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Martin G. Pomper Russell H. Morgan Department of Radiology and Radiological Science Department of Rheumatology Johns Hopkins Bayview Campus March 6, 2009 [Male] Exciting to welcome Dr. Martin Pomper to our division. Marty is a professor in the department of radiology. He is a graduate both of his M.D. and Ph.D. training in biochemistry at the University of Illinois Urbana-Champaign. And interestingly when he left the state of Illinois he arrived in Baltimore in 1990, he hasn't left the place since. After pursuing his training in general radiology, he also pursued additional training in nuclear medicine and neuroradiology. He really epitomizes multi-disciplinary collaborations, novel techniques in imaging but he's always had an interest in rheumatology because the July 1990 internship class included Antony Rosen so they crossed paths for many years. And you see Marty's wearing his Osler tie. So we're excited that you're here to bring us up to date and to learn from you this afternoon. Slide 1 ("Translational Molecular Imaging") Thanks a lot Alan. Yes, I wear my Osler tie, even though I was only an intern I didn't do the other two years but I think that that one year kind of justifies it [Laughing] for those of you who've been through it. Anyway I want to apologize that I'm not going to be talking a lot about rheumatology. Although I have a strong interest in rheumatologic disorders and have a couple of papers with Dr. Helman from back when I was a resident hopefully starting a molecular imaging project with Dr. Dmitri pretty shortly. But everything that I'm going to discuss is perfectly applicable to rheumatology because molecular imaging really just kind of looks at things at the cellular molecular level and it doesn't really matter what organ system you're looking at. Slide 2 ("The evolution of diagnostic imaging") So by way of intro I'll say that diagnostic imaging is really evolving quite rapidly. So anatomic techniques like CT and MR which still form about 95 percent of what we do clinically everyday in the clinic are being kind of transferred into functional modalities where we're doing a lot more CT perfusion studies and MR perfusion and functional MR imaging and diffusion tensor imaging and etc. etc. These are functional applications of a formerly anatomic technique. In fact, angiography, so as a neuroradiologist I used to do a lot of cerebral angiography, we're really doing you know a little bit less of that and instead that's being taken over by these functional applications of CT and MR.

Hybrid technologies where we combine the anatomy of CT and MR with the functional techniques of PET and SPECT, which I'll get into in more detail, also a very big area now in molecular imaging research. And I say in the future, although that's in quotes because that's happening now, we hope to perform molecular imaging which is just looking at cellular and molecular processes in vivo non-invasively, no probes are put in. So that you can really get a nice picture of the physiology in a completely unperturbed environment and that's one of the main benefits of doing imaging to study these kinds of things. And for that we're going to use all the modalities, which are overlapping and complimentary to one another. Slide 3 ("Molecular imaging: a multidisciplinary enterprise") So here at Johns Hopkins we have two centers, in fact, one of the reasons that I'm going to be talking mostly about oncology today as opposed to rheumatology is that they're funded by the National Cancer Institute because the NCI kind of got on board with molecular imaging about 10 years ago. And they've really been promoting it through these centers. So we're fortunate here, we have two centers, one is the in vivo cellular and molecular imaging center that's lead by Zaver Bhujwalla who is an MR biophysicist and what we do there is we look at what's going on around the University. We have experts in each of these different areas and we try to extend what they do, improve on what they do with imaging, using some of these different techniques to study cancer. We also have what's called a small animal imaging resource program. This program is a little bit more oriented toward nuclear medicine as opposed to the ICMIC, which is more towards MRI. It's got three different nodes of activity where we have a physics group that develops hardware and software for imaging pretty much just small animals. We also have a chemistry group, about a dozen or so chemists that develop new probes for imaging. And a molecular biology group that generates molecular genetic reporter systems for imaging, looking at signal transduction cascades and I'll talk about how we do that. You know most molecular imaging research frankly is pre-clinical. But you know we're hoping to translate it and I'll show you how we can do that in the context of this talk. So between these two centers, which are actually very collaborative with each other we've got about maybe 50 people or so that are doing this at the Homewood Campus, I mean at the East Baltimore Campus. Slide 4 ("Utility of molecular imaging for clinical medicine") So why are we talking about this at all? Well what molecular imaging can do, couple things. It's an expensive thing to do to develop these probes, you have to have chemists, physicists, lots of equipment. So you're not going to use this necessarily as a screening test like a mammogram, that kind of thing. But what you can do is you can detect changes that occur in tissue very early.

So if you have a patient that has a certain disease and you want to see whether or not your drug is working you can sometimes tell within hours or maybe a day or so whether that's happening by looking at the changes in physiology that are going on. Also you can predict who may actually respond to a certain therapy and I'll talk about how you know we can do that in a little bit. But you know we can take advantage of a patient's particular phenotype. So for example, if a patient has breast cancer and you want to know whether or not to put them on Herceptin which is a monoclonal antibody, you want to make sure that they actually have you know a phenotype that expresses HER2 new receptor. And we can do that with molecular imaging. So we can predict who's going to respond to these therapies. All the major drug companies have in house molecular imaging programs, at least pre-clinical, some of which are actually clinical. And they do that because you can do pharmacokinetic, pharmacodynamic studies with various drugs. I just mentioned that you can pick the right kind of patients for your clinical trials. Okay, and you can even calculate how much of your drug gets to a particular target site you know using these techniques. So I'll talk about all of that in a minute. Slide 5 ("Molecular imaging modalities") Now molecular imaging is a biology driven enterprise in that we're really just interested in the biology. So if you know Dr. Petri came to me and said, "You know we want to study lupus. You know what can we do to follow these patients, we're going to treat them with you know such and such a drug. You know what do we do?" Slide 6 ("Positron emission tomography - PET") Well we have chooses as to what we can do. We can use either you know optical, nuclear MR ultrasound. You know in that case, since it's mostly CNS oriented you know we could do you know PET scanning and look at inflammation in the brain using a molecular imaging tracer and just sort of follow that over time which is what we're going to do. But these techniques are not exclusive of one another but they're really complimentary. So you could do different things with them. They have different capacities for being translated from rodents to patients. They also have different sensitivities. But it depends on what you want. Ultrasound has very good temporal resolution. So if you've ever seen a fetal ultrasound you can see the baby moving. I mean that's like real time. Okay, you can't do that with the nuclear techniques because you have to accumulate radioactivity counts on your detector and then generate the image. So you don't have that same temporal resolution. You also don't have the kind of special resolution you get with MRI, which could be 100 microns. So these all kind of balance each other out which is why multi-modality is kind of an important way to go.

