1

              U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
                          PUBLIC HEALTH SERVICE

                      FOOD AND DRUG ADMINISTRATION
              CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
                INTERNATIONAL ASSOCIATION FOR BIOLOGICALS
          NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
                     NATIONAL VACCINE PROGRAM OFFICE
                        WORLD HEALTH ORGANIZATION
                                  - - -

             EVOLVING SCIENTIFIC AND REGULATORY PERSPECTIVES
               ON CELL SUBSTRATES FOR VACCINE DEVELOPMENT
                                  - - -
                                WORKSHOP
                                  - - -
                      WEDNESDAY, SEPTEMBER 8, 1999
                                  - - -

                      The workshop was held in the Plaza
          Ballroom, Doubletree Hotel, 1750 Rockville Pike,
          Rockville, Maryland 20852, at 8:30 a.m., Harry Rubin,
          DVM, and Martin Myers, M.D., Co-Chairs, presiding.
          PRESENT:
                HARRY RUBIN, DVM            Co-Chair
                MARTIN MYERS, M.D., PhD     Co-Chair

                NAOMI ROSENBERG, PhD        Session Chair
                HENRY PITOT, M.D., PhD      Speaker
                ALEX VAN DER EB, PhD        Speaker
                JAMES McDOUGALL, PhD        Speaker

                                                                          2

          PRESENT:  (continued)

                STEPHEN BAYLIN, M.D.        Speaker
                WALTER DOERFLER, M.D.       Speaker
                JAMES COOK, M.D.            Speaker
                SATVIR TEVETHIA, PhD        Speaker
                FRANK SISTARE, PhD          Speaker
                MICHAEL FRIED, PhD          Speaker
                SANDRA RUSCETTI, PhD        Session Chair

                CLIVE PATIENCE, PhD         Session Chair
                LEONARD EVANS, PhD          Session Chair
                DAMIAN PURCELL, PhD         Session Chair
                PAUL JOLICOEUR, M.D., PhD   Session Chair

          ALSO PRESENT:

                DAVID ONIONS, PhD

                                                                          3

                                I N D E X

                                                          Page

          SESSION 2:  Mechanisms of Neoplastic Development
          and Neoplastic Cells Tumorigenicity:
          Implications for Cell Substrate Development

          Introduction: Session Co-Chairs                    5

          Animals of neoplastic development                  5
          Henry Pitot, M.D., PhD

          Multistep carcinogenesis, Harry Rubin, DVM        21

          Transformation by DNA viral oncogenes             45

          Alex van der Eb, PhD

          Hit and run transformation leading to             61
          carcinogenesis, James McDougall, PhD

          DNA methylation and epigenetic mechanisms of      75
          carcinogenesis, Stephen Baylin, M.D.

          A new concept in viral oncogenesis                89
          Walter Doerfler, M.D.

          Role of nonspecific NK/macrophage cell host      108
          responses in assessing tumorigenicity,
          James Cook, M.D.

          Role of CTL host responses and their             125
          implications, Satvir Tevethia, PhD

          Transgenic animals that might be useful in       137
          identifying unsuspected oncogenic factors,
          Frank Sistare, PhD

          SESSION 2:  Panel-Audience Discussion            155

          SESSION 3 (Part 1) Viral-Viral and
          Viral-Cellular Interactions

          Introduction                                     229

          Generation of MCF retrovirus as a model of       233
          viral-viral and viral-cellular interactions
          Sandra Ruscetti, PhD

                                                                          4

                                    
          INDEX  (continued)                              Page

          MuLV packaging systems as models for estimating/ 248
          measuring retrovirus recombination frequency,
          Clive Patience, PhD

          In vivo and in vitro effects of mixed retrovirus 264
          infections, Leonard Evans, PhD

          Cross-species pathogenesis of replication-       277
          competent retrovirus in immunosuppressed hosts,
          Damian Purcell, PhD

          Pathogenesis of defective retroviruses,          296
          Paul Jolicoeur, M.D., PhD

                                                                          5

     1                    P R O C E E D I N G S

     2                                        Time:  8:00 a.m.

     3                CO-CHAIRMAN MYERS:  On behalf of the

     4    National Vaccine Program, welcome.  Our first speaker

     5    this morning will be Henry Pitot of the University of

     6    Wisconsin, and he's going to start talking about

     7    animals of neoplastic development.

     8                DR. PITOT:  Hopefully, this thing works. 

     9    Does it?  Both Harry and I chose to speak down here. 

    10    I guess the other speakers can decide whether or not

    11    they can see the slides from up here or down there.

    12                I think my function this morning was

    13    basically to try to cover in about 15 or 18 minutes

    14    most of the more commonly used animal models for

    15    cancer development, and probably without further ado,

    16    I'll just start with the first slide, which is, I

    17    think, familiar to most of you.

    18                It's probably the most widely used animal

    19    model for neoplastic development.  It's, obviously,

    20    used by the regulatory agencies, and some people have

    21    considered it what might be considered the gold

    22    standard of carcinogenesis.  

    23                Unfortunately, it has a lot of drawbacks,

    24    as those of us that work in experimental systems know. 

    25    But looking at the literature, in fact, probably most

                                                                          6

     1    of the data on whether or not a chemical is

     2    carcinogenic really comes from this sort of data that

     3    you see here, which really is based on studies that

     4    went back many years, actually to the 1930s where

     5    animals were given a carcinogen for an extended period

     6    of time until tumors developed.

     7                In this particular system there's

     8    actually, as you can see, a fairly standardized type

     9    of thing which allows one over a two-year period to

    10    determine the development of neoplasm.

    11                Now nice as it might look, there are a

    12    number of problems, and I don't have the time this

    13    morning to go into all of the difficulties.  But one

    14    of the more interesting facets that's come out of this

    15    whole series is this table which I borrowed from Dr.

    16    Ames' publication several years ago, which basically

    17    demonstrates, looking at a series of some 380

    18    different chemicals that were tested, the relationship 

    19    between whether or not they were carcinogenic or

    20    whether or not they were mutagenic.  

    21                I think you can look at that table and

    22    immediately see that perhaps one of both the advantage

    23    of this chronic bioassay and the disadvantage is that

    24    there are a variety of chemicals which, obviously, are

    25    mutagenic and carcinogenic, which one might expect,

                                                                          7

     1    but there are also a large number that are perhaps not

     2    mutagenic but still are carcinogenic.

     3                So this type of system has led us to the

     4    finding that we can do certain things with it, but

     5    clearly we cannot begin to study mechanisms and

     6    looking at dissecting the whole process of

     7    carcinogenesis. 

     8                So as a result of that, of course, one

     9    goes back a number of years, actually to the 1940s

    10    when some of the earlier studies on what might be

    11    called, what we call today at least, multi-stage

    12    carcinogenesis were carried out.  

    13                This slide is just a classic experimental

    14    slide which shows the studies that were done

    15    originally by Barren, Blum and Schubik and others in

    16    the 1940s which basically was done on the back of a

    17    mouse, painting with a chemical, then administering a

    18    material which at that time was an irritant called

    19    croton oil, but today we call a promoting agent,

    20    eventually ending up, if the format of the system is

    21    appropriate, with neoplasm.

    22                Now what I mean by appropriate is that

    23    there is first an initiating agent, this material that

    24    is given on the back of the skin, followed by a

    25    promoting agent.  If you reverse the process, it

                                                                          8

     1    doesn't seem to work, at least in most instances.

     2                If you, for example, try just the

     3    promoting agent, nothing happens; and the interesting

     4    experiment that was done by Bautwell some 20 years

     5    later was that, if you change the format of the

     6    administration of the promoting agent, then you do not

     7    get neoplasm.

     8                So here was a system which allowed for

     9    carcinogenesis to occur.  The endpoint of this

    10    particular system is seen in this slide, which is the

    11    typical papilloma of the mouse.  It is not a malignant

    12    neoplasm.  It is benign, but it is useful in this

    13    particular system, which really tried to understand

    14    what might be called the latent period of

    15    carcinogenesis.

    16                So this system was used, actually, for

    17    about 30 years, during which time the chronic bioassay

    18    developed. This system never became really important

    19    with respect to regulatory agencies or anything of

    20    that sort, but it was used primarily in academic

    21    circles in trying to understand the whole process of

    22    carcinogenesis.

    23                It was in the 1970s that Dr. Carl Pareno

    24    at the Argon laboratory actually first sort of broke

    25    the ice by demonstrating that other tissues besides

                                                                          9

     1    mouse skin showed this exact same phenomenon -- that

     2    is, of a stage phenomena in the development of

     3    neoplasia.

     4                Dr. Pareno's system, which is sort of

     5    shown very briefly here, was to administer a

     6    carcinogen -- in this case, this is actually a

     7    modification of his which is done in our laboratory --

     8    with a mitotic stimulus followed by the administration

     9    of some promoting agent.

    10                This material, this promoting agent, which

    11    did the same thing as the irritant in the skin system,

    12    now had become sort of a much more well recognized of

    13    what we call today promoting agent.

    14                Over the years since Pareno's

    15    demonstration, there's been sort of a parallel

    16    development of both the mouse skin system and the rat

    17    liver system, which is what Pareno studied, in

    18    addition to -- and I won't have time to name them all

    19    here -- probably a dozen other different tissue

    20    specific multi-stage models of carcinogenesis.

    21                So it's not just unique as it was a few

    22    decades ago just to the mouse skin.  It clearly goes

    23    well beyond that.

    24                So I'll spend just a few minutes talking

    25    about the liver system, simply because that's what's

                                                                         10

     1    done in our laboratory.  I think potentially it can be

     2    utilized perhaps to go on to other model systems as

     3    well.

     4                One of the big advantages in the liver

     5    system is that we think, and there's certainly

     6    reasonable evidence to argue, that one may identify

     7    initiated cells.  That is the very first step in the

     8    development of neoplasia.  This is just a histologic

     9    section which shows in the middle of this section a

    10    single cell which now expresses a gene, the piriform

    11    of glutathionase transferase, which normal hepatocytes

    12    do not express.  

    13                We have found this to be a very useful

    14    what is called a marker for identifying initiated

    15    cells and, as you'll see, these cells then under the

    16    influence of a promoting agent go on to clonally

    17    develop into small colonies in the organ --  this is

    18    all done in vivo -- expressing the same particular

    19    genetic component.

    20                Now this -- It turns out not only is the

    21    expression of this particular gene in these cells

    22    abnormal, but a whole series of others which have been

    23    studied over the years by many, many different

    24    laboratories, either as markers or trying to

    25    understand what actually is going on during this

                                                                         11

     1    process of promotion.

     2                As a result of that, looking to mining

     3    years of work and many, many experiments, one can come

     4    up with a series of conclusions for the effect of

     5    promoting agents.  

     6                Now I'm sure in the discussion we can

     7    spend an awful lot of time on various other aspects,

     8    but I'm going to home just in on these two

     9    characteristics which I feel are probably the most

    10    critical aspects of these agents which cause a

    11    selective enhancement of cell replication basically of

    12    initiated cells.  That is, they cause the replication

    13    of these cells different from the normal cells.  They

    14    will also cause normal cells to replicate, but the

    15    initiated cells they cause much more effectively and

    16    selectively.  That was shown several years ago by

    17    Farber and others.

    18                The other aspect that they also do has

    19    been shown recently by Dr. Schulte Herman in Vienna. 

