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WSCS 2015: The MSC Paradigm, Failed Industrial Clinical Trials, and Cellular Pharmaceuticals

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WSCS 2015: The MSC Paradigm, Failed Industrial Clinical Trials, and Cellular Pharmaceuticals

The Use Of Cryopreserved Allogeneic Mesenchymal Stromal Cells (MSCS) For Treatment Of An Array Of Immune And Tissue Injury Disorders Has Become Medical Practice In Many Jurisdictions. The Enthusiasm For Their Use Builds Upon Compelling Phase Ii Clinical Trial Data Published By European Collaborative Groups In The Past Few Years. Notwithstanding, A Large Multicenter Phase Iii Clinical Trials Conducted In The Usa Examining The Use Of Industrial Msc Products For Gvhd, Colitis And Stroke Fail To Meet Their Primary Clinical Endpoints Of Effectiveness When Compared To Placebo. It Is Worthwhile Taking Pause And Reflecting Upon The Apparent Discrepancy Between Encouraging Animal Pre-Clinical Data, The European Academic Experience And Industry-Sponsored Studies. This Presentation Will Focus On Articulating Plausible Failure Analysis Focusing On The Potential Variables Affecting Mscs, Potential Remedies And The Lessons Learned Informing The Cellular Pharmaceutical Field At Large.

Video Transcript

PRESENTER

Jacques Galipeau, Md, Frcs (C), Emory University School Of Medicine


Good afternoon folks and my iPhone tells me it's 3 p.m. sharp since we got the cleaners here and I'm sure you want to go and enjoy what hot light line night life Atlanta has to offer afterwards I want to I don't wanna be the one between that and you are evening so I'd like to thank you for the invitation to camp this week today gentle medicine meeting and I just said my talk is going to be something different owner lot of people are cheerleaders for the field then the glasses that consistently have fulled I'm going to be looking at it from the perspective of classes maybe a half empty but with a smile on my face because I think there are remedies for the field to move forward so that being said I'm preaching to the apostles here everybody's a believer in cell therapy and number of clinical trials now these are not clinical trials of meat but they don't bone marrow transplant I'm talking about experimental cell therapies and this is from a terrific data base that Alex email cure aids on his website.

He hasn't had the 2015 data by the invite you to take a look at that website now the USA leads the field china's very enamoured with the cell therapy platforms and you have a bunch of other players again this is data that at least see curated and using what's app published in clinical trials dot gov and the king of the field arms akimbo stomal cells by these are the most popular non-traditional cells in clinical use really still in exponential growth few cells are very hot of course especially with the advent the card these cells everybody wants to cook some cards in their garage to give to somebody and of course there is the clutch of all the other cells that are playing it down here so embassies.

I think if we're Donna be looking at sell out there that's fairly mature they could inform us of a pathway to follow that's the platform we should be examining now for those are not familiar with them is in came stomal cells there's really two buckets of stem cells in your bone marrow hematopoiesis stem cells which give rise RCD 34 sorts of crude marker here I saw the blood products this is the first time we'll be talking about him adequate stem cells and then you have MS a couple cells you gotta understand are very rare nucleated cells in your bone marrow one out of a hundred thousand to maybe one out of a half-a-million is amusing coma stomal cell so the notion that you can yank out a gallon Aero thinks the Embassy's out of that use that up front is really not practical now are any Kaplan coined the term's income all stem cell in the early nineties because when you take these cells and you put them in culture in their Poetry dish very simply they stick to plastic excels float off.

He started proliferating fibroblasts just going go without you having to do anything so in the IRRI prior to synthetic cytosine was very easy to grow most drama cells in Vito and what was discovered is if you had you know either area TGIF bed or vitamin C you can tweak them into differentiation into bone roof at or countryside precursors hands them Syngamy progenitor cancer and early on in the field a quarter century ago everybody thought this would be the cat's meow to bone repair it was all like bone repair stuff but what has been discovered since is that there are MSC like cells over your body now people get their knickers in knots about Caesar stem cells are not stem cells and seized from fat too different from Boehner.

I think they're all six degrees of separation I lump them together as Manlike so and the bulk of the embassy like cells outside of your bone marrow are almost certainly parasites now parasites are cells that populate to basically you have your capillaries you know your arteries arterioles capillaries and you have to oppose capillaries Venuses the capillaries venues is where you have these parasites and USA macro phages and these parasites can also be harvested and stomal vascular fraction from fat and you put that culture is all parasites people say they get him to cease from liver or brain or whatever it's all parasites and the very much behave like embassies when you culture expand them in Vito some more prevalent and what also has been we've learned over the years is initially peopled saw that huge scored in MS ease into tissue.

