HOUSTON– History will come to regard the teen years of the 21st century as the time the immuno-oncology wars got going….first with Pyrrhic, expensive and commonly quite toxic CAR-T work. As the field matured, grew more comfortable with itself and began appreciating its powers, the clinic saw the advent of adoptive T-cell transfer into cancer patients with the cells coming from donors (CAR-T cells are autologous) and being entrained ex vivo to stalk a set of tumor-associated antigens (CAR-T goes after but one antigen).

But the term “wars” would also enlarge to mean something more than clinicians fighting cancer. It would be recognized to mean aggressive inventive start-up immuno-oncology firms engaged in combat for investor dollars and clinical victories with each other. We contend that at this juncture, surely no company has won investor dollars so auspiciously as Marker Therapeutics ($MRKR) has. We’ve discussed Marker’s reverse merger origins in the Texas Medical Center complex of the Houston gulf, Marker’s key founders being Baylor College of Medicine faculty as well as alumni of great laboratories and corporate contenders in immuno-oncology.

As the 2018 holidays drew near, we grew to sense editorially than no company warranted our attention and our investment more strongly than Marker, and we spoke at length with CEO Peter Hoang. At Hoang’s invitation, we spent several days in Houston during February 2019 both visiting its headquarters and attending with company officers Marker presentations at the 2019 American Society for Bone Marrow Transplantation meeting in Houston.

During the next weeks, BioPub will feature a series of interviews with Marker officers.

Marker CEO and President Peter Hoang was CEO and President of Marker predecessor company TapImmune. He served as Senior Vice President of Business Development at Bellicum ($BLCM) and as Managing Director of Innovations at UT MD Anderson Cancer Center. He has extensive investment banking experience for tony firms like Deutsche Bank, JP Morgan and Oppenheimer. Hoang earned an MBA from UCLA’s Anderson School of Management.

Hoang

So back in 2004, when Juan [Marker chief development officer Juan Vera, MD] started doing this, the therapy was created in 24-well plates, which are little tiny plates, probably a centimeter in circumference. See, in a 24-well plate, the limitations on the culture were pretty severe.

In those old-fashioned plates, what happens is that you’ve got this really very shallow plate, right? When you’re culturing the T cells, they settle to the bottom of the plate. So you fill this plate with culture medium, right? Oxygenation happens through the surface and so if you get a plate that’s too deep, if you end up putting too much media in, then the cells become hypoxic. They don’t grow because they just lack oxygen. All the oxygenation happens up here so that it puts a real limitation on how much media you can put in.

For our applications, we have to use lots of cells. We need to. But if you culture lots of cells, they end up using lots of culture medium. They burn through glucose, they produce lactate, pH starts to drift……the culture medium gets worn out quickly and you’re constantly having to refresh it. So culturing things that way gets very very manually intensive. And it’s a system that crashes readily.

Depending on what you’re seeding density of the cells are, they end up using a lot of media; they go through glucose, they produce lactose and they basically deplete the media so you’re constantly refreshing the media as these cells grow and as the cells grow they use media more and more quickly.

KSS

My entire PhD was done in cell culture, seriously, that was literally everything I did all day for four years. Manually intensive indeed….

Hoang is speaking of the tedium of old-fashioned ways of culturing cells, which not only tear through nutrients and liberate acid but also must be kept strictly aseptic, as culture medium is an excellent growth forum for bacteria and fungi. While cultured cells are generally incubated with added antibiotics, bacteria can learn to resist those. Antifungals are less readily used because they tend to be cell-toxic….and to come to the culture room in the morning and find that weeks of cell culture work has been ruined overnight by fungal contamination is the bane of the cell scientist’s existence.

Marker scientists found themselves up against a number of serious issues: a need to incubate lymphocytes ex vivo for extended periods of time, and keep them nourished. Lymphocytes are not adherent, but settle to the bottom of their container. But during this culture period, the cells are “snogging,” having intimate interactions with selected tumor antigens and selected agents like interleukins to train the cells to go after target antigens….and cannot be disturbed. How to keep them fed, oxygenated, and undisturbed? It was a tall order. And Hoang emphasizes that any sort of agitation of the culture can derail the efficacy of the process, which is about entraining donor lymphocytes to become professional assassins that seek out and kill cells bearing selected tumor antigens.

Hoang

So it’s really easy to crash this culture and it’s really hard to produce an adequate number of cells because you know it’s very very manually intensive. So in 2007, we actually called a group of mechanical engineers up in Minnesota at Wilson Wolf. They are a small company, and John Wilson, the CEO and the hundred percent owner, was actually the guy who picked up the phone. We ended up spending almost an hour talking about the problems. And this is how Juan became a co-inventor of the G-Rex device. [Vera has collaborated extensively with Wilson Wolf.]