Our group tends to focus on the radiopharmaceutical based techniques because to my mind they represent right now the best way to translate new probes from rodents to the clinic and I'll explain why in a minute. And also they've got high sensitivity for detecting molecular phenomena. So MRI, very good special resolution, but for detecting molecules you really need micromolar concentrations at the very lowest and usually millimolar concentrations. Well as we can see, nanomolar or even picomolar concentrations with the radiopharmaceuticals. So just to make sure we all know what we're talking about a PET is kind of the premiere translational molecular imaging technique in my mind right now. And the way it works is you've got a positron emitting nucleoid that's attached to a molecule of interest that homes to a certain process. This is FDG, which is used for looking at cancer for Alzheimer's disease, looking at glucose metabolism. So the F18 achieves stability, it decays by emitting a positron which is just a positive electron that collides with an electron about a millimeter away from the decay event to create these annihilation photons that are detected in coincidence around the patient. You then combine that with CT and then you get the current state of the art, which is the PET CT. And here you can see FDG in the brain, the heart and this patient happens to have cutaneous lymphoma. Now the other thing to keep in mind is that we're not detecting the decay we're detecting the annihilation. So we're really limited in resolution to about a millimeter or so. And the physicists tell me that if you correct for non-collinearity of you know these photons coming off and for the positron path range you might be able to get down to half a millimeter resolution. So for pre-clinical work that's important to know because if you want to use a mouse model of EAE for example and study the brain with an inflammatory agent you have to realize your resolution is going to be on the order of some of the substructures in the mouse brain. Slide 7 ("Why radionuclide techniques translate readily: the tracer principle") So why do these techniques translate so readily? Well here's an example, Carfentanil binds to the u opioid receptor in the thalamus. If you give 5 mCi, which is a standard dose of high specific activity, meaning high radioactivity for little mass to a 70 kg person, you give them 5.5 x 10-6 mg/kg. Okay, so if you have an ear infection you take, I don't know, 500 mg of Amoxicillin three times a day. Right, that's millions of times higher than this. This is always subpharmacologic. So there's no affect that you get from the drug. So you know we do that everyday in the PET center and we get images like this and the patients are fine. But it turns out that Carfentanil, if you give 2 ½ mg per kg of this IM that's what they use on the Discovery Channel. Okay, this dose would actually be lethal in a human being. And that's not a very big dose. So you know what's interesting about these radiopharmaceutical techniques because radio activity is so easy to detect you can give you know lethal compounds basically that you know can get FDS approval and we can just get them right into the clinic. The toxicity studies are much easier than they are you know for drugs certainly and for other imaging agents. Slide 8 ("Outline: translational molecular imaging")

("Outline: translational molecular imaging") Slide 10 ("Outline: translational molecular imaging") Slide 11 ("Outline: translational molecular imaging") Slide 12 ("Outline: translational molecular imaging") So I'm actually going to talk about four of our more mature projects all of which have somewhat translational component. Okay they are all cancer related. But again, they can you know the same principles can apply to rheumatology. So I'm going to talk about these mechanism based imaging agents for looking at prostate cancer. This is kind of more of an exercise in how you even begin looking at a project in molecular imaging. So how do we even know where to start or you know develop a probe and I'll go through that process. And then bioluminescence imaging I want to talk about that because this is a big area pre-clinically for testing new drugs, a lot of companies are using that. Talk about a project that we're doing in collaboration with Bert Vogelstein and how you can take kind of an unusual idea like this and translate it by having it enhanced with imaging. And then, another project where we can turn imaging into therapy. Slide 13 ("Prostate cancer: why image?") Slide 14 ("Prostate cancer: why image?") Okay, so first for prostate cancer, this is kind of the slide that shows what's on the background of significance or the significance section of my grants. Although this is really why you'd want to study prostate cancer, but the reasons you want to image it, number one, hopefully none of you had a prostate biopsy. I haven't had one yet. But they're not very comfortable and biopsies are inherently prone to sampling error. Because you can you know get many different biopsies in both sides of the gland, you could still miss the cancer. Okay, that's number one. And the other thing is that when you use ultrasound to detect where the cancer is you're only looking at a portion of the gland. Imaging gives you the whole thing all at once if you're going to image directly the prostate. The other reason is that patients that have their prostates removed they still you know return a year or two later, their PSA level, the blood test starts going up, you want to know where that recurrence is happening. Is it in the calvarium, is it in the surgical bed etc. If you don't need that information you don't need to image. But if you want to be able to treat this thing then generally imaging is indicated. So that's why you'd want to image it. Also for staging, you know a patient comes in with a high PSA you want to know, do they have widely metastatic disease or not. So you know there's a lot of reasons that you want to image. Slide 15 ("SPECT Imaging with ProstaScint")

Now there is a mechanism-based technique for doing that now. Okay, of course we do bone scans and that sort of thing but if you want to you know look at the actually tumor. The process then is a monoclonal antibody that was developed about ten years or so ago. And this slide really just illustrates that monoclonal antibodies do not make very good imaging agents. Okay they are big molecules, you know 55,000, 100,000 molecular weight. And they tend to circulate in the blood for a long time. You have to actually get a concurrent blood pool image and subtract that out. You have to wait five days. You have to use a radioisotope that doesn't have very good imaging characteristics. In any event, not very good. This is supposed to be where the tumor is in this pelvic lymph node and you know we don't really use this agent here. It's not that easy to interpret. Okay, but it is an example of a mechanism based agent. Slide 16 ("Prostate-specific membrane antigen (PSMA): a marker for prostate cancer and tumor neovasculature") And the reason I mention it is that it binds to a very valuable target which is known as the prostate-specific membrane antigen, which his a marker for prostate cancer and also pretty much all solid tumors will express this in their neovessels except prostate cancer. So if you can develop an imaging agent for this, like the company that developed antibody did you can maybe use it for a lot of different indications. Slide 17 ("A 'conventional' target: PSMA") Now it turns out it's a very interesting target because there's an enzymatic active site on the outside of the cell. Now processing tends to bind to an internal epitope so the cells have to be dead for process to gain access or the antibody to gain access. So if you can design agents that bind out here then you might have something better. So the most important aspect of molecular imaging research is to find the right target and we thing this is a good target. So you know there are probably other targets in rheumatology that you might want to think about. I consider this a conventional target because we're treating PSMA as if it's just a receptor that's hanging off the cell and we're going to image it, it's kind of an easy problem and I'll talk about non-conventional targets in a minute. Anyway, so what it does is as you're developing an imaging agent you want to look at you know the function of this thing and what it does is it basically cleaves an acetyl-aspartyl-glutamate to glutamate and N-acetylaspartate. So by knowing the mechanism you can start thinking about you know ways of imaging this. Why this happens in the prostate is not clear. It also happens in the brain and it happens a little bit in the GI tract. But this enzyme has a fairly limited expression, which also makes it a good target. In other words, it's not all over the body. So an imaging agent will not have a lot of background activity.