They would trance differentiate into that tissue replacement all of that is bunk they don't do that what Ms sees do is the secreted factors and again are any Kaplan likes to whine that them and the factors fall basically into two buckets anti-inflammatory and tissue repair so isolation of embassies I'm a haematologist and oncologist you know you have those dimples the back right here so freezing like the dentist does not go in with a Trojan under local anaesthesia in my office I could pull out 20 CC'sAmero without a lorry are put a band aid you can walk out of the office and will play go easy as that you take that twenty CC's Aero and you put thepetrovic I spoke of and having just 10% metal calf serum in DMEM Ralph or whatever you want the MSC stick to plastic they start growing and they grow and grow up they have a phenotype which is a clutch of cell surface markers which are non-specific a lot of mesenchymal tissues express these 1057344 probably if you want to see if the most precise reductionist cell surface marker.

I would suggest his CD 271 which is a low-affinity nerve growth factor which probably enriches the most the clonogenic precursors what's important is adorned expressed to the 45 which is that ban him out to put a marker again when you grow cells in Vito at first monocles will stick to plastic you want it gone except monocles and embassies and they don't express he cam one which is an endothelial cell marker and again the picture dishes what they look like mouse human actually most of the most difficult than missus to grow embassy some humans monkeys pigs dogs horses they grow very easily so I describe the procedure and you're Donna ask you why is it a miss these really are a solution looking for a problem to fix because one property in one property only and it's this one is if I take twenty ccs of your bone marrow and I put it in culture within two to three weeks I can generate a gram of yourselves tend to the nine sounds.

I know relatively what I'm doing and especially he used to play say I can generate a kilo of yourselves so these cells even in mature adults have a really robust proliferative capacity in vitro as opposed to see skin fibroblasts little poop out after a week in there becomes very quickly so you grow these and you can have a cell pharmaceutical so here is a perfect solution looking for a problem to fix now there are two types of problems are fixed agenda medicine new modulation I'm Donna focus on immune modulation of the first piece of data DCMS he's a fascinating field because the first clinical trial done in people was done without any animal dealer it was done just on the in Vito basis the first real meaningful animal data was actually published by here and there speciality journals was sponsored by a Cyrus yonder and what they did was basically modulation they a monkey they took skin from monkey a and put it in the form our monkey bee and skin is the most immunogenic wish you could transplant car across.

The barrier and if you do that after seven days of peace is Donna fall off if you gave him a cease to the monkey it took eleven days to follow so this was the first piece of Caesar really immune modulator in Villein a simian model you know Helen launched a thousand fish ships the paper that launched dozens of clinical trials is this one looking overseas for graft-versus-host disease so Katrina LeBlanc is an empty PhD bone marrow transplant at the KarolinskaInstitute in Sweden here we go there she is and she published a case report the Lancet in modern medicine published case reports now I'm back journals is virtually impossible that's really old school but the reason she got away with it is the following its nine year old boy was dying of graft-versus-host disease after allogeneic bone marrow transplant so if I think overheads marrow and I put it in you and there's a level of mismatched.

The T cells from a donor Merrill attack your liver your skin that's called graft-versus-host disease thirty percent of patients get that good goodly fraction will die from the gut involvement in particular to hear this young boy had another transplant in the open circles are stools richer circuit have diarrhoea and a black circle are liver dysfunction and what Katrina did and then Sweden they can do this because they have this dispensation so they can do crazy staff if your somebody is like dying there's no option that they can just say I'm Donna do I think's Donna work so she went to the boy's mom collecting moms marrow group moms and misses a tackle identical to be technical and gave the boy moms embassies added those two million cells particularly here there was a response there is a relapse of you age.

She gave a second those seventy days after transplant the boy went to complete remission of graft-versus-host disease they eventually relapse and he died but this is what show remarkable because when you have a great for GVHD a death sentence and this was truly Lazarus effect so this was followed by a series of academic sponsored phase 2 studies largest of which again Katrina here and lancets fifty-five patients are large multinational grouping in Europe and we're getting terrific response rates seventy percent of patients regarding either a complete or partial responses graft-versus-host disease truly astonishing multiple phase 2 studies replicating that their field was on fire so the logical next step is it completely logical industrialised so the Daily Show you initially was I P they came out of KingsWestern which will eventually made its way to a Cyrus and their the idea was they would take a bone marrow from young college kid.

I presume given time to 200bucks I presume and grow from their website this from their website 10,000doses from each donor and they would freeze ass off and they could ship those through the East Dakota Cancer Center and all they had to do there's that the frozen cells saw them the way you with fresh frozen plasma hang it and give it to the patient and they would treat all sorts of the ailments I'm I'm sorry to see that is not treating male pattern baldness but that's a different discussion so completely logical however in 2009 they issued a press release now this is now six years ago about the protective face three randomised study where they failed to meet their primary endpoint of getting complete remission agraft-versus-host disease and steroid refractory patients so in their hands in a series was no better than a sugar pill they've never published a paper they presented this as an abstract a demeaning that's so I can't comment on the rest of the other that they don't how was analysed.

