Thank you so much, everybody. Thank you for coming this morning at 7.15. There's such a beautiful island out there, and you're all here, and I just can't thank you enough. So the title of my talk is the ABCs of Antibodies. And you know, the ABCs of Antibodies, you might think that this is just a nice jaunt down immunology trails. But after we get through the ABC, and then there's the other antigens to class two molecules, DR, DQ, DP, DQ-alpha, DP-alpha, and then there are the thousands of polymorphisms associated with these various antigens, and various antibody characteristics like subclass of Ig and also strength. And there are also, let's not forget, non-HLA antibodies, which I'm only going to hint to on this one slide, a class of antibodies that are not directed to HLA antigens, but to autoantigens, which we have been studying in various places in our research and development, and also recently, the STAR review that came out that Dr. Levine is going to talk about more. So you might be on immunology trails, but after all of this, you're in a really scary, thick forest, so I just want to come here and unpack some of the things we know about antibodies, how we test for them, and how we can help you define, how the laboratory can work to help you define what are safe antibodies to cross. So these are the learning objectives. We're gonna go through HLA structure and function. We're going to go through antibody detection methods and some of the caveats there, saturation, dilution, and C1q. We're going to look at donor-specific antibodies and graft rejection and antibody testing for accurate virtual cross-matching and physical cross-match prediction. So this is the major histocompatibility complex, and it is located on the short arm of chromosome six, and it is, does this work? No, does this work? Yes, if you can see that all the way out there. This is the class one region with genes for HLA A, B, and C, and the class two region with the genes for DR, DQ, and DP. So class one genes, when they are translated, are a single polymorphic chain with multiple domains, alpha one, alpha two, alpha three, stabilized at the surface of the cell with one molecule of beta two microglobulin, while the class two genes, when translated, are a heterodimer of an alpha chain and a beta chain. Both of these chains are polymorphic, which is why you know about antibodies not only to DQ but to DQ alpha. So polymorphisms, they are located all over the HLA antigen. In this figure, what we're showing here are an HLA DQ molecule and a DR molecule, and in yellow are the polymorphic residues, and they're all over, but the majority of them are in the upper domains in the part of the molecule that is most well-known for holding the antigenic peptide so that it can show peptides to the immune response. So polymorphisms, those are the differences in amino acids between patient and donor, and they are, this allosensitization will occur after pregnancy, blood transfusion, transplantation, or through the use of tissue allografts for vascular reconstruction, and bacterial and viral infections can also boost the alloimmune response or the immune response. So there are multiple mechanisms of allorecognition, and they are termed, I mean, simply termed, the indirect, direct, and semidirect, but there's a lot of intricate differences between these methods, and I'll just go through the indirect real quick. So in this slide, donor cells are noted in blue. So this is the donor endothelium at the bottom expressing donor HLA, and this HLA is shed and then can be taken up by a recipient B cell, processed and presented in the context of the recipient's own HLA, and then presented to T follicular helper cells, or CTL, causing this B cell to further mature and make antibodies. When you think a little bit more about direct allorecognition, and this is a very high-level example of what this looks like, here we have a donor, this is a kidney, let's just pretend for today it's a lung, and we have one cell on this organ that is enlarged here, and it's expressing HLA class one and class two molecules. So a CD8 T cell will recognize HLA class one on the surface of these cells and produce perforin and granzyme. CD4 T cells will recognize class two on the surface of the cell and produce effector cytokines, such as IL-2 and interferon gamma. And they'll also provide help to CD8 T cells and B cells to further ramp up the immune response, causing these cells to become even more mature and more active producers of these effector molecules. And then B cells will be activated to produce antibody, which will ultimately bind their targets, enhance, enhance, enhance. And so once those antibodies bind to their targets, their specific HLA antigen targets, there are a lot of multiple mechanisms through which immune cells become activated and enter the tissue. So right here, we have a bit of donor vasculature, which is surrounded by endothelial cells shown in pink. And the activated endothelium are shown in the brighter pink. So activated endothelial cells express effector molecules, such as P-selectin shown here in blue, which along with the constant region of the antibody, which is affixed to HLA class one in this case, will tether monocytes through their FC receptors and cause them to bind here, enter the tissue and mature into macrophages. Also, FC gamma receptors in K cells tethered to the constant region of HLA antibodies in similar ways and enter the tissues become activated. CD4 T cells recognize HLA class two on the surface of the cells. They can become activated here and enter into the tissues. And then let's not forget complement. Complement, so if you have two antibodies to HLA right here, and they're close enough in proximity to bind one molecule of C1q, it can result in the activation of the complement cascade, which results in deposition products on the surface of the cells, such as C4D shown right here, and split products which are soluble, called anaphylatoxins, such as C3A and C5A, which can actually further serve to increase the activation of monocytes. And finally, HLA antibodies which bind also result in intracellular signaling