NIH CLINICAL CENTER GRAND ROUNDS
Episode 2009-008
Time: 1:04:31
Recorded March 4, 2009

UNDERSTANDING AND ENSURING THE QUALITY OF CELLULAR AND GENE THERAPIES
Dr. Keith Wonnacott
Chief, Cellular Therapies Branch, Center for Biologics Evaluation and Research, FDA

THE USE OF MOLECULAR ASSAYS TO ASSESS THE POTENCY OF CELLULAR THERAPIES
Dr. David Stroncek
Chief, Cell Processing Section, Department of Transfusion Medicine, NIH Clinical Center

ANNOUNCER:  Discussing Outstanding Science of the Past, Present and Future – this is NIH Clinical Center Grand Rounds.

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ANNOUNCER:  Greetings and welcome to NIH Clinical Center Grand Rounds.  We have two speakers for you on today's edition.  Dr. Keith Wonnacott, chief of the Cellular Therapies Branch, Center for Biologics Evaluation and Research at the Food and Drug Administration, will talk on the topic, "Understanding and Ensuring the Quality of Cellular and Gene Therapies."  He will be followed by Dr. David Stroncek, chief of the Cell Processing Section at the NIH Clinical Center's Department of Transfusion Medice, who will discuss "The Use of Molecular Assays to Assess the Potency of Cellular Therapies. 

We take you to the Lippsett Ampitheater at the National Institutes of Health Clinical Center in Bethesda, Maryland, where Dr. John I. Gallin, Director of the NIH Clinical Center, will introudce our first speaker.

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GALLIN:   Good afternoon and welcome to Clinical Center Grand Rounds.  I think we have a special set of talks today.  I'm very pleased to introduce our first speaker, Dr. Keith Wonnacott, who is chief of the Cellular Therapies Branch in the Center for Biologics Evaluation in the Food and Drug Administration.  His topic is “Understanding and Ensuring the Quality of Cellular and Gene Therapies.”  Dr. Wonnacott's branch is responsible for the FDA’s chemistry and manufacturing and control review for all cell therapies -- a broad class of products that include stem cells, allogeneic, pancreatic eyelet cell, immunotherapies, cancer vaccines, xenotransplantation products, and tissue engineered products.  Clearly, in the current trends of modern medicine.  His branch is also responsible for review of medical devices used in the processing and storage of cellular products.  Dr. Wonnacott who, has published several articles and book chapters on the regulation of cell therapies, joined the FDA in 2002 as a microbiologist and has been in his current position since 2006.  He received his Ph.D. and held a post-doctoral fellowship in microbiology and immunology at the Pennsylvania State University College of medicine and undergraduate degree from Brigham Young University.  Welcome.

WONNACOTT:  Thank you, Dr. Gallin.  Thank you for giving the FDA an opportunity to participate here in Grand Rounds.  We're excited to share some of the stuff that we're doing both in terms of the regulation of cell therapies, cell and gene therapy and also the science that we're doing.  They asked me to put financial disclosures and I have none to disclose but I have some obligations I decided to put there. 

My objectives today are to describe important FDA safety concerns related to cell and gene therapy and understanding the challenges and opportunities of developing cell and gene therapy and describe the FDA science intended to facilitate cell and gene therapy development.  This is the cell and gene therapy-centric view of the FDA to give you a feel for where I'm at.  The FDA regulates devices, drugs, veterinary medicine and food, and biologics.  I'm in the Center for Biologics in the Office of Cell Tissue and Gene Therapy, the other product offices include blood and vaccines.  I'm in the Division of Cell and Gene Therapy.  And in my division we do the manufacturing review for INDs, manufacturing and testing.  My office in general as was just mentioned regulates a large number of products, not just cell and gene therapies but cancer vaccines and xenotransplantation product, tissue and tissue-based product, combination products such as tissue engineered products and devices used for cells and tissue processing and storage.  To give you a flavor for the activity of cell and gene therapy -- INDs -- I put this schematic up.  What you can see is the activity in the blue bars which is cell therapy has been relatively constant over the last five years or so and gene therapy also has been relatively constant.  And -- but it's a very active field.  There's over 100 -- approximately 150 new IND applications every year. 

I also put this slide up to just say that this field is exciting but it's a field of the future.  Currently we have only one licensed or approved FDA product in the cell therapy arena and that's the cart cell product made by Genzyme. 

The FDA mission statement is that the FDA is responsible for protecting the public health by assuring the safety, efficacy and security of human and veterinary drugs biologic products, medical devices, our nation's food supply, cosmetics and products that emit radiation.  And the FDA is also responsible for advancing the public health by helping to speed innovations that make medicines in foods more effective, safer and more affordable.  So I wanted to put this out there to emphasize it's not just to protect but it's also to advance public health.  We accomplish that mission through a number of different mechanisms including research, policy development, inspection and enforcement, application review, communication, collaboration and outreach and surveillance. 

