Cells： from Robert Hooke to Cell Therapy - Professor Carl June

Excellent. So we come to, unfortunately, the last two speakers of what's been an amazing two days. So the next speaker is my very good friend, Professor Carl June, who's at the University of Pennsylvania, Pennsylvania, I should say, in USA, used to genetically modified T-cells in the treatment of cancer. Can we please give Carl a big welcome? Thanks very much, Carl. Thank you. Thank you, Chris and Ian and Johan for a great learning experience for me to see this session today. So I'm disclosing now that I have a sponsorship by Novartis, and then I'll discuss off-label use of CTL-019. So this was introduced in part by our previous speakers, but cancer is now realized to be a disease of genetics with abnormal pathways. But finally, there's been another pathway, which is that this cancer cell, in order to evolve, has to invoke tolerance to it so that it's not rejected. And so that's why we now have this field of emerging immuno-oncologies, where you heard from Ben Jakobson describe checkpoint therapies and adoptive cell transfer, or ACT, as I'll refer to it. And it began with vaccines, but now, really, these two have become the dominant modalities. And then adoptive cell transfers comes in three flavors. There's the so-called tumor-infiltrating lymphocytes, and I'll mention, and then gene transfer methods involving CAR T-cells and T-cell receptor T-cells. And I reviewed this recently with some colleagues. So the TILT therapy is more logistically complex. It happens in patients who have a surgically accessible metastatic lesion that then is grown in the lab. And this is now open, you know, in a phase three trial in Europe. Robert Hawkins is leading it here in the UK. And it's had sort of a, it was a first trial to be started, this TILT approach. But it's had, it's been basically the comeback kid in that now. With genetic testing of tumors, the neo-antigens are created by mutations, can be targeted by TILT. So it's one way to achieve the kind of therapy that Ben showed you with natural T-cells. So far, it's only worked routinely in melanoma, but there are some striking cases in other kinds of tumors such as biliary tract cancer that have been reported. The other approaches start with the blood, and so therefore logistically more simple, and then involve gene transfer to insert CARs or T-cell receptors. And then the cells are then manufactured and given to patients. So we began our leukemia trials in 2010. And if you search on clinicaltrials.gov, as of about two weeks ago, there were 77 trials ongoing now with CAR T-cells. Most of them are in the US. And a lot of the basic science for CAR T-cells was done here in UK and Europe, and yet they only have eight trials open. And I think that's a regulatory issue, not science. And China has gone from having no trials to 20, and I'm sure will outpace the US shortly. So there's geographic disparities of where these trials are, but it's gone from a small handful of people doing these five years ago to now where it's an emerging industry. And I'm going to then dwell on what CAR T-cells are and some of the clinical results. These are some of the major issues facing the field, which is a long-term persistence of these CARs required. They're referred to as a living drug. When you put these genes in, they stay in the patient and through various mechanisms. And how long should we have these persist, for instance, is one issue that's open. Another is how genetically to modify the T-cells. And there's a number of approaches such as lentiviral vectors, retroviral vectors, and things like transposase, sleeping beauty, and even RNA electroporation, which we have trials underway with. So there's a number of ways to genetically modify T-cells. And then what kind of T-cell do we want to use? There are a number of subsets of cells, and I have focused on memory stem cells. But there are other kinds of cells such as effector cells, and I'll briefly talk about that. And this is the same point that Ben Jacobson raised, which is how can we combine checkpoint therapy with CAR T-cell therapy, which our preclinical data and others show are very synergistic. So CARs didn't just start. The first one was reported in 1991 by Art Wise at the University of California. And it was a synthetic molecule made to actually test the signal transduction properties of the T-cell receptor. So he fused the intracellular zeta chain of the T-cell receptor with an extracellular domain of CD4. And this then retargeted cells to HIV, because CD4 is the receptor for HIV. And we did the first CAR trial starting in 1997. And so this was then expressed with a retroviral vector. And we treated about 40 patients with that, with HIV, and found that it was safe. And we found that the cells had persisted in those initial patients treated beginning in 1997 now for more than a decade in the patients with an average half-life of greater than 17 years. So very long-term engraftment in those patients and no safety. And then the field evolved with grafting of antibody or antibody fragments onto the CAR signaling domains and then ever more complex signaling domains to add in