pathways resulting in anti-apoptotic cell survival and proliferation pathways leading to fibrosis and allograft damage. So we really know a lot about antibodies and about all of their multiple mechanisms and how they are creating graft damage in many different ways. And if we take just a long road back, and we look back into 1969, when Patel and Terasaki published this seminal paper in the New England Journal of Medicine, they wrote, the presence of preformed cytotoxic antibodies against the donor appears to be a strong contraindication for transplantation, of course, in kidney at that time. So let's fast forward to today and look at two very recent papers. The one on the left is from the Toronto group, and the one on the right is from the Heidelberg group. And what they did in this paper over here is they sorted out the patients into three different categories, either virtual cross-match negative, virtual cross-match positive, but flow cross-match negative, and virtual cross-match positive, flow cross-match positive. And in their roughly nine or 10 year experience here, they showed that there was no difference in the probability of allograft survival by sensitization cohort in lung transplant patients. And so this is very nice, very nice work here. In this side over here, in the Heidelberg group, now both of these groups are also showing their use of various desensitization protocols with pheresis IV and ATG, or different types of pheresis and IVIG and Ritux here. In this case, 62 patients were transplanted with a positive, that were positive for donor-specific antibody over a range of strengths. And I think that this right here is very much the crux of these two papers. What is the strength of the antibody that the patients were transplanted across at the time of the transplant? Out of these 74 patients, the paper was a little bit difficult for me to interpret, but I believe that the majority of these donor-specific antibodies in this group were weak. In this side, there was a range of strength. There was a median of 4,000 with a range up to 7,000, but there were also seven patients transplanted across a cytotoxic positive crossmatch. And in this group, 23% had negative MFIs. So 23%, although they crossed donor-specific antibody, they were negative. Do I have enough time? Am I doing okay? Okay, great. So 23% were negative. And I just want to make a suggestion here that the purpose of, as we all know, the purpose of this desensitization therapy is to bring down the strength of that known donor-specific antibody because we do know that hyperacute rejection is possible after lung transplant, and we want to try to mitigate negative effects. So how do we assess for the strength or risk of a preformed HLA antibody to provide accurate virtual crossmatch and physical crossmatch prediction? This is some example here of the single antigen bead test raw data. The single antigen bead test, this is our most sensitive and specific test for identifying HLA antibodies throughout the course of the transplant patient's entire course, pre-transplant and post-transplant. It is a Luminex bead-based test with 100 beads on the panel that are charged with different values of red dye. And when mixed with the patient's serum, if there is antibody that will bind, it will bind to the antigen on the bead. And then we identify it with a detection reagent and read it out on a Luminex machine. You can see here this patient has a cascade of HLA antibody strengths. Each one of these bars represents a different HLA antibody. And some of them are very strong, and some are weak, and some beads are negative. So there is a limit of saturation in this test. There is a dynamic range. If you have an antibody at 12 to 15,000 and an antibody at 20,000, they might as well both be 20,000. You can't tell the difference. The scale here goes to 20,000, so we don't know if these antibodies are strong or if they're really strong. And there are also some caveats. I just mentioned saturation, that's strong or really strong. But there's also interference in what some people, and even myself also at times, call prozone. So what is saturation? Saturation is occurring when the strength of the antibody is so strong that it's precluding all of the antibody from being able to bind to the bead. And so here we have an MFI of 12,000. But if we dilute that serum one to 10, we still have an MFI of 11,000, so we know that this antibody is very, very strong. And in my experience, an antibody that's still this strong at one to 10 dilution will not be amenable generally to a desensitization-based desensitization therapy. I should have said a pheresis-based desensitization therapy. So that is saturation. In this slide, I'm going to talk about inhibition or prozone. This is a patient serum, and the patient had a real range of MFIs in their serum at undiluted. But when we diluted at one to 10, a subset of antibodies, which seemed rather weak and innocuous, actually became much stronger at dilution. And we also have found that sometimes patients' samples tested at NEAT might have MFIs here, and at one to 10 could go down, but at higher dilutions go up. This is pretty rare, but it's going to happen in the most very highly-sensitized patients. This is why longitudinal testing is very helpful. And it's pretty prohibitive to do a dilution series on every serum. Wrap it up, okay. And so we can use a C1q test right here, which identifies all of these very strong antibodies. Two minutes, okay. So I just want to go through a case study. This is a 60-year-old female listed for her first lung transplant. Her underlying disease was idiopathic pulmonary fibrosis, and at initial evaluation, she had a CPRA for all antibodies at 99%, and all of those at one to 10 was 87%, and C1q positive 73%. Her sensitizing events included at least one pregnancy. So when I first reviewed her data, I saw that at dilution, many of her antibodies actually diluted to negative or weaker. It indicated that she would be a candidate for phoresis. However, our program opted to use daratumumab, which is an anti-CD38 monoclonal antibody and has multiple mechanisms for targeting