    20    They selectively inhibit the programmed cell death of

    21    cells.  So these two selective actions, actually, I

    22    would propose -- and we can discuss it -- actually can

    23    explain virtually all of the actions of promoting

    24    agents.

    25                Now, of course, one easily says what is

                                                                         12

     1    the molecular mechanism of this.  I think at the

     2    moment we really don't know.  We certainly have a lot

     3    of ideas about what's going on, but there's still a

     4    lot of work to be done.

     5                You can also see in this system, perhaps

     6    unlike the mouse skin system, that it is possible to

     7    quantitate the development of neoplasia.  So if one,

     8    for example, administers a -- initiates cells by just

     9    a single dose, a very small dose of a carcinogen, you

    10    can see you increase the number of these putatively

    11    initiated cells by three orders of magnitude, which

    12    perhaps is not unexpected.

    13                Also notice that there are a certain

    14    number which occur spontaneously.  Then if you promote

    15    and cause these to develop into these small colonies,

    16    only about one percent of these develop into that; and

    17    although I don't have the quantitation on there, one

    18    can also go on into neoplasm and, in fact, of this

    19    perhaps one-tenth of one percent of these cells

    20    develop into neoplasm.

    21                So it allows one in a model system to

    22    quantitate the various stages and really get an idea

    23    of how effective the various stages are.

    24                Now to do this, there have been model

    25    systems developed -- this is one of them -- where you

                                                                         13

     1    can actually dissect each of the three stages.  One

     2    can initiate with this very small dose of a

     3    carcinogenic agent, an initiating agent, which by

     4    itself will do nothing.  

     5                One can then promote, as shown with the

     6    blue line here, with a promoting agent.  Then

     7    somewhere down the line after these focal lesions,

     8    these small lesions, have occurred, one can administer

     9    another agent which Bernie Weinstein coined the term

    10    progressor agents, which actually then causes during

    11    this period the development of malignant neoplasms.

    12                So during this period from the

    13    administration of the initiating agent to this point,

    14    basically you have these focal lesions.  The cells are

    15    different phenotypically.  They are undoubtedly

    16    different genetically, at least from point mutations

    17    and other things, but they are not different

    18    cytogenetically.  They have perfectly normal

    19    cytogenetics.

    20                It is in the stage of progression that the

    21    cytogenetics of cells become abnormal, as we all know. 

    22    Not only do they become abnormal, but they

    23    continuously get worse.  That is, they continually

    24    evolve in what is called evolving karyotypic

    25    abnormalities.

                                                                         14

     1                From this, both this model and others,

     2    again one can determine certain characteristics.  I've

     3    just listed some of the consequences of this major

     4    factor of the stage of progression.  Four of them

     5    here, gene amplification, gene deletion,

     6    rearrangement, and a very effective way, certainly

     7    cells that are in the stage of progression are much

     8    more effective at accepting external genetic

     9    information by transection mechanisms than are normal

    10    diploid cells.

    11                Consequences of -- You might say the

    12    functional consequences of this particular phenomenon

    13    can be seen in this slide, just some of them.  There

    14    are probably a number of others.  I've listed a few of

    15    these here, and probably for the discussion of this

    16    particular symposium, one of the most important ones

    17    is this particular part right here.  

    18                It is well known that cells in the stage

    19    of progression lose the expression of the MHC

    20    determinant which, of course, makes them a very

    21    difficult target for the immune system, because there

    22    will be no interaction between the MHC system and the

    23    T-cell receptors.  

    24                I'm sure that will be discussed in many

    25    other components here, but it does allow one by using

                                                                         15

     1    this multi-stage phenomenon, then to dissect this

     2    component into its various units and actually try to

     3    determine changes which occur at each of the different

     4    stages.  

     5                Finally, and I've taken this from an

     6    article by Ray Tennant from the NTP, the more modern

     7    models of carcinogenesis are transgenic models, either

     8    transgenesis itself by adding genes in the standard

     9    transgenic mechanism or by gene targeting of

    10    knockouts.

    11                What I've listed here are three --

    12    actually, two of the more common ones, the upper two

    13    here, the T-53 knockout animal, both the homozygote

    14    and the heterozygote, and the so called TGAC animal in

    15    which the viral Harvey ras gene has been associated

    16    with either a Zeta hemoglobin promoter or also, more

    17    recently, with a keratin promoter, such that one can

    18    actually do many of the skin tumor painting without

    19    worrying about initiation, because basically one

    20    already has initiated cell populations.  So promoting

    21    agents actually under this system become complete

    22    carcinogens, in quotes at least.

    23                Just briefly, looking at the upper one,

    24    the P53 deletion mutation, this is taken from an

    25    article that was recently published.  This just shows

                                                                         16

     1    the tumors that develop in these animals, and you'll

     2    notice that -- these are mice -- that the normal

     3    animals spontaneously, at least out to this number of

     4    weeks, develop very few spontaneous neoplasms.  But

     5    as; you might expect, in both the heterozygotes and

     6    the homozygote P53 animals, the spontaneous

     7    development of neoplasia is really extremely high.

     8                This creates a problem perhaps when one

     9    actually is trying to determine mechanisms, because it

    10    is rather difficult to add to the system something to

    11    perturbate it, while at the same time the system is

    12    spontaneously developing a very large number of

    13    neoplasms.

    14                On the other hand, if you look at the

    15    neoplasms that are produced in such a system, you can

    16    see that there seems to be a somewhat, at least

    17    quantitatively, different spectrum.  Perhaps in the

    18    homozygotes deletion animal, lymphomas are the

    19    predominant component, whereas in the heterozygotes,

    20    one can see that the lymphomas still are a significant

    21    component of it, but both the soft tissue sarcomas and

    22    osteosarcomas, which are really a very small part here

    23    -- these soft tissue tumors become very prominent.

    24                Now there are -- This is not the least in

    25    this long list of modern models.  This just shows you

                                                                         17

     1    -- Some of you in the back probably can't read this,

     2    and it's really not that critical.  It's not complete

     3    by any means.  There are a whole series of transgenic

     4    models which are now developing.

     5                The difficulty is, if, of course, one

     6    wants to study the development of a specific neoplasm,

     7    it may be extremely useless.  On the other hand,

     8    because the animal is programmed for certain things,

     9    it becomes perhaps difficult to study the earlier

    10    stages in the development of neoplasia and, rather,

    11    what one will be looking at is the later stages

    12    themselves.

    13                So just to sort of sum up, what I've shown

    14    you are three basic models:  This chronic bioassay

    15    which is perhaps, from the regulatory standpoint, the

    16    gold standard, but from the investigatory standpoint

    17    it has many problems; the multi-stage model which

    18    allows one to look and dissect at the various stages

    19    of neoplasia some of its mechanisms and its

    20    characteristics; and finally, the newer transgenic and

    21    knockout models which, certainly in specific areas,

    22    can answer specific questions, but probably they have

    23    to be geared and tooled to do this, and it seems that

    24    they will probably be most significant in answering

    25    questions in this final stage of neoplasia -- that is,

                                                                         18

     1    of progression.

     2                So I'll stop there, and hope I kept on

     3    time.

     4                (APPLAUSE)

     5                CO-CHAIRMAN MYERS:  We have time for a few

     6    questions.  If you would come to the microphone and,

     7    as we are transcribing the meeting, if people could

     8    identify themselves, both by name and institution.

     9                DR. LEOWER:  Johannes Loewer.  Has ever

    10    DNA of neoplastic or normal cells been tested as

    11    initiator or promoter in these -- If so, what was the

    12    outcome?

    13                DR. PITOT:  I'm not sure of all the

    14    experiments.  I know it was tested once in mouse skin,

    15    but probably not appropriately.  It didn't work.  But

    16    I guess that, certainly, if one can use in -- as we'll

    17    turn in the next discussion, in cell culture, one can

    18    clearly by transection of DNA get a situation where

    19    one can get -- I'm not quite sure what the stage one

    20    is dealing with, but certainly a transformation.

    21                DR. FRIED:  I'm Mike Fried, ICRF, London. 

    22                You said in the beginning that certain

    23    carcinogens were not mutagenic.  What is the

    24    mechanism, how they work, if it's not a genetic one?

    25                DR. PITOT:  Well, we can spend another

                                                                         19

     1    three hours on that one.  I'll give you my opinion,

     2    and I'm sure it can be discussed otherwise.

     3                I showed you that in the putatively

     4    initiated cells in the liver, there are a number of

     5    spontaneous lesions.  We well know that spontaneous

     6    carcinogenesis is very characteristic of all mammals

     7    probably, more so of some than others.

     8                So one could make the argument that the so

     9    called non-genotoxic carcinogens which are not

    10    mutagenic in the chronic bioassay or in the Ames

    11    system and other systems are, in fact, promoting

    12    agents.  What they are doing is they are causing the

    13    development of spontaneously initiated lesions through

    14    promotion and then spontaneously into progression.

    15                That's a fairly simplistic answer, but I

    16    think it follows the facts.

    17                CO-CHAIR RUBIN:  I want to call attention

    18    to some recent work by Zarbell and Tilley with -- I

    19    think it was NMU carcinogenesis in the mammary gland

    20    of rats where they found that this so called mutagenic

    21    agent, in fact, was causing mammary cancer, mammary

    22    epithelial cancer.  But it turned out that all the

    23    tumors had a Harvey ras mutation in them, and that

    24    Harvey ras mutation preexisted in the mammary

    25    epithelium, and the agent -- you could call it then a

                                                                         20

     1    promoter, but in effect what it did was select with a

     2    clonal expansion of those clones in the mammary gland.

     3                DR. DOERFLER:  My name is Walter Doerfler

     4    from Cologne in Germany.

     5                Isn't it a problem perhaps with the

     6    transgenic model as people are now finding applying

     7    the DNA array technology that, when one does a

     8    knockout, perhaps quite a number of functions are

     9    altered, not at all exclusively the functions that you

    10    are knocking out, but other functions.

    11                So the interpretation must become

    12    extremely complex.

    13                DR. PITOT:  I certainly agree.  I think

    14    that the use of the gene targeted and transgenic

    15    animals may be very useful, but I think it's new

    16    enough that we really don't know all of the problems

    17    that may occur.

    18                AUDIENCE PARTICIPANT:  In your multi-stage

    19    approach, how specific to the species that you are

    20    testing, the fact is, particularly related to the MHC

    21    expression, it is clearly published -- the regulation

    22    of the expression differs from species to species.

    23                So, therefore, what could be changing

    24    expression in your rat model may not be applied to

    25    humans.   You know very well that expression of MHC

                                                                         21

     1    genes is all over the place.  It's up-regulation,

     2    down-regulation.

     3                So how can you apply this overall and

     4    generalize your results?