My talk today will focus mostly on application review and some of the scientific research going on in my group.  So if you look at product development, the product development starts in the pre-clinical phases and moves into the clinic for phase 1, 2 and 3 trials which in the case of cell and gene therapies require an IND.  And then it can go on to licensure which would be accomplished through a license application.  And when an IND is submitted, the primary focus of the FDA is safety.  The regulations say that our primary objectives in reviewing an IND are through the investigation to ensure the safety and rights of the subject.  As the product proceeds through development, especially in the later phase development before licensure, there's an additional emphasis of applying the quality regulations in a way that are appropriate to the stage of investigation and making sure that we can have a correct evaluation of safety and efficacy of the regulations say in phase 2 and 3 to help ensure the quality of the scientific evaluation is adequate to permit an evaluation of the drug's effectiveness and safety. 

So one of the things that I asked myself when I first got to the FDA was, well, what is a safety concern to the FDA for a clinical trial?  And how do people know what the FDA is concerned about in terms of the safety?  So we started a few years after I started, we -- a group of us got together and said hey, we can kind of go back and look and see what are the safety concerns with cell and gene therapy products.  So we have a unique access to all the letters that go to the IND sponsors for cell and gene therapy.  We took all of those letters and looked at the ones that pertain to people who are going to go on clinical hold which means that they have issues they need to resolve before they can get into the clinic.  And looked at all the reasons, the safety reasons that prevented them from immediately going into the clinic.  We analyzed the data and tabulated what were the common reasons for clinical hold.  We found that a number of different things.  I don't want to go through these lists point by point but I just -- suffice it to say that there's a lot of reasons, and the reasons are common across INDs. 

For example, one of the major reasons that a clinical protocol is found to be deficient is because the eligibility criteria is not adequate or appropriate for the intended use of cell therapy.  In terms of manufacturing we find most of the safety concerns revolve around either the viral and -- the viral safety of the product or the reagent or the testing of the product at the end with sterility being the most common reason for manufacturing, most common manufacturing deficiency.  For pre-clinical, it ranges from they didn't do any studies to support the research, the clinical research, or they didn't provide the data or there's some other problems with the design of the study or other problems like that. 

So what this did is it kind of confirmed to me that many of the issues that IND sponsors have are common.  And I don't have a slide to demonstrate this but I thought for cell and gene therapy that one of the reasons was that we had a majority of academic sponsors who probably don't spend a lot of time or have a lot of help in the regulatory arena.  But we found that it was -- it held true for a lot of the small companies that do cell and gene therapy research as well.  So we -- part of the reason we published the paper was to try and get the message out there, that hey, these are the concerns that people ought to be thinking about and I put this slide up here to just emphasize that there are a lot of resources available for sponsors to get the information they need to address all of the concerns that are listed.  And there are many guidance documents and other documents available on the Internet on the FDA website, sometimes it can be difficult to navigate but I have some specific documents as a reference at the end of my presentation that you can look at. 

Another thing is, you can communicate with the FDA before you send in your submission.  There's informal discussions that many reviewers like myself are willing to have and there's also a formal mechanism of requesting a pre-IND meeting.  Then lastly, remember that there's a difference between a grant application and an IND.  We want detailed information.  There's no page limit.  In fact, the more information the better.  We want all the relevant data and information included. 

So that kind of sets the stage for what we look for in terms of safety testing.  And as the -- a cell or gene therapy product moves into later phases of development then characterization of the product and a better understanding of what's going on in the clinic so that you can control the quality of the product and relate the potency of the product to what's going on in the patient becomes very important.  In terms of what this means for lot release testing there's a number of tests that we require that every IND sponsor perform on their cell gene therapy products before it can be administered to patients outside of the typical safety testing and these are required in the later phases of development and they include identity testing, which is defined as an attribute that identifies a product.  And distinguishes it from other products produced in the same facility.  Purity testing. which is relative freedom from extraneous matter in the finished product, and potency testing which is defined as the specific ability or capacity of the product to affect a given result. 

With cell and gene therapy products these are very difficult issues to grapple with.  For example, when when you have a product derived from an apheresis, a patient’s blood cells, there maybe multiple cell types contributing to the mechanism of action.  You might have the NK cells, the T cells and B cells all working together and if you want to call your NK cell product, an NK cell product, that's fine.  But what if only 50% of the cells are actually NK cells?  What are the rest of the cells?  Are they impurities or are they contributing cells to the efficacy of the product? 

And so here are some of the additional challenges that we think about when we see cell therapies and gene therapies come in.  One of them is that there's -- it's often a small lot size with a limited sample volume for testing.  For autologous therapies the lot size is one.  Often there's a limited shelf life.  There's limited availability for starting material for product, process and test method for development.  There's patient-topatient variability and cellular heterogeneity, there's multiple potential mechanisms of action.  That's lack of reference standards, complex manufacturing schemes with lots of different reagents.  There's an inability to terminally sterileize cell and gene therapy products.  Having said that we see a lot of advantages.  I'm sure that many of you as clinicians also see advantages to cell therapy.  These are exciting products with great potential.  The cells are themselves dynamic.  They have the ability to migrate, proliferate, differentiate and respond to their environment.  Both in vitro during processing and in vivo after administration to the patient. 

