Podcast Transcript

David Brühlmann [00:00:25]:
Welcome back to Part 2 with Jitendra Kumar of Entos Pharmaceuticals. We’re picking up the conversation on proteolipid vehicles and the Fusogenix platform, moving deeper into the practical and strategic questions behind delivering genetic medicines with a truly novel delivery vehicle. You can check here Part 1. 

Expect candid perspectives on process development, analytics, regulatory strategy, and what this technology could mean for patients where existing approaches fall short. Let’s get back into it.

Another part that’s absolutely vital is the regulatory aspect. Now, your platform is novel. It’s a novel delivery mechanism, unlike lipid nanoparticles (LNPs) or, let’s say, AAVs. How do you approach the regulatory side and these discussions with the agencies? What is your strategy there?

Jitendra Kumar [00:02:34]:
As a company, our strategy has pretty much been to let the science talk. We go in with all the data and all the information that we have. Obviously, you have to complete your GLP toxicology studies. The regulators are well aware of where we stand. In terms of the process, you also have to explain, in our case, that we have this membrane protein. What are its limitations? What acceptance criteria are we using? And how are we ensuring that the quality is maintained for safe use?

My experience with the regulatory discussions, especially with Health Canada, is that they are open to new technologies. They want to see innovation in the field. They encourage those efforts. Their job is to make sure the technology is safe. If we can demonstrate that we meet all the criteria laid out in the guidelines—or, if we don’t, that we have a sound scientific explanation for why our approach is still appropriate and valid—then those discussions become science-driven. Because we are the ones developing the technology, we have the scientific understanding behind it. So we have to communicate that knowledge clearly. That’s my philosophy: your science has to speak louder than your claims when it comes to regulatory discussions.

David Brühlmann [00:03:39]:
And does this also include a solid analytical characterization package?

Jitendra Kumar [00:03:45]:
Of course. I mean, you have to have a good analytical package. That is how you ensure that you are meeting all the required criteria.

David Brühlmann [00:03:52]:
And how different is that package for the FAST protein compared to an antibody or other more standard recombinant proteins?

Jitendra Kumar [00:04:03]:
The thing is that the FAST protein is not our API. Our API is the nucleic acid. The FAST protein is one of the components on the surface of our PLVs (proteolipid vehicles). Having said that, we still have to meet all the required criteria until it becomes part of the PLVs. We have to demonstrate that its presence is what drives the delivery. So there are certain assays that we have to perform to ensure that the protein is present on the particle surface. We also have to ensure the stability of the protein itself because, from a regulatory perspective, it functions as an excipient.

Because it’s part of the envelope, not the core of the drug product (DP) or drug substance (DS). Our API is the nucleic acid. Having said that, we have performed all the necessary studies, including viral clearance studies, to ensure that the protein is safe and meets all the criteria that would apply to any other recombinant protein.

David Brühlmann [00:04:51]:
So that means that even though your PLV is not the API itself, you still need extensive characterization because you’re delivering it into patients.

Jitendra Kumar [00:05:06]:
Yes, we do. And I think the same is becoming true for all novel lipids as well, where the FDA is asking for full characterization of any novel lipid that is not already included in their database. I think that’s a fair request. Even if something is considered an excipient, if it’s novel, the regulators want much more information about it.

David Brühlmann [00:05:24]:
Now I’m curious, Jitendra, you’ve made quite a bit of progress. What still needs to happen before you obtain full approval, and how long do you think that will take?

Jitendra Kumar [00:05:35]:
Obviously, Phase I and Phase II are relatively short. At the moment, the product that we believe is closest to the market is our leptin gene therapy for lipodystrophy. We hope to start Phase I early next year. Then, before ultimately submitting a Biologics License Application (BLA), it will probably take another eight to nine years at least.

As you mentioned, one of the things we still need to address is scalability. That’s where our Cytiva bioreactor experiments came in. We wanted to understand how we could scale up, whether there is any loss during scale-up, and whether we could further improve production.

In terms of the BLA, we obviously need much more robust analytical characterization. One advantage of working with a membrane protein is that we have to maintain it in a detergent solution, which naturally creates an environment where many viruses cannot survive. That actually becomes an advantage rather than a disadvantage. For many other biologics, you need a dedicated detergent treatment as one of the viral inactivation steps. In the case of membrane proteins, they already require detergent to remain functional in the micellar environment.

Most of what is required for the IND also applies to the BLA, but the BLA simply requires much more comprehensive studies and much stronger analytical data. One more thing that I forgot to mention is the importance of finding new analytical technologies that can support your regulatory filing.