     5                DR. PITOT:  I think that you see the sort

     6    of thing that you're discussing in the human also in

     7    the rodent, not only in the rat but the mouse, and the

     8    same sort of system.  So that you will find neoplasms

     9    that will up-regulate the MHC components, others that

    10    will down-regulate them. 

    11                Unfortunately, that it will happen in the

    12    same neoplasm, that some cells will up and some will

    13    down.  So, therefore, you're going to be faced with a

    14    problem, I think, from the vaccine question actually

    15    of trying to get cells to up-regulate, and there are

    16    mechanisms to do this, so that some of the vaccines

    17    may well be able to work.

    18                I think it is applicable to all different

    19    systems, and certainly the human is perhaps the best

    20    example.

    21                CO-CHAIRMAN MYERS:  Thank you very much. 

    22    Our next paper will be presented by our Co-Chair, Dr.

    23    Harry Rubin, Professor of Molecular and Cell Biology

    24    at Berkeley, and he's going to talk about multi-step

    25    carcinogenesis.  

                                                                         22

     1                CO-CHAIRMAN RUBIN:  As most of you know

     2    may know, our speaker was supposed to be Larry Loeb. 

     3    He was supposed to be the first speaker.  Larry Loeb

     4    suffered a back accident trying to climb into a cave,

     5    which he shouldn't have done, with his grandson.

     6                We tried to find at a late date some

     7    alternate speakers.  It was too late to get them to

     8    come, and so as the designated chairman I volunteered

     9    to speak.  That will explain the rather primitive

    10    state of some of my displays, which were -- Some of

    11    them were made at four o'clock this morning.

    12                So I would like to talk about --

    13    basically, about the transformation of cells in

    14    culture, both the transformation to the neoplastic

    15    state of primary cells, if you like, of normal cells

    16    obtained from the animal and in some cases even from

    17    humans, and then into permanent cell, the highest.

    18                The first observation that was made in a

    19    semi-systematic way about the transformation of normal

    20    mouse cells into sarcoma cells beginning with fiber

    21    blast was made, of course, by Wilton Earle of the

    22    National Cancer Institute, beginning in about 1943,

    23    and working together with Sanford they investigated

    24    this problem for many years.

    25                Of course, as many of you know, at first

                                                                         23

     1    what they were working with was carcinogen induction

     2    of transformation in mouse cells, but they found that

     3    their controls were transforming at the same rates as

     4    the carcinogen treated cells were, and that started

     5    the whole study of spontaneous transformation of cells

     6    in culture.

     7                Probably the earliest really systematic

     8    investigation of the transformation of mouse cells in

     9    culture, although that's not what it was called in the

    10    particular paper, was by Todaro & Green in 1963,

    11    Journal of Cell Biology, and a similar systematic

    12    investigation was carried out by Paul Kraemar and

    13    associates in New Mexico with Chinese hamster cells.

    14                What I've listed here are the basic -- not

    15    necessarily the sequence of changes, but an indication

    16    of a variety of changes that occur during spontaneous

    17    transformation of rodent cells in culture.  So let me

    18    just run down them.

    19                You take the mouse fibroblast, in the case

    20    of Todaro & Green, and while they multiply fairly well

    21    when they're first put in cell culture, the rate of

    22    multiplication decreases with every passage.  They go

    23    through a crisis.  If you're transferring a small

    24    number of cells -- in that case like three times 105 -

    25    - you may, in fact, lose the culture, in a sense like

                                                                         24

     1    you lose human cells eventually.

     2                If you have enough cells at each transfer,

     3    every third day you go through a crisis period, which

     4    means it looks like the cell culture is dying out, but

     5    then there begin to appear variance in the culture,

     6    and gradually you get an increase in cloning

     7    efficiency.  You get an increase in the rate of

     8    multiplication back to the original rate and perhaps,

     9    in some cases, an increased rate.

    10                Then you find that the growth -- the cells

    11    start to multiply to a higher saturation density.  

    12    Roughly about the same time, they will grow in low

    13    serum concentrations or lower serum concentrations

    14    than they would grow in before.

    15                Then later on at a later step the cells

    16    will grow in suspension in soft agar or methyl

    17    cellulose.  This is very often associated with an

    18    ability to produce tumors in the animal, but not

    19    always.  It is not an invariant accompaniment of

    20    ability to produce tumors.

    21                Anywhere along these lines -- and we'll go

    22    into this in a few minutes -- you begin to get

    23    transformed colony morphology and transformed foci in

    24    the culture.  Some of these things such as the

    25    transformed foci or production of tumors are spoken

                                                                         25

     1    about in a qualitative way when, in fact, they're

     2    quantitative, progressive increases in the neoplastic

     3    transformation of the cells.

     4                You see that, I think -- In particular,

     5    this was shown by the group that worked with Chinese

     6    hamster cells at Los Alamos, Kraemer  & Kramm,

     7    etcetera.  They found that their hamster cells went

     8    through many of these stages, but as they were going

     9    along, they were continually testing them for ability

    10    to produce tumors in the isologous animals, in the

    11    Chinese hamsters.

    12                What they found, that even though all

    13    these changes were occurring -- there were tissue

    14    culture representations of changes in the behavior of

    15    the cells after they had gone through their crisis

    16    also -- they would not produce tumors, and that was

    17    the case even where every cell in the culture had

    18    shown chromosome aberrations.  After the 20th passage

    19    in culture, every cell in the culture was

    20    chromosomally abnormal.  

    21                They were still not producing tumors when

    22    injected back into Chinese hamsters, but about the

    23    40th or 50th passage they could produce tumors, but

    24    they could produce tumors only under certain

    25    conditions.  

                                                                         26

     1                The particular conditions were (a) to use

     2    10 million cells -- a lot of cells -- and instead of

     3    inoculating the cells directly subcutaneously into the

     4    hamster, they were inoculated into gelatin sponges

     5    that had been implanted subcutaneously.  Under those

     6    conditions, they produced tumors of the gelatin

     7    sponge, which up to that time didn't worry the hamster

     8    too much, but in fact they spread into the rest of the

     9    hamster after that.

    10                Then with further passages when chromosome

    11    aberrations really became quite severe in all of the

    12    cells, the tumors could be produced by direct

    13    subcutaneous inoculation, and even later tumor

    14    production would occur with smaller numbers of

    15    inoculated cells.

    16                Now the cells would also change -- this

    17    was shown first, I think, in the mouse -- would change

    18    after they produced the tumor in the mouse, if you

    19    take the tumor out of the mouse, and the tumor

    20    required the inoculation, let's say, of a million

    21    cells.  Once you got the tumor, you could produce a

    22    tumor with 1,000 cells.

    23                So that there was continuous progression. 

    24    What you see here is really a complexity of

    25    progression, that each one of these stages that we're

                                                                         27

     1    talking about is, in fact, an indication of another

     2    change in the population of cells.

     3                Could I have the next transparency?  Well,

     4    this I did draw at about 4:30 this morning, and

     5    they're from memory.  So if someone has a better

     6    memory than I do, please inform me.

     7                This is taken from Todaro & Green's paper

     8    in 1963, which is real classic.  What's shown in these

     9    top two graphs here is a plot on the horizontal axis

    10    of the number of passages of the cells in culture, and

    11    the increase in the cell population every three days,

    12    n-zero being the number inoculated.  Sometimes they

    13    used n-1, that if they counted cells at one day and

    14    then they counted them in three days when they passage

    15    them.

    16                At first, you could start out with a

    17    population that would increase about eightfold in

    18    those three days, doubling every day, but with each

    19    successive passage the cells would multiply slower. 

    20    You saw a lot of pathology in the culture. 

    21                Then at about the tenth passage it looked

    22    like the whole culture is going to die out.  This is

    23    in passaging three times 105 cells, which at least at

    24    that time was thought to be a low inoculant of cells. 

    25    Then with further passages variance appeared.  That

                                                                         28

     1    could be distinguished morphologically from the

     2    original cells.

     3                They continued to grow faster and faster,

     4    and they reached a plateau, which was a relatively low

     5    plateau.  These cells would not produce tumors in

     6    immunocompromised mice, for example, which is usually

     7    said to be the case, but in fact if you wait three,

     8    four, five months, very often a tumor will appear.

     9                Again, when you get that tumor and

    10    reinoculated it into mice, it will produce a tumor

    11    very fast.  So all that time these cells have been

    12    incubating in an occult stage in the animal, and

    13    finally a variant appears there, just like it appeared

    14    in cell culture.

    15                Now if one increases the concentration of

    16    cells at every passage -- and this is a 3T12 passage

    17    which means 12 times 105 or 1.2 times 106 cells were

    18    passaged at each passage; three simply means they were

    19    passaged every third day -- then you get a decline in

    20    the rate of multiplication of the cells, but it's a

    21    much shallower decline, for one thing.  Although

    22    there's something that resembles a crisis, it does not

    23    appear to be a very serious crisis. 

    24                Then you start to get the upswing in the

    25    rate of multiplication of the cells, and they end up

                                                                         29

     1    multiplying faster than these -- maybe not much faster

     2    -- but what they do do is multiply to a much higher

     3    saturation density.

     4                These cells, when inoculated into mice,

     5    will now produce a tumor fairly quickly.  The only

     6    difference here is that these cells have been passaged

     7    at four times higher concentration than the 3T3 cells. 

     8    So there's something about the higher population

     9    density of the cells that furthers the transformation. 

    10                In fact, a few years later in 1968-1969

    11    Aaronson & Todaro found that if you took cells

    12    directly from the mouse, start passaging them in

    13    culture, and inoculating them back into mice every few

    14    passages, that the higher the density of the cells

    15    that you made your passages at, the quicker the tumor

    16    would start to show up in the mouse.  So there was

    17    something either selective or inductive about high

    18    population density in inducing tumors.  It is

    19    obviously the selective aspect that you select for

    20    cells and that you grow at high density.  

    21                Now I want to say a word about human

    22    cells.  This is a paper by Smith & Hayflick in 1974,

    23    and Hayflick can correct me on this.  I'm sure I've

    24    got some of it wrong.  Again, it's from memory.

    25                Basically, this was an experiment with

                                                                         30

     1    clones of human fibroblasts.  I think it was the WY38

     2    line.  So what you're looking at here is the

     3    proportion of cells that go through a certain number

     4    of divisions in culture.

     5                So I think it's widely thought that human

     6    cells will go through 50 divisions and sort of fall

     7    off the end of the cliff.  That's based on work with

     8    large populations of cells, but when you study clones

     9    and their capacity to continue multiplication in

    10    culture, the capacity drops off in some clones right

    11    away, and right at about roughly the tenth passage or

    12    so -- I can't really give you an exact figure -- about

    13    half the cells have lost the ability to multiply.

    14                So it looks like the loss of capacity to

    15    multiply is a stochastic event.  It's a random loss of

    16    capacity to multiply.  What you appear to end up with

    17    is the last surviving clone.  That's the clone that

    18    goes 50 plus or minus ten divisions in culture.

    19                That result was basically confirmed by

    20    Peter Rabinovitch of Seattle in 1983.  He used a new

    21    technique for labeling the DNA of the cells,

    22    gromodeoxyuridine label.  He worked with whole

    23    populations of cells, and he found that the percentage

    24    of human cells that were cycling decreased linearly

    25    with the number of divisions that the cells went

                                                                         31

     1    through in culture.