Multiple cell types and mechanisms of action can be involved in a single product.  The therapeutic outcome can be curative and permanent.  The targets may not need to be fully defined such ads tumor antigens where you're taking a whole tumor lysate.  Autologous cells -- autologous cells are matched.  The cellular product characteristics may inform you about the patients themselves so you take the leukophoreses product and before you re-administer it to the patient the product itself will tell you something about maybe disease status or potential for response to the therapy.  The cells can act locally as well as secrete factors systemically. 

So because we see the opportunities, we want to be able to address the challenges.  And so at the FDA there is an active research program that is involved in trying to address some of the challenges with cell therapy development, cell and gene therapy development.  Some of the things we recognize as needs are needs for improved evaluation methods.  They need to be more rapid with smaller product sample used for each test.  And increased quality and quantity of the data generated by the tests.  There needs to be a better understanding of the products and their in vivo function so we can understand appropriate controls and parameters for processing, testing and use of the cell therapy -- and cell and gene therapy products. 

Here is just – here’s an example.  First example I would like to share of some research going on in our division to address one of the challenges.  This involves looking at the microenvironment for cell therapy development, and specifically in this example, in vitro.  So one of the labs, Dr. Steve Bower runs, looks at B cell development and he found that he grows -- the pre-B cells on a stromal layer.  In a normal culture when you put the pre-B cells into culture and add IL-7 they will proliferate.  But if you then remove the IL-7, it causes the cells to either differentiate or undergo apoptosis.  But he found that if he identified a factor DLK-1 that was associated with abnormal B cell development and when he created stromal cells with reduced DLK-1 expression and put his pre-B cells into culture, they would expand in culture.  When you looked at these cells by flow cytometry, by cell surface markers, you really could tell no difference between these cells and these cells.  But when you remove the IL-7, there was no differentiation and no apoptosis.  Obviously indicating that altering the stromal cell had a profound effect on the B cell development.  And the role of DLK-1 was confirmed in knock-out mice which had altered B cell development and function. 

So it's just -- as I said, stromal cells can alter the cell product in a way that is not related or revealed in lot release testing.  So another example of research that's being done is research done by Dr. Jerry Marty who has used B cell clones as early markers for chronic lymphositic leukemia development and what we know is that the vast -- what we found is the vast majority of patients with CLL have a precursor state from six months to six years before the development of clinically recognized leukemia.  He's developed methods to be able to detect these -- this precursor state of monoclonal B cell lymphocytosis. 

So by flow cytometry you can take patient blood and analyze the light chain restriction of the kappa or lambda expression in existing B cells and in a normal polyclonal patient you see a great variety in kappa and lambda expression but in monoclonal B cell lymphocytosis you see a much more limited repertoire of kappa and lambda expression.  And when you further analyze patients using the immunoglobulin gene rearrangement from heavy chain V gene by PCR you can see that in monoclonal B cell lymphocytosis you already are starting to see clonealty in the cell population which goes on in chronic lymphositic leukemia to be almost completely clonal population of cells which is different from the diverse repertoire that's seen in a normal patient. 

So what you have is the ability to detect in simple assay of flow cytometric analysis of a patient cells that the monoclonalty which may potentially predict progression to CLL.  It can also be used as a measure of cell product safety which can be applied in a product testing setting your product is, for example, bone marrow or peripheral blood stem cell transplant. 

So another example is work that's being done in Dr. Peri’s lab.  Just want to take a second to acknowledge that Dr. Peri was going to originally give this talk and he sends his regrets, he wasn't able to make it due to a -- something that was rescheduled, a prior commitment that got rescheduled.  But work that has been done in his lab has demonstrated that you can use microarray to assess the quality of cell substrates.  And these experiments were done to kind of look at how can we look and identify how cells in different environments are able to produce gene therapy vectors.  So we used 293 cells that were seated or measured at different densities and transvected with an adinovirus vector and looked for -- and then it was analyzed to see what the vector expression was going -- would be from these cells after two days of culture.  What was found is that after two days in culture cells that were only 40% confluent at the time of infection produced a very high yield of infectious virus but the more confluent the cells were, the less viral vector could be produced from them.  And with the overconfluent cells being very low in their expression. 

So when we looked at the different confluencey levels by microarray, what we found was clustering of genes that was different between the 90% confluent state and the overconfluent state which could then be used as a marker to assess the overall conditions of the cell culture and whether they would be conducive to producing high-quality and high quantity viral vector in a manufacturing setting.  This becomes important in a bioreactor setting where you can't just visually look at confluence by microscopy. 