I haven't published it so this is a bit of a bummer now after societal of Athersyscause I love bob these rules moved on but anyway they have another MSC like product difference to me PCs its technology from Catherine 4429 miss you like so universal donor they claim they can grow a million doses from a single donor in April of last year they tested their product in subjects with ulcerative colitis which is an autoimmune disease a large bow and it didn't work they did not meet the primary endpoint of efficacy April was not a good month for this company is in April 2015 this year they tested their product adult subjects with acute stroke and they were giving it a stroke and they were asking improve the outcome of stroke and the answer it did not they did not meet their primary endpoint of efficacy and stroke so here we are I haven't shown you a single piece of mouse dealer now.

I did a PubMed this morning as I like to do so before Clarke'Como stem cells and mouse you'll get nine thousand seven hundred and fifty-three hits I'm not picking up this number I have published dozens of people be demonstrating that embassies in marine models will have not only as a signal a statistically significant but substantial there on a wide array of immune inflammatory disorders this has been replicated ad nauseam and multiple labs across the world academic and industrial she had this overwhelming way of preclinical animal data that says this should work you have faced two academic trials in Europe predominantly showing a strong Shinto yet the industrial studies failed field now I'm Canadian moved to USA six years ago I was a lil have you as my grandson come with me boo too I had to write a bunch of NIH grants so you're writing grants and you know I'm an industry expert rightly.

I am so very and you know you get this pink sheets and you know this like a getting a double open hand slap across the face and you say Galilean you're smart you know all these studies are negative you know move along you know you something else this is a waste your time so I sat in front of a piece of paper with my pencil and I was figuring what is it that I can say to these sceptical quarrelsome stiff neck tonight reviewers I would possibly convince them that Mr C's are dead that there's actually a rational explanation that can be hypothesis driven and remedied understanding why the industrial studies yet the academic preclinical animal data works and here are four points I'm going to bring up for discussion today first donor variants I told you after six when Dorner.

I told you that a Cyrus R under 10,000 doses so let's talk about a bit of cell biology ms season this occurred tune from everything written by the amphibian number years ago you see a gazillion pathways I miss his work when you see that immediately you're sceptical because that means that nobody knows how it really works but the one thing you need to remember is this interferon gamma stimulation of embassies in Vito substantially deploys their immune modulators properties they become profoundly immune suppressive when the interferon-gamma activated there in Li Li is probably the RosettaStone to understand how the pico there's certain well understood mechanisms of action by which well-documented suppressor cells I T regs in the critics cells can attenuate the immune response and that's what they're talking about 10 the jet bad carcass to which makes prostaglandin and ideal one.

What we did is we took in his seized from normal human donors before and after interferon gamma stimulation and looked at the irony of these genes and we found that Ms season ever make I'll 10 under any circumstance ever make a bit of TGF bet abut it's not a breakaway by interferon-gamma but both cocks to an ideal one or massively up-regulated at the RNA level not ideal one we went onto show by Western blot you need to do that the protein actually goes up and virtually absent and culture expanded resting embassies which is this Stephanie companies in Jack and interferon-gamma activated ideal goes out so how do we test functionally this activity so we do what's called T cell suppression assay sunrise on the assets done.

I'll take 10 ccs of your blood and then I put in the centrifuge and then it separates if you put a bit of vicar you get the red cells and neutrophils in the bottom and you get at the interface lymphocytes and monocles that's called the Buffy CO and if you look at the Betty code by flow optometry what you have here despite his sights can afford to scatter about monocles and lymphocytes up for 21 so I buffy coat is a commish this what you have in fractionated blood all the granulocytic those are removed.

We do is we take these T cells and we had diabetes which is something you buy commercially hasn t cd3 EQC 28 crossing city CRI Cote d macro molecule and it's cool that he sells interests are proliferating sort of mimicking immune activation and if you look at the cells with a diagonal CFC and they're not stimulated high but if they're stimulated as this daughter cells split 248 16 so on so forth it dilutes the dying and you get this big comet tail here so we looked at the embassies of a bunch of normal human donors number 303 to 311 and if you had when I miss cheaper 380 cells activated completely blocks t-cellproliferation you see this is proliferating cells block an issue diluted out the mess he's 21 for 27 T-cell the T-cell sort of overcome the MSG effects are growing but if you look at this middling ratio 129 you'll notice that donors 3088 is still very suppressive yet the donor 305 is much less so which suggests that maybe more suppressive horrendous so there you go why is that so what we did is we took three of four normal volunteers donors grew their Missy's there's no idea protein in resting cells we stimulate them with interferon-gamma if you had to be the TNF alpha jacks up the effect.