     2                Now if you look at published literature

     3    also, it looks as though human cells will not undergo

     4    spontaneous transformation.  There are many ways of

     5    eliciting long term growth in culture, but there's

     6    been some unpublished work, the senior author of which

     7    was a cytogeneticist, Tamara Ignatova, was working in

     8    Marguerite Vogt's lab at the Salk Institute a few

     9    years ago -- unpublished work using Li-Fraumeni cells,

    10    which are unstable -- relatively unstable human

    11    fibroblasts.

    12                Again, what she found was that, if you

    13    kept the cells in a condition of confluence, which as

    14    we all know results in contact inhibition of the cells

    15    -- but I think it's not often appreciated, there's

    16    also a good deal of cell death that occurs at

    17    confluence -- then after a month or two of leaving the

    18    cells intact in a confluent layer, you start to see

    19    large scale chromosome rearrangements and indefinite

    20    growth.

    21                So these cells apparently have undergone

    22    some kind of genetic changes involving chromosomal

    23    changes that are fairly easy to see that results in

    24    indefinite growth.  That is, in effect, spontaneous

    25    transformation.  Unfortunately, as far as I know, this

                                                                         32

     1    work has never been published, and I think there's

     2    some disagreement among the group that's done it about

     3    aspects of it should ultimately get published.

     4                Do I have another?  That's it?  Well, I

     5    want to turn to some slides now.  I want to deal now

     6    for a few minutes with a cell line, but what I'm going

     7    to say about this cell line, I think, can be

     8    generalized to cell lines in general.

     9                So what I want to talk about is the NIH

    10    3T3 cell line, which is famed in modern molecular

    11    genetics as the first line in which there was

    12    demonstrated a transformation of an animal cell line

    13    by DNA extracted from a human tumor, the EJ bladder

    14    carcinoma.

    15                A problem with that original finding --

    16    there are many problems with the original finding

    17    which three laboratories reported back in 1981 -- was

    18    the fact that again, if you take the original line of

    19    NIH 3T3 cells and you let them sit at confluence for

    20    a couple of weeks, that you start to see spontaneous

    21    transformation.

    22                In fact, probably the outstanding

    23    characteristic of Jane Hill, Todaro and Aaronson's

    24    original line of NIH 3T3 cells is the ease of

    25    spontaneous transformation.  All you have to do is

                                                                         33

     1    leave the cells for two weeks at confluence and

     2    transfer them once, and foci start to show up

     3    throughout the culture.  I'll give you some examples

     4    of that.

     5                So again there's something about

     6    surprisingly enough from conventional genetic thinking

     7    where one thinks that at the highest rate of mutation

     8    or genetic change is during cell division when DNA is

     9    being replicated.  Actually, it appears that the

    10    highest rate of chromosomal change of mutational

    11    change -- we're not sure which, or maybe all of them

    12    together -- occurs when the cells are inhibited at

    13    confluence.

    14                You might ask why.  Well, there's

    15    something else which I already inferred, that there is

    16    a considerable amount of cell death when these cells

    17    or many cells are left at confluence for an extended

    18    period of time.  So there's considerable damage done

    19    to the culture.

    20                So let me show you a few slides of the

    21    kind of observations that one makes.  Let's go back to

    22    the first one.  Okay.

    23                What you see here is a culture of NIH 3T3

    24    cells which ordinarily would look like this, a mono-

    25    layer of cells, nontransformed cells, but when left

                                                                         34

     1    for a couple of weeks or if left for a couple of weeks

     2    and transferred, you get a high density population. 

     3    This is a transformed focus where the cells are no

     4    longer in any regular arrangement.  They're criss-

     5    crossing.  You can't identify individual cells,

     6    because they're piled so thickly.  

     7                This is an unstained slide, which was one

     8    of the first observations we made back in about 1988-

     9    1987, which surprised us that the cells which people

    10    were talking about as being used as targets for

    11    transformation by the Harvey ras oncogene would

    12    transform by themselves.  

    13                Now another feature is, when one looks at

    14    independent transformations -- and now we're getting

    15    to another point in transformation.  So you start out

    16    with a single culture, and you start splitting it, and

    17    you let each one of them go to confluence various

    18    numbers of times.  You get independent

    19    transformations.

    20                If you look at the foci in each one of

    21    these dishes -- these are the cuts from individual

    22    dishes -- in a rough sense, the foci, which are these

    23    thick aggregations of cells on a background of mainly

    24    monolayers of cells -- each of the groups of foci look

    25    different from one another.

                                                                         35

     1                So you can't simply talk about

     2    transformation of cells by producing foci.  These --

     3    If you collect the cells from any of these foci and

     4    inoculate them into mice, they'll produce tumors, and

     5    the tumors in the mice will all look like sarcomas. 

     6    But in culture where you can find a distinction in the

     7    appearance of the cells, each one of the independent

     8    transformations is different from every other one,

     9    which means that the genetic changes that are

    10    occurring in these cells are different in each one of

    11    these cases.

    12                So there are genetic changes, chromosomal

    13    rearrangements, deletions, etcetera, that are going on

    14    in these cultures that cause that kind of variation,

    15    which is reminiscent of what pathologists were telling

    16    us way back in the Thirties and Forties, that no two

    17    tumors look exactly the same.

    18                Well, okay, now I want to show you want

    19    will happen with the NIH 3T3 cells if you culture them

    20    in a certain way.  So what you see here are a series

    21    of cells that have been through what we call a primary

    22    assay, which means two weeks at confluence, and every

    23    two weeks they are transferred to become confluent

    24    again, and this is a tertiary assay, and this is after

    25    eight such sequences.

                                                                         36

     1                You start out with a culture that has no

     2    foci.  By the third round of confluence, even when you

     3    place only 1,000 cells on a background of

     4    nontransformed cells, you see these thick foci.  By

     5    the eighth passage at confluence, even 200 cells are

     6    producing about 100 foci.  So that's a high rate of

     7    transformation.

     8                Here is a culture derived from this same

     9    initial culture, but transferred originally about 100

    10    times at low density.  You go through the same

    11    operations, and you see much less evidence of

    12    transformation.

    13                So there's continuous selection and change

    14    going on in these established cultures, and one cannot

    15    rely on them by saying, well, they're going to remain

    16    the same as long as we continue passaging them.  It's

    17    very dependent, just like the original transfer of

    18    cells directly from the mouse -- dependent on

    19    population density.  Even the established lines depend

    20    on population density.

    21                If you look at another criterion of what

    22    was happening to these cells, in these cultures if you

    23    took the undiluted cell populations, you got a very

    24    rapid rise in saturation density, which remained

    25    roughly constant after that; whereas, in this case

                                                                         37

     1    even by the seventh round of confluence, there was

     2    only a minimal increase in saturation density.

     3                Another important aspect of these

     4    observations, contrary again to what you might expect

     5    -- You might think the cells which are becoming

     6    transformed would certainly grow more at high density. 

     7    If you would grow them at low density, would they grow

     8    any faster?  Quite the opposite is true. 

     9                Actually, at low density these cells grow

    10    slower and slower, which is another indication that

    11    what confluence -- extended confluence is doing to

    12    these cells is damaging them, and apparently damaging

    13    their DNA.  The problem is that occasionally in some

    14    of the cells, that particular type of damage results

    15    in transformation.

    16                Now if you take the original line of NIH

    17    3T3 cells and clone it out,  what you see here is

    18    three different clones.  We've done this with very

    19    large numbers of clones, but these are illustrative of

    20    the heterogeneity you get within any single

    21    population.

    22                So this clone 1A actually started to show

    23    even in the first round of confluence very tiny dense

    24    foci.  By the second round, it was showing these huge

    25    foci.  Then we had to dilute it out, and it continued

                                                                         38

     1    to produce large foci.

     2                This clone of cells transformed more

     3    gradually, and here you can see progressive

     4    transformation within a clone.  At first, light foci,

     5    broad foci are produced.  Then denser foci begin to

     6    show up, and finally you get foci with this clone that

     7    are just about the same roughly as this clone.

     8                You take clone 4B here, and in spite of

     9    many rounds of confluence, here you seem to get one

    10    light focus, but in the sister dish which was being

    11    transferred and not being fixed, apparently were no

    12    foci.  Even after five rounds of confluence, there's

    13    only a minimal amount of transformation.

    14                So what this means is every time you are

    15    working with a mixed population or an uncloned

    16    population, it's always a heterogeneous combination of

    17    clones that behave differently from one another in

    18    transformation.

    19                Okay.  Now I want to talk about one other

    20    point.  I tried to emphasize the point that an

    21    important aspect in transformation, both of primary

    22    fibroblast or at least explants directly from the

    23    animal, and in established cell lines is high density,

    24    is contact inhibition of the cells.

    25                I might mention parenthetically, it's now

                                                                         39

     1    been clearly established in bacteria, which is always

     2    our great sounding board for genetic change -- Warner

     3    Arbor who won a Nobel prize working with bacteria that

     4    led to very important discoveries found that, if he

     5    left a culture, several cultures of bacteria sitting

     6    around for 17 years so they hadn't grown at all during

     7    that time except the first day that they were sitting

     8    there, that there were enormous chromosomal

     9    rearrangements that occurred even in a single

    10    chromosome of bacterium.

    11                So cells that are being starved, in

    12    effect, are  great candidates for genetic change, both

    13    in bacteria and apparently in animal cells.

    14                Now what I'm going to illustrate in this

    15    last pair of slides is something that came as a great

    16    surprise to me.  Having worked with cells for about 50

    17    years now, I thought it was pretty hard to surprise

    18    me, but these cells are pretty clever.

    19                It was this.  We were working with this

    20    subline of NIH 3T3 cells which was very difficult to

    21    transform by keeping them at confluence, and we

    22    decided, well, we ought to look at what happens with

    23    clones of those cells to see how heterogeneous they

    24    are.

    25                What we found was quite a shock to us. 

                                                                         40

     1    Here we have two clones -- they're representative

     2    clones, clone 1A of this resistant line and clone 2E. 

     3    Then the parental population, which we're calling

     4    large A prime here.

     5                Again, we go through one round of

     6    confluence, and then this is the fourth round of

     7    confluence and the sixth round of confluence.  The

     8    shock was that, while the parental culture -- which

     9    is, after all, made up of all of these clones -- was

    10    exhibiting very little, if any, transformation or the

    11    barest minimum, the clones were showing a lot of

    12    transformation.

    13                So what does this mean?  I won't go into

    14    this point here.  So we quantitated that on the next

    15    slide.  This is the last slide.  And we made a scale

    16    of transformation, which is shown here.

    17                So none of them transformed to the extent

    18    of producing these really thick foci, but they would

    19    produce distinguishable foci, and they would produce

    20    foci even on a background of nontransformed cells.

    21                Just look at this first panel here.  We

    22    don't have to go into the second one.  It's sort of a

    23    cumulative observation of relatively heavily

    24    transformed cells -- that is, this type here -- or an

    25    accumulation of those plus more moderately transformed

                                                                         41

     1    ones -- these here -- or more moderately and lightly

     2    transformed ones.  