plasma cells. So this is what the patient looked here like at initial eval, and she started on a course of daratumumab every one to two weeks. For multiple weeks, this took a couple of months, you can see that there was a cyclical effect. Let me see, here we go. At one to 10, you can really see that there was a cyclical effect of antibodies, of the strength of antibodies over time with her treatments, and at one point, she had to have ECMO placed with plasma phoresis, and that really brought everything down. So the patient then received an offer, but of course, on the date of the offer, this was not the data we had. This was the data we had. So her antibody strengths at the time of the virtual cross-match were quite weak. One DSA to A24 at 3,000 and one to DR4 at 4,000, and this was a great match for her, a four out of 12 mismatch and no additional DSA. The program accepted this offer for her, and when we tested her antibodies on the day of the offer, those two antibodies were very strong at 25,000 and 14,000. They were C1q positive and positive one to 10. There was a negative cytotoxic cross-match and a positive flow. They proceeded to multiple rounds of phoresis pre-transplant which brought her antibody strengths down. You can see that here. Her cross-match strengths came down, and post-transplant, they also drew a serum. It was like within the same 24 hours. Those antibody strengths were very, very weak, and the cross-match now was negative. So how did the patient do just rounding out the bend? It's been a year since her transplant. Her antibody to DR4 is negative, shown in red, and her antibody to A24, it persists, but it's relatively weak and manageable, and she's doing quite well. I just want to thank you all for your time. I gave a lot here in 15 minutes. Thank you very much. In lung transplant patients, we can use HLA antibody testing to identify any of these things here and probably even more, and crossing preformed DSA is manageable with desensitization therapy and accurate antibodies assessment. Thank you very much. Thank you. Thank you. Thank you so much. We're going to hold if we have time. I don't think we're going to have time for questions, but if we do, we'll be probably at the end. Thank you so much, Dr. Hickey, and Dr. Nerula now is going to speak to us about sensitization, and we're looking forward to a great talk. Hers will be better, so wait for it. Thank you so much. I want to thank Dr. Nerula and Chess for inviting us all to speak about antibodies, and Dr. Nerula is going to talk about what happens before transplant, what is a sensitized patient, and how does that affect listing and transplantation, and we're going to go a little out of order, but we're going to talk now about what is the evidence of antibodies and the association post-transplant. I have no disclosures, but if you find yourself on the windward side of the island, this is Kailua Beach and probably the most beautiful beach in the world, and also one of the, if not the, largest kite surfing beaches in the world. Beautiful. So again, thank you, Dr. Hickey, for a beautiful journey through antibodies, discussing really what they are, and really telling us in 15 minutes a whirlwind of how we get to where we're going. Dr. Nerula was going to talk to us very soon about sensitization and what happens before transplant. I'm going to take it kind of home before Dr. Cochran talks about treatment. We're going to talk about graft injury and lung transplant. So it's been well-established, mostly from the kidney world, obviously, over 30 years of knowing the negative association between HLA, DSA, or donor-specific antibodies, and solid organ transplant. Associations with increase in rejection and graft loss, as well as decreased survival, both of the graft and the patient. Kidney, heart, and now coming up, liver. We have no, we are no different. In fact, we kind of knew the association of antibodies and graft function in lung transplant way before even the term donor-specific antibodies was coined, and a lot of this, I think some of the Duke people are here. Duke and Toronto really started the effort way back when, along with others, to show us that antibodies in general have a association with both high-grade and persistent severe rejection, as well as bronchiolitis obliterans syndrome, and decreased survival. Now, back then, there was not the wisdom or the experience with donor-specific antibodies. So often, you would think some of these are going to be donor-specific, and we just did not term them that. So it may be just that the age of this, those age of the papers. But as HLA-DSA became present in lung, just as in other organs, we did see the associated graft dysfunction and decreased survival. And it's been seen in multiple papers for the last 15 years. Not only in terms of outcomes, we know that in terms of risk factors for CLAD, chronic lung allograft dysfunction, we know that both donor-specific antibodies and AMR itself are independent risk factors for CLAD. In fact, it's the primary, in my mind, maybe if you're a hammer, everything's a nail, but in my mind, two of the most primary risk factors. But that's along with other immune injury like ACR and LB. But then there's other non-immune injuries, and it also includes primary graft dysfunction, infections, and GERD. And everyone in this room knows this. I don't have to show this slide too many times, but we know that the association with CLAD really is one of the worst associations that we could have. So CLAD is highly associated with DSA. Not only is it associated with worse outcomes, we also know that DSA is not that uncommon after lung transplant. You're gonna see when you look in the literature a very large spectrum of frequency of donor-specific antibodies being present. And that's not that they're not there or they're there for some people and not for others. It's really probably a reflection of how we monitor patients, how often you're testing for it. If you don't test it, you're not gonna find it. But also, it's going to be how the lab, and Dr. Hickey can talk about this too at the end, how the lab is looking at which assay