George Netto, MD (Host): Hello, everyone. And thank you for tuning in. Today's episode is our 10th episode of P Cubed. I wanted to take this opportunity to thank the staff behind the scenes. Those include RadioMD and our staff here in Penn Pathology and Lab Medicine who really have collectively made this podcast series a success.

And today, we're going to focus on a topic that hopefully you share with me the excitement about, CAR T therapy. And everyone probably is familiar that CAR T therapy objective, main objective was to treat cancer, started with liquid tumors and now, hopefully, with solid tumor. But increasingly, applications beyond cancer are becoming also critical, and that includes autoimmune disease and the topic of today's conversation, which is the potential role of CAR T therapy in facilitating kidney transplantation and opening the door for patients who are not prior such methodology candidates for or easy-to-find grafts for kidney transplantation.

To discuss this topic, I picked another one of our rising stars, Dr. Vijay Bhoj, from our department, and he's an Assistant Professor of Pathology and a member of the star team in Transfusion Medicine and Cellular Immunotherapy under Carl June and Don Siegel and many other pioneers. So, welcome to this episode, Vijay, and thank you for accepting my invitation.

Vijay Bhoj, MD, PhD: Oh, thank you. It's such a pleasure, George, to be here. And thanks for the invitation to talk about what we've been doing.

Host: Excellent. So, we're going to use as a platform to the discussion your recent first author pilot report on a phase I study that is multi-institutional. I'll let you describe it to the audience in a second, where the publication was just out in New England Journal of Medicine. So, congratulations. I'm sure this is going to come a long way, playing into your candidacy for promotion soon. I never had a first author IN New England, but I'm so happy I'm working with people like you. So, let's talk about that a little bit, and give us a little background on why you did the study. What was the challenge that you thought such an approach could address?

Vijay Bhoj, MD, PhD: Yeah, yeah. I'm happy to talk about this. You've already alluded to it, George. We were applying this novel immunotherapy, cellular immunotherapy, for an application that hadn't been done before, and namely transplant, organ transplant. So, the challenge in organ transplant, of course, is immune rejection. Your immune system now, you know, foreign organ, foreign tissue comes into your body, your immune system, not knowing any better, is going to see that in foreign, potentially dangerous, and attack it and try and reject it. And so, this can happen very quickly in the wrong pairing of a recipient and donor, and I'll get into it a little bit more in detail. The rejection can happen very quickly, within hours, minutes to hours of an organ coming into the body, and that's called hyperacute rejection. So, what causes that?

We know from decades of prior work that there are patients who, during the course of their life, are pre-sensitized to other people. And the way that occurs is through exposure to other people's cells or tissue. There's primarily three ways that can happen: through a prior pregnancy or pregnancies in women or exposure to other people's cells through blood transfusions, and then the last being prior organ or tissue transplant. You know, they've gotten a previous kidney or heart, and it's worked for a number of years and finally stopped working, most commonly due to immune rejection.

Through any of those exposures or combination of exposures, patients have mounted immune responses. And really, we're talking about antibodies. Antibodies are those proteins that are supposed to protect us from infections, but they can also, of course, mediate, as we know, diseases like autoimmune diseases or transplant rejection. So, there's a subset of patients who have been pre-sensitized through this previous exposure and harbor antibodies.

If I may give you some broad numbers, just to put it in context, at any given time in the US, there's maybe about ninety to a hundred thousand patients who are waiting for a kidney transplant. Of that subset, of that cohort, about three thousand or so patients have antibodies at very high levels against the potential donor pool. When I say at very high levels, we actually quantify that. We do a lab test in our department, as you know. It's a standard clinical test. The result of that lab test is called a CPRA, a panel reactive antibody or calculated panel reactive antibody, and it goes from zero to one hundred.

Zero means you have no antibodies against anyone in the donor pool. You can get an organ from just about anyone. A hundred means you have been pre-sensitized through all those exposures I described through your life and have antibodies against virtually everyone. And so, that makes it, as you can imagine, very hard to get a compatible organ. If we took an incompatible organ to which you have antibodies and put it in you, you would undergo this hyperacute rejection. The antibodies would bind to the organ right away and cause rejection.

And so, for that about three thousand patients who are very highly sensitized, meaning their percentage is at the level of 99.9 or greater, basically one hundred, they're not going to get a compatible organ very easily.

The data says, and other people have done this, following these patients, they get an organ offer on average about once every decade. So, that doesn't mean they actually get transplanted. They get an offer which has to then be evaluated to determine, okay, are we going to accept that and go ahead with the transplant?