So what they came up with was this. [Hoang puts a G-Rex canister on the table in front of us.] Basically a cell culture device where there’s a gas-permeable membrane at the bottom of the chamber. So oxygenation happens through the bottom. You can seed all the cells here and put as much media as you want in and the cells still get oxygen through the bottom. Oxygen comes in, carbon dioxide goes out through this gas permeable membrane. Because of the chamber height, there’s relatively an unlimited amount of media that you can replenish if needed. But this media is especially important if you end up increasing the number of cells that you see and what we find is that in this therapy, probably not surprising to you, that the seeding density is actually super important. Getting a seeding density of a million cells per centimeter squared, for example, is only the beginning.

We are joined by Juan Vera, MD, and by Tsvetelina Hoang, PhD. Vera trained originally as a surgeon in Bogota, Colombia, but later cultivated strong bench research interests in T-cells and immuno-oncology after coming to the US. Prior to Marker, he worked extensively with both Celgene ($CELG) and bluebirdbio ($BLUE), helping those firms innovate novel CAR-T cell therapeutics. “Lina” Hoang is Marker Vice President for Research and Development. Bulgarian by birth, she met future husband Peter while both were undergraduates at Yale. Hoang’s PhD is from Johns Hopkins, and she is former director of translational research at Bellicum ($BLCM). She was on the faculty of  UT MD Anderson Cancer Center, where she worked in the laboratory of Nobel laureate and Anderson professor James Allison, PhD.

Hoang

So this is the G-Rex that we use. This is a G-Rex 5….. they come in all shapes and sizes.

So if you look at what we do, basically we are creating tumor-infiltrating lymphocytes we they need to grow 50 – 100 billion T-cells. The seeding density’s actually really important, you know. We are truly recapitulating a kind  of ex-vivo lymph node in the chamber because it’s the crosstalk that’s important. Juan’s the important one here, you (Juan) could take over. I’ve started on crosstalk and we talked about avoiding agitation of the cells…..

Vera

Yeah, sure. So, actually my background, I’m a medical surgeon and I came to Baylor in 2004, and one of the initial things that we were working on was in the generation of antigen specific cells. So Cliona Rooney, PhD, who is one of our scientific co-founders actually was training under Alan Rickinson, who is actually the person who developed the method to generate ex vivo EBV specific T cells as basically from the same laboratory where the Epstein Barr Virus was actually characterized. So Cliona Rooney was the first person that use T cells specific for EBV to treat EBV related post-transplant lymphoproliferative disorders (PTLD), right?

And when I initially came to Baylor, I was fascinated by the fact that this type of therapy was actually very effective in the clinic to treat PTLD without any side effects. One of the things that I was really shocked was why not every single patient received this type of therapy and why it was so complicated. Well, after spending a few months in the lab, it became very clear that the limitation is a technical one. So the way that we used to expand the cells in the lab, it was a very manual manner, right? So in one hand, you have a therapy that is effective, you have a therapy that works in patients that have no alternative care, and that is very safe, right? But when you actually see it step by step, how the cells have to be manufactured, what you actually have is a major set of obstacles.

Let’s say that I want to manufacture cells from you. So what I do is I take your blood, I take it to the lab, and I separate out the T-cells and begin propagating them. Basically you will have an operator that is examining the culture, the T cells growing in vitro, and you’re basically propagating those cells manually using  transfer well pipettes.

Hoang

KSS is an MD PhD. Formerly an MD Anderson professor.

Vera

Okay, got it. So basically, what that means is that while you can actually effectively reproduce that method at a small scale, at the moment you basically start expanding to large number of cells, the same process that is viable at a small scale becomes completely unfeasible…. right? So when I started working with that method in the lab, I was like, OK, well, this is actually fascinating, you have a method that works in the clinic, but you have this very simple practical constraint that will really limit the impact of this therapy worldwide. So I was basically trying to figure it out what would be an easy solution for that particular problem. One of the things that I was basically taught early on when I went to the lab is about this crosstalk Peter was mentioning a moment ago, the relationship between the antigen presenting cells and the T cells. You actually want a close interaction because you are relying on the T-cell synapse to allow the presentation….and moreover there is cross talk that takes place at different levels. So basically, you have the crosstalk between antigen-presenting cells and T-cells. But you also have crosstalk between T-cells and other T-cells, right? So the way that we used to grow cells was actually in a 24-well plate. And you basically want to have those cells very close together, you see, in order to allow that crosstalk, that dialogue, to take place. There’s a reason that in that method you cannot extrapolate to large surface areas where basically, there is no opportunity for T-cells to enter in cell-to-cell contact. And you just cannot use, for example, wave cytoreactors where things are constantly being jostled and in motion. With that, you’re not allowing for this gentle procedure to actually take place. The cells have to be calm and whisper to each other. So I was basically looking for an alternative and I came up with this interesting device [G-Rex] for the production of “conditioned” T-cells.