they're using. So definitely there is that issue that we'll talk about in a little bit. So not only is it important, but we also need to know when to test. And really, post-transplant, we can find antibodies at any time. Dr. Narula's gonna talk a lot about sensitized patient, which happens before transplant when there is sensitizing event that occurs, including pregnancy, platelet transfusion, or others. Even if a patient had another transplant after a lung transplant, that can also be a sensitizing event, as well as decreased immunosuppression and others. Those are things that can occur after transplant as well. And those usually occur as a de novo antibody, which usually occurs between two and three, or after two or three months after transplant. Prior to that, in fact, in the first couple weeks after transplant, you can have what's called a pre-existing or pre-formed antibody that were there as from the sensitized patient. Between those two, between the pre-formed and the de novo, there is the option of having a memory antibody pop up, and that's usually between three weeks and less than three months. So really, all throughout the spectrum of transplant, from pre-transplant all the way until clad or decreased survival, you have the ability to make antibodies. And even along with the different kind of mechanisms that you can develop antibodies, antibodies are different. They're not one-size-fits-all. Different DSA features can really help us kind of assess risk in terms of the patient in the graft. So these are a lot of different types of characteristics or features of antibodies, and some of them pretend worse outcomes than others. For example, if a patient has a de novo antibody versus a pre-formed, usually it's a little bit worse of a prognosis, as well as patients who have a persistent or recurring antibody even after you treat them, that's also a poor prognostic factor. Complement binding, if it's C1q positive or C4d positive, these patients also have worse. When we talk about strength, we really haven't talked a lot about MFI versus titer, but we know that the higher the strength, the harder it will be to treat. Just like Dr. Hickey showed us, when those patients had very high levels of antibody, it was hard to clear, even after diluting. So these kind of features or characteristics, we can actually use them to help develop different roles of how we use antibodies in our practice. So yes, of course, we're gonna use it to develop kind of a diagnosis of antibody-mediated rejection. But we also can look at it in terms of immune monitoring throughout the progression of the patient's life, and then look at what Dr. Cochran's gonna talk to us about after we treat patients with whatever he's gonna teach us to treat them with, we're gonna know what the treatment response is. So many, many roles, and I wish we could spend an hour going through all this, but we're just gonna talk about the diagnosis. And one thing I just wanna point out from this slide is that in different organs, in their guidelines or in their consensus, the presence of a circulating antibody is definitely important, but it's not universally required to make the diagnosis. And lung is no different. In the ISHLT consensus back in 2016, we introduced an initial set of diagnostic features, and those features became criteria. Circulating donor-specific antibody, allograft dysfunction, histologic features, positive C4D staining, and then exclusion of other graft dysfunction. Those features helped us really develop this uniform clinical definition and criteria. That definition was a working definition. It was meant to be changed as experience increased. That was there to define risk, understand the true incidence, and differentiate phenotypes, so it'd be easy for us all to work together in clinical trials. As you see, the five different characteristics then became the criteria, and DSA is obviously one of them. And this is in a little bit bigger picture to show you that when you have, I'll just go back one step for those who don't know this chart very well, is that if you have all five of these criteria, you have definite AMR. Reportedly, if you have less than one, it's probable, and less than two, possible. You can actually only have one of these criteria and have possible AMR. But I want you to take a look at the red boxes, which are negative. And as we were discussing before, those are different diagnoses, a probable and three possibles, that the circulating DSA is not even there. So it's not required to make a diagnosis. So remember that one. So the last seven years have been kind of up and down in terms of really figuring out all of us as a group. How do we use this definition? What is the criteria? Are we settled with this definition, or do we need to make changes? Well, I think we know that from the clinical aspects, the clinical criteria, the clinical characteristics, the pulmonary function test, and imaging, we know that there's a pretty, those criteria, oops, sorry, those, what's happening? It's telling me to go faster. Hurry up. Anyways, all of those have a very low sensitivity in terms of the diagnosis. When you look at histology, which should be the biggest and brightest and gold standard, we know also that those lesions can occur in other entities. And so in viral infections, in ACR, severe ACR, and even ALI after transplant, all have the same type of lesions that can occur in AMR. So there's overlap diagnoses and very difficult interpretation variability. So all of them don't look great. What about DSAs? That's probably pretty significant, right? Well, no. Even in DSA, there's challenges with it as a criteria. Marked variability, as we discussed between centers, really variable thresholds in their assay, and when you call an antibody an antibody. What about when the DSA is not detectable? We talked about that. There may not be an assay available, or you may not have that assay. But the other one is this presence of non-HLA antibody, as Dr. Hickey has described to us. All of us aren't checking for it, but could it be there? Do you have a patient who has the scenario of AMR, histology looks