One other application of microarray is using microarray to identify tumor genicity.  And -- or the -- as sort of -- as a diagnostic for cancer cells and comparing normal console to HNSCC tissue, we found that there was a definite differentiation between the two tissue types that can be confirmed by QT PCR, RT PCR and tissue staining.  So microarray could be useful in characterizing disease or whole tumor vaccine.  I just throw these out there as a couple of examples to show that we are very interested in the science behind the product testing and the product development. 

One of the things as we think about microarray in particular as an example of a method that could be applied, as a powerful tool that could be applied in the cell manufacturing setting, we understand the pros and cons because we have scientists who are experts in the method here in our center at the FDA.  And it allows us to not only take that science and apply it to a manufacturing setting but it also allows us the expertise to properly evaluate and review products when this kind of data is submitted to us. 

So I would just like to conclude by saying the FDA mission is to protect and advance public health and the regulatory review and scientific research contribute to our ability to ensure the safety and quality of cell and gene therapies.  And CBR is proactive in researching and supporting enabling technologies for cell and gene therapy development.  I would be happy to -- here is a number of references that will are relevant -- a number of references that are relevant to cell and gene therapy IND applications.  I would be happy to email a copy of the slides to anyone who wants them.  You can just send me an email and I'll send you a PDF version.  But I would like to finish by thanking all the people that have been involved in the research.  Dr. Peri and his lab.  Jennifer, Oscar and Steve Bower and Jarred Marty and individuals in their lab.  With that, I'll end.  Thank you   

[Applause]

GALLIN:  We have time for a couple of questions if there are any.

WONNACOTT:  It was crystal clear.

GALLIN:  It was crystal clear.  Thank you.  And it clearly is a nice forerunner for our second talk which will discuss further some work using some of the cell therapy approaches.  And David Stroncek from the Cell Processing Section in the Clinical Center’s Department of Transfusion Medicine will present his lecture, “The Use of Molecular Assays to Assess the Potency of Cellular Therapies.”  Dr. Stroncek's section manufacturers a variety of cellular and cell therapy products for patients seen here at the Clinical Center.  Before he was named the chief of the Cell Processing Section in 2007 he was chief of Transfusion Medicine's Laboratory Services section for 11 years.  Earlier in his career he served as the first medical director of the National Marrow Donor Program.  Dr. Stroncek earned a bachelor's degree in chemical engineering from the University of Minnesota and an M.D. degree from the same medical school.  He completed an internship and residency in internal medicine at the University of Minnesota Affiliated Hospitals and completed a hematology and oncology fellowship there as well.  His research focuses on the use of gene and microRNA expression analysis for the assessment of cellular therapies.  He's done pioneering work in the fields of unrelated marrow donor transplantation and the mobilization and collection of peripheral blood stem cells for allogeneic transplantation.  He's also an expert on neutraphil antigens and transfusion-related acute lung disease.  So welcome, David.

STRONCEK:  Thank you, John.  So as everyone else who works at the NIH and FDA, I have nothing to disclose.  So today I'm going to explain some of the nature and scope of the work we do in the cell therapy laboratory.  Discuss importance of potency testing of cell therapies and the limitation of some commonly used assays.  Then I'll describe the emerging role of cell and gene or gene expression, microRNA expression and analysis and assessment of cell therapies. 

Our laboratory really, most therapies we use in our laboratory are developed by research labs here in the intramural program.  And the development of these protocols are really intended to go right to treat patients in the clinical center.  But these therapies can't go directly from the research lab to the bedside because typically a lot of processes used in the research lab aren't appropriate for using in a clinical laboratory.  So one of the missions of our laboratory is to scale up these clinical therapies and to modify them so all reagents used and all supplies used are appropriate for use in -- for clinical therapies. 

Another purpose of our lab as we scale up products is really to develop new reagent supplies and instruments to facilitate the scale of these therapies.  As you can imagine, a number of therapies are pioneered at the clinical center, so our laboratory has been creative in developing bags used for cultures of cell therapies, instruments used for watching and isolating cells, and instruments even for analyzing these therapies.  What we've also done somewhat but not as well is to develop better ways to assess these therapies.  And this seems to be the new frontier in our field.  There's a number at this institution there's a number of people on the forefront of developing molecular assays and using them both in the research setting and clinically.  So our recent focus has been on to develop better assays. 

As Keith mentioned there is a number of aspects of assessing cell therapies.  The one that we have been focused on is potency testing.  And the reason for this is, it's really critical to cell therapy, the more technical definition -- one definition is potency testing is a measure of a critical biological activity within a complex mixture by qualifying activity in a biologic system meaning does the product do what we expect it to do?  These again, the assays that assess this obviously should be and in vitro type assay be it an animal model or a cellular model.  But these tend to be complex to do.  Measuring potency isn't the same as does the protocol work or does the therapy work, because you can have a cell therapy that really does what you expect but there could be other reasons for why that protocol didn't work out or that therapy, be it related pre-conditioning regimens or there's something different about that particular patient. 