The Ideal Protein goes up if you normalised a loading control you do a ratio what you find is you can normalise I'm out of ideas on what we found is that which was not surprising is the more I deal with terrorism or t-cell proliferative your there's a good correlation so why does this matter and me that if you're a very good idea inducer and besides use a very poor idea inducer year or Cyrus used his cells to grow ten thousand doses there be there in much more trouble than if they had used yourselves so that's where donors selection bias may have played a role industrial studies please remember the academic clinical trials the academic centres when they go in this Eastern donor they grow at most 10 doses 10 out of a donor the year Cyrus product they grow 10,000 doses of a donor sole's hang onto that the notion there so the other thing also that we found that if you activate interfere with interferon-gamma before Hannah told you they were good I D O and the completely blocked the bill Timothy cells to make interferon gamma and not only different gamma block the T cells are making.

I'll to as well and TNF alpha and you see activated T cells embassies were quite different gamma work even past and this just a different way of activating the sales began showing that it different gamma activation arguments there noon suppression and it's believed that if you inject him this season the person and they see interferon-gamma because a person trained that's how the Embassy's get really jazzed up as for blocking t-cell proliferation among st others and again these assays have been there I'm part of the ICT the embassy committee so these concepts have in mind in a couple of position papers about how to interrogate the immune plasticity of embassies so how does this work so here's our NEC can really like this read out because you know what you should see my grad students by grass roots walk up to me you Dr. Gallup I want to do a gene chip in to 50,000geez.

I said, I don't want to hear about that he shot me to see 500 days go up to300 jeans go down I have no idea what it means are in a Sikh is much simpler because you choose your target genes and quantitative who looks at RNA levels so what we did is we took him is seized from normal a normal subjects and we interrogated 47 genes that we chose is because in the published literature people have claimed that these useless290 left police some sort of role in the immune modulation by embassies so we looked at these cells and after they had been exposed to activated T cells in vitro which would be the physiological source of interferon-gamma and what happened to the gene expression and you'll see here some genes go upin expression quantitatively luxuriously ideal when you see blue is low and red is high.

I for your convenience I boxed hear those genes that really get jacked up after interferon gamma activation of embassies and again I D Ois a very important in June blocking agents were there a lot of jeans here they're important for regeneration HGF is important for regeneration IL six is there Jeff is there you'll notice PDL windows up as well as a blocker so what we did to be even more reduction is rather than using activated T cells without took Ms seized from normal volunteers a bunch of them and my little things appear there is he here before and after interferon gamma activation now they would just add to the recombining interferon-gamma now so it's just you two front gammas and what we found was again we had massive app regulation of these genes was the last time showing.

This business has been published it now and there are other genes that were published literature that people claimed were important in there in fact but in our hands we actually found the gene was down regulated by interferon-gamma but still you have a goodly list here of reasonable effect or pathways involved in the immune modulation me generation are particularly by different on gamma and this has real effects on activated T cells when you call culture embassies and activated PBM sees CPMC for example make a lot of aisles 13 but when they sort of tango with embassies the aisle 13 goes down simply file seventeen.

I'm showing you hear all this cytosine made by T cells go down significantly when they tango with NBC's network tradition are listed here and here are other genes that are actually produced proteins are produced by MS season when these see activated T cells got up regulated so again there's this cross time so I told you that when we do these asses with PBMC peripheral blood mono nuclear cells purified from a fire call you don't have pure T cells its T cells and monocles together and people sort of kind of pooh-pooh themonocyte so they're just like you know they're like watching the show they're just sitting there watching the show so we ask that question do monocles play role in the apparent immune suppression of embassies in Vito so what we did is we actually remove the monocles from the DMC's and inner suppression assay in you and what we found was that if you add embassies 2 p.m..

Caesar's lymphocytes and monocles you have suppression but if you ruled themonocyte the suppressive effect is markedly attenuated this suggests that the sides are not non specific bystanders a billion direct role in MSC mediated suppression in Vito and indeed what we went on to show was at MSC directly talk to minus sites and the man decides develop an end to macrophage phenotype and the monocle stem cells start producing a lot of now this is really important because this is work published by a group of the NIH as she had this terrific model sepsis she takes mice slices open their belly she tightens up their coalition stick the needle through it she shows up the mouse the bacteria from the colon seep into blood they get blood poisoning sepsis the most I if she gives the mice embassies I V.

The most don't I really clean reader what she and this is it a near the survival curve septic mice with embassies septic mice without embassies but if I clearly she depleted my semana signs before giving the Embassy's the therapeutic effect was completely lost and what she showed was imaging on the left I'll walk you through it when you inject a missed season at helping of amounts to the same thing as injecting in misusing their first capillary bed encountered will be the lung capillaries Messi's micro gram belies and everybody knows that 99.99% the cells go to the long the stick around for one to three days then they go away in minuscule fraction may redistribute at sites of information but the bulk of the cells are not what she found was that the MS easy eat grass where the capillaries in venues on and start interacting with them on our sites about half of them sort are kissing the mountainsides.