     3                So the combination of all of them after

     4    five rounds of confluence had involved about 80

     5    percent of the clones that we had.  At that time,

     6    shown on the scale at the top, the parental culture

     7    had shown no transformation at all, in spite of the

     8    fact that it was made up of thousands of those clones.

     9                So could I have the lights, please?  So

    10    the right panel is a repetition of the same

    11    experiment.  What that says to us, that when you get

    12    a heterogeneous mixture of clones from the parental

    13    population, there is some kind of mutual protection

    14    against transformation.  

    15                We later learned that back in 1981 George

    16    Post had found something very similar to this with

    17    melanoma cells in the mouse, that parental populations

    18    retained their consistency of behavior; whereas, the

    19    clones obtained from those populations underwent a

    20    great deal of variation.

    21                What we have to think about in that case

    22    is what that means for progression in the animals,

    23    because the general model that Henry Pitot was

    24    presenting to you, and general model that Wallace

    25    Clarke, the great melanoma specialist, has emphasized,

                                                                         42

     1    that in many systems in humans, including the

     2    formation of melanomas, I think, of liver cancers, of

     3    colorectal cancers, of skin cancers, all seem to go

     4    through a sequence, let's say, -- colorectal is

     5    probably the best known these days -- of the formation

     6    of foci of cells developing into polyps, into

     7    adenomas, all of which are of monoclonal origin.

     8                It's from those monoclonal, benign tumors,

     9    whether they are moles or adenomas or warts or, in the

    10    case of the liver cancer models in animals, these

    11    altered hepatic foci, that the next step is most

    12    likely to occur.  

    13                So that's a new parameter that we have to

    14    deal with where we get an association between the

    15    changes in the cell culture reflecting, we think, the

    16    effect of clonal expansion in the organism.  Thank

    17    you.

    18                (APPLAUSE.)

    19                DR. ONIONS:  Harry, I think you've proved

    20    that you don't need much warning to --

    21                CO-CHAIRMAN RUBIN:  Oh, I didn't go over. 

    22    That's a first.

    23                DR. ONIONS:  Is there time for one or two

    24    questions?

    25                DR. COFFIN:  John Coffin, Tufts.  What is

                                                                         43

     1    known, if anything, about the molecular changes that

     2    are associated with these different colony

     3    morphologies, for example?

     4                CO-CHAIRMAN RUBIN:  If you're asking me

     5    what have we done, nothing.

     6                DR. COFFIN:  Well, what is known

     7    elsewhere?

     8                CO-CHAIRMAN RUBIN:  Well, let me tell you

     9    something negative.  This is work Stuart Ansen did

    10    with the NIH 3T3 cells before we ever got them.

    11                So he encountered this spontaneous

    12    transformation as well.  So he tested the spontaneous

    13    transformation by extracting DNA and doing the

    14    classical transfection experiment into nontransformed

    15    NIH 3T3 cells.

    16                He found essentially none of them were --

    17    the DNA of none of them was able to transform more

    18    rapidly than as spontaneous transformation went on. 

    19    So there's no indicating, at least, that the Harvey

    20    ras gene had mutated there.

    21                What we know from classical genetics of

    22    cells in culture, actually, was done with other cell

    23    lines that was independent of transformation is that

    24    when you get chromosome rearrangements or deletions, 

    25    like the deletions that you get in loss of

                                                                         44

     1    heterozygosity where you lose real chunks of the

     2    genome, is you end up with cells that will grow slower

     3    than the original cell.

     4                If you get point mutations, it's very rare

     5    that that slows down the rate of multiplication, at

     6    least in the thymidine kinase gene, which has been

     7    looked at.

     8                So we think that the correlation that we

     9    see of reduced growth rate at low density of the

    10    transformed cells parallels the classical genetic

    11    findings, and it's likely that what we're seeing is a

    12    lot of chromosome rearrangements and deletions in the

    13    cell, but we don't have any proof of it.

    14                DR. COFFIN:  If I may raise another

    15    question.

    16                CO-CHAIRMAN MYERS:  Make it short.

    17                DR. COFFIN:  Yes.  Can you separate the

    18    slow growth property from the transformation property

    19    by forcing rapid passages to transform cells, for

    20    example?

    21                CO-CHAIRMAN RUBIN:  Yes.  Well, you always

    22    then select for faster growers, and it varies with

    23    whatever population you use.  In one population you

    24    can select from the faster growers, and they turned

    25    out to be less transformed, and in another population

                                                                         45

     1    you don't.  But you can do it.  

     2                Not all cells that grow slower are

     3    transformed.  That seems to be a more general finding

     4    that involves the whole population.  It's only a

     5    subset of those, presumably particular chromosomal

     6    rearrangements, deletions, etcetera, that result in

     7    transformation.  But a large proportion of those

     8    changes result in a slowing down of the growth of the

     9    cell at low density.  Okay?  Thank you.

    10                CO-CHAIRMAN MYERS:  Thank you.  Our next

    11    paper is the transformation by DNA viral oncogenes by

    12    Dr. Alex van der Eb from Leiden University.

    13                DR. VAN DER EB:  So as you have heard last

    14    night and you will hear in the coming thoughts during

    15    this meeting, diploid human cells have a finite life

    16    span in vitro.  They divide a certain number of times

    17    and then they stop dividing.

    18                This property limits to some extent their

    19    usefulness for the production of viral vaccines or

    20    production of viral factors for gene therapy.  

    21                Now cell lines, continuous cell lines, are

    22    immortal and are, therefore, more suitable in certain

    23    aspects.  However, there may be certain risks

    24    associated with the use of continuous cell lines. 

    25    Therefore, it would be helpful if we would be able to

                                                                         46

     1    immortalize diploid human cells without transforming

     2    them.  

     3                So far the only means of immortalizing

     4    diploid human cells is by -- reproducibly

     5    immortalizing is by transforming them with a DNA

     6    virus.  Whereas spontaneous transformation or

     7    spontaneous cancer is a multi-step process which

     8    requires a large number of different gene mutations or

     9    changes or alterations in genes that accumulate over

    10    the years, transformation by DNA virus apparently

    11    seems to be a one-step event.

    12                This is due to the fact that the viral

    13    transforming genes are -- or gene is a multifunctional

    14    protein that alters simultaneously a number of

    15    different regulatory pathways in the cells.

    16                I would like to discuss briefly

    17    transformation and immortalization by DNA tumor

    18    viruses, and particularly focus on SV40, an adenol

    19    virus, and also say a few words about HPV, but that

    20    will be more extensively dealt with by Dr. McDougall

    21    later during this meeting.

    22                If I can have the first slide, please.

    23                This slide shows the SV40 large T antigen,

    24    which is the major transforming gene of the SV40

    25    virus, and it also shows the three main transforming

                                                                         47

     1    domains in this gene.

     2                On the righthand side you see the P53

     3    binding sites which overlaps with ATPase binding site,

     4    which is very important for transformation by SV40. 

     5    A second site is more to the left, and that coincides

     6    with CR1, although CR2, the conserved regions 1 and 2,

     7    also is important.  That is a site that's responsible

     8    for binding of the RB protein and the RB family

     9    proteins as well as the coactivator B300 or CBP.

    10                In addition -- So this is the second

    11    important part for transformation, and in addition a

    12    third part is the N terminus which is the DNAJ-like

    13    protein -- like domain which resembles the DNAJ

    14    proteins of E. coli that have an important role in

    15    molecular chaperons for -- in conjunction with HSB

    16    proteins.

    17                How the DNAJ and terminus of SV40

    18    contribute to transformation is still unclear, but it

    19    is clear that P53 binding and inactivation of the RB

    20    proteins or P300 really very importantly disrupts the

    21    main growth control pathways in the cell.

    22                Adenovirus transforms basically in a

    23    rather similar way, and the next slide shows the

    24    adenovirus E1A gene, which is the major transforming

    25    gene of adenovirus.  As you can see here, there are

                                                                         48

     1    two again conserved regions here.  One is CR2 which

     2    are important for transformation.  

     3                The third region, CR3, is not important

     4    for transformation, but another region, the N terminus

     5    which is not conserved among adenoviruses, is on the

     6    other hand again essential for transformation.  

     7                So the N terminus, CR1 and CR2 are

     8    important for transformation of primary cells, as well

     9    as association to cellular proteins.  The N terminus

    10    and CR1 are responsible for binding to P300 and CBP as

    11    well as the P400 coactivator which is similar to --

    12    more or less similar to P300, but as CR2 and CR1 are

    13    important for binding to the retinoblastoma protein

    14    and the related pocket proteins, so again basically in

    15    a rather similar way compared to the SV40.

    16                Now the adenovirus E1A gene confers such

    17    a strong growth promoting effect on cells that these

    18    cells apparently -- certainly, if one has primary

    19    cells in which E1A is expressed to a reasonable

    20    extent, that these cells respond by activating their

    21    P53, and this will lead to either growth arrest or

    22    apoptosis.

    23                So to counteract this growth arrest or

    24    apoptosis, the E1B region is necessary.  So you see

    25    here the E1A region with the two proteins -- with the

                                                                         49

     1    two RNAs of proteins that I just showed you with CR1

     2    and CR2, as well as the N terminus which is located

     3    here.  But the E1B region calls for two proteins, a

     4    large protein and a small protein, and these are

     5    essential for neutralizing the apoptotic and cell

     6    cycle arrest activity of the E1A region.

     7                So E1A and E1B are needed in order to

     8    transform cells, because of the effect of E1A on P53.

     9                Therefore, it is almost impossible to

    10    transform -- obtain E1A transformed cells alone, and

    11    very few cells -- and those are rodent cells that are

    12    obtained that are transformed by E1A alone -- express

    13    E1A to very low levels.

    14                HPV, the human papilloma viruses,

    15    basically transform again in a similar way.  Next

    16    slide, please.

    17                The two transforming genes or papilloma

    18    viruses are E6 and E7.  E6 targets P53 and causes its

    19    degradation, but E7 again targets the other growth

    20    regulatory proteins, PRB, the RB protein and its

    21    family members as well as probably P300, but that

    22    we'll hear later in more detail.  So they resemble

    23    again the adenoviruses which have basically the same

    24    properties.

    25                Now not all these cells react in the same

                                                                         50

     1    way to these transforming genes or viruses.  Next

     2    slide, please.  Here I show you SV40, how SV40

     3    transforms cells, human fibroblasts, diploid

     4    fibroblasts or epithelial cells and keratinocytes.

     5                Fibroblasts are very efficiently

     6    transformed by SV40.  Immortalization, however, is

     7    very rare, at least 10-7 events per senescent cell,

     8    and there is a pronounced extended life span.

     9                Epithelial cells, on the other hand -- not

    10    on the other hand, but epithelial cells more or less

    11    at the same weight are responding more or less at the

    12    same rate as SV40.  There is morphological

    13    transformation.  There is also rare immortalization. 