The other important thing about potency testing is it's required to be performed on all lots of particular -- that are manufactured.  For us, for a drug company a lot tends to be thousands or millions of doses of medication.  For us a lot is one particular therapy for one particular patient. 

The types of assays used for potency can be broken down into biologic assays and analytic assays.  The best assays are the biologic assays.  These tend to be the best ones of those are the in vivo animal models but the animal models are really difficult to develop.  They're really not practical to do on a real time basis when each lot is used for one patient.  Other assays involve cell-based assays.  Again, these are a little more practical but still difficult to do.  So as a result a lot of laboratories have been using analytical assays including Elisa assays, flow cytometry, ELI spots, flow cytometries and other assays are moving into quantitative real time PCR. 

Our particular laboratory is centralized.  This summarizes some of the products we make.  They include hematopoietic stem cells, dendritic cells, lymphocytes, NK cells and gene therapies.  Among these products such as hematopoietic stem cells we deal with mobilized bone marrow cord blood, isolated CD34 cells and soon isolated CD34 cell also deplete CD 19 cells.  We deal different types of lymphocyte products including manufacturing of T-2 cells and depleting alloreactive cells.  We make dendritic cells for several protocols and they're loaded some with peptides, be it tar peptide to treat prostate cancer.  We also have P-53 transduced dendritic cells to treat melanoma and some protocols include tumor lysate.  We're making expanded NK cells and soon to make expanded NK cells.  We treat chronic granulomatous disease with April peripheral and CD34 cells and skids with CD34 cells.  The point of this we do a lot of different -- we make a lot of different products and it's just not practical to have specific in vitro and in vivo assays for potency testing.  We have been using flow cytometry to really be the backbone of our potency testing, individual investigators for their protocols often will get cells from us from the final manufactured product and do additional testing but it's usually not a formal part of our potency testing. 

For that reason we've gone -- we think there's room for new assays for potency testing.  The reason why we think this is important, really have robust assays for analysis of these therapies is several fold.  One, these therapies are quite complicated and have multiple functions.  For an example, a hematopoietic stem cell needs to once it's transplanted needs to migrate to the site of engraphment, it needs to start to engraft and expand.  Then differentiated to several types of cell lines.  Dendritic cells need to migrate to the site of activity.  Often they need to mature there and present antigens and present co-stimulatory molecules.  Part of the issue with these multiple functions is that when we start to look at these cell types we may not even know what the critical function is.  This might be particularly true for dendritic cells and things like NK cells.  We think we know what's critical about it but many cases it's really not completely understood.  Next there's a limited time to analyze these materials and the amount of material we have available to analyze the cells are limited. 

So the methods we have been exploring to look at cell therapies are gene expression profiling and microRNA expression profiling.  We selected these methods because we feel they have several advantages.  One, we were using global platforms so we can simultaneously measure expressions of thousands of genes and hundreds of microRNA.  We can do this too with a very limited number of cells, anywhere from 10,000 to a million cells required and we can perform a full analysis.  Some of the -- there are some disadvantages to this -- these assays.  And one is transcription doesn't always reflect protein expression.  The platforms we use are not yet standardized.  They're changing rapidly and improving rapidly but still, there's a number of different platforms available and they're used slightly in different ways and different laboratories.  Finally the assays right now tend to be rather lengthy.  They take a couple of days to perform.  But we feel that as work progresses on these platforms and we identify genes and sets of genes and microRNA important for assessing cell therapies, new platforms will be developed and standardized and the time to perform these assays will really decrease.
Now I would like to show you a couple of examples how we've used these assays to assess cell therapies.  The first we looked at we tried to address the question on can peripheral blood monocytes be stored before they're processed?  And this is kind of new issue.  Years ago this wasn't a problem because cells would be collected at one center, processed at the same center, typically just down the hallway, and then transfused to the patient that was probably a couple of floors above or below the cell processing lab.  In that situation there's no need, the processing can happen very quickly.  There's no need for a lag time or storage of products.  With unrelated donor transplant development 20 years ago, we started collecting cells at one site, transporting them across states, countries and even halfway around the world to be processed at another site and transplanted there too.  And the need for transportation and storage has grown even more, because now we have specialized centers of processing.  So collection might occur at one center, be transported to a second where it's processed, then transported to a third center where the cells are given.  So this creates a need to have these transportation from one site to another can take as long as a day or two so there is a need to store cells for up to 48 hours. 

The model we used is peripheral blood mononuclear cells.  We selected that because we make quite a few dendritic cells.  The way we do that is peripheral blood mononuclear cells are collected by apheresis.  Those cells are treated to sort out the monocytes.  The monocytes are treated for three days with IL-4 to make immature dendritic cells.  The design of this study we collected an apheresis product, split it in half, we processed one-half immediately to make for dendritic cells and the second half we stored for 48 hours and then we performed the leukoforation, isolated the monocytes and made dendritic cells.  We compared the fresh and stored PBFCs. 