The other half the monocle actually phagocytosis the MSC so she went on to show that this in a C-minus I cross dock was really important because if you wanna sites doesn't work if you use a mouse that I'll 10 knocks out through their monasteries can't make it doesn't work and she showed that you don't miss easy if the Express cards do is important because of their cocks to knock out and prostaglandin it didn't work either so she came up with this scheme I like this model is almost moused now know you meant that Ms sees will operate the lead parks do if there is stimulated with LPS or interferon-gamma talks to express the gland in which then activates damn mama size knickers make I'll tend that's how prime she comes up with this model which makes a lot of sense that she's an asset suggesting.

Mr C's convert the long to an immune endocrine gland making out there so this cartoon we published summarises ten years worth of work you know I'm no brings tears to your eyes that ten years brought this but I interferon gamma will operate it ideal one in human mouse doesn't do it which is a vitally important Vito conversion of defence against Iranian regime bhakti cells and monocles and we have shown that ideal plays a role in Roman sites to introducing him to we've recently published at its own game activated she's also make one which also blocks T cells and I would propose the following model that embassies produce Cox who actually talks to them produced Cox two cocks to makes prostaglandin.

I'll 16 HGF which can directly talk to monocles and also lead to increased Nile 10 production so this whole thing that nobody knows any misuse how they work is bunk we know exactly how they're going to work we have a very good sense we're going here so the other big fish in the early days of the MSC field at least the industrial narrative was embassies are magical there just magical because I can't think you're in a cease and I could give him to everybody in the room and nobel-winning you reject them privileged there's something special about them especially their feed off their feudal embassies you can give him across species their immune privilege is that truth is that may play a role in why they don't work because when this artist me ten thousand doses and they're giving kudos a week for four weeks in a row are people developing immune reaction against.

If you go treat Crown's disease you're not going to get anybody by giving a couple of doses is going to be a leg Remade or anything else once a month Russia life will you get immune sensitises it possible premises to get to even be immunogenic will argue if you do flowcytometry embassies like any somatic tissue express MHC class one it'll be a praline different gamma resting embassies do not make him a chi class too but if you stay with the different gamma they make MHC class to class 1class to your telling me they're immune privilege they make it Missy class 1class to which is what at the gym presenting cells do they will not a prickly Costa military molecules see dat CD 8601 already showed that to test the hypothesis are privileged we did a simple experiment black six mice in bob's he buys are completely MHC mismatched completely homogeneous measurably use a black to white icon to make this simple for you to track these cells in vivo used a gene therapy trick we grew a messy from a black mouse and Jean engineer them to make a complete embassy don't make Depot but you knowIpoh that's a drug Lance Armstrong you know shot up so this but can't go up well when you have a pope reducing in a season a mouse you can track.

If I miss your cell I by measuring the hematocrit over time as if she's go away the council go down so what we did was here if you take any cease-fire black sixmiles have been genetically engineered to make you go and put them in a black 6recipient decision X the same as at August as your own team adequate of the mice the red cell chemicals from normal to say hi and stays there for a long time mice can tolerate I am adequate they won't get a stroke if you do use the exact same black 6 embassies making EBO and put them in a complete mismatch BobC mouse to get us burden of increasing red cell but it drops down over time back to base line so we figured all well maybe we messed up let's give another dose so we gave another dose what happened is again this is spaghettibloxorz each individual mice the increase was last in the duration or shorten it.

We gave a third hardly scored any effect at all this is a classic pattern of a low immunisation embassies are not immune privilege don't let anybody convince you about that now and transfer orthodox transfusion medicine will inform that you know when you get transfusions you gotta go red cells which don't express MHC molecules or platelets express MHC class one and it's well known that if you received random donor match platelets that about 40% of people eventually get our communities within 28 days so it's well described that when you infuse in a person I MHC class one mismatch sells a goodly fraction.

We'll get our immunise now other groups have now replicated the data we've published in fifty showed that actually don finney went a step further because you actually injected in his season the brains of monkeys' brains are supposed to be immune privileged like your eyeball type of thing even though he did that the monkeys developed an immune reaction against Mitt smashed in a season putting their brains so reputed transfusion of unmatched Missy's may lead to an alloy mineralisation I didn't say will lead mainly culture expansion not going to dwell upon this because I told you the industrial studies didn't work now talking about talked about donor variants we don't know how they chose their subjects or if they use the right parameter to select the donors.

I just showed you that our cells can get them you directed how culture expansion sot he five those forces 10,000 receive a million dollars paradigm and then I'm not making these numbers up because five doses with academic centres do is Cyrus a million is a three assists so when you do in Vito expansion people passage and this and that let's just say that most typical clinical doses are grown up within about two to four weeks to generate a couple of doses out of a donor and anything beyond that is whatever you want to the question we asked was our replication ship them is seized equivalent.