co-stimulation. Because the first cancer trial done with CARs was reported in 2006 from the Netherlands. And it was a so-called first generation CAR that just had the zeta chain. And the cells did not persist in the patient. So it's different than in HIV. Likely the tumor microenvironment is more immunosuppressive in cancer than in non-cancer conditions. So this is the CAR that we currently have in clinical trials with a 4-MB co-stimulatory domain. And another that's in advanced trials in the United States is the CD28 co-stimulatory domain. And we've been studying those now. And this is work by Omkar Kualakar in my lab to ask if we can change metabolism of CAR T-cells. So T-cells go through cycles. They can go from a naive resting cell to then an effector cell where they are great at killing targets. And then back to a memory cell that can persist for the rest of your life. If you then get reinfected, these cells can revert back to this stage. And they eat different food. At this point, they're basically sugar burners. They're all glycolysis. And to make their energy, they're very energetically demanding when T-cells are very motile. And at the memory stage, they live off mitochondrial ends of fatty acid oxidation and Krebs cycle. And we've been measuring this in CAR T-cells with the so-called Seahorse assay, looking at oxygen consumption rates where we can, by various mitochondrial inhibitors, measure oxygen consumption. And what we have found is that if you have a CAR that expresses CD28 signaling domain, it is an effector cell that lives off oxygen, glucose, and makes a lot of pyruvate and lactic acid turns immediate orange very rapidly. And if you look by confocal microscopy, they have virtually no mitochondria. And if you have a cell that has the same retargeted CAR but now has a 4MDB signaling domain, it's full of mitochondria. And so it has turned on mitochondrial biogenesis. And you can actually count these. So you can visually tell what signaling domains the CAR has by this simple assay of counting the mitochondria. So this is a new property of CAR T-cells, and that is that you can reprogram their metabolism as well as their specificity. And we think this can help have cells that are destined to have short persistence in patients or long persistence. And this may explain the results of our clinical trials where we've used this 4MDB signaling domain. And as I'll show, we've had CARs persist now in cancer patients for more than five years. And we believe that's because they have the metabolic capacities that you see with memory cells. So we treated our first patients with a second generation CAR in 2010. And the first three patients had responses. And we just now have published a five-year follow-up of that. And there were 14 patients on the trial. And the overall response rate, this is advanced refractory chronic lymphocytic leukemia, which is the most common leukemia in the U.S. And we had an overall response rate of 57% with four CRs and four PRs. And so now we can look back and see what are the characteristics of the responding to the non-responding patients. And we have measured these CARs in the patients, both in the blood and in bone marrow, with a PCR assay that detects the 4MDB sequence adjacent to the TCR zeta sequence, which is not that way in the natural human genome. So the PCR is specific to the transgene that's integrated in at one or two vector copies per T cell. And this is on a log scale. So what you could see is that the CR patients, and the x-axis is out to a year, have a very high spike of CARs 10 to 20 days after the CARs are infused. At this point, in this patient, essentially 100% of the peripheral blood is CAR cells. And then what happens is a target goes away, the patient went into remission, and also his normal B cells, as I'll talk about. And then what happens is they refer to having memory levels in this patient now five years later. And that happened to all the patients who you can see are arrayed with the CRs on top. But the non-responding patients have CARs engraft, but the frequency is about a thousand fold less than it is in the responding patients. So we think it's a numbers game. If you don't have enough effector cells, there are patients that we initially treated all had between five and seven pounds of tumor. And so a single infusion was able to eradicate that, but it required substantial proliferation. And now we can look if there is durable anti-leukemic effects. And this is using next generation sequencing for the gene that gets mutated in CLL is the IGH, the receptor, B cell receptor. And initially these patients in peripheral blood and bone marrow were nearly all one rearranged receptor. So 99.76, for instance, in patient one. So you get a pack bone marrow. And then we've looked out here in his case to three and a half years in the bone marrow. And with very deep sequencing, we've looked at a million cell equivalents. We can't find that receptor anymore. So we think that the patients have had the leukemic clone eradicated. It was the same in all four patients. It's still possible they could have late relapses as Ben Jacobson pointed out. It would likely be an