So my, academic background actually is in the generation of genetic engineering T-cell products. I worked on CAR-T cells and was basically trying to come up with a better method to concentrate retrovirus. There was this interesting device that was being manufactured in a small company, which basically has a dialysis membrane. So it’s a cell culture compartment that is divided into cell culture and reservoir of nutrients, connected to the outside  through a dialysis membrane, so that basically you have nutrients going one direction, but the cut off is a particular molecular weight, so that then whatever recombinant proteins or viral particles come from the product get trapped. So it’s a very clever design, right? So I contacted this company, and it just happened that the person who answered the phone was John Wilson [Wilson owns 8.2 million shares of Marker, as of Feb. 14 filings, via reverse merger] who was the CEO of the company and an engineer as well. At that time, I didn’t actually know that he was the CEO and we start talking and I explained “OK, well, what I’m trying to accomplish is this…. and I need to know their mechanical standard details of the dialysis membrane to see whether it will work for my purpose and when we were talking, I said, Okay, well, I mean this will be very good and I will try it, but if you really want to solve a problem in the field that perhaps is not really fully recognized yet, we need to do something for T-cell culture, we need to do something to address that particular constraint and that was an interaction in 2007. So since then, we basically developed many different series of iterations from prototypes to desk concepts to now basically a product that really has transformed how antigen specific cells are manufactured.

KSS

So this device in front of us is really what Marker’s using now for antigen entrainment?

Vera

Correct. So you’re probably aware of the process of tumor infiltrating lymphocytes (TILs) that Steve Rosenberg had generated. [Steven Rosenberg, MD, PhD, is an immuno-oncology luminary at National Cancer Institute, and his work informed the clinical aims of Lion Biotechnologies ($LBIO), now Iovance ($IOVA).] Now, I think that there’s no there’s no doubt in anyone’s mind that TILs actually work right, I mean if you actually look historically the track record of TILs versus chimeric t cell receptors, TILs have been out for quite some time.

KSS

Yeah, he led the advent of CAR-T by several years it seems.

Vera

Correct. So TILs…. I think it’s something that has been around for a while, and in my view actually did validate the use of T-cell therapy in the context of solid tumors, something that CAR-T cells actually haven’t done yet. Now the biggest limitations of TILs was a complex manufacturing process and if you actually look the way that Steve Rosenberg and many of the CAR-T cells of the TILs groups are manufactured, it’s actually using the G-Rex. Now I think that one of the limitations that we have is that sometimes we get put in the same category as the CAR-T cell companies and people don’t necessarily understand what we do.

KSS

Just so you know, BioPub as a website, we’ve been kind of  negative on CAR-T from the beginning. We think it’s an excrescence….. its extreme, horrible downsides like CRS, the expense, the immunodepletion… we’ve said that in so many words leading up to initiating coverage of Marker. You know, “We’ve been telling you the downsides about CAR-T for a long time and this, Marker, is the company that comes in to really save the day.”

Hoang

So you know, it’s really interesting. We were just in roadshow meetings last week. One of the observations that was made by an institutional investor was that almost everybody who has invested in the company from an institutional perspective has been fairly evangelical. They’re all going out to other firms and saying, “Hey we love Marker, you should too!” The pushback though is from new institutions, and the first thing they do is they look and they say, the CR rates, “How can this possibly be true? You’re telling me you’re getting great responses, more durable responses, it’s 10 times cheaper, and there’s no toxicity and you get this level of CR. I don’t believe it!” It’s skepticism right? Our institutional friends, though, say this: When they look at the length of ongoing continuing complete remission for patients, they say, “I don’t even think about it in the same context as CAR-T, what I think about it is this is almost no different from hormone therapy for a patient coming out of transplant, which is that our infused conditioned cells do no harm, you can afford to give them to every patient in remission from their last therapy….. every patient should get these cells.”

Yes, who shouldn’t?…. right?

Actually, I have to say that a lot of the patients that we have in our solid tumor program have been physicians, because some of the people that actually learn about this therapy, that truly know about it….. you’re basically talking about oncologists at the medical center. Nothing’s available in the way of treatments they actually want and believe can work,  and they come and basically asked for a single-patient protocol to actually be treated. I mean, who wouldn’t actually want to receive maintenance therapy that is non-toxic?

As an old CAR-T guy, I always have a tendency to chase sexy, active, bulky disease, right? But, you know, the first thing that I acknowledge —and this is one of the things that I tried to emphasize—-was that you to look at this as a differential therapy in its own right. This can be used in a maintenance therapy setting, basically consolidation therapy setting where you can’t use a CAR- T. You know, this is a differential therapy in its own right and what I do acknowledge is that while it’s less sexy in a lot of ways, the far bigger market for us long term is setting up, right?

To be continued…..

Disclosures: KSS has a long position in $MRKR but no other firm named in this column. No advice or solicitation to trade in $MRKR shares is intended, and KSS will avoid trading in $MRKR for 7 business days after this column appears. Neither BioPub nor its editors has a pecuniary relationship with either $MRKR or its officers. Copyright 2019 KSS, Allan Lee and BioPub. May not be reproduced without permission.

Corrigendum: Because of a discrepancy in saved versions of this manuscript, where the version appearing on the writing software differs from the version on the website, a revised version correcting many errors and transcriptional lapses was uploaded to the website by KSS at around 19:45 on 15 April. If you read an earlier version and something didn’t quite make sense, have a re-read please.