Just messinTennessee's so in essence is a phenomenon that is called he flicks limit he likes a guy in the sixties that took cells from foetuses and ask of how many times will these double and the limit is far too kind of fifty-two doubles now to expand actual 52 use of very large number but that's sort of kind of the limit unless this has become transformedHeLa cells and that's a different kettle of fish so you take these days 0 you split em you they grow you receive them a low-density they grow you receive them low-density they did grab it so as you go on eventually after see forty days in serum and serum that's only way we can generate senescent cells using serum is much harder actually if you would like to see but that's a different discussion they stop dividing and there're markers for that's in essence the cells are but gal positive.

You do flow optometry their big fat cells who hear words with him this season but he was engaged so you know the big fat round cells are senescent cells little spindly once reserved replication and they're all G00 big fat that is positive and there are virtually indistinguishable by the phenotype with it sells so shitty 1944 all the ICT markers are identical but we can't suppress T cells in Vito so this is a slightly more sophisticated readotra t-cell proliferation CFC deletion interferon gamma been activated T cells proliferate to make it different gamma you had fifty Massey's they block over this he had suggested indices senescent embassies actually stimulate T cell proliferation and this is just in trying here so innocent embassies have an intact phenotype if you grow your cells as you want to generate the gazillion doses you won't be able to tell them apart unless you interrogate her directly they haven't altered transcript own and ensure that the immune suppressor properties are messed up and there is some clinical data that suggested.

The late passage embassies are not as good again this is out of Sweden and the camera web cam they've seen so many patients or GV age and they had sort of late passage production runs an early passage production runs and again this retrospective but patients received a missed season were more lead pastor production runs the outcome was not as good as early passion show again is soft the retrospective data that suggests making five doses of a donor is probably better than making ten thousand doses of its owner ok so if there's one thing I want you to remember at the end of this conference on a Saturday afternoon is this Mrs are like sushi who here or go buy sushi for their partner and say you're flying back tomorrow from Chicago ok I'll put the shoes in the freezer for you and him are you consolidate needed nobody would think about that any scene graduate student whose PHDs depend upon your in experiments for them to graduate using embassies injected in the animal never ever use frozen cells.

I mean they could cause of the freezer put him back in culture make sure the cells are growing and they're perfect and I don't know about the students near allowed this tomy lab they read books to their cells to come in on Sunday to changing media to make sure they're perfect they harvest the cells fresh to put him on nice they run to the animal facility the injected cells to the mice 99.9% of the published preclinical data in animals use so we went from I told you nine thousand seven hundred and fifty-three papers themselves announced virtually all which sells to clinical trials were what do we do reproduce these cells massive expansion almost certainly also nelson you harvest your freezer and then how it's done is they thought the bag at the bedside and their thoughts cells are delivered I V to the subject within four hours with the question.

We asked was I thought sells the same as life so if you take so straight out of the freezer there's a readout for dead and dying cells its p/enxn and Ms easy human embassies you'll have a lot of dead cells and dying cell she had like cells and that's okay it's kosher the FDA tells us that you can inject a product in humans as long as no more than 30% of the cells are dead and that's ok now these are cells are kept in culture continuously of course are we more live cells and S&;P eyes very much more sensitive and try pan blue because this is indistinguishable by the way to the question we asked was our thoughts cells as good as life sells these are the exact same as season of course you anticipate the answer is of course not so here you have T cells are proliferating.

If I add fresh life it perfects happy embassies they completely suppress t-cell proliferation if you take cells straight out of the freezer and put them in the petrols activated T cells they worked no better than spit now you can do more sophisticated experiment where you do a dilution of a mystery to PBMC ratio live resides cells in live cells are always superiors suppression trial will catch up so they're not totally dead but they do they're clearly blunted in their ability to reduce block user proliferation in vitro and what we found was you talk to the Shroud cells straight up the freezer this is a missile proliferation in Vito control I'm sorry this is T cell proliferation in Vito or control if you had sought in this season told you they don't work but if you take the exact same thought in a season put him back in culture for a day for them to sort of recovery emotionally from the thought you know me like the film aliens you know they come out of crime freezing dark coffee huh they completely recover their immunosuppressive properties within 24hours so that the freezing there's nothing wrong with the freezing it's all about the phone and we repeated this remarkable donors' why is that so we basically user interferon gamma tree.

So if you take so Street of the freezer new stimulated with interferon gamma you look for a readout up regulation of IdealProtein thought cells immediately post doc can up regulate idea well as if you put American culture for a day or week we can why so bad chemistry interferon-gamma biased receptor cells are still had the interferon-gammareceptor phosphorylation estate 1 which the least DNA and up regulation of ideas on a good staff so I showed now what we found was that followed cells have a blunted fossil that one response to basically the machinery downstream of this which doesn't work no more than American culture it recovers why he cares thought cells undergo heat shock response so you will look at heat shock proteins in three different volunteer donors and we look at a time course these heat shock protein 70.