immune sanctuary site. That's what happens in bone marrow transplant. Usually the leukemia is eradicated where it starts, which is in the bone marrow. But if they do have relapses, it's usually in a sanctuary site. So where we've measured it, the clone has been eradicated by a sensitive technology. And so do these cells remain functional? Their car cells can be a living drug. This shows our patient two who at a year, a thousand days later, we can detect the car on the surface because it's an antibody and we can stain it with an anti-idiotype. And this patient had a mixture of CD8 and CD4 cars, mostly CD8 here. And they remain functional. He donated blood between days 50 to a thousand days after treatment. And when we put them on leukemia targets, they either have CD19, so take his peripheral blood and directly incubate with either CD19 positive targets, which is what the leukemia expresses, or cells that don't have AML targets that don't have CD19. We get specific degranulation, as you can see. And so the cells remain functional, at least a subset. And as of July this year, we had treated now 180 patients with various B-cell malignancies, initially CLL, and then we began with ALL in 2012. So our follow-up's longest in CLL. And both pediatric and adult, we have had a higher response rate than that 57% rate that I showed for CLL. So that's a major surprise. We now have enough power to say that the response rate is higher in a disease with a much more rapid cell proliferation kinetics, ALL, compared to CLL, very different tumors, and yet we have a 90% CR rate in ALL. And that's what we published. The survival of relapsed refractory ALL otherwise is about 10% to 20% at two years. So we have very promising results so far. Our first patient treated was Emily Whitehead, who was a six-year-old. She's now 10. And here, shown here, she was invited to the White House in January of 2015 because of all the attention that came. But that's when President Obama and Francis Collins, NIH director, announced the Precision Medicine Initiative. And he invited her to the White House to be in the front row on that. And this is the case of Emily. So she happened to be the first patient on our trial. We had pediatric ALL and adult ALL trials. And she got no chemotherapy. She had had an icarose-deleted chemotherapy, a genotype that's not responsive to chemotherapy. And all the chemotherapy she'd had had left her lymphopenic but with a packed bone marrow for tumor, which you can see here. These are sequential bone marrow specimens and blood. She had all of her IgH were of one dominant clonotype. And then you can see the clearance of those after it took six months until they were not detectable any longer. So she's now more than three years out with no further treatment except B-cell replacement therapies. All of our patients are getting gamma-globulin replacement therapy if they have persistent B-cell aplasia. So we can now compare CARs. There are three in advanced development in the U.S. This is the one being developed by Novartis that came from my lab. This is the one developed at the National Cancer Institute. No, yes. And then this is the one being developed at Memorial Sloan Kettering and being tested now by Juno. And so they reviewed these. They have different single-chain FVs. They have different affinities. It's hard to compare results because no single, the variables haven't been held constant between cell manufacturing. The CAR signaling domains, ours, as I mentioned, has 4-MVB. These others have CD28. So they're both second-generation CARs, but different designs, different affinities. And they're introduced into the T-cells with, in these two cases, retroviruses and ours with an HIV-based lentivirus. And the expression has been very different in the published trials. So a short duration, about a month with those two, and now more than actually five years on our first patient that we treated. But the response rate in ALL has been the same, about a 90% CR rate. ALL rapidly goes into remission with these CARs. But in CLL, it's quite different. As I showed, we've had a 57% response rate, and there were no responders with this CAR. So it appears that you need durable persistence in CLL, and it takes longer to get complete remissions, as I'll show, than in ALL, which, in some cases, can be in a complete remission within two weeks. Okay, so we have had some resistance, and they're different in the different diseases. In ALL, as I mentioned, in adults, we've had about a 90% CR rate. But we've had 15 patients who relapsed, and 10 of those relapsed with loss of B-cell aplasia. And so we think their B-cells came back. So we're now treating with repeat infusions if they lose engraftment. And then we've had 10 cases, I'm sorry, we had five relapses that were early B-cell aplasia loss, and then 10 with loss of CD19, so their target loss. And for that, we're developing other CARs on B-cell lineages so that they'll get a combination. We haven't had target loss in CLL. So in ALL, they can lose CD19, but in CLL, it's rare. So I mentioned we had four cases with CRs with the CLL cases in this array, but one of them was looked to be an outlier, and