    14    It's not completely clear how much their extended life

    15    span is, but I believe that there is an extended life

    16    span again in these epithelial cells.

    17                The situation is different for adenovirus

    18    E1.  Forget E1B.  This is an old slide where I thought

    19    that I had E1A transformed human cells, but this is

    20    not the case.

    21                Fibroblasts or epithelial cells appear to

    22    be surprisingly resistant to transformation by

    23    adenovirus.  Frank Rijm in 1974 has done many attempts

    24    to transform human embryonic retinal cells with DNA of

    25    adenovirus, and he was initially completely

                                                                         51

     1    unsuccessful until he found one clone, and this clone

     2    gave rise to the well known 293 cell, and that is

     3    really the only clone that he has seen transfected

     4    with adenovirus.

     5                Fibroblasts similarly do not show

     6    transformation, and I mean now transformation

     7    according to the focus assay.  You can introduce by

     8    transfection the adenovirus E1 genes into human cells

     9    where they are expressed, but we have the impression

    10    that expression is lost after a while.  There is no

    11    morphological alteration.

    12                These transformed cells -- and I'm talking

    13    now about 293 cells -- They immortalize probably after

    14    short crisis periods.  Efficiency of immortalization

    15    is not so clear, because there's only, as far as I

    16    know, only one example.  It's not so clear whether

    17    there is an extended life span.

    18                So we then switch to embryonic retinal

    19    cells, because we had heard from work of Phil Denimore

    20    and others that neural cells, cells of neural origin,

    21    could be transformed more easily also in the rate

    22    system, the rodent system; and we switched to

    23    embryonic -- primary embryonic retinal cells, and they

    24    can be transformed quite efficiently by adenovirus E1,

    25    although still at relatively low numbers compared to

                                                                         52

     1    transformation of rat kidney cells, for example, but

     2    it is reproducible.

     3                Also there is a high frequency apparently

     4    of immortalization, and there is no apparent crisis,

     5    which is rather surprising.

     6                I will not say much about HPV E6 and E7. 

     7    There is little interaction with fibroblast.  There

     8    may be reported very rare immortalizations by E6, E7,

     9    but epithelial cells at keratinocytes are immortalized

    10    more frequently, and apparently there is no distinct

    11    crisis.

    12                So transformation of human cells by SV40

    13    has been studied most extensively.  So I will briefly

    14    turn now -- go back now to SV40.

    15                If diploids and diploid fibroblasts -- If

    16    diploid fibroblasts are transformed by SV40, then a

    17    number of changes occur, and these are depicted here

    18    in this slide.

    19                The normal diploic fibroblasts grow to a

    20    certain saturation density during a number of

    21    population doublings, and then they stop dividing, and

    22    an irreversible arrest, growth arrest, which is called

    23    senescence, and it has been mentioned before.

    24                No immortalization will ever occur, as far

    25    as I know, from these senescent cells.  They do not

                                                                         53

     1    die, but they sit there just sometimes for many

     2    months.

     3                If you transform the culture at this stage

     4    for a -- with SV40, then the saturation density

     5    increases, and the transformed cells seem to ignore

     6    this senescence arrest phase and just go on for a

     7    number of -- for a large number of passages sometimes,

     8    which causes an extended life span.  However, in the

     9    end the cell -- the whole protein will die and enter

    10    into so called crisis where the cells -- the

    11    transformed cells really die, unlike the situation in

    12    senescence.

    13                Only in a very few cases, very rare cases,

    14    an immortalization will occur after a shorter or

    15    longer period, and this will etherize to a cell which

    16    is the same as the SV40 transformed cell before the

    17    crisis, but now they are immortal and can grow

    18    indefinitely.

    19                So what is the basis of the appearance of

    20    the senescence and the crisis?  That has to do with

    21    the telomeres.  In contrast to the germ cells which

    22    have telomeres active in these cells, the normal

    23    somatic cells are telomeres repressed and have no

    24    telomeres activity.

    25                So during population doublings, t he

                                                                         54

     1    telomeres are lost, become smaller and smaller until

     2    a certain stage is reached, which may be about two-

     3    thirds of the length of the telomeres, approximately

     4    two-thirds, but certainly not all telomeres have been

     5    used up, and at that stage the cells start senescence.

     6                This senescence is called M1 or mortality

     7    phase 1.  If you, in contrast, inform with SV40, again

     8    the M1 senescence is ignored, and the cells continue

     9    dividing, and also the telomeres become shorter and

    10    shorter until they are almost completely disappeared.

    11                That coincides with the crisis which is

    12    also called mortality stage 2.  So the M2.  If there

    13    is no immortalization, the cells -- all cells will die

    14    here.  Immortalized cells have now in most cases

    15    active telomerase.

    16                So this is then the extended life span

    17    between senescence and crisis, and it's also this

    18    period which is characterized by the onset of

    19    chromosomal abnormalities.  So there are -- and that

    20    effect already was also seen in the normal cells. 

    21    When they approach crisis, there is chromosomal

    22    rearrangements.  There are dicentrics formed, and so

    23    on.

    24                That is probably due to the fact that

    25    during senescence the P53 pathway, P53-P21 pathway, is

                                                                         55

     1    activated for some reason.  So what happens during

     2    senescence is that at a certain critical length of the

     3    telomeres -- we don't know exactly what the signal is

     4    -- will cause activation of preexisting P53.  There is

     5    probably not more transcription of the P53 gene, and

     6    this P53 causes accumulation of active P53 which

     7    causes activation of transcription of the P21 gene,

     8    the WAF-1 gene, which in turn is an inhibitor of

     9    cyclin/CDK and inhibits the cell cycle.  So this

    10    triggers apparently a certain minimum length of

    11    telomeres.  

    12                Now catalytic subunits of the human

    13    telomerase has been isolated.  The question can be

    14    raised would it be possible just to introduce

    15    telomerase in cells and immortalize them in diploid

    16    cells and immortalize them in that way? 

    17                Indeed, there has been reports that this

    18    is indeed the case.  Dr. Hayflick has already

    19    mentioned an example yesterday.  Also Dr. McDougall

    20    will probably talk about this later, but as Botnar and

    21    coworkers have also found, that fibroblasts as well as

    22    retinal epithelial cells become apparently immortal

    23    when the human telomerase catalytic subunit is

    24    introduced into the cells.

    25                Rather, Piono et al. showed that mammary

                                                                         56

     1    epithelial cells as well as keratinocytes do not

     2    immortalize just with only introduction of the

     3    telomerase, and this works that immortalization is

     4    obtained when, in addition, the E7 gene is added to

     5    the cells.  That, I think, will be discussed later in

     6    much more detail by Dr. McDougall.

     7                So this means that, if you inactivate the

     8    RB, then that would lead to immortalization.  This may

     9    be due to the fact that there is apparently a third

    10    type of senescence, and that third type of senescence

    11    is called N0 and occurs usually before M1.

    12                It is known that, unlike fibroblasts that

    13    can be found a long time, 50 to 60, 70 or sometimes 80

    14    population doublings, depending on the cell strain and

    15    also depending on the lab, I have the impression, that

    16    many other cells when taking a tissue culture form

    17    human tissues will not grow very long, and after ten

    18    or maybe 20 passages they just stop dividing.  This is

    19    long before the telomeres have reached the size, the

    20    minimum size which corresponds to the senescence, M1

    21    senescence.

    22                Now Weinberg has suggested that this M0

    23    state is due to a kind of physiological stress and is

    24    caused somehow by suboptimal growth conditions that

    25    occur in vitro.  So introduction of telomerase in

                                                                         57

     1    cells that still have to undergo an M0 may not work,

     2    because M0 is cause for physiological stress and has

     3    nothing to do with the size of the telomeres.  On the

     4    other hand, cells that have no M0 but only an M1,

     5    there it may work, but this is all theory, and we'll

     6    see if it is really true.

     7                So the last slide then summarizes here for

     8    the two senescent stages that occurred in certain

     9    cells, including the mammary epithelial cells but

    10    probably many more cells, and that is that M0 occurs

    11    after -- in the case of mammary epithelial cells,

    12    after about 20 population doublings, and may be

    13    activated by physiological stress.

    14                It's controlled by the RB P60 pathway, and

    15    can be bypassed by HPV.  It must be E7.  This must be

    16    E7, right?  It's wrong.  Jim, it's wrong.  It is E7. 

    17    I'm sorry.

    18                This was made just before I left, and

    19    there are more mistakes here, as you can see.  The

    20    computer did a trick and did not exactly what it was

    21    supposed to do.

    22                Anyway, it's bypassed by HPV E7.  One, on

    23    the other hand, may be triggered by certain extent of

    24    telomere shortening, and which is actually the mitotic

    25    clock, and is controlled by the P50-CP21 pathway, and

                                                                         58

     1    that is bypassed by HPV E6.

     2                M2 is called crisis leading to cell death

     3    and is probably caused by extensive telomere

     4    shortening.  It has nothing to do with all these

     5    things.

     6                So I think I'll stop here.  Thank you.

     7                (APPLAUSE.)

     8                CO-CHAIRMAN RUBIN:  We can have a few

     9    questions now.

    10                CO-CHAIRMAN MYERS:  Could we have the

    11    lights, please.

    12                CO-CHAIRMAN RUBIN:  Lights, please.   

    13    Could we have the lights?  Yes, thank you.  

    14                DR. HAYFLICK:  Hayflick, UCSF.  Unless I

    15    misunderstood your introductory remarks, there are

    16    indeed two other ways in which one can transform

    17    normal human fibroblasts to an immortal cell

    18    population.  Namely, we have done this with exposure

    19    to cobalt 60 radiation, producing a cell line called

    20    SUS M1.

    21                It's also been done with chemical

    22    carcinogens, producing a cell line called KMS T6, in

    23    addition to H terp which you've mentioned.  I also

    24    should mention that, contrary to popular belief, there

    25    are several publications in the open scientific

                                                                         59

     1    literature reporting spontaneous transformation of

     2    normal human cells.

     3                For those of you who would like those

     4    references, please contact me, and I'll be happy to

     5    supply  you with them.  Thanks.

     6                DR. VAN DER EB:  Thank you.  Yes, you can

     7    immortalize -- transform or immortalize human cells

     8    also by radiation or chemical carcinogens.  I think

     9    this is often quite difficult.  You have to expose the

    10    cells many times, as far as I know at least, to the

    11    chemicals or to the radiation, and it is a lot more

    12    easy just to take SV 40 large-T for fibroblast.  But

    13    you are completely right, yes.

    14                DR. HAYFLICK:  Well, the reason I

    15    emphasize that --

    16                CO-CHAIRMAN RUBIN:  Can you get to the

    17    microphone?

    18                DR. HAYFLICK:  The reason I thought it was

    19    worth emphasizing is that the thrust of this meeting

    20    is the development or the consideration of immortal

    21    cell populations other than those that might be

    22    currently used for vaccine production.

    23                Two of the obvious choices, if one wants

    24    to avoid the overt introduction of viruses or their

    25    fragments into cells, is by the use of radiation or

                                                                         60

     1    chemical carcinogens.  