For the first part of the study storing cells for 48 hours at 4-degrees but we had no problems with the leukoporation, they worked well.  We had similar yields and similar purity.  I'll show you the analysis of the final products.  This is gene expression profiling of lymphocytes, peripheral blood stem cells, monocytes and the dendritic cells we made.  As you can see the cells clustered into groups.  The first group is the isolated lymphocytes.  Then there's a group of mixed peripheral blood mononuclear cells and the isolated monocytes really wasn't good separation but then the dendritic cells were all separate on the end of this heat map.  The important thing is if there was a big effect with storage we'd expect to see these different types of cells segregate by fresh versus 48 hour stored.  That didn't occur suggesting there really wasn't much difference with storage in these cells.  What we then did is looked at the 17,000 genes and looked at how many genes were differentially expressed between fresh and stored PBMCs and there were some genes, there were 273.  Similarly there were differences between fresh and stored lymphocytes and fresh and stored monocytes.  In fact we had quite a few genes differ between fresh and stored monocytes.  We had 711.  But surpisingly when we looked at the dendritic cells there were really only three genes whose expression differed, those genes weren't really very important functionally. 

So our conclusion here is that though we see changes in lymphocytes and monocytes over 48 hours of storage when we stimulate these cells to make immature dendritic cells those changes go away and the final dendritic cell product is no different.  So we do -- we try and process everything fresh but if needed now we will store peripheral blood monocytes overnight before we process them. 

Now we want to focus on potency testing we have done with hematopoietic stem cells.  Classically the model to look at hematopoietic stem cell potency has been a nod skid mice remodel, a repopulation study.  This is a transplant model that works great but takes several weeks to complete.  It's really not practical for a clinical lab.  As far as culture assays, LPCICs are a great assay but they take several weeks to perform and are not practical.  Culture in methyl cellulose is easy to perform but it's not very quantitative and not very helpful.  For analytical assays we still do total nucleated cell counts.  Believe it or not that's the gold standard for assessing umbilical cord bloods at this time.  New assays come along measured L to D hydrogenase expression not used much in the gold standard for many years has been measuring CD34 counts and that's what we currently use.  The problem of looking at CD34 counts it's a heterogeneous population of cells.  A small portion are primitive stem cells but we have cells that express different types of antigens be it CD34 and CD 38 positive or negative or CD 133 positive or negative or HLA DR positive and negative.  So these are at various stages of maturity and going down different lineages. 

So the CD34 population really is very heterogeneous.  This creates some problems when looking for potency because we know CD34 counts don't mean the safe if we're measuring in bone marrow versus umbilical cord blood versus peripheral blood stem cells.  They mean different things.  We have been able to use CD34 counts as a marker of potency because most of the products we collect are mobilized peripheral blood stem cells and we mobilize those cells with GCSF.  But that's changing.  Even though GCSF has been our standard for mobilizing stem cells for many years, there's some problems with it.  First there's a lag in mobilization, GCSF has to be given for three to four to five days before stem cells are mobilized.  That's expensive and inconvenient for patients.  The mobilization is variable.  Generally works really well but one to 5% of patients not enough stem cells mobilized for effective transplant.  If enough stem cells are mobilized on average clinical applications demand more and more stem cells.  In addition there's an occasional rare but serious adverse effect associated with GCSF administration and there's also persistent fears about possible long term adverse effects of GCSF administration. 

As a result people have been looking at new mobilization agents for many years, none have been really very effective until AMD 3100 or PROLIXA 4.  This agent has been recently licensed to use with GCSF to mobilize stem cells in autologous setting.  This works by completely different mechanism of action of GCSF.  GCSF mobilizes stem cells by down-regulating CXCL-12 and release releasing a proteolytic enzyme.  AMD 3100 works by the disrupting the binding of CXCR-4 and 12, as a result mobilization is almost instantaneous.  Following either subcutaneous or IV injection stem cells are mobilized in four to six hours.  This is a real advantage of using AMD-3100. 

This just shows a slide that shows a mobilization of stem cells with AMD-3100 given at time zero, this shows four different doses the peak mobilization occurs anywhere from 6 to 8 hours depending on the dose.  For 40 micrograms per kilogram is a licensed dose which has been licensed and people are using.  The real interesting thing about AMD 3100 is what happens when you use it with GCSF.  This slide shows people with five days of GCSF.  This is the fifth day at time zero, they have a fifth dose of GCSF.  The stem cells mobilized 70 million per liter.  When you give -- on the 5th day if you give one dose of AMD 3100 mobilization almost doubles, goes up to nearly -- well, about 125.  If you have five days of GCSF and 3100 mobilization doubles to over 150 million per liter. 

So there seems to be something special about using these drugs in combination and that mobilization is much greater than using either agent alone.  As -- from the other slide the dose of AND 3100 alone would have mobilized 50 here.  So there's a real appeal of using these in combination. 