that was patient 10. And we now have an interesting lesson from him. So if we plot the PK, which is the CAR and blood again, as I showed, this is what it looks like in our CLL cases. This patient, you can see, had a much later emergence of his CLL cells. So when you get to a thousand copies per microgram genomic DNA, that's essentially all of your peripheral blood is a CAR. So this is when they have cytokine release syndrome and tumor lysis syndrome, and then they decay down to the memory levels that I said by going down several orders of magnitude. And so that patient was an outlier that way. And this is what happened. The patient is now 81 years old. He had massive CLL with a 15-centimeter mass around his aorta and packed bone marrow, leukemia, and circulating cells. But at day 28, when we first assessed for response, he was a non-responder. He still had a packed bone marrow. But on day 45, he presented to our hospital, developed fevers, and then had fulminant cytokine release syndrome and tumor lysis syndrome 50 days after we had treated him. He required hospitalization, intubation, and then we treated with tocilizumab, which blocks IL-6 signaling, and that reversed that. And he's now in complete remission. You can see at two months after treatment, he still had persistent lymphoma mass. And in about a year, which I'm not showing here, he remains in remission for three years now. So how did that happen? It turns out we sorted his cells on day 28. So we sorted the CAR-positive cells. And on day 28, they were all 23B beta T-cell receptor families. So we sorted the CAR-positive cells. They were CD8 cells and a variety of different T-cell receptors, all of them presented. But on day 51, at the time when the tumor was being eradicated, he only had one T-cell receptor, and that was B beta 5.1 present. So a clonality here. And I won't show all the data, but it turns out his CAR inserted in chromosome 3 and into the gene called TET2. And I put this in here for Irv Weissman, who has studied this. So TET2 is demethylated cytosine, methyl cytosine. And he had an inactivated, his insertion of the CAR is right between exons 8 and 9 of TET2, and it functionally inactivates one allele of TET2 in this patient. And the stem cell field has shown that TET2 increases renewal of stem cells, although it's not oncogenic by itself. And so our patient may have had, what we don't know yet is whether this was a passenger or a driver or not. So one cell cured that patient. I think we can say that definitively from the data that I showed you. And massive proliferation. So he went from one cell that wasn't even detectable on day 28, or in his infusion proctors, a very rare cell. And it had to have had more than 20 to probably 30 population doublings because at the time of tumor lysis, he had approximately 10 to the 11th CAR cells in him. So the principle is to redirect cells and then make them have a proliferative capacity in the patient and not have to grow them all in a lab. You don't need a lot of cells. You need one good one, I think is the message. So that's an outlier. That was one out of 180 patients. But it taught us an important lesson. And we're going to look at intentionally disrupting TET2 now and testing that. I'm not saying that's going to be something that goes right in the clinic, but that's a concept. So we've also now looked at the treatment in myeloma with these CD19 CARs. And myeloma starts from mutations, often translocations, in a B cell that's CD19 positive. It goes through a state of so-called MGUS myeloma, a gammopathy of unknown significance, and then evolves into the disease that's unfortunately usually incurable, progressive bone erosion, with that CD19 negative. And when you talk to a hematopathologist, although we thought maybe it might be CD19 dimly positive because the CARs are very anti-cell receptor engineered cells, are enormously, they can see very low levels of target, as Bent pointed out. And so we thought maybe there could be also, you know, like epithelial mesenchymal transition in the cells. And there's some data that may go back and forth between a CD19 positive state and a 19 negative state. And people have reported finding the clonal B cells that have the same rearranged receptor in blood, as you can see in the marrow, in the malignant plasma cells that are 19 negative. So perhaps we could target a precursor cell or dim level expression, and that's why we started a trial. And so it's a pilot study looking at patients in the U.S. who have advancing myeloma receive an autologous stem cell transplant. And then we required that they had to have progression, and then we gave them a second transplant with the same malfulant conditioning, and then an infusion of the CAR. And the idea is to measure then the time of progression to see if there's a remission inversion. And so it's a pilot study of 10 patients, of which we just enrolled the number 10 patient. And we're asking, do we have the same endpoints of safety and feasibility, such as cytokine release syndrome, and engraftment of the CAR cells, B cell aplasia, and remission inversion. And so we just reported the first two patients on this