     2                DR. VAN DER EB:  I know from cells that

     3    are spontaneously immortalized at least and also

     4    clearly transformed that they have lost quite a lot of

     5    things like P53 and also the RB pathway.  So what you

     6    do then is basically the same as what occurs or

     7    happens in spontaneous transformation, I think.

     8                CO-CHAIRMAN RUBIN:  One more question.

     9                DR. BERKOWER:  Ira Berkower from the FDA.

    10                One concept that we've been struggling

    11    with is the notion of cells that are more malignant

    12    and less malignant, more transforming and less

    13    transforming, with the idea that if vaccines could be

    14    made in less transformed cells, that will be safer.

    15                Does that make any sense in terms of these

    16    molecular mechanisms that you're studying, that there

    17    would be a more transformed phenotype?

    18                DR. VAN DER EB:  What is a more

    19    transformed phenotype?

    20                DR. BERKOWER:  Or a more malignable

    21    phenotype?

    22                DR. VAN DER EB:  This is difficult, I

    23    think.  If I see it now -- If I look at the

    24    literature, then I have the impression that if you

    25    take immortalization as the important final step that

                                                                         61

     1    you want to reach, that then HPV genes may be more

     2    suitable.

     3                I have the impression that they are -- The

     4    cells look less transformed at least than in the case

     5    of SV 40, for example, but also in the case of adeno. 

     6    But there's very little I can say to that, I think. 

     7    It's so difficult.  What is immortal?  Which cell is

     8    more transformed than the other?

     9                You would have to compare things like

    10    tumorigenicity.  I should say that cells transformed

    11    by viruses are often not yet immortalized, I showed

    12    you, but also if they are immoral, they are not yet

    13    immediately tumorigenic.

    14                So maybe tumorigenicity would be more

    15    important than just looking at the cells, how they

    16    grow.

    17                CO-CHAIRMAN RUBIN:  Okay.  We'll have to

    18    move on to the next speaker, who is Dr. James

    19    McDougall from the Fred Hutchinson Cancer Research

    20    Center at Seattle, Washington.  

    21                DR. McDOUGALL:  Well, as Alex has already

    22    pointed out, the two things that I would rather talk

    23    about are human papilloma virus and telomerase

    24    immortalization.  But when Andy called me -- Andy

    25    Lewis called me -- he said that somebody else had

                                                                         62

     1    dropped out of talking about hit and run, and so I got

     2    lumbered with the job.

     3                It's interesting.  It's interesting, but

     4    it's controversial, without doubt.  I'll just put my

     5    first slide on.  In the olden days, and there are a

     6    few of us that were there in the olden days, one of

     7    the most interesting studies that was going on in

     8    terms of looking at how viruses might transform cells,

     9    and particularly how they might contribute to human

    10    cancer, was the belief that herpes viruses might very

    11    well be responsible for, for example, cervical cancer.

    12                There were good reasons for believing

    13    this.  For example, serology gave very good evidence

    14    that women with cervical cancer had high levels of

    15    antibodies for herpes simplex, and it became clear

    16    that this was herpes simplex Type 2.

    17                So as time went on, it looked reasonable

    18    to try and sort out whether or not this virus had

    19    fragments, had subgenomic fragments that could

    20    actually transform cells.  That was one of the studies

    21    we concentrated on in the early 1980s.

    22                The studies were carried out mostly in rat

    23    cells or hamster cells, and lo and behold, we were

    24    very pleased to find that we could actually transform

    25    these cells and, if we grew these cells up from an

                                                                         63

     1    initial transformation by exposing them to herpes

     2    virus DNA and to fragments of herpes virus DNA -- and

     3    I'll show you the specific fragments in a minute --

     4    that those cells would then produce tumors in their

     5    rat or mouse host.

     6                So this looked very good, and we felt we

     7    were on the track of what might be the key genes that

     8    were involved in the development of an important human

     9    tumor, in this case cervical carcinoma.

    10                So over the years there were a lot of

    11    examples of how herpes viruses might perhaps have a

    12    hit and run type of effect rather than a similar

    13    situation that we see with the adeno viruses and SV 40

    14    and human papilloma viruses where it's clear that very

    15    specific fragments persist in the cells that are

    16    either immortalized or transformed by those.

    17                I just put up a series of papers that all

    18    refer to the hit and run phenomenon related to herpes

    19    viruses.

    20                So if we look at the herpes virus genome -

    21    - and here is the genome along the top here, and what

    22    I really want to bring your attention to are these

    23    fragments here that map in this region of the genome. 

    24                We initially show that this one called

    25    morphological transforming region 1 would carry out

                                                                         64

     1    that transformation of both rat and mouse cells at a

     2    reasonably efficiently level, almost at the same level

     3    as SV 40 would transform these cells.

     4                Subsequently, there were other studies

     5    which identified two other regions which also were

     6    capable of transforming, in this case, rat cells -- in

     7    both of these cases, rat cells.  

     8                Now that was somewhat surprising in that

     9    there would be more than one region of this genome

    10    that would actually transform or immortalize cells

    11    but, of course, this is a very large genome anyway. 

    12    So that that might not be too unreasonable.

    13                So we continued with a large series of

    14    experiments to try and track down exactly what the

    15    region of the genome might be that was producing this

    16    transformation effect.  Unfortunately, we were able to

    17    make smaller and smaller fragments of this MTR1 region

    18    until we got down to a region of that DNA which, in

    19    fact, was too small to have its own open reading

    20    frame, and immediately one has got to start worrying.

    21                If there's not an open reading frame, what

    22    in fact, is affecting the cell?  Is there a protein

    23    being produced?  In this case, clearly not.  So we

    24    began to worry what the mechanism might be in this

    25    situation.

                                                                         65

     1                So one of the mechanisms we thought about

     2    was that there might be just random insertion of

     3    sequences into the host cell genome.  So we looked at

     4    these MTR1 fragments, and particularly the MTR1

     5    fragment of herpes simplex virus type 2, and by

     6    sequencing that we were able to show there was a

     7    structure in there very similar to a bacterial

     8    insertion sequence in its structure, and felt that it

     9    was highly likely that this was perhaps integrating

    10    and then excising randomly from cells, and this might

    11    in fact have a mutagenic effect upon the cell.

    12                Now interestingly, we were able to take

    13    HSV1 and take a similar -- the identical region from

    14    the genome of HSV1 and look at the sequence and

    15    structure of that.

    16                What we found was that that structure,

    17    although for half of this loop, is pretty well

    18    identical.  The other loop did not pair sufficiently

    19    to make an insertion sequence-like structure.  That

    20    region of HSV1 will not transform cells, whereas this

    21    one will transform cells at a reasonable efficiency.

    22                So this seemed to be a reasonable

    23    suggestion, and perhaps we should look at, in that

    24    case, mutagenesis by this fragment, the MTR1 fragment. 

    25                Now while we were doing these experiments,

                                                                         66

     1    we also realized that there was another herpes virus

     2    that we should look at, and that was the human

     3    cytomegalovirus.  The genome is shown up here.

     4                Again, the region that we were

     5    particularly interested in was this early region, the

     6    immediate early region of the genome, which is

     7    transcribed very early after infection by the virus. 

     8    So one of the experiments we did was to try and work

     9    out whether we could actually, first of all, transform

    10    cells with this region, and the answer was yes, and

    11    this was repeated in two or three different labs.

    12                Then we wanted to find out, if we used

    13    very sensitive PCR experiments, could we in fact

    14    identify regions of that CMV immediate early region

    15    present in cells that were transformed by CMV.  By

    16    going through two rounds of PCR to pick this up, we

    17    were able to in fact identify a fragment from CMV that

    18    persisted in some of the cell lines, but really in a

    19    very low percentage.

    20                Most of the cell lines, just like the

    21    herpes cell lines, had lost the DNA that we

    22    transfected into them.

    23                Again, when we looked at the transforming

    24    region of CMV and made lots of deletion mutants of

    25    that region -- so we started off with a region here

                                                                         67

     1    which was quite a reasonable sized piece of DNA, 2.9

     2    kilobases, and just made deletions of this all the way

     3    down.

     4                Again, to our surprise, we could get right

     5    down to here, and we were still capable of

     6    transforming cells with that region.  So again, this

     7    suggested to us that maybe we were looking at the same

     8    situation with CMV that we had seen with herpes

     9    simplex type 1.

    10                In fact, in that very small region of CMV

    11    DNA, again you find this same structure present, and

    12    in most cases that structure was lost from the cells,

    13    that it had been put in by transfection, and we

    14    actually carried out not only transfection with DNA

    15    but made retroviral constructs and put that region in,

    16    could again show that we could transform cells with

    17    that region, but that it was lost from the cells on

    18    passage and in tumors produced by those cells.

    19                So the DNA clearly had been present there,

    20    but had been lost fairly on in the process, despite

    21    the fact that these cells are capable of making

    22    tumors.

    23                Obviously, one of the ways to look at this

    24    is that maybe these viral DNAs are acting in some way

    25    as mutagens, and there was certainly a history of the

                                                                         68

     1    fact that herpes virus infection would produce damage

     2    to chromosomes.

     3                I must qualify this, of course, by saying

     4    that in general herpes virus is lytic to cells, and so

     5    you don't expect cells to survive.  However, in most

     6    herpes virus populations there's a lot of defective

     7    virus present, which might very well allow some cells

     8    to survive in an infection.

     9                We could also show experiments done in Zur

    10    Hausen's lab in Germany that you could find mutations

    11    in host cell genes after transfection of herpes virus

    12    DNA-N.  So clearly the virus is acting as a mutant,

    13    and this is just a demonstration of some thioguanine

    14    resistance with a number of these herpes virus

    15    transforming regions, showing that there is in fact a

    16    mutation frequency that is measurable in these

    17    experiments.

    18                So this immediate early region in CMV

    19    became a region of interest to us, but we really

    20    stopped those experiments because they had been

    21    started out of an interest in cervical carcinoma, as

    22    I initially said.  We stopped those, because human

    23    papilloma viruses raised their beautiful heads, and

    24    obviously we now know that this is the prime

    25    initiating cause of anogenital carcinomas.

                                                                         69

     1                So we really stopped looking at this, but

     2    about ten years later it gave us a lot of pleasure

     3    when Tom Shent suddenly discovered the idea of hit and

     4    run, and reported in the literature that they've used

     5    immediate early genes of CMV and had essentially come

     6    to the same answer, that these regions would produce

     7    increased foci, increased transformation of cells, but

     8    in fact the DNA was then lost from the cells, and so

     9    this again looked like a hit and run phenomenon.

    10                I'll just go past this one for the moment. 

    11    And the type of experiment that came from Tom Shent's

    12    lab, which were published in 1997 in PNAS, was that we

    13    heard from Alex van der Eb already about the effects

    14    of adenovirus E1A, and what they showed was that if

    15    you combined E1A with the CMV early regions, E1 or

    16    preferable the E1 and E2, you could get a much higher

    17    number of foci produced in those transfected cells

    18    than if you had just E1A alone, and they showed this

    19    in a number of different experiments.

    20                So clearly, the CMV region, which was not

    21    then retained in these cells, was nevertheless

    22    contributing to the transformation of the cells, the

    23    same sort of picture that we've seen earlier.