great, everything looks great, but there's no DSA? Well, this is a slide from a kidney working group in the Banff working group in 2021. And really what it showed was that same scenario. People looked and felt like they did have AMR, but there was no DSA. At that time, when this survey was put out, only less than 20% of the people who were involved in the survey said, hey, we wouldn't test for non-HLA antibodies. We would test for non-HLA antibodies. So most people were not testing for non-HLA antibody in that specific situation, which would be probably the most important situation to check it in. 48% didn't even think they had room, they didn't even have the ability to do it. And 82% of patient people said, hey, if you get it and it's positive, I'm not gonna change my management. So these are things to think about. I think our whole community in transplant hasn't fully embraced the idea of non-HLA antibodies. However, there was great work done by the STAR group in 2022 that we'll talk a little bit about. Currently, there are testing available, marketing. You can get anti-H1 antibodies, MICA, as well as some of the endothelial cell antibodies. So you can get it. It's usually a send-out in very few centers. I think UCLA is one of them, and I think Duke is one of them, that you can order these antibodies. However, more to come. There's emerging assays. I think there's 40 to 50 new targets being evaluated in different organs. That's not ready for prime time, but really may help us in terms of really elucidating some of these antibodies. There's probably a lot more, but it takes a long time to get to that. Think how long it took us to get HLA-DSA. And now think of how long it's going to get us to be on board with that. This is the article from the Star Journal meeting. And basically, what they did was they broke it down into different organs. And you can see that heart and lung, and they mixed us with heart, so lung is even less, is that there's hardly any evidence, in fact, to even make a recommendation for lung. There's better data in the other organs. However, what they said, quote, there's no trials to date that really study the impact of non-HLA antibodies. And is there even enough data to tell us that antibodies even mechanistically contribute to graft injury? So even they're like, hey, it's good, but we still have problems, but the test is not universally available. There's no standardized reagents. And there's really a lack of data on clinical validity and utility. So really, if you don't, just because you can get it, should you, especially if you don't know what you're going to do with it afterwards, until it's a little bit better study, unless it's in a research situation. So really, we have a lot of challenges, especially with antibodies. And so can we use the challenges we've just talked about to improve our diagnostic strategy with AMR? Well, for DSA, we can actually reflect on the need for better collective understanding from all of us, get a little bit more on board with a standardized evaluation. We need to reflect the need for more standardized monitoring in a larger cohort. Sorry, it really wants me to get done. DSA really reflects also the need for additional and adjudicated diagnostics. I just want to go through one thing, is that we did talk about diagnosis of AMR. Let's talk a second about monitoring. Do you monitor? How do you monitor? Nobody really has a good, consistent approach. And I think it is important for us to do that, just because of the decisions for therapy, as well as how we're going to do research together. So it's 2023. Are we settled with this diagnosis? Are we ready? Everything's perfect? Do we need to make changes? Well, I think at least in the antibody world, we have a lot more questions to ask that haven't been answered. And I think we need to work through that. And even, we're lucky, because in the whole 2016 paper, it says that this paper was made to enable the refinement of different concepts that we have to find as we move forward. So there is the opening for new data and experience. And the second iteration of the ISHLT consensus statement is still in progress. And hopefully, by ISHLT, we'll have some news for you. But we're going to work on that. And so thank you so much. We will move on. But hopefully, some of the changes that we are going to make in the second iteration will make it easier for us to move forward together. Thank you very much. And I'd like to introduce Dr. Cochran. I'm going to introduce Dr. Cochran. He's going to teach us everything he knows. He's world-known about antibody-mediated rejection therapy. And we're so lucky to have him. So thank you. Thank you. So good morning. Getting back to the title of the session about the complexity of antibodies and lung transplantation, I'm going to talk about the therapeutic options we have when it comes to these. I have no financial disclosures. However, every single drug I mentioned will be discussed in an off-label manner. That's part of our problem in this domain is that we have nothing approved for it. So just understanding the complexity of treatment and discussing the literature, somewhat limited literature, that validates the treatment. So the complexity of it. We have all these different choices, some with OK evidence, some with little evidence, some with hopefully developing evidence. So the first treatment that we have is plasmapheresis, or the plex of complexity, and IVIG. This is a direct way of clearing antibody out, the antibody that's causing your AMR. IVIG replacement is nonspecific replacement to prevent a rebound in the antibody that you have cleared. However, this is also part of our limited data. It's well accepted. Most centers are doing plasmapheresis IVIG replacement. However, in the plasmapheresis guidelines, plasmapheresis for AMR is a level three of evidence because there is not really evidence out there. There's not really literature out there by itself saying that plasmapheresis and IVIG is effective. So hence the blank slide with no literature. The first agent that has been used historically is rituximab, which is an anti-CD20 agent. Dosing is