So what we did was we looked at NAAG and you can do a retro synthesis and get back to compounds that are going to be even higher affinity and more stable than the natural substrate usually doesn't make a very good imaging agent. Slide 18 ("Urea-based PSMA inhibitors") Okay, so then we developed these ureas with Alan Kozikowski at the University of Illinois. And functionalized them for imaging. And these are all very low molecular weight and the way you know that we've done it is just by looking at the natural substrate. But since then we've seen the crystal structures. Slide 19 ("PSMA: three domains – dimer") So Cyril Barinka, one of our collaborators at the NCI Frederick has crystallized the protein. We know the three domains contribute to the active site. We know that it's a dimer when it's on the cell surface usually this kind of work doesn't help all that much in developing new imaging agents. Usually, you know, drug companies have already kind of given us a good lead, but in this case having the crystal structure was extremely helpful, as I will discuss. Slide 20 ("S1 binding pocket: arginine patch") And if you look more carefully here at the enzyme you can see that there are a bunch of molecules that we've made that are now overlapping there. This is a computational docking study. And you can see here that there are two different pockets really to the enzyme. There's one pocket where everything overlaps and that has to be a certain structure. There's another pocket that's a little bit more open to taking larger substrates which is good because that's where we can put our chelaters and other things you know toxic moieties, other things that we can put out there for imaging. Slide 21 ("[11C]DCMC: radiosynthesis") But before we knew any of that, you know back in 2002, we just took the natural substrate and we made it into a urea which is more stable and we put a sulfhydryl group out here. Because what that does is it attacks this iodomethane which is carbon-11 methyl iodine and then we were able to make this compound, which we consider a direct probe for PSMA. Now this has a 20-minute physical half-life, carbon-11, so it's really just for proof of principle. So you have to be very efficient. You have to synthesize it in a couple of minutes. You're going to have to purify it, sterilize it, inject it into the patient and you know image etc. all within about an hour or so. Also this wouldn't even be useful to make at Hopkins and then send it over here to the Bayview Campus because it'll all be gone by the time it gets here, it has a 20 minutes half- life. This just shows the tracer principle visually. So when we synthesize this we have to purify it so we inject it onto an HPLC column and here you can see the mass peaks coming off and this is the radioactivity peak, we collect the radioactivity which represents this molecule and you'll see that

there's really no mass associated with it. So we're giving the patient radioactivity and very little mass and that's why we can translate this to the clinic. Slide 22 ("Picture") Then we take our small animal PET scanner, we have a whole array of these instruments. Everything for a human except for mice and rats. So you've got MR and PET and SPECT. You know x-ray machines, irradiators. So we have all that over there and it's very useful because these animal models are becoming more and more relevant. The motivation for doing this is now we're got all these knock-ins, knock-outs, transgenic animals that are worth studying. They weren't there ten years ago. Slide 23 ("ATLAS PET imaging of PSMA with [11C]DCMC") So we get a scan like this, this is a coronal image and here we have out PSMA producing tumor. So we're on mechanism here. And we have about a 12 to 1 target to non-target or tumor to muscle ratio, which is actually pretty good. The process then has about a 2 to 1 target to non- target ratio. This is already you know improved that because this is a low molecular weight agent. Much better pharmacokinetic profile than an antibody. Slide 24 ("Urea-based PSMA inhibitors: [18F]DCFBC") Then we developed fluoro 18 version because this has a 2-hour physical half-life as opposed to 20 minutes. This we can actually ship around the eastern half of the United States and other centers can use it. And most importantly the National Cancer Institute is interested in F18 because you know you can ship it around and they'll actually pay for the toxicity studies, which are about $300,000.00 to do for a new compound. Even though we know it's not toxic you still have to you know go through the motions and prove it. Slide 25 ("Mouse whole body pseudo-dynamic scan") And then with a compound like that we get images like this. And what this is, this is a mouse that has two tumors, one under each arm. These are PC3 prostate tumors that don't normally express PSMA. But one of them has been engineered to express the target and the point behind the slide twofold, number one, it shows how with one mouse you can tell if you're on mechanism or not with your imaging agent or with your drug. You know in the old days we'd have 28 mice, we'd have to sacrifice them like every you know 15 minutes or 20 minutes and then we'd get time activity curve. But now we can just do one or two mice and we can tell if we're on mechanism. The other thing is you can see that unlike the antibody these low molecular weight agents, you know there's a little bit of live uptake. This is actually specific binding in the kidney because mice have PSMA in their proximal tubules but there's not a lot of background activity. So this will make for a pretty good imaging agent. It's excreted into the bladder. So if you want to look at the prostate we have ways of getting around that bladder activity. But also the main indication will be for patients that don't have a prostate.

("Cancer Imaging Program") So this is one where the Cancer Imaging Program they've got a very nice program called the Decide Program where they pay for the toxicity. They're paying for the toxicity on this and this agent is about to enter the clinic. The other nightmare that I'm learning about is you know we have the toxicity, we can synthesize it, but now we have to synthesize it according to good manufacturing practice. Okay, and that's really a nightmare. Because we've had this compound for two years and you know we have to do it ourselves because the company that licensed our patent on this is not interested in the PET, they're just interested in the SPECT agents. So we have to do it ourselves, which is not easy to do. Slide 27 ("Additional PSMA-based ligands: halogens") Slide 28 ("YC-I-37") Slide 29 ("YC-I-37") So in the meantime while you know one or two of our students are working on that we you know went ahead and took advantage of the crystal structure and you know figured out how long we need this linker here, you know what kind of moieties we could put out here. And you know again by doing these docking studies you can kind of pick up maybe a couple other interactions with this iodine. And you know we know what residues that this is going to interact with and we can get very high affinity agents. And now we start getting around maybe 20 to 1 target to non- target ratios. And again this is specific binding in the kidneys and very little background if you let things kind of wash out. Slide 30 ("[125I]YC-VI-11") And we have other agents that you know don't bind to the kidneys very much but instead just bind to the tumor. So after a day or so with this I 125 label compound we get very high target to non-target ratios with essentially no background at all. So you know we're encouraged by those kinds of studies. Slide 31 ("The single amino acid chelate (SAAC) concept") The single amino acid chelater concept was developed by Sangeeta Ray and her colleagues. She's now a research associate in our group, but she did this in graduate school. And the idea is that you could take a molecule like this and put it into a peptide synthesizer and you've got this tridentate chelate there where you could put a technetium so you could incorporate technetium into any peptide that you want. Slide 32 ("General strategy for labeling PSMA-based ligands") Okay, it's a really nice idea but what we use it for is for linking up to these PSMA ligands, these Ureas. And what we can do is we could put you know technetium out there for imaging, we