So, what that distills down to for these patients is a very high morbidity and mortality rate with a five-year survival of maybe 50%. So it's close to many cancer cohorts. It's pretty bad for them. Life on dialysis is not good. Quality of life is not good, but also lifespan is shortened. So, we're trying to get these patients to transplant, and the way we sought to do that was to use this cellular therapy to get rid of those offending antibodies, get that percentage down from 99.9 or a 100. That's sort of the big picture.

Host: That is a beautiful mini lecture on transplantation and hyperacute rejection or humoral rejection. Just to clarify, those antibodies, many of them are directed toward HLA, right?

Vijay Bhoj, MD, PhD: That's correct. That's correct. I didn't mention that. That's important.

George Netto, MD (Host): Yeah. No, I'm sure that people realize that. So basically, we're looking at 3% to 5% of recipients sitting and waiting decades, at least five to 10 years, which is very risky, just because they are among those who have a lot of antibodies against a lot of HLA types. And the way to clear the list to be offered a graft is you have to have some compatibility in that regard; and hence, they're going to be incompatible for a lot of donors. And you're trying to solve that problem.

A poor man pathologist like me, despite being CP boarded, forgot everything about transfusion medicine. I would say, "Why not, do apheresis? Take all these antibodies out. What's wrong with that? Why was this not happening, and why did you need this new methodology? Explain to us.

Vijay Bhoj, MD, PhD: Sure, yeah. So, apheresis is part of my bread and butter clinical work. As you know, I'm an apheresis doc. So, I'm a proponent. But in this instance, for these patients, it's just not effective on its own. And the crux of the problem with apheresis as a monotherapy, mono strategy, if you will, is that it doesn't get to the root of the problem, the cellular source, the B cells and the plasma cells that are continuously making the antibodies.

So, you need additional modalities. And the field has tried that with something like rituximab, an anti-CD20, to deplete the cells. But even that doesn't do a very good job because it doesn't do a good job at tissue-level depletion of those immune cells. That's where the CAR T cells we think have an advantage.

Host: So, here we go. So, how would it work? What would the CAR T target?

Vijay Bhoj, MD, PhD: I can back up to what you in your introduction alluded to, that CAR T cells had their first successes in treating liquid malignancies, what we call liquid malignancies, which are leukemias and lymphomas. And those malignancies were all stemming from the B-cell lineage, right, the immune B lymphocytes, and working very well. And now, we have regulatory approval for several of those products. They target two antigens. Depending on the type of malignancy a person or patient has, they'd get one or the other.

The first most successful, the earliest approved set of CAR T-cells targeted CD19. So, that's an antigen on all B-cells. When B-cells get activated, they start to mature. They make memory B-cells, and they make plasma cells. That's the terminally differentiated cell of that lineage, and it's the plasma cells that actually secrete these antibodies that we've been talking about. When a B-cell becomes a plasma cell, the CD19 starts to go down, and a second marker becomes expressed called BCMA, B-cell maturation antigen. So, we now have in our armamentarium CAR T-cells against BCMA.

So with the two of them, you cover that whole lineage of humoral immune cells from B-cells to plasma cells, right? Going to antibody production. So ultimately, our approach was to combine both CAR T-cells. We had done preliminary work and data from our group as well as others suggested that really it's the combination of the two that might be needed to be successful, in doing what we were trying to do. But I think, you know, it's early days, and we need more data to say, "Could we use just one of them? Would one CD19 alone or BCMA alone do the trick?" It's still an open question.

Host: Excellent. So, you're trying to cover the whole journey of a B cell being sensitized and then maturing and then into plasma cells and leaving some memory. So, you wanted to cover all bases with two different clones or population of manufactured CAR T at both ends. And so, I guess you make that cocktail and inject the patients.

But it's like an opportunity, not every day our audience hear about CAR T, to explain how does it work? Is there a phase before you inject the CAR T? I know you make the CAR T in the lab. But before that, it's not like you inject them directly into the patient, right?

Vijay Bhoj, MD, PhD: Absolutely. I can walk the audience through what a patient would go through on our trial. That, as you know, George, from work being done at Penn and elsewhere in the world, that manufacturing process is changing very rapidly as this CAR T field advances. But the way we carried out this trial, and it's an ongoing trial, is the way CAR T's generally started. So, it starts with the patient's own cells, right?