shown there. You can use it weekly for four doses or at a larger dose twice in 14 days. And the adverse drug reactions are really infusion related. The graphic there is the limitation of rituximab. So as a CD20 blocker, the CD20 is located on the pro-B cells, the pre-B cells, and the immature B cells. However, our problem in AMR is the plasma cell, as that's the cell that is generating the antibody. So really, rituximab may just be preventing a future AMR or future DSA from developing, but it's not actually doing anything to your current AMR episode. So there is literature on its use presented here. So there's a case presentation, a case series with four patients. These are lung patients that got a combination of rituximab in combination with the plasmapheresis and IVIG. And at the end of their six-month follow-up, there was one patient that actually cleared DSA, one patient that cleared class 1 DSA, but not class 2 DSA, and two patients that had died. Essentially, I would assume did not respond to the rituximab therapy. The other series is nine patients with rituximab in combination with plasmapheresis and IVIG. Overall, the MFI decreased after the rituximab therapy. However, again, six of the nine patients died in follow-up, and all of those patients had developed BOS or RAS prior to their death. And three patients were alive at the end of follow-up, and all of them had BOS at the time. So I think this was one of the early agents that we had, but I would move that this is an agent that we should not be pulling off the shelf first line for treatment of AMR at this time. We have other agents, as shown in the word cloud at the beginning. Bortezomib is an agent that has been used in AMR. This is the, it's a reversible proteasome inhibitor. Its indication is multiple myeloma, so all of our use is off-label. The dosing is four doses over two weeks, and adverse drug reactions are neuropathy and GI intolerance. Most of those are limited if you give Bortezomib subq versus the IM injection that it is labeled as. So the use of this, there's a first use in lung transplantation after kidney has used it for a while. This is a patient that received it on post-op day 15. They were positive for HLA-DR13, and at post-op day 60, there was a significant improvement, as noted by the authors. Next is a 15-month-old girl who had AMR unresponsive to IVIG and rituximab one month after the Bortezomib C3d, C4d cleared, and the PRA had a significant decline. And then the third mention is in a 65-year-old with IPF treated with repeated courses of methylpred, plasmapheresis, IVIG, and rituximab, then received three doses of Bortezomib instead of the four doses, and was noted to have an improvement at four weeks after those three doses. Carfizomib, also a proteasome inhibitor, this one is irreversible, so perhaps it has a longer-acting benefit than Bortezomib, but there are no studies that compare the two to each other, also indicated for multiple myeloma. And then this is six doses over two and a half weeks, and the doses are all in sequential days. Adverse drug reactions, acute kidney injury, which is pretty profound, which I'll talk about in just a moment, and then bone marrow suppression. So the literature in lung transplant, the first one was 14 patients with at least probable AMR that received pheresis and IVIG, and then it was said that they augmented that with Carfizomib. So 10 patients responded to the Carfizomib. 12 patients received the full course of Carfizomib, two of which developed AKI. And the authors noted that the non-responders had a longer time from the DSA development to Carfizomib treatment, and those patients had an increased risk for progression to CLAD, being non-responders. The other study in lung transplant looked at 28 patients. 50% of patients had class II. 50% patients had a combined class I and class II antibody. 57% of the patients died at a median of less than a year after Carfizomib, and more than a quarter of the patients had acute kidney injury. There was recovery with this, but still you're seeing that this is a significant amount of kidney injury that you're dealing with and complicating your treatment of AMR. Dr. Hickey mentioned Daratubumab that they used pre-transplant. There's little literature post-transplant, but I'm encouraged by this agent just on its mechanism. It's a monoclonal that binds to CD38, which is on the plasma cell, so direct agent to the plasma cell, approved for multiple myeloma. The dosing is important to note here. So it's 16 milligram per kilogram, IV weekly times eight weeks. And I say it's important because we'll look at that in the next couple of studies, adverse drug reactions, pneumonia, diarrhea, and peripheral edema. So 16 milligram per kilogram times eight weeks. So if you look at the first study in lung transplant, they looked at patient 12 months status post-lung transplant for definite AMR, who had recurrent AMR after carfizomib, plasmapheresis, and IVIG. They used Daratubumab at eight milligram per kilogram instead of the full dose, times six doses instead of the full regimen. And then they had recurrent AMR where they used Daratubumab and carfizomib and had a durable DSA clearance and were AMR free for six months follow-up. So the graph at the bottom shows the blue arrows are the Daratubumab doses. So you see the failure of the initial plasmapheresis and rituximab on the left part. Then they used the six doses at the lower dose and there was a response in the DSA over time. Another study looked at Daratubumab use. In a patient who was three years after lung transplant, the patient received plasmapheresis, photopheresis, thiamo, rituximab, and they cleared their AMR. I think that did not clear their AMR, I'm sorry. And then they used Daratubumab at the correct dose but at a different frequency. And then they showed a PRA and DSA significantly decreased and did not return in their seven month of follow-up. So anyone who after this talk is intrigued by Daratubumab, I encourage you to use the proper dose and the correct number of doses instead of going renegade. And perhaps with that we can develop a body of evidence, a body of