could put rhenium for therapy, that's a therapeutic nuclide or some of these are actually inherently fluorescent like the bisquinolines because they're highly conjugated they're inherently fluorescent. Slide 33 ("Urea chelates") So again using molecular modeling we've optimized the chain link and the hydrophilicity of this particular chain and we get very good images with this. This is on the order of 50 to 1 target to non-target ratios with this molecule. And it's also convenient. The workhorse of nuclear medicine is not PET. Okay, that's kind of the premiere, you know the Cadillac technique. The workhorse is technetium and that's because you can generate technetium using a generator and any nuclear medicine department, in fact, every nuclear medicine department has technetium as part of their armamentarium. So that's why a technetium agent is so desirable and it's also very convenient, you inject it, you know the patient can you know sit around or go to lunch, come back a few hours later and then get the image. So that's kind of where that stands. Now the translational part comes in that the company, Molecular Insight, that licensed our patent, they went ahead and developed a slight variation of one of our iodinated compounds and actually gave it to patients. So they've done about half a dozen patients. Of course this patient – you know we were supposed to do the first patient here at Johns Hopkins but the IRB, right, so but at Duke they got it right through like that so the first couple of patients were done at Duke unfortunately. So now they get all the glory of doing that. Slide 34 ("Urea chelates ") But what this shows is how there's less uptake in the kidneys as I showed compared to the rodents. You've also got a metastasis here in the lumbar spine, another periaortic metastasis that could not be identified by any other method really. And you know a little bit in the bladder. So you could see how you know this might be a good way to image prostate cancer if the patient had more widespread pelvic mets etc. So that's kind of the first translational project. Slide 35 ("[123I]MIP-1095: First-in-man for metastatic prostate cancer") Slide 36 ("PSMA = glutamate carboxypeptidase II (GCPII)") I'll also mention that what's interesting is if you look at the mechanism of your agents rather than just stick to an organ system. Okay, you can see how there's more utility than you originally intended. So PSMA turns out to be the same as an enzyme called glutamate carboxypeptidase II which was discovered by Joe Coyle back in the late 1980s when he was here at Johns Hopkins. And they showed that there's disregulation of this enzyme in schizophrenia and another group down at Georgetown showed that if you inhibit the enzyme ameliorate some of these symptoms in the PCP model of schizophrenia. So you know you see a paper like that and you wonder, "Gee, maybe we can use our prostate agents for looking at that?" Slide 37 ("PSMA/GCPII in the CNS")

So the first thing we did was we took a radiolabeled version of one of our PSMA ligands and this is an in vitro autoradiogram. We proved binding specificity of our agents by first blocking the receptor and then treating with the radioactivity and seeing no uptake. But nowadays you've got all these transgenic knock in, knock out etc. mice so we see very little uptake in the homozygous knockout and then a little bit of gene dose affect here. This is the wild type. So between these two studies we believe that we can actually look at the brain using our prostate agents. Okay, now these are in vitro studies because the compounds are very hydrophilic and they don't get across the blood brain barrier. Slide 38 ("Frontal Cortex – Grey Matter/Entorhinal Cortex") But we can also look at human brain specimens and compare you know schizophrenia to normal etc. and we found that this enzyme, and this is the first time the protein has actually been studied. People have done in situ hybridization look at mRNA. But here we can see that there are differences in certain brain regions between normals and schizophrenics or between schizophrenics and bipolar disorder. So now you know we feel that this is a good target for psychiatric disease. So the next step is to try to get compounds that don't have all these carboxylate groups, don't have all these charges. They can get across the blood brain barrier. So we're modifying these compounds in an effort to do that. And that's actually very challenging. In fact, I'm a neuroradiologist is kind of my clinical work. I do a day a week as a neuroradiologist but you know most of our work is moving a little bit away from that. If we want to develop new probes because of the blood brain barrier, okay, it's just so hard to get things in. They have to be low molecular weight, they have to be you know certain polarity, a certain hydrophilicity etc. But anyway, that's kind of where we are with that project. Okay the other projects go quicker. Okay, than that first one, that was an exercise in how you find a good target and start developing probes for it. And once you have a probe now we can study you know prostate cancer, we can study schizophrenia potentially once we get it into the brain. We can look at different aspects of these diseases. So that's kind of where the fund begins. Slide 39 ("Design of PSMA/GCPII inhibitors") Slide 40 ("Outline: translational molecular imaging for oncology") Slide 41 ("'Unconventional' target: hedgehog signaling cascade") Okay, so bioluminescence imaging is for looking at unconventional targets like signaling cascades. So a signaling cascade here at Hopkins described by Phil Beachy is being worked on in about eight different labs. Okay, and that's the hedgehog signaling cascade. This is a developmental pathway whereby if there's a mutation in one of these proteins, this is a sonic hedgehog ligand then you get a kind of disregulation of this pathway and then eventually