trial. Both of them have had very prominent anti-tumor responses, but the first one had very severe cytokine release syndrome and had gotten 360 million CARs. So we then modified protocol and then have now been treating with 50 million. And the second patient had no side effects except hypogamma glottal anemia. But the first patient had very severe cytokine release syndrome. And myeloma cells can make themselves IL-6. And IL-6 is what we think is mostly involved in the cytokine release syndrome. And this is what happened to our second patient. She had an IGA myeloma, as shown here in our first transplant. Two grams of the plasmacytoma of the pair protein were here in the blood, and she didn't respond to 200 milligrams of melphalan. Then got lanolinamide and a variety of other cortisone drugs added back in. And then she came on to the second trial with a myeloma facing her bone marrow. This is CD138 staining and bone marrow failure with a kind of thrombocytopenia. And then she'd had nine lines of prior therapy and had genetically the worst kinds of every bad biomarker you can have for myeloma. And this is what happened in the second transplant. She'd now gone to five to six grams of her pair protein, which is a marker of tumor mass in myeloma and secreted by the myeloma cells. And she got a dose-reduced melphalan autologous stem cell transplant. That's because she had stem cell damage, organ damage from the first transplant. And then, so the real difference here is that she got CAR cells infused, and she's now been in a molecular admission for more than a year. And her platelet counts, and she now has a normal blood count. So a very striking response for all previously administered forms of chemotherapy failed. And I'm not showing the data. It was published in the New England Journal that by looking as hard as we can, we can't find any CD19 in her baseline myeloma cells. So 99.97% of her myeloma cells were CD19 negative and had no transcripts for CD19. She had .05% of her cells had detectable transcripts. So we had to have an indirect effect here. And we don't know yet the mechanism. One could be this paper by Shalipur. This is from Mike Caron's lab that was in Nature recently, where he showed in prostate cancer models that if you target nonmalignant B cells in the tumor microenvironment, it enhances the response to chemotherapy. And so it's possible that within melphalan, we got more mileage out of it by targeting normal B cells. And another, it's also possible there are subsets or that this transitioning back and forth to a CD19 positive state. There's a recent paper showing that in humans, there are really four major subsets of plasma cells, and two of them do express CD19. So it may be possible to treat some CD19 negative malignancies with CD19 CARs. So this is a slide from Chris Mason. And we are in this point where we're, I think, you know, where this whole meeting really is, you know, going from the three models of our current healthcare of pharmaceutical industry and devices and biotech to, you know, to making a fourth pillar, which will include cell therapies. I don't know how long, right now, our main issue in that of Novartis is a so-called N of one. And I'm sure that you have all discussed that. It's worth the effort if it works, is what I would say. And right now, N of one is the only way we've been able to get long-term engraftment of cells, unless you do a full myeloblata transplant. So, but I do think that in some point, we will have third-party cells where mobile engineering cells will be able to have off-the-shelf cells. In the U.S., the model for genetically engineered cells is that it's going to be done in central manufacturing plants with full pharma control. I think till therapies will happen maybe more peripherally in sort of a Red Cross-like model at the source, a more point of source where the patients are. And so, in summary, you know, what I've tried to show is that multiple factors affect the efficacy of these engineered cell therapies, of which one is the synthetic biology aspects of how you modify the cell, either with CARs, TCRs, et cetera. The sky is the limit now, especially now with CRISPR technologies and so on that will allow us to target many different aspects of cells to change their trafficking, for instance, what lymphocyte subset to use, and then how to actually manufacture the cells. All those are important to the final outcome. So, I've shown that durable remissions occur in CLL. We've had durable remissions in ALL for three years now, and in promising results in myeloma via an unexplained mechanism at this point. And now, in the one experiment, showing that at least in some cases, a single cell is enough to eradicate a very large burden tumor. A lot of people are involved in these studies. I'd just like to highlight Bruce Levine, who's worked with me since 1992 to make the cell manufacturing technology. David Porter, who's taking care of all our leukemia patients. And Michael Malone made the 4BB core in my lab. And Stephen Grupp and his team at Children's Hospital, who have taken care of all the pediatric leukemia patients. And I'd like to thank you for your attention.