One project we carried out with the National Institute for Nanotechnology (NINT) over the last year involved using cryo-EM to characterize our particles. It’s a fantastic technology. You can visualize many aspects of the particles. You can see the cargo, the lipid bilayers, and one of our goals before the BLA is to visualize the FAST protein on the particle surface as well. That would provide direct visual evidence that it is driving the delivery mechanism.

Coming back to my own experience, when I joined the company, one of the tasks assigned to me was performing electron microscopy (EM). I received training and carried out extensive TEM and AFM (Atomic Force Microscopy) work to characterize particle size and morphology.

Another point I’d like to add from an analytical perspective—and most people in the field already know this—is that every analytical technique has both strengths and limitations. For example, when we use Dynamic Light Scattering (DLS), which is still the gold standard used in many FDA submissions, it provides one type of information. Then you move to TEM or cryo-EM and perform essentially the same particle size analysis, and you notice differences.

Those differences are not necessarily because the particles themselves are different in size. With DLS, you’re measuring the hydrodynamic diameter (or hydrodynamic radius). With electron microscopy, you’re imaging particles on a grid. During sample preparation, the particles become dehydrated, and because of that drying process they can shrink slightly. So when you’re comparing analytical methods, you need to understand both the advantages and the limitations that each technique brings.

David Brühlmann [00:08:24]:
Yes, this is absolutely key. And there is often not a one-size-fits-all strategy or method.

I’d like to circle back to your career path. You have spent significant time working on unmet medical needs in oncology and neurodegeneration. How does that now apply to what you’re doing today, developing PLVs (proteolipid vehicles)? What are they offering to new patient populations that currently have limited or no effective treatment options?

Jitendra Kumar [00:09:00]:
I will start my answer from a different place. Long back, when I first joined as a summer student in India, I went to an institute called the Indian Institute of Pulses Research. They worked on lentils, and I was in a laboratory doing tissue culture. It was a great experience. I was there for two months. The head of the laboratory, Dr. N. P. Singh, and I were talking, and I asked him, “You’re doing a lot of work here, but I don’t see that many publications coming out of the lab.”

One thing he said has stayed with me. He said that publications matter, but what matters more is the real impact. We were developing drought-resistant crop varieties for farmers in that particular region. What we were doing would directly help those farmers achieve better yields, even under drought conditions. That is something I feel I have carried with me unconsciously throughout my career. Many things matter, but ultimately it’s about how you contribute to humankind and how you create real impact.

Coming back to my own career path, I have been very fortunate. As I mentioned, I worked with Professor Harald Schwalbe. He is an amazing scientist who has done outstanding work in NMR spectroscopy. That is where I started working on an unfolded protein. One of my projects focused on studying the residual structure of the prion protein.

The hypothesis behind the project was that because this protein mysteriously unfolds and then misfolds in the brain, there must be residual structural elements within the protein chain that drive this process. If we could identify those residual structural elements, they could become potential targets for drug development. It turned out that although the hypothesis was reasonable—based on work we had done previously with other proteins—even in a highly unfolded state, the prion protein still aggregated. So my project evolved from studying residual structure into understanding the transition from the unfolded state to the misfolded state.

I used NMR spectroscopy together with electron microscopy (EM) at the Max Planck Institute of Biophysics. I was fortunate to interact with researchers there. Werner Kühlbrandt is one of the leading pioneers in cryo-electron microscopy (cryo-EM), and interacting with his group was an incredible experience. That journey sparked my broader interest in structural biology, protein-protein interactions, and protein-small molecule interactions.

Many researchers there were working on different projects, and we also had industrial collaborators. Overall, it was a fantastic scientific environment in Frankfurt. At the same time, it made me increasingly curious about the biological side of these diseases, which eventually led me to my postdoctoral work. I moved to Edmonton, where I still live today and now consider home.

Working with a neurologist gave me a completely different perspective because now I was looking at diseases from a clinical and medical standpoint. The project I worked on involved isolating infectious particles from infected brain tissue. These were not human samples. One project focused on chronic wasting disease (CWD), the prion disease affecting deer and elk populations. We obtained those brain samples, isolated the infectious particles, and studied their infectivity using animal models. I became heavily involved in developing field-flow fractionation methodologies during that work.

Another project involved designing molecules capable of sequestering amyloid-beta (Aβ) aggregates, following a similar scientific concept. That project motivated me even more because I could clearly see its potential to translate into therapies that might directly benefit patients.

The peptides we developed were effective at sequestering those aggregates, and the goal was to move toward therapeutic development. I became involved in discussions with several groups here, hoping we could move the project forward. Unfortunately, that did not happen. After that, I moved to another laboratory that focused on protein-drug interactions. So I have had quite a diverse scientific journey.