So, a patient, once they get enrolled in the trial, they meet their eligibility criteria, they come in for a visit to our apheresis center at our hospital at Penn. We collect their white blood cells through leukapheresis. So, that's a set of technologies where we can harvest blood from a patient, separate the blood into the different components, like the plasma is separated from the red blood cells. And for this purpose, we take the middle layer, which is the white blood cells, the lymphocytes, the T cells, which we're going to turn into CAR T cells.

And then, the rest of the blood is returned to the patient all in real time in about an hour and a half procedure. Those cells are then sent to a lab, also within our department, to make those CAR T cells, right? So, we use a lentiviral vector to transduce them, to deliver the gene encoding the CAR. And so, for this trial, we made two products for each patient. One is a CAR-19, and the other is a CAR BCMA. So, these manufacturing processes happened in parallel over the course of about, let's say, ten days or so. And they were frozen, and we do some release criteria testing, and it has to pass various criteria to be safe to put back into the patient.

Once that passes, the patient is brought back in to the hospital to get some level of chemotherapy, if I can say it in broad layman's terms almost, to make space for the incoming T cells to engraft. So, they get a little bit of chemotherapy. And then, a few days later, an infusion of the two CAR T cell products. And that happens over, you know, just a few minutes, again, usually in our own apheresis suite.

Host: That's wonderful. So, of course, the term CAR is chimeric antigen receptor T. So, when you're saying you're transducing, you're putting the gene for the B-cell receptor CD19 or BCMA in each one. You're transducing cellular engineering into the T cells. And now, that T cells carry this on its surface. So, it can attack the antibody to this antigen. So, it attacks CD19 and attacks BCMA.

Vijay Bhoj, MD, PhD: Correct.

Host: And in chemotherapy, in this case, these patients don't have a neoplasm, it's just to lower the immune response. So in general, in malignant applications or autoimmune, it's the same. You do that just to lower make space, like you said.

Vijay Bhoj, MD, PhD: That's right. That's right.

Host: So, you did this. This is a multi-institutional study from what I gather, and this is your first two patients. So, let's get to the good news. What happened to these patient? So, these are patients who started with 99.9%, meaning these guys are ready to reject any graft you give them because they covered all HLA types, right, against it.

Vijay Bhoj, MD, PhD: Yeah. Yeah. If I may, before I answer your question, just to emphasize that truly, as you've said, this is a team effort, multi-institutional, if I may just name them to give credit where it truly is due, this is a trial that's sponsored by our colleagues and friends at NIAID, N-I-A-I-D, so at the NIH, fantastic partners. And the three academic institutions are Penn, NYU, and MGH. And truly, you know, we get on weekly calls to discuss progress of every patient, truly weekly. So, everyone is engaged, and it's a team effort.

So, your question, what happened? So, we, of course, did each patient sequentially per the FDA mandate. This was a phase 1. So, safety is the main focus here. So, our first patient was treated. Now, it's been well over a year After the infusion, I described to you the process of CAR T manufacturing up to the point of infusion. So, let's call infusion day zero. At day zero, he got the two CAR T cell products, CD19 and BCMA, and we followed him. There's a little bit of detail.

About a month after his CAR T cell infusion, we brought him back to our apheresis center to do what you mentioned, the plasmapheresis. So, we gave him five treatments of plasmapheresis. Why did we do that? We did that because if the CAR T cells work, they kill the cellular source of the constant production of these antibodies. But the antibodies that were already in him circulating from just, you know, days, weeks before they were produced, they would have a half-life. It would take them weeks or months to gradually come down, even though there was no new production. So, we wanted to hasten that process, remove all the circulating antibodies. And then, see if we were successful, the antibodies that declined from our removal shouldn't come back because we've killed the source. If we were unsuccessful, they should rebound within just a couple of weeks, be right back at his, baseline, and he would remain untransplantable. We were really happy to see that they stayed down. it was about three months after that CAR T infusion that we felt comfortable that they've stayed down durably. They haven't, you know, come back and so on that we could now start delisting antigens from the UNOS national database and say, "Okay, these antigens to which he used to have antibodies that were unacceptable, we wouldn't accept an organ with those antigens, now we are okay." And the offers started coming in. He ultimately got four organ offers. And we chose one of them, and he received a transplant. He is now about 15 months post that kidney transplant and doing very well.

The second patient went through the same thing and is about 11 months post his kidney transplant and doing very well and was featured in The Inquirer in Philadelphia. And, You know, he talks about it making a big difference to his life. He can now travel, see friends and family, not be tied to his dialysis center three days a week, sitting there for hours.