literature that will support the use rather than kind of clouding the use of it. Tocilizumab, another agent is an IL-6 receptor blocker approved for rheumatoid arthritis. This is dosed at eight milligram per kilogram once every four weeks. You have some bone marrow suppressions and GI perforation that can occur with this. There's the new study here that just came out about three or four months ago in lung transplant. So this was 18 patients. Half the group received Tocilizumab in combination with other therapies versus a non-Tocilizumab group who received other therapies in combination with it. So Tocilizumab showed improved DSA clearance and a lower DSA rebound, so encouraging. However, there was a persistent decline in FEV1 in the Tocilizumab group versus the non-Tocilizumab group. And so we've treated the antibody but we haven't actually had an improvement in the FEV1 which I would say mixed results here. It was mean of six monthly doses of Tocilizumab but some of these patients received up to 12 months of Tocilizumab. So this is mixed results. I'm not jumping at using this one right away. The lower study is a kidney transplant study who failed the standard of care. They got eight milligram per kilogram. The authors did not note how many doses that was. Similar to the study above, the serum creatinine decreased in both the Tocilizumab and the standard of care groups. The red line is the Tocilizumab plus the standard of care and the y-axis, sorry, is MDRD. So you see the kidney function didn't actually improve. In fact, it actually drifted down with Tocilizumab group. So similar to the FEV1 not improving in the above lung study, the kidney function didn't improve and actually declined over time with Tocilizumab. So work in progress with this one. The next agent is Clazikizumab which is another IL-6 binding agent, different binding than Tocilizumab. It is not approved in the United States. It has some use in Europe. The dose is 25 milligrams subcutaneously monthly times three and there's some transaminitis that can occur with this. So this is a kidney study, but there's nothing in lung yet. So they had 10 patients that got Clazikizumab monthly times 12 months and then extended for two and a half years for patients who had an improvement. So they showed a stability of GFR over the 24 months with a mean decrease in DSA. 14 infections which were respiratory infections which they called seasonal. So if you're thinking lung transplant patients using Clazikizumab in the future and respiratory infections, think of seasonal respiratory infections. Edema and pyelonephritis. Eculizumab, another agent, it's a complement inhibitor. It's approved for HUS, myasthenia gravis. The dosing is weekly. This is a very expensive drug and I would encourage people to really consider its use before pulling it off even though it's kind of glamorous to use, I suppose. These are kidney transplant studies. There are three here that were all terminated either due to lack of efficacy, didn't reach significance. And the third one there was withdrawn by sponsor. So apparently not encouraging in use in AMR from that standpoint. The lower one is a heart transplant study where they gave Eculizumab at the time of transplant and over the nine months of use after, there was a 20% rate of AMR2 in the patients that received Eculizumab. Not sure what that would have been without the Eculizumab. So if you are considering Eculizumab but don't like that, there are other complement inhibitors that are being investigated right now. Very limited, well, no literature. They're enrolling in studies. And then if you add to the complexity of AMR treatment, you have other investigational agents, Fostamatinib, which is recruiting, Felzartumab, which is another CD38 agent. So this along with theratubibam may be possible and then Inflimidase, which has been approved in Europe for desensitization in kidney transplant. So thank you for listening. If we're tight on time for questions, you can email me with any questions you have. Thank you. Thank you. Thank you so much, Dr. Cochran. That was a great journey. Now we reintroduce Dr. Narula, who is going to talk about sensitization and we save the best for last. So moment of truth, it's working now. Thank you. I apologize for the technical issue and too soon. Okay. All right, it's working, great. So I'm gonna be talking about the highly sensitized pre-transplant patients and I would be dividing my talk into who, why and what. The why is missing from title, but we'll talk about it. I'm Noreen Narula and I recently joined the Lung Transplant Department, University of Kentucky. So fortunately, unfortunately I had no disclosures. Moving ahead. So the objectives that I have include acknowledging that pre-formed DSA are still considered barrier for lung transplantation, risk stratifying each recipient and expanding the donor pool, that should be our goal and provide equal opportunity to undergo transplantation for all these highly sensitized recipients. So moving ahead, who is sensitized? So sensitization means before transplantation in which your HLA antibodies are circulating in the blood. In 2007, UNOS approved a proposal by the histocompatibility committee to use CPRA determined by the antibody specificity. This calculation is derived from HLA frequencies and the reference database represents the percentage of actual organ donors who express more than one of the unacceptable HLAs. For example, if you have a candidate with a CPRA, calculated PRA of more than 80%, this would be expected to be incompatible with 80% of the donors. Now there is no calculated CPRA threshold for sensitization lung transplant, but many centers use more than 50% as a threshold for defining sensitization and more than 50 to 80% for defining highly sensitized. So coming to why, why are we discussing this? So poor waitlist outcomes are associated with sensitized recipients, shorter post transplantation survival and potential graft loss. An analysis by the UNOS found that a CPR of more than 25% was an independent risk factor for death after transplantation between 87 and 1997, but not between 1998 and 2005. What