What I have learned overall is that whether you are studying protein structure, RNA structure, or DNA structure, molecular structure fundamentally drives biological function. One of the key challenges I try to address in my current role is particle size. Whenever you’re delivering LNPs or any other nanoparticles, they have to travel throughout the body.

That movement depends on diffusion, and diffusion depends heavily on particle size. If you can produce smaller particles, they generally diffuse more efficiently. To make smaller particles, you need to understand how your mRNA is packaged—how it folds and how you can compact it more efficiently—so that the surrounding envelope is also more compact.

Another exciting area of research that many groups are now pursuing is engineering the protein corona surrounding nanoparticles to achieve more targeted delivery. That is where my previous background becomes particularly valuable because I can apply my knowledge of structural biology to help design and optimize these delivery particles.

David Brühlmann [00:13:49]:
This has been great, Jitendra. What additional question should I have asked?

Jitendra Kumar [00:13:54]:
I mean, I have listened to your podcast. It’s mostly focused on CMC, and I have worked on the CMC part, but my major role is really around this membrane protein. So I could talk much more about that, although it’s closely related.

Having said that, the biggest thing is that it allows efficient and stable direct fusion with the cell membrane. People often don’t realize that there can be a delivery method where your particle fuses directly with the cell membrane and delivers the cargo straight into the cytosol. Regarding additional questions, I honestly don’t know. It’s up to you. If there’s anything else you have in mind regarding the work I’ve mentioned, I’d be happy to discuss it.

That peptide I designed for sequestering amyloid-beta (Aβ) aggregates is still very close to my heart. If I ever get startup funding, that’s the project I would pursue. Having exposure to industry, I now understand one of the biggest challenges. The problem with virtually every neurodegenerative disease is diagnosis. By the time we detect the disease, it has often progressed to a stage where it’s very difficult to reverse.

Having said that, the peptide I worked on in 2013–2014 was very effective at sequestering Aβ aggregates. The way I see it is that if we could fluorine-label that peptide—which would allow MRI detection—we could potentially achieve much earlier diagnosis. The advantage is that the peptide not only detects aggregation but also sequesters and helps clear the aggregates.

So you would not only detect that aggregation is occurring, but you might also be able to remove it. From an industry perspective, however, I found that the concept wasn’t considered exciting enough unless we first demonstrated proof of concept in animal models. That requires a significant amount of funding. So there are limitations everywhere.

David Brühlmann [00:15:24]:
You’re right. Early detection is key. Hopefully we’ll find ways to make that happen.

Jitendra Kumar [00:15:32]:
Another point—and whether it’s a question or not—is the difference between academia and industry. One of the biggest lessons is learning where to stop. Many exciting scientific opportunities appear along the way, but you have to make difficult decisions because you need to focus on what is feasible for your company and for the available time and resources. There are many scientific possibilities and many different paths that may eventually lead to the same result.

But you still have to define which path you are going to follow. Even when we work with our technology and change a few formulation components here and there, we often obtain mixed results. At some point, however, we still have to commit to one direction.

David Brühlmann [00:16:08]:
Yes. Focus is key. What is the most important takeaway from our conversation, Jitendra?

Jitendra Kumar [00:16:16]:
In my opinion: Find the right partners. Build a strong network. And understand both the strengths and the weaknesses of your technology. Knowing your strengths is important, but understanding your weaknesses is equally important because those are the areas you need to address as early as possible and continuously improve.

David Brühlmann [00:16:31]:
Excellent. Where can people get hold of you and learn more about your technology?

Jitendra Kumar [00:16:37]:
People can reach out to me on LinkedIn, and they can also visit entospharma.com. That’s the easiest way to learn more about the technology and get in touch with us.

David Brühlmann [00:16:46]:
There you have it, Smart Biotech Scientists. I’ll leave the links in the show notes. Please reach out to Jitendra and his team. Thank you so much, Jitendra, for being on the show today, for sharing your passion, and for the work you’re doing to advance drug delivery. Thank you so much.

Jitendra Kumar [00:17:02]:
Thank you, David. Thank you for the invitation. It was a pleasure to talk with you and share my journey.

David Brühlmann [00:17:06]:
Novel delivery platforms demand new playbooks. Jitendra Kumar has shown how process decisions, analytical strategy, and regulatory thinking all come together when there is little regulatory precedent. From Phase I trade-offs to the key questions every CMC leader evaluating non-viral delivery systems should be asking, this was a masterclass for anyone building in this space. Thank you for tuning in today. If you found this episode useful, please leave a review on Apple Podcasts or your preferred podcast platform. See you next time.

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