So what do we know about, what's common thoughts about AMD 3100 in the clinical field?  This is -- I think what most people know about it is it -- AMD 3100 enhances CD34 mobilization, decrease thee number of days of apheresis required to collect stem cells, this enhanced mobilization makes apheresis collection more predictable.  Enables more autologous patients procede towards transplant.  It does result in prompt engraftment.  In general, it's well tolerated but experience is limited.  I just show this because it actually comes from a monogram by the company.  But I think this is what most clinicians, even transplant physicians have a lot of things they're dealing with.  So I think this is their understanding of AMD 3100.  While it's correct, it only talks about CD34 mobilization.  Our thought is, there's different mechanisms of mobilization by GCSF and AMD 3100 so these CD34 cells may differ. 

So the questions that have come to us, one question was, can autologous AMD 3100 CD34 cells be used in place of GCSF mobilized stem cells for gene therapy?  We had that question.  And we are waiting for this question but it hasn't come yet but can AMD 3100 mobilize PBCSC used for allogeneic transplant.  If so we wonder what dose of CD34 to give, the same as GCSF?  And will the outcomes differ from transplants using GCSF mobilized transplant. 

To address this, we teamed up with Bob Donahue from NHLBI who has a nice animal model for mobilizing stem cells.  So we gave rhesus monkeys GCSF and AMD 3100 or combination and mobilized stem cells.  And we also because rhesus monkeys don't mobilize well with GCSF we also used a combination of GCSF and SCSF.  These mobilized peripheral blood stem cells were collected by apheresis and isolated by using CD34 monoclonal antibodies.  We then analyzed those cells by gene expression profiling and the first thing we did is we compared the CD34 positive cells and negative cells.  As you would expect the hematopoietic stem cells had a different signature of expression as the peripheral blood leukocytes.  Within the CD34 positive group you see a number of genes share expression across all the different types of mobilized cells.  So there's no difference, there's a number -- as you would expect, commonnalties in expression of genes in these types of cells.  What's interesting is you see this cluster down here and you see some -- this cluster gene expresses more in G cells mobilized by GCSF and SCSF approximate you see a cluster mobilized more with people given AMD 3100. 

So what we did is we looked at the -- just the genes differentially expressed in the hematopoietic stem cells.  This is what we found.  We found that not unexpectedly that the three monkeys that were given GCSF and SCSF clustered together.  But not unexpectedly the AMD 3100 mobilized stem cells from the same monkeys CD34 cells clustered separately.  And but what was really unexpected was the G AMD plus GSCF mobilized cells formed a separate independent cluster shown here.  And you can see that there's some genes that will only express by GCSF positive cells.  These were expressed by AMD 3100 cells but not GCSF mobilized.  Then you have this set of genes only expressed by AMD plus GSCF mobilized cells. 

The mobilization protocol mobilizes different populations at CD34 cells.  How do they differ?  We looked at this a number of different ways.  We looked at which genes were in each one of those clusters.  We looked at pathway analysis of differentially expressed genes.  We looked at genes most expressed in each of these types relative to the other.  And what we found is that the GCSF mobilized CD34 cells tended to show more neutraphil and monocyte macrophage dendritic cells markers.  Those mobilized by AMD 3100 showed more D cell t-cell mass cell markers.  And a couple of markers of progenitor cells, not stem cells but committed progenitors.  With AMD 3100 mobilized cells we saw higher expressions of B cells and T cells and a few committed progenitor cell markers. 

We also looked at microRNA and the data was interesting.  We did see a microRNA 155 of expressed most in GCSF mobilized cells.  This is an important marker of activated dendritic cells.  Again, GCSF mobilized cells.  This time we saw the marker expressed by CD34 cells, microRNA 126 and 10A were expressed in the GCSF mobilized cells.  Wasn't anything unique about the AMD 3100 mobilized cells in terms of microRNA but an A plus GCSF we saw two microRNA markers of T cells and one of T cells and pre-B cells.  So again, this is consistent with T and B cells being mobilized by AMD 3100, neutraphils and macrophages by GCSF and we're not sure which protocol mobilizes more primitive stem cells. 

So getting back to those original questions.  Based on these studies consist we still -- can we use AMD 3100 mobilized CD34 cells for gene therapy?  Probably.  There's no reason to say we can't.  What dose of CD34 cells should we give if we do collect AMD 3400 mobilized cells?  We really don't know.  We will need clinical data to assess this.  Will the outcomes differ between allogeneic transplants using GCSF and AND 3100 mobilized cells?  This suggests it will.  Engraftment rates may be different particularly with T and B cells. 