overproduction of this transcription factor known as gli. And if you have over production of gli patients go on to develop glioblastoma or medulloblastoma or prostate cancer or pancreatic cancer. Slide 42 ("Needed: reporter gene/reporter probe system for imaging hedgehog signaling") So this is a big target for the drug companies to be able to modulate this, to turn this off. So wanted to be there with an in vivo model so that they could test there drug. And you know we started doing that and you know the way you do it is a little bit complicated slide here, but it's not rocket science. So to do molecular genetic imaging to look a signal transduction cascade you do just what molecular biologists have been doing for 40 years. You just use a reporter gene. Okay, so instead of you know if you want to look at your gene of interest you just link its activity to the reporter. The only difference here is is that the reporter rather than being GFP or beta- galactosidase instead it's a thymidine kinase protein that can be detected using an external imaging device, like a PET scanner. Okay, so you produce thymidine kinase whenever gli is turned on. The thymidine kinase then phosphorylates radioactive nucleoside analogs, like FIAU which get phosphorylated and then trapped in the cells and then you can image them. So the way you do it practically is you take your gene, like this, this is a reporter gene, it's under the control of gli, so if gli is active you turn on – this is actually a little bit complicated, it's a tri-fusion reporter gene. So if gli becomes active you turn on three different proteins, firefly luciferase for bioluminescence imaging, red fluorescent protein for cell sorting and then thymidine kinase for radiopharmaceutical based imaging. So any cell that has this gene in it that is active in terms of hedgehog is going to turn on these three proteins and then you can use these imaging devices to detect those proteins. Okay, it sounds a little you know 30:24 Rue Goldbergese but it tends to work. Slide 43 ("Multimodality imaging of the hedgehog signaling cascade in experimental brain tumors") Slide 44 ("Bioluminescence imaging of the hedgehog signaling cascade in experimental brain tumors") So for example, this mouse has a U87 tumor that has that gene in it. If U87 produces gli you turn on the reporter, you inject luciferine into the animal and then the luciferine reacts with the firefly luciferase that's being produced by your reporter gene and now you're basically glowing with hedgehog signaling. So you're looking at hedgehog there. The next step is to come in and turn that light off with various drugs at Merck for example or Pfizer may be producing to prove you're on mechanism. Now the reason that that's a good thing

to do is, number one, it's a totally realistic situation. You're giving the animal the drug, it's not in vitro etc. The other thing is you can turn that light off potentially without having to kill any of the cells. So you don't have to wait for the tumor to shrink. You don't have to get a caliper out; you don't have to use lots and lots of animals over a long period of time. You can tell if you're on mechanism within 20 minutes sometimes. You turn that cascade off, the light goes down. That was the idea. Okay, but that's not how it worked out. And that's because bioluminescence imaging which is being used by drug companies for that exact purpose, all the companies that I've seen have bioluminescence programs they have little research groups, that's all they do is this kind of in vivo mouse imaging. Slide 45 ("Bioluminescence imaging") But it's complicated. It's not like I showed you with the PET agent where you just kind of bind to your receptor, you light it up, and you look at it with a PET scanner. For this you've got to have a tumor that's producing the reporter gene that produces firefly luciferase, you then have to inject luciferine, you then have to have ATP and oxygen around. So there's all this other stuff going on. Slide 46 ("Treatment with a hedgehog antagonist, HhAntag-691 increases BLI light output") So you may not be reporting on what you want to report on. We want to report on hedgehog. Okay, so you have to keep that in mind. And it turned out that when we first gave the first compound we tried this HhAntag we knew that this was going to turn that light off because it's antag, right? So it's going to turn it off. But it actually made the light brighter. So we give it to the animal, we see the tumors start to light up brighter. Not good, right, for the drug company. But you know we kind of figured out why or our hypothesis was that HhAntag was inhibiting a pump that normally pumps luciferin you know out of cells. And if it does that then more luciferin can get into the cells and react with the firefly luciferase and then you get more light output. Slide 47 ("Treatment with ") Slide 48 ("D-Luciferin, the substrate of firefly luciferase, is also a substrate of the ABCG2/BCRP MDR pump") So you know we found that that happened in intact cells and extracts it didn't happen. So then the goal was to figure out you know what that pump was. And when we figured out and make a long story short we tried a bunch of different specific pump inhibitors but the only ones that really gave that high light output was a compound called fumitremorgin C which is specific for the ABCG2 breast cancer resistant protein pump.

And shown here in an in vivo situation just on the right side, this is a prostate cancer line. If you inject vehicle here along with your luciferine and then you do bioluminescence imaging you know nothing really happens. But if you inject an inhibitor of ABCG2 you get that increase light output. So this really throws off using bioluminescence imaging for drug development unless you can account for the presence of ABCG2 in your cells that you're studying. Slide 49 ("D-Luciferin is an ABCG2/BCRP substrate: implications for bioluminescence imaging (BLI)") So this is the most common method for looking at transgene expression in vivo. The luciferine is the most common substrate. So you have to account for the presence of this protein but more important they don't call this breast cancer resistant protein for nothing. Okay, this is a protein that does confer resistance to chemotherapy, because it's a pump, it's one of these efflux pumps. Slide 50 ("Screening the Johns Hopkins Clinical Initiative Library") So what's nice is the bioluminescence imaging there's not a lot of background etc. so it makes for a very nice screening test. So we can take cells and put them in these 96-well plates and we can basically just add all sorts of compounds to them and see which ones cause the light to increase. If they do we know that they're inhibitors of ABCG2. So it's a screening test for new ABCG2 inhibitors. There's no clinical ABCG2 inhibitor that's ever been used in patients because they tend to be a little bit toxic. So you know we took Jin Liu's compound library that perhaps you've heard of and we tested lots of compounds. We've got about 60 of them that really increase that light output. And these are all FDA approved. So now we can that these FDA approved compounds, give them with standard cancer chemotherapy and then inhibit that ABCD2 and get more of the chemo into the tumor. Okay. And they're all FDA approved so we should be able to do it quickly, if that's possible. So that's kind of how we took a basic imaging study. We just wanted to look at hedgehog signaling and we turned it into a little you know drug development program. Also a lot of these compounds are related. So we can get a pharmacophor on the molecule and then develop the ultimate ABCG2 inhibitor. And also, it turns out ABCG2 is what confers the side population phenotype to cancer stem cell. So if we have a way you know something that sticks to these ABCG2, an inhibitor, then we can you know maybe take out that stem cell population. So those are all kind of thoughts that we're throwing around. Slide 51 ("Development of PEG-Prom mediated imaging systems/I. Experimental lung metastasis model of human melanoma") Also another thing about molecular genetic imaging I want to mention is that if you've got a good system, like for example, there's this PEG promoter. This is the progression elevated gene