A&;B are massively a particularly immediately apropos thought is this song is that a physiological see so it's really acute cell injury and reverses and as soon as he goes back to normal the cells are proliferating so basically he charged proteins are owed to metabolism with p53 is to DNA and homing in Geneva so to ask that question in and again no none of these me in Vito tests foreshadow this we took a unanimous seized from men injected them to tell being a black six mice and we can interrogate told you inject Sylvan all the images so we looked at we fished our human Emma seasons along by killing myself a day later together laundry PCR for human DNA so if you inject live cells in the mouse twenty-four hours later you kill them I was it's very easy to detect DNA this number of PCR six cycles on PCR machine the more cycles you need the less DNA there is a year has lots of human DNA in the long and it disappears after 48 hours and we can't find it anywhere else if you inject the exact same time this season the exact same donor right after thawing we actually cannot detect any human DNA in the lungs of them.

I SAT 24 hours the same as if we had injected salt water and we looked at all the other tissue in the mice spleen bone marrow the kiddies noting the cells are cleared why one explanation is the following so when you free cells the cells undergo a process called vitrification descriptive the cytoplasm becomes my class that will actually completely deeper liberalise your action cytoskeleton that was described a quarter century ago to hear this is fellow you do in staining live in a seizure trypsinized you just receive them down and they have very beautiful cytoskeleton is if you take so straight up the freezer.

You let him sit on plastic the cytoskeleton is all messed up and is actually produced in quantity so basically when cells are there at the side of his deep memorised basically that's a bag of broken bones there's an impact on the bio distribution so when we did we took live cells and treated them as I totally Cindy which is a fact in the polymerisation doesn't kill the cell and we did bio distribution studies with these cells this completely replicated suicide accolades in these cells were not human cells were not be covered one nice so these are alive they were not charged so there is a plurality of things together effect fossils now getting Katrina bunks group showed another phenomenon is actually fascinating but Meneses the bottom of the Petri ne dish and you had whole blood on top in misses with the sheik regulation.

Missy's expressed tissue factor and haematology geeks here in addition factor but blood coagulation if you do the same thing with sodomy sees the clot formation is way more vigorous caught in a season the sheet cladding just showed that thought in his season new Vito are susceptible to complement mediated lysis live cells much less so the thing we found was that if you take embassies live and thought you had activated T cells and activated T cells with diabetes and start making perforce and granzyme they just go crazy and this sort of lose their stuffing the media live cells are quite tolerant to activated T cells thought cells get licensed activated T cells in Vito and for example here live cells when Emma seaport to T cells we still it can recover a lot of his season you use the same ratio and side cells much reduced their when MSU perform activated T cell slot of indices all the cells are gone so there's nothing wrong with freezing nothing it's all about the fine now I think the concept and sharing with you.

I'm focused on embassies these concepts I believe are true of any somatic cell has been utilised the cell therapy approach if you're you have a small company in all your IP is based on I'm Donna make this up you know he paddocks cells to treat Parkinson's disease I'm taking the time and all the data in mice were using live cells but then the NBA types your company said life sales forget that's not deploy a ball less reason it will be a business model around frozen cell but you haven't convinced yourself you haven't shown yourself that the frozen product behaviour the same as alive especially if you're going too far it is where I invite you to verify that because you can go down a road that would be very disappointing so thought cells undergoing heat shock response are susceptible to tease out killing acceptably compliment medial.

I cystic dilation they have abnormal membranes that's why I think they are cleared also in defeat in vivo and in accelerated way they have an abnormal cytoskeleton but other than that the perfectly fine there one lesson here is that a twenty-four to forty-eight our culture completely rescues the phenotype so to the NIH I said I have hypothesis driven remedies to what I think ills embassy lakes cells in their utility as a cell pharmaceutical for immune disorders to the problem of donor variance in the city one may consider using tautologous cell when feasible no forget those 4753mouse peepers most of the times embassies using the mouse models in genetic back-to-back six bucks it about see so that I get our guests culture expansion that advocate low passage European style no more than five to 10 doses I understand you wanna make $10,000.

I get them but there're reasons why I'm that may not work and I think the money shot is really to avoid a freezer burn at the low-hanging fruit so this informed clinical trial that we initiated category using a colleague ass Marin Drive MSC he's brash for Crohn's disease where we did it was scarce collision a single dose two million five million 10 million cells pretty low so well tolerated and I&;I signal we only could give windows because the FDA was so impressed that fresh cells with different from everything else they thought it was an entire new seller pharmaceutical and they did not allow us for a free human study to do mufti dossed which will follow later from this trial will be presented at the ICT in Singapore in May2016 I invite you to go pee.

