised by this result; it was unexpected. That is what made us think perhaps, if clotting five is really so decreased, maybe this receptor does something to the blood brain barrier. 
But then we had the challenge of importing the mouse and starting on the in vivo experiment, which takes 8 months to complete. It takes a lot of time to breed the animals and such. So after 8 months of hard work, we were so satisfied to see in the mouse, there was also a decrease of CLDN5, and not only was there a decrease -- there was also a leak of the blood brain barrier. So it was serendipitous, in a way. It all started with an extra control that gave us the idea to do this experiment.

Now in terms of the challenges, I think the biggest challenges still lie ahead of us. Turning this lab discovery into a treatment in humans is still going to take time, and a lot of effort. And a lot of learning on everybody's part. But we're excited to proceed.
 

Going forward as both a professor and a founder, where does the uncertainty lie when you look ahead?

Well we have opened the blood brain barrier, so now the question is what lies behind? It requires expertise in a lot of different neurological and neurodegenerative diseases that I do not have. I know a lot about blood vessels, but not enough about these diseases. It required setting up collaborations and getting all the necessary expertise to understand what can be targeted, and how to do it. So there's a lot of work ahead of us.

We're thinking to test this in a clinical setting, brain tumors would probably be our first indication. Brain tumors, such as glioblastoma, have a median survival rate of a few months, even with chemotherapy and radiation treatment, which is the current standard of care. And that standard of care has not changed in the past 15 years. So once we have a clinical grade antibody, which costs $5 million to make, we were thinking that the first clinical trial would likely be in a glioblastoma patient. This offers interesting possibilities to run a phase zero or phase one type trial in a human, to check if chemotherapy delivery is increased by our antibody, and to what extent, and that there is no toxicity. That would already be a huge step forward. And then afterwards, the other challenge is to find suitable agents that can be efficacious on the other side of the blood brain barrier. It is not enough to show you can get agents across, you need to know if it is active and whether it does what you wanted to do. That's lots of exciting research.
 

Have you been approached by the developers of any drugs looking for an appropriate delivery mechanism such as D2B3?

We're discussing with a lot of venture capitalists and also pharmaceutical companies that have drugs that have efficacy against CNS diseases, but cannot cross the blood brain barrier.