promoter that my collaborator at Columbia, Paul Fisher, developed and what we've done with that is we've basically just use this promoter to drive an imaging reporter. And the reason why I do that is is the PEG promoter is only expressed in cancer according to my collaborator. So you know we took these gene constructs and we were able to you know formulate them into a cationic polymer which took about a year or two to do, inject it intravenously and in you know mouse models of melanoma or models of – I think we had one of breast cancer. We only get expression of this gene in the tumor. So now we have kind of a genetic construct that will home in on the metastasis etc. And that's just another example of using molecular genetic imaging for this case pre-clinic. Slide 52 ("Development of PEG-Prom mediated imaging systems/II. Experimental lung metastasis model of human breast cancer") Oh, this is the breast cancer, also lung mets here for breast cancer. You can see very nicely how the CT, the mouse CT scan shows that. So that's molecular genetic imaging. Slide 53 ("Outline: translational molecular imaging for oncology") Last two projects are a little bit related. They use the same reagents but I'll talk about this. This is one of my favorite projects with Bert Vogelstein. So Dr. Vogelstein who most of you know is – he's at a point in his career where he just wants to cure cancer and he doesn't care if you have to put bacteria in patients to do it. Okay. Slide 54 ("Heterogeneous oxygenation of tumors") So you know he's got more papers in nature and science that I'll probably ever publish. But now he just wants to get to the point. So what the idea is here is taking advantage of the fact that tumors when they outgrow their blood supply become somewhat hypoxic. So what he wants to do is add anaerobic bacteria to the situation and these are injected intravenously as spores. They go all over the body. But they only germinate in the hypoxic core of tumors because there's nowhere else in your body that's hypoxic it turns out. Unless you have an abscess of something. But you know that's kind of the most hypoxic area. The idea is the bacteria get in there and they consume these tumors from the inside out. Okay. It's kind of a crazy idea. But it's not a new idea. It's not his idea, actually. Like most good ideas it was probably from either the post-war era or the early 1970s at the NIH. But what he brought to the program was looking systemically at all the different anaerobes that you could find and there are other groups trying to do this, like at Sloan Kettering they're using like staph or strep species. Slide 55 ("Bacterial strains tested") It's kind of a crazy idea. But it's not a new idea. It's not his idea, actually. Like most good ideas it was probably from either the post-war era or the early 1970s at the NIH. But what he brought to the program was looking systemically at all the different anaerobes that you could find and

there are other groups trying to do this, like at Sloan Kettering they're using like staph or strep species. But Bert systematically looked at everything you could buy and found that it was really clustered novyi (type A) that got in there and really went to town on these tumors. Slide 56 ("Effects of C.novyi in vivo") So all I remember from pathology is that blue is bad. So the blue is the tumor and the pink is normal liver. So you give these spores and then overnight the spores will germinate and they will start munching away and lights the tumor and then if you do a gram stain you can actually see the bacteria in there. Slide 57 ("Clinical need: to image the bacteria as they home in on tumors") Now the only problem with this therapy, particularly for clinical translation is the fact that the C. novyi tends to be lethal. [Laughing] So you're going to have to do something about that. And what they did was they inactivated the lethal toxin. So now they have clustered novyi non-toxic. Okay, and this is not toxic anymore. Slide 58 ("Bacteriolytic therapy: potential for molecular imaging") And this is kind of the stage where they come to us and they say, "Okay, you know we want to put bacteria in patients. We want to prove that we have this under control. So let's image it somehow. How do we image it?" So I suggested, well why don't you put one of these reporter gens in there, into the bacteria, and then you can kind of follow around the bacteria using the radiolabeled you know FIAU. Well, Bert said, "No, we can't do that." And I said, "Why not?" He says, "It's too complicated. I can't you know explain." But the bottom line is you don't need to do that. And that's because of an extremely capable graduate student that was in Bert's group, Chetan Bettegowda, whom some of you may know. He's actually a – I think he's a PGY-4 neurosurgery resident now. But when he was doing you know useful things like trying to cure cancer you know and in the lab he found that you could actually take the stuff that we normally use for imaging, which is again, in these tracer does, if you move them up to pharmacologic doses you can actually kill C. novyi. Slide 59 ("Tumors can be imaged with radiolabeled FIAU, once bacteria have homed to them") Now FIAU was not developed as an anti-bacteria agent. It was actually developed as an antiviral agent to treat AIDS patients with hepatitis and actually was lethal to some of these patients unfortunately back in the early '90s and was totally you know forbidden to be used anymore. But you know we can use it for imaging again because of this tracer principle.

So when I saw this curve I knew that if you can kill the bacteria with FIAU you can image it with radioactive FIAU. In other words, the bacteria have the same thymidine kinase or very homologous one to the viruses for which this was designed and it'll turn that over and accumulate in the bacteria. So if you take a mouse, and this is kind of a dismembered mouse here, tumor, blood, intestine, kidney whatever. If you take the mouse you inject him with radioactive FIAU you don't see any uptake in the tumor because FIAU does not bind mammalian thymidine kinase. It is not a tumor imaging agent. Okay. It binds to you know these bacterial and viral TKs. On the other hand, if you give the bacteria, the C. novyi, it goes in, it munches away on the tumor. You then give your radioactive FIAU and then that's going to give you an indirect read out of where the tumor is because it binds to the bacteria. So you can see that here. Here's a mouse. You can even see that there's gas being produce as a bacteria and go to town on that tumor. You then inject your imaging agent and that only goes to the bacteria. Okay. It's not in the brain. It's not in the lungs. It's just in the bacteria and you can use that kind of imaging information to show the IRB or the FDA that you kind of have this under control. Okay. [Inaudible Male Question] In the spleen. [Inaudible Male Question] On here. Yeah, there's – certainly this – but this is actually, the FIAU is actually going to be metabolized so you're gonna see it in the spleen, you're gonna see it in the intestines, etc. And you know almost to a similar extent is you see it in tumor. But the key is where the C. novyi is. Okay. So this is just normal – these are just metabolic organs. This stuff is always going to go to those organs. Slide 60 ("Mechanism of FIAU Uptake") And in fact on the next slide, you can see that it goes – you know you get a little dehalogenation here, you get some of the oral pharynx, it's in the liver. So that's just the FIAU. Okay. But the bacteria are you know pretty much only localized in one place. And the main thing was where the bacteria are going. So here if you knock out the thymidine kinase, you can see that you can't image anymore. It's proving that that is indeed the mechanism of the uptake. Okay, here's the wile-type, here's the knock-out. So we looked at these different nasty bacteria. Unfortunately TB does not have a thymidine kinase, nor does HIV. But there are a lot of other organisms that do. You know a fungi and viruses. Slide 61 ("Graph")