So in conclusion, we found that the composition of stem cells is dependent on the mobilization protocol.  Stem cells mobilized by combination of agents was not simply a mixture of mobilized cells mobilized by each agent separately.  And CD34 counts might not accurately reflect the potency of stem cells mobilized by different agents.  Future directions we plan to continue similar studies but we do have some protocols initiated where we're expanding NK cells and giving them clinically.  We're working with the investigators to save products we like to correlate gene expression microRNA expression profiles of these cells given to the patients with clinical outcome to try and come up with a good potency assay for those.  We also -- there's a big initiative in the Clinical Center to produce bone marrow stroma cells and use them clinically.  Part of the problem with using bone marrow stroma cells is they aren't good markers.  So when we change conditions to improve the manufacturing, it's difficult to assess whether or not these products have been changed short of using in vivo assays and we're using gene expression profiling to assess that.  I would like to thank my collaborators who did all these studies and those who were instrumental in setting up assays an designing these studies an helping us out and I would like to also acknowledge the support of Harvey Kline and John Gallin in the Clinical Center.  Thank you.     

[Applause]

QUESTION:  So safety being one of the main topics of the lecture, what can you say about the -- I guess the risk of the donor subject through both of the drugs and what are the long term effects of those sum to those treatments?

STRONCEK:  Interesting question.  There's been a lot of concern about GCSF given to healthy subjects and there's a couple of small studies to suggest temporary genetic changes in lymphocytes.  But despite that, the national donor marrow program has been closely watching all of their donors that have gotten GCSF and they have been followed almost 10,000 donors for more than ten years.  They really haven't seen any increase in any kind of malignancy.  So looks like at least for a long-term risk of leukemia or cancer, GCSF isn't the problem.  The other types of things like ruptured spleens happen really rarely.  That's not -- there are -- we don't like to see them happen but it's not something that prevents us -- it happens so rarely, doesn't prevent us from using the drug.  AMD 3100 we really don't know what that's going to do.  Yeah, it's been studied but maybe it's only given to a thousand people.  Once we start to give it to tens of thousands of people, there maybe risks for that drug that we wouldn't expect to see with small studies.  And giving both drugs in combination, again, is yet another issue and we may see clinical risks of the donors that are different in giving either drug alone.  So I think what we're going to see is a slow beginning to use the AMD 3100 in allogeneic patients under studies hopefully under IND and get some of that data and if it approves to be safe, to use it in more patients and clearly not to use it in unrelated donor settings until there's been more adequate clinical experience.

QUESTION:  Thank you for two very interesting presentations.  Could you tell us a little more about the nature of this compound AMD 3100?  Where is it derived?  Is it a synthetic chemical or biologic?

STRONCEK:  It's a biocycline, developed to try and treat AIDS because CXCR-4 is a major receptor for HIV -- where HIV virus enters the cell.  When they were screen it they noticed patients given the drug had increase in white cell count.  Somebody investigated what happens to stem cells.   

QUESTION:  I was wondering if you did any correlations with in vitro bioassays in the three different mobilization protocols and how that compared to your microarray analysis.

STRONCEK:  That's a great question.  We haven't but Dr. Donahue continues to -- he continues to use AMD 3100 in his animal models.  And he's beginning to acquire some of that.  It's a critical issue.  Besides just doing the expression profiling, it's really helpful to have the clinical data because that's ultimately what we have to correlate it to.  We think that in the CD34 -- in hematopoietic transplant most people are probably using far more cells than they need so we don't think we change drugs to AMD 3100, we're going to see engraftment, it's just going to take some clinical time to see if it's fast or slower.  Then it would be ideal to try and correlate that with gene expression profiling.  As far as hematopoietic stem cells, I think a better model for us to study would be almost umbilical cord cells where there's a real problem with delayed long term engraftment.  We're right at the edge of not having enough cells but we done do a lot of cord blood transplant so we haven't been able to get clinical material to look at gene expression profiles of cord blood cells and correlate them with engraphment.

GALLIN:  Thank you to both speakers for a wonderful session.     

[Applause]   

(Music fades in, under VO)

ANNOUNCER:   You've been listening to Dr. David Stroncek, chief of the Cell Processing Section at the NIH Clinical Center's Department of Transfusion Medice, discussing the topic "The Use of Molecular Assays to Assess the Potency of Cellular Therapies."  We began today's podcast, recorded March 4, 2009, with Dr. Keith Wonnacott, chief of the Cellular Therapies Branch, Center for Biologics Evaluation and Research at the Food and Drug Administration, who spoke on the topic, "Understanding and Ensuring the Quality of Cellular and Gene Therapies." You can see a closed-captioned videocast of this lecture by logging onto http://videocast.nih.gov -- click the "Past Events" link, or by clicking the "View Videocast" link for today's podcast at the Grand Rounds podcast page at www.cc.nih.gov/podcast/grandroundpodcasts.html.  The NIH CLINICAL CENTER GRAND ROUNDS podcast is a presentation of the NIH Clinical Center, Office of Communications, Patient Recruitment and Public Liaison.  For more information about clinical research going on every day at the NIH Clinical Center, log on to http://clinicalcenter.nih.gov. From America’s Clinical Research Hospital, this has been NIH CLINICAL CENTER GRAND ROUNDS.  In Bethesda, Maryland, I’m Bill Schmalfeldt at the National Institutes of Health, an agency of the United States Department of Health and Human Services.