Host: How rewarding. And I can see it in your tone and your eyes. I work with you every day, and I know how excited you are about this project, and rightly so. And it's just a tremendous example about how pathology is intertwined not only with cancer and not only in the backseat, but really at the frontline of patients and therapeutics.

So, what a role model you are to our young pathologists. So clearly, those CAR T cells are going to continue to live in the patient. My understanding and from the examples in cancer that they exist there even years and years sitting and waiting on the lymphoma to come back and attack it. Is that the case in this setting? So in other words, do some of the plasma cells—you keep saying we killed the source—do they come back later or you don't know yet?

Vijay Bhoj, MD, PhD: Yeah, that's a really important question, especially in this therapy and to set the contrast with our applications in cancer, as you rightly point out, we don't want them to persist. As opposed to cancer, we want these CAR T cells to deplete those B cells and plasma cells thoroughly and then go away. We want the CAR T cells to sort of die so that the new B cells and plasma cells can come back. Why is that? Because B cells and plasma cells, simply stated, are protective, right? That's why we've evolved to have these lymphocytes to protect us from infections and cancer and so on. So, we don't want to leave these patients persistently immunocompromised with no new B cells and plasma cells and antibody production.

You know, God forbid we have a new pandemic, we want them to be able to make antibodies against new things that they would encounter in their environment. So thankfully, in the patients we've treated, it's sort of following the example that the field is seeing in autoimmunity with CAR T cells, the CAR T cells don't last that long.

In cancer patients, a subset of patients will have them for years. But generally speaking, and we don't entirely know why this is, we have some ideas and lots of people are interested in investigating this, the CAR T cells last for a few months and then die. And then, you get new B cell regeneration. That's what we're seeing.

Host: But hopefully, you don't get the same B cells that are going to produce because now your patient has HLA, you know, that initially this patient, the recipient was sensitized to. So, those clones or it's just the reconstitution that is miraculously not attacking the new graft?

Vijay Bhoj, MD, PhD: Yeah. So, you're getting into some really fun transplant immunology here.

George Netto, MD (Host): Maybe I should be on the next paper, please. Maybe I'll finally have a New England.

Vijay Bhoj, MD, PhD: It is actually where we're moving. So, I'm working with, of course, Dr. Ali Naji, who's a transplant surgeon and immunologist at Penn. He's the senior investigator on this trial and grant. And we are working to use CAR T cells now to prevent the emergence of new antibodies post-transplant.

So, you know, I've talked about just now with you about getting rid of established preexisting antibodies, but can we use CAR T cells to prevent the development of new antibodies once the allograft comes in, once the transplanted organ comes in?

So to really answer your question again very more clearly, we think that our CAR T cells have really wiped out the B cells such that we're not getting return of those preexisting clones. We are not seeing antibody rebound in these patients to the donor antigens. So, that's good. We think there may be an opportunity to use this technology to induce long-term tolerance to the graft to trick the immune system into thinking that the graft is actually self, to not attack it.

Host: Then, that could be that second part. I know a kidney transplant is different than liver transplant. But that second part then can be expanded to other organ transplantations where you get rid of immunosuppression, or at least part of the immunosuppression strategy. And this is exactly why, like you said, it worked in SLE, right, in autoimmune, because these clones don't come back after failures of different therapeutic approach in autoimmune disease. There are some tremendous success stories where patient "are cured" for years just after the injection with CAR T. What exciting field.

As a cancer pathologist, it's very hard for me to get excited about non-cancer stuff, but this is actually arguably more exciting. So, I love it, and I hope our audience enjoyed that, and I'm sure they learned a lot about a topic they don't think about every day. I thought CAR T is something that started at Penn, and Penn has led the way and shared it with the world. So, I'm glad to see that you guys are taking it now to different new applications.

And Vijay, you're such a promising star or already fulfilled star, and I wish you the best. And thank you again for taking the time. I know you're on call today. So hopefully, a fellow is not going to come and grab you off the camera.

Vijay Bhoj, MD, PhD: No, I'm okay.

Host: Thank you very much.

Vijay Bhoj, MD, PhD: No, such a pleasure to be in this department, being able to bring these to patients. Really, Penn's a great place to do that. Thanks, George.

Host: Excellent. Thank you again for listening, and I hope you join us for the next, 10, 20, 30 upcoming episode. The podcast has enjoyed the success. The big part is, like I said, the staff working on the production and the terrific guests that were accepting our invitations, and more to come. See you next time.