("Bacterial strains imaged after i.m. injection") Now what's interesting is that you know this is E. coli. This isn't clustered novyi. So I asked Chetan to take a look at the sequence for all the TKs, for all the bacteria he could find. And it turns out that these bacterial TKs are fairly homologous to one another. So we thought that you know maybe we can actually image bacteria. Slide 63 ("Mechanism-based bacterial imaging: infected prosthetic joints") So you know we developed a new way to image bacteria and we thought the first indication for that would be to look at infective prosthetic joints. Because orthopedic surgeons are often fraught with patients that have painful – and the problem with patients having a painful prosthetic joint. And they don't know if it's infected or if it's just loose. And those are going to have two very different therapeutic you know outcomes. Slide 64 ("State of the art: MRI") So in order to determine that we can use our bacterial imaging technique and this just shows you know the current state of the art is just to use MRI. And all I try to show here and this is kind of what I do clinically is look at these spine MRs. But basically, what this shows is that this patient that has discitis and osteomyelitis you'll see some enhancement around the disc. This patient also has enhancement around the disc but they just you know had surgery about 15 years ago. Okay. So it's a very sensitive technique but not very specific but the technique that we have actually just looks at bacteria because you have to have living, breathing bacteria to metabolize at that FIAU. Because again, FIAU doesn't bind the mammalian thymidine kinase. Slide 65 ("rFIAU Scan") So we took about half a dozen or ten patients and these were you know mostly patients with staph aureus and we had path proof on all of them. And we were able to show using PET CT with I-124 labeled FIAU. Slide 66 ("Image - Infected Knee Prosthesis") So you can go from pre-clinical with I-125 to clinical with I-124 and this is an infected knee prosthesis. And this is just kind of an infected knee. And then we've got other infections here that we've been able to look at. So you know we start out looking at bacteria at cancer therapy and now we have you know a new way to look at infection, which is a problem. There's not a lot of good ways to look at infection. Right now the state of the art in nuclear medicine is to use a tag white blood cell study where you take the patients white cells, you label them with technetium, you reinject them to the patient, hopefully that's the correct material you're reinjecting, it's not some other patient. And you know so there's problems with that. But this just looks at the bacteria.

("Image – Arthritic Joints") This is just to show that you do not see uptake in bad arthritic joints that are aseptic. Okay. So there's no bacteria there, it's inflamed. So you don't really see uptake. Slide 68 ("Control subject with DJD") This is just a prosthesis that you're looking at the beam hardening artifact from a metal prosthesis on this coronal PET CT scan. So it's specific for infection and not inflammation. Slide 69 ("Outline: translational molecular imaging for oncology") Slide 70 ("Imaging human herpesvirus lytic infection in mouse models of lymphoproliferative disease") Okay. And last which is related to the other project a little bit. Is work we're doing with Rich Ambinder, who's an oncologist. And what Rich is interested in is he's interested in tumors that are associated with viruses. So tumors are becoming increasing associated with various infectious agents, as you know. It turns out that a number of tumors are associated with the Epstein - Barr virus in particular. The one that causes mono. So lymphoma, nasopharyngeal carcinoma, gastric cancer, Kaposi's sarcoma. These are all you know associated with gamma-herpesviruses to a certain extent. So what Rich wants to do is, he wants to take advantage of that fact by you know normally the virus lies dormant within the cancer cell. If you give a pharmacologic agent like SAHA or some of this histone deacetylase inhibitors you can actually wake the virus up and get it into it's lytic cycle. When it wakes up and becomes lytic it lysis the cell that it's in. And it tends to reside in the tumor cell. Okay and that way you kill the tumor. Now it turns out that only a minority of patients will respond to this therapy and also the therapy is not trivial it's somewhat toxic. Okay in other words it's you know a little bit neurotoxic, GI distress, etc. and it's also expensive. Slide 71 ("Imaging lytic gene induction: EBV associated lymphoma – EBV-TK is similar to HSV1-TK") So the idea, here's where the personalize medicine comes in. How can we predict who's going to respond to this kind of therapy? Okay. So I asked you know, "What happens when the virus goes from latent to lytic you know are any genes turned on?" And it turns out by great good fortune that the Epstein - Barr virus has a thymidine kinase that is turned on in going from latent to lytic. It needs the TK to do that.

And the goal was to show that we can actually image the Epstein - Barr virus TK like we could do the viral and the bacterial TKs just using that same probe the FIAU that we've been talking about. Slide 72 ("Imaging bortezomib-induced lytic gene induction") So the first step was to just take cells that are constitutively expressing the EBV-TK and we showed sure enough we can image them using radiolabeled FIAU. But more importantly if you take a TK or an EBV associated tumor like a Rael lymphoma and you inject FIAU you can't really see anything in the tumor because again it doesn't bind to mammalian TK. But if you then wake the virus up using bortezomib. And this was discovered in a high-throughput screen using that same library that Jin Liu has at Johns Hopkins clinical drug library. If we wake up the virus the TK is produced and now we can image it with FIAU. So you can imagine a patient with a nasopharyngeal cancer coming in on day one and getting a baseline image, you then give them bortezomib, they come back the next day and if their tumor lights up then you know that you can continue them on this therapy and that they have the right phenotype to respond. Okay. Now all of these mice respond because these you know Rael tumors we know are EBV associated but not every nasopharyngeal cancer. You can do a biopsy and determine that too. But you know this is kind of a non-invasive way to do it. And also, you know can be done repeatedly etc. So that's kind of where the personalized medicine aspect comes in. And you know we've shown that not only can we look at lymphoma but even gastric cancer you know different species of lymphoma can be imaged using this technique. But more importantly if you look carefully you'll notice that the I 125 FIAU once we wake that virus up and produce TK it's really sequestered for you know a fairly long time in the tumor. And you know it goes away from you know pretty much all the other organs. So it really kind of hangs out in there. When you see that you think about a therapy, right, so you have long contact time with your drug. So what's nice about radiopharmaceuticals is you can go from I 125 you add six I 131 and now you've got a therapeutic nuclide. Okay, because I 131 gives off beta particles. And what's nice about beta particles is they're kind of like a hand grenade. They're very indiscriminate about what they kill. So you get a bystander effect. If you have a tumor not every cell has to express your thymidine kinase and you'll still get you know more cells killed than just the ones expressing it. So you know we talk about bystander effects with chemotherapeutic agents and gene therapy that have to get through you know from one cell to the next but here we don't really need to do that because these beta particles are remitted a certain distance away from the original cell and usually a couple cell diameters. Slide 73 ("Ex vivo biodistribution: EBV-TK(+) tumors with [125I]FIAU")