does this mean? Do we have more advancements in HLA antibody detection methods or we know how to deal with it? So this is a study that was published in 2015 in Journal of Heart, Lung and Transplant. You can assess from the graph A and B that when you compare the not allosensitized recipients with the allosensitized recipients, there was no significant difference between either freedom from CLAD and then when you compare allosensitized individual with a CPR of more than 50% to an individual with CPRA one to 50%, again, there was no significant difference between freedom from CLAD. Moving ahead, you have graft survival. Again, not taking too much time, there was no significant difference whether these recipients were highly sensitized or just sensitized. Okay, this happened again. All right, talking about the listing process. What we do and what some of the other programs do is at the listing, we screen all our patients for preformed HLA antibodies and then repeat this antibody testing every three months while the patient is on the wait list and two to four weeks after any potential allosensitizing event. Our center's histocompatibility lab defines this HLA antibody positivity as reactivity with an MFI of more than 2,000. This cutoff is used for antibody detection before and after transplantation and then we compute the CPRA. We define allosensitization as any HLA antibodies, either historical or current with an MFI of more than 2,000. We accept donor lungs if there's a virtual crossmatch and it's compatible with all previously identified antibodies. At the time of the transplant, there is another direct CDC crossmatch in all recipients. Coming to what, what's next? So what I and what there are some papers out is helping to risk stratify the recipients. You can risk stratify the recipients in three broad categories. Talking about the first, you have high CPRA, low titer and C1q negative antibodies. These patients are considered a little difficult to transplant since identifying a donor with acceptable antigens is not common. But what I would like to focus is, we should not just focus on the pathology of the HLA antibody, but also on the physiological aspect of the antibody too. That means you have to look at the HLA titer and the C1q or the complement positivity or negativity that's associated. So what are the options that these recipients have? What can we offer them? You can either wait for a donor with acceptable antigens, that's what some of the programs do follow and you wait for antibody avoidance. Or the second is you go ahead with a transplant, you cross one to two weak antibodies and you monitor these patients very closely with the essays. The second group is high CPRA, moderate titer antibodies and you have a C1q positive or a complement positive recipient. These high CPRA patients are a little bit more difficult than the first group to transplant. And then some of the therapies that we can offer them includes PLEX, either pre-transplant or intraoperative or some programs offer post-transplant too. What is the thought process behind it? There are some therapeutic PLEX can remove moderate titers of these antibodies and reduce the IgG antibody titer as well, which may lead to a state of low titer and C1q negativity. That's what the thought process is behind this. This is one study that showed in which they had four cycles, this recipient received four cycles of PLEX and IVIG. As you can well see, I will try to use this up here. So you have class one and you have class two antibodies up here. After receiving four cycles of PLEX and IVIG, the MFI dropped by 77% for class one and it dropped by 76% for class two. And you can also see that this previously positive beads have also dropped in the amount of number. So looking at this, we could say that safely if you have this second type of group and you offer them pre or intraoperative PLEX, there is a chance that you can make them C1q negative and decrease the amount of antibody titers. The third group is that we always fear about is our recipients with high CPRA, high titer and our C1q positive. Unfortunately, most of the time we avoid antibody, avoidance is the way to go. We avoid transplanting them or we wait for a very, very long time. In certain scenarios based on clinical status, some patients can be transplanted. How do we do that? We combine weak antibodies with low titers and restrict the crossing to a maximum of two unacceptable antigens while also implementing perioperative interventions and again, closely monitor those DSAs after transplant. These kind of recipients require several desensitization cycles before we observe a decline in the titers. So to club everything together, you assess a donor offer based on DSA titer and the C1q status. The sensitized recipient, you calculate the CPRA and the HLA antibody titers. Check for the C1q and then club them into either low titer, C1q negative, that means a negative DSA and C1q negative. This donor is acceptable, you move forward. Second is moderate to high titer, C1q positive. Moderate titer, C1q positive, you can offer them IVIG Plex pre-transplant, intraoperative Plex. High titer, C1q positive, we spoke about them. We have limited options but the answer is never know. We can work on it as Dr. Cochrane told us about the further therapies that are available. Almost done. Then we have a take home message. Individualized strategy is the way to go. Each candidate is different. Consider the patient's clinical status. Specificity of HLA antibodies important but don't forget the physiological nature of these antibodies. Desensitization before a transplant, why is it important to know? It's important to accurately predict the total time required for treatments. It can help schedule the OR time for your surgery and risk stratification can expand donor pool and improve the graft outcomes. So I know we're limited on the time but this is my email. Please feel free to reach out with any questions. Thank you.

antibodies

transplantation

graft rejection

antibody-mediated rejection

treatment options

sensitization

antibody titers

research

highly sensitized individuals