I was super intense dairy and I think he was in the audience here somewhere Thank You KY from spirits this is also informed clinical trial led by a Munich a net Walter we're here we're using auto logous marrow derived cells from patients that had this tablished graft-versus-hostdiseasetomorrow after they have GBH using at all against Merrill to treat that we now have three patients treated so it's feasible to use a college sells fresh and we're basically asked me a scientific question just been ultimately when we do an effective and effect a study where the primary endpoint is advocacy if we do achieve a substantial and point result then.

If you see the difference then you can go back to a thought product and try to reverse engineer it to achieve the same in points so what's the future the field I propose to you and miss you version 2.00that the universal donor embassy platform has a role to play in that's where the acute injury syndromes where a colleague of cells are simply not practical or feasible stroke heart attack acute respiratory distress syndrome radiation exposure there's no time for two to three weeks to grow your cells it has to be off the shelf and I'll immunisation doesn't matter because it takes two to three weeks to get out and these diseases the outcome please itself on the first weeks let you know I think you need to resolve the riddle post our cell injury and the other thing is and that's not going to feel is the notion of augmenting the embassy functionality prior to transfusion.

I told you that it different gamma arguments activity dependent for the embassies to get activated sort a kinda all their own in vivo y not pre activate them in the best tradition a controlled manner you could have an enhanced pharmaceutical I'm totally different game could be other means of augmenting the cells all the way up to gene enhancement and of course decipher the mechanism action so I think for a cute injury syndromes were time is of the essence and you need to act within three weeks and no long-term use anticipated that fit I mean fresh crab meat trashy can be culture rescue bellies fifth allogeneic cells is the way to go and maybe even pre activated our genetic sounds for chronic inflammatory immune disorders I'm talking about and mass krone's you know those ailments a year dissipate that you're Donna need even this regular probably the optimal platform to buy assets in your favour with the Italian embassies.

I'm unbiased adipose your bone marrow-derived I think it's a sort of the same difference honestly which brings me I know there's very industry driven so I'm Donna make a few points about self-help industry in the sustainable pricing strategy in particular having to do with the MSC world in particular everybody knows about them was the first fda-approved cell-based vaccine technology in the us-and they went bankrupt was at the year last year so they won the battle of FDA marketing approval yet they lost the war of business sustainability and why is that so it's a combination of two things price in substantial effect and now people have prochymal Richard from a serious which has now been acquired by mesoblast by the way I have no conflict of interest.

I have nothing to do with either outfit they got approval for use of the park I'm a product that's your Cyrus indices for pediatricgraft-versus-host disease in Canada New Zealand so I'm Canadian Army haematologist I go back what's the mile and the largest paediatric BMT Center in the Eastern Canada's and Montreal's changes to the hospital my friends and I asked him since has been approved candidate for a paediatric DBA job how many of the children with GBH in your practice here two largest eastern Canada operation have been treated so how many have been treated as a hundred kids safety kids 50 ok you're bold0 not at your own bang on not a single child has been treated so it's been approved you can say why am why has no kids treated because and this is not my words this on the net you know.

I gotta love the net the transcripts of the 2013 Q-one discussion Osiris quoting check Mills now the new head of Serb blah prices are we look at $200,000 for a course of therapy in the paediatric setting sothey wanted two hundred thousand dollars for a course of therapy in the paediatric setting their product can tell you in Canada that's a non-starter especially if you have a product that's ruggedly to demonstrate that's better than a sugar pill so I would say a word of caution and realism is if your start up and your business model and half a million dollars per course at room by the way they have a million boxes with Novartis would like to charge of the current peace all products marketing approval are you Donna charges that you have to get that C arty if you allow yourself to death pretty much and if you're the reason that then on product failed we're asking$93,000 per course of treatment for a product for men with prostate cancer and these survivals advantage on the average was 4.1 months so unless and he was typically significant that's where they got FDA marketing approval so a word of warning.

I think you have if your Donna moves for with the platform I think you have to move forward with your the best foot did you get a say well you know what I thought our generic product expanded yards it might be good enough just to make it even if you do that may not be enough for you to win the war at the end of the day so I think there's a role for academic health centers on an academician at Emory and I think the role of academic health centers is that we need to be thought leaders because we can say whatever the heck we want from the audience is and I think we need to innovate in sustainable I think that's the key you know if it's two hundred thousand bucks and nobody uses its high evidence-based medicine evidence-based medicine cell therapy in the academic health centre in space.

I think we have a role to play 2d risk industrial deployment and that's what we're doing at our centre the Emory personalising new therapy centre we're helping start ups to don't have all the infrastructure do engage in force in human studies and we're motivated we think so therapies actually a very interesting so based platform I haven't touched the pipette man in years this is the team at emery cloth at work and Cold play good friend of mine the Canadian are brought to south with me when I came here six years ago tragically died prematurely at the young age of 42 this summer so this talk today is a dedicated to his memory and these are the sponsors of much of the data presented today and for those are interested the MSC the premier embassy meeting is the international media cell therapy next meeting in Singapore in May so if you're interested there you go thank you much for your attention.

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