("Lytic activation and the bystander effect for enzymatic radiotherapy") Slide 75 ("Bortezomib-induced enzyme-targeted radiotherapy (BETR): dose response") So if we look at thymidine kinase producing tumor with FIAU you know we can actually you know these are lymphomas by the way. This is referred to as bortezomib induced enzyme targeted radiotherapy. So you know we can keep those lymphomas at bay if we increase the dose we can actually kill those tumors. And interestingly if we have tumors that are comprised of 10 percent TK producing cells as opposed to 50 percent we get a similar cell kill here which is sort of indicative of that bystander effect. So you don't need every cell producing it. And more significantly in my opinion even diseases like gastric cancer that as you know don't have a lot of good therapies can be treated with this particular technique. So we have a clinical trial that we're going to start in Singapore because that's where all the nasopharyngeal cancer, gastric cancer etc. happens to be and a smaller trial here to start testing this you know in patients. Slide 76 ("Bortezomib-induced enzyme-targeted radiotherapy (BETR): tumor response") Slide 77 ("Acknowledgments") So with that I just want to thank some of the people involved. Particularly, Ron Mease, who's our chief chemist and that kind of oversaw the chemists that worked on the PSMA project and Yiamao Zhang who did the bioluminescence imaging and collaborated with John Laterra's group and then of course Bert's group with the bacteria. And Rich Ambinder and De-Xue Fu for the viral study. And these are some of our funding agencies here. But these are all pretty much expired now. So I don't know if I'll be giving this talk much longer but [Laughing] with this stimulus package, right, that's going to save everybody. Okay, so anyway, thanks for your attention and if there's time I'm happy to answer questions if you have any. Thank you. [Applause] Are there questions, Dr. Rosen. [Male Question] So I'm wondering how [Inaudible] issues of diagnosis, issues of activity, issues of [Inaudible] and I'm wondering how onerous it is because it's clearly a beautiful [Inaudible] the way he presents it, but I'm wondering if going from A to D is relatively easy to generate or is this likely that 99 percent fail and 1 percent don't?

Well this is probably one of those 99 percent fail [Laughing]. No, you know these projects have really only been going on – all these projects since you know 2004, I would say. So I think that if you pick the right targets and you know you've have people like Bert Vogelstein etc. working – see one of the beauties about being here and the reason I've been here since 1990 is because of you know you guys. So all these projects are very collaborative, very interdisciplinary. We have all the equipment for doing this kind of work. We have the chemistry group for doing this kind of work. We're trying to develop a translational molecular imaging center here in Bayview, you know a couple of hundred you know yards from here. So I think that you know all we really need unfortunately – we certainly have plenty of ideas. We the money. Okay. Because it's a very expensive enterprise. So just you know to run a group that does this kind of stuff is you know is several million dollars a year. So as long as we have that then I mean it's not difficult at all. So usually the way that these projects get started is somebody will ask me to you know help them out with imaging and they I'll see if they can have a post doc that they can designate to do it. Okay, usually they don't. Right, because post docs have their own projects already. So then we have to try to hire a post doc just for that project. If you have a dedicated post doc or a dedicated grad student I mean you can really get anything done. The sky is the limit as far as I'm concerned. [Female Question] You know about Antony's point though, in our diseases it's – I mean in tumors and bacteria you have somewhat unique molecular targets. Whereas in types of diseases like ours or maybe GI diseases it's probably more an issue of intensity or magnitude of a particular gene expression or molecular expression so it might not be you know how much inflammation versus how little inflammation. Yeah, I understand what you're saying. So you don't have you know a nice you know like a cell wall or something that you can go after. But I think that as long as if you're studying these different signal transduction cascades and if there is a gene that doesn't have to be vastly over expressed. If you've something that's over expressed you know 4 to 1 and we can probably tell the difference between a patient with that disease and the patient that doesn't have the disease. You know if you know if it's even somewhat modest like that. But in rheumatologic disorders – I mean one big area – I showed you the infection stuff but we also have tracers for looking just at inflammation okay that we're trying to validate now. We're using if the first indication is HIV then the second indication is gonna be SLE. But I think that you know there are certainly ways to do that. And in fact, I think you know there are other agents I didn't even discuss. There are optical probes that work by fluorescence resonance energy transfer that are activated when they're near you know some sort of a proteolytic enzyme. Are there proteolytic enzymes that are up regulated in rheumatologic disorders? Right. So I mean you know you can do that kind of stuff. I just didn't show that because we haven't done it yet.

[Inaudible Male Question] [Male] We will. [Male Question] If there's a phenotype [Inaudible]. Right. So just think about is there an enzyme in your disease of interest? We can develop a probe. Usually we start with an antibody and then work our way down to the low molecular weight agents. Or you know there may be existing probes that we can use. So we have all the imaging equipment too. So it depends on how motivated you are to do it. It has to be like a really good target. If you're motivated to do it we can do it. Yes, sir. [Inaudible Male Question] Right. Yeah. That's just an inflammatory marker. Yeah, you can look at you know inflammation, it's not hard to look at. [Male] Closing comments from John Carrino our friend and colleague in musculoskeletal radiology. [John Carrino] Yeah, I think I know most everybody here, but I'll just briefly introduce myself. I'm the section chief for musculoskeletal radiology at Hopkins. I came here about two and a half years ago from the Harvard system and we developed a clinical service on the East Baltimore Campus. We have a clinical service that should start here in July. But I'm here today to who our commitment and support for rheumatology in the research area. I asked to have Marty give this talk because I think this is a fantastic area for us to collaborate with on the research side. And so as part of our commitment we're also building our research infrastructure. As Marty said, you need some people to make this happen. So I want to introduce one of my research associates, Dr. Antonio Mushadow, I'll have Antonio stand up. He's a research associate with me. He's working on an RO1 I have for interventional MRI. He's a Brazilian trained radiologist but also has experience in molecular imaging. He came from University of Chicago this past summer. So he would be that person who could help facilitate these projects or kind of develop these ideas. He's also starting to work with Marty on some of the ideas that he has. So I think this is one area where we can really work together to develop outstanding unique kind of research program. And I just wanted to – I don't get here as often as I'd like nowadays. In July that's going to change. We have new hirees coming in that will help us balance the workload. So you'll see me a lot more in the next academic year.

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