    24                The other picture that they also found was

    25    that there was an increased mutation frequency,

                                                                         70

     1    looking at APRT in this case and HPRT in this case,

     2    that again if you put these immediate early genes in -

     3    - and you can do this on their own or in combination

     4    with E1A again -- you could demonstrate that there

     5    clearly was a mutation frequency resulting from the

     6    transfection of this DNA into, in this case, rat cells

     7    or mouse cells.

     8                So, clearly, there's no question that

     9    these small fragments of viral DNA that can transform

    10    cells most likely act as mutagens and are lost from

    11    the cells upon passage of these cells, but the cells

    12    retain their transformed phenotype.  That's probably

    13    the most important thing to remember out of this.

    14                So in conclusion for this talk, I'd just

    15    like to make these points:  That it's clear that

    16    subgenomic fragments of herpes simplex virus and

    17    cytomegalovirus can transform cells in vitro.

    18                Now these experiment are not conducted in

    19    human cells, but in rodent cells, as I've already

    20    said.  It's clear from a number of experiments that

    21    have been carried out now in certainly more than one

    22    or two labs that that virus DNA, which is capable of

    23    transformation, may not persist over the long term in

    24    the transformed cells.

    25                Although you can detect early on -- If you

                                                                         71

     1    put a complete open reading frame in, you can detect

     2    the expression of viral proteins.  You only see these

     3    early, and they do not persist, again suggesting that

     4    there is not only a loss of that sequence but perhaps

     5    a loss of sequence over time out of these transformed

     6    cells.

     7                Lastly, it's clear that these viral

     8    transforming regions can indeed be mutagenic, shown in

     9    a number of experiments both by our labs and by Tom

    10    Shent's labs.  So that still leaves the possibility

    11    that herpes viruses can be responsible for some -- the

    12    development of some tumors, and there are still

    13    experiments being looked at and some that have been

    14    published showing that, even in cells that are

    15    immortalized by human papilloma virus, the presence of

    16    some of these morphological transforming regions

    17    results in a more rapid conversion of those cells to

    18    tumorigenicity.

    19                So hit and run may seem an old phenomenon,

    20    but it's not dead.  Thank you.

    21                (APPLAUSE.)

    22                CO-CHAIRMAN RUBIN:  Questions?

    23                DR. KUNG:  Hsing-Jien Kung from UC-Davis.

    24                I'm just wondering, do you have some

    25    hypothesis whether this is due to insertion of

                                                                         72

     1    mutagenesis or due to the fact it knocked out P53,

     2    make the genome just generally unstable; therefore, it

     3    takes its own course.

     4                DR. McDOUGALL:  Well, I don't think P53 is

     5    not found in these cells.  So I still believe the

     6    hypothesis, that what you're seeing is integration of

     7    this and then a random excision of it is probably the

     8    reason, and that the integration is random, the

     9    excision is random, and probably the way to get at

    10    this, if we wanted to go back to these experiments, is

    11    really to do much more detailed sequencing of these

    12    regions and look at the flanking areas on any DNA that

    13    might still be there.

    14                It is very clear that this DNA is

    15    generally lost from these cells.

    16                DR. KUNG:  But you do have some residual,

    17    right, in some of the recent experiments?

    18                DR. McDOUGALL:  Some of the experiments

    19    with CNV there is some material that remains there,

    20    and we could go back to that and ask those questions,

    21    yes.

    22                DR. FRIED:  Did you --

    23                CO-CHAIRMAN RUBIN:  Identify yourself.

    24                DR. FRIED:  Fried from ICRF.  Do you take

    25    the region of that secondary structure that you

                                                                         73

     1    pointed to, and did that stop the mutagenesis?

     2                DR. McDOUGALL:  No.  What we did was just

     3    to use the HSV1 region as a control.  So we didn't use

     4    APH -- We didn't do anything to the HSV2 or CMV

     5    regions.  We just used the HSV1, which is, as you see,

     6    a tail.  It does not produce a nice insertion

     7    sequence.  We used that as our control.

     8                DR. FRIED:  And in Shent's experiments,

     9    were there any specificity to the type of mutations

    10    that were formed, based changes or --

    11                DR. McDOUGALL:  I confess, I don't

    12    remember the answer.

    13                CO-CHAIRMAN RUBIN:  We go to the back now. 

    14    Someone has been waiting there.

    15                DR. KRAUSE:  Phil Krause, FDA.  To your

    16    knowledge, has anybody ever taken MQR regions and

    17    injected them into nude mice or something like that

    18    and looked for an in vivo tumorigenicity endpoint?

    19                DR. McDOUGALL:  Not to my knowledge, no.

    20                DR. COFFIN:  John Coffin.  Stem loop

    21    structures such as you drew must, of course, be very

    22    common, you know, kind of DNAs, and actually -- I

    23    mean, is this effect specific for herpes virus DNAs or

    24    have they been studied so much more than almost any

    25    other DNA of similar kinds or complexities, if you

                                                                         74

     1    take bacteria for HDNA or --

     2                DR. McDOUGALL:  Well, in the bacterial

     3    systems, right.  I don't know of many studies in this

     4    sort of viral system, and I don't know of any that

     5    have been done very recently either.

     6                DR. COFFIN:  So, in fact, it could be that

     7    all kinds of DNAs may carry regions --

     8                DR. McDOUGALL:  I wouldn't be at all

     9    surprised.

    10                DR. COFFIN:  And it's just the herpes

    11    viruses that have been looked at.

    12                DR. McDOUGALL:  It just happens that that

    13    was a good candidate.

    14                CO-CHAIRMAN RUBIN:  One more question, and

    15    we'll have to move on.

    16                DR. BROKER:  Jim, has anyone taken

    17    synthetic stem loop structures or is it specific to

    18    the particular sequences that these various herpes

    19    viruses that are produced?

    20                DR. McDOUGALL:  Well, in a way, that's the

    21    same as John's question.  I think the answer is no. 

    22    AT least, I don't know of any experiments like that. 

    23    As I say, we got hopelessly diverted by papilloma

    24    virus.

    25                CO-CHAIRMAN RUBIN:  Could you please give

                                                                         75

     1    us your name?

     2                DR. BROKER:  Oh, Tom Broker, UAB.  A good

     3    choice, Jim.

     4                CO-CHAIRMAN RUBIN:  We'll have to go to

     5    the next speaker.  Thank you very much.  

     6                The next speaker is Stephen Baylin, Johns

     7    Hopkins University School of Medicine, who will talk

     8    about DNA methylation and epigenetic mechanisms of

     9    carcinogenesis.

    10                DR. BAYLIN:  Thank you very much.  I'd

    11    like to thank the organizers for inviting me.  As I

    12    hear some of the talks going before, with respect to

    13    the area our work is in and our group works on, I

    14    think of another kind of change other than the classic

    15    genetic changes in DNA that we've been hearing about,

    16    which is also heritable and involves DNA probably

    17    through chromatin structures which are variable.

    18                In terms of contribution to oncogenesis

    19    right now, I think one of the interesting aspects of

    20    that which has emerged is that process as it regards

    21    epigenetic gene silencing during all phases of tumor

    22    progression.  If I can have the first slide, please.

    23                Now for those of you who don't work in

    24    this particular area of epigenetics, let me just

    25    quickly remind you that in the eukaryotic genome and

                                                                         76

     1    certainly in humans and rodents, essentially the only

     2    place that we methylate our DNA is at the base

     3    cytosine and only at those cytosines that occur 5

     4    prime to a guanosine.

     5                We talk about the CPG dinucleotide as the

     6    substrate for this event.  Now it's an event which is

     7    catalyzed by what up until recently was thought to

     8    just be one mammalian enzyme, certainly DNA methyl

     9    transferase.  

    10                There are at least two others now that

    11    have activity, and this is going to probably emerge as

    12    a very important part of the story in tumorigenesis,

    13    the interplay and the roles of these different DNA

    14    methyl transferases.  But they each catalyze a

    15    reaction in which acidenosine methionine is used as

    16    the methyl donor group, and the methyl group is placed

    17    on the 5 position of the ring of cytosine.  

    18                The other important aspect of this for

    19    tumorigenesis which is reduced to real simplicity here

    20    in terms of what's being understood about methylation

    21    and its role in the genome is that in the eukaryotic

    22    genome the CPG dinucleotide has been drastically

    23    reduced over evolution to a small fraction of its

    24    predicted frequency.  

    25                So most of the genome has a reduced CPG

                                                                         77

     1    content.  However, in most of the genome there is a

     2    high percentage of those CPGs that actually get

     3    methylated in rodents and humans.  So we have heavy

     4    methylation, as shown by the yellow dots, in most of

     5    the genome.  

     6                This methylation may play various roles,

     7    one of which may be to participate in chromatin that

     8    is transcriptionally repressive for repeat elements in

     9    areas of unwanted transcription in the bulk of the

    10    genome.

    11                Now the exception to that type of

    12    distribution occurs in and around about half of the

    13    genes in our genome where the CPG dinucleotide

    14    frequency maintained at its predictive level, and

    15    these are the so called by Adrian Byrd and others CPG

    16    islands.

    17                For most genes -- say, for inactive genes

    18    on the X chromosome of the female and selected

    19    silenced alleles of imprinted genes, these CPG islands

    20    are maintained free of methylation, and this is

    21    thought to be a permissive state.  It doesn't matter

    22    whether the gene is actively transcribed or not, but

    23    it's a permissive state for active transcription of

    24    the gene.

    25                What's being more and more frequently

                                                                         78

     1    recognized in the cancer cell genome is an increasing

     2    list of genes in which this balance is disrupted, such

     3    that in the face of loss of methylation from large

     4    regions of the genome other than the promoter regions,

     5    CPG islands around an increasing list of genes

     6    actually have become methylated.

     7                Whether it's the cause or the result is

     8    still being worked out.  This correlates with a

     9    chromatin organization around that gene which is

    10    unfavorable for transcription of the gene.

    11                Now the impact for this on carcinogenesis

    12    and tumorigenesis, I think, can be looked at from the

    13    types of genes where this has now been defined.  For

    14    those genes which are known tumor suppressor genes by

    15    virtue of the fact that when they're mutated in the

    16    germ line of families, those families have inherited

    17    forms of cancer.  

    18                About half of those genes, as shown in the

    19    yellow, in somatic forms of cancer, noninherited

    20    cancer, have had some frequency now shown and well

    21    worked out of this promoter region CPG

    22    hypermethylation.  

    23                I would point to you that one of the most

    24    frequent times when this is seen is in the cyclin DRB

    25    pathway for the P16 gene, and now in a number of

                                                                         79

     1    experimental systems, including mammary epithelial

     2    cells, loss of M0 that we just heard about, at least

     3    experimentally and probably in several natural tumors,

     4    may be in association with hypermethylation of this

     5    cyclin dependent kinase inhibitor gene P16.

     6                So it can be a very early event in

     7    carcinogenesis and a very important one, as shown by

     8    just that one gene involvement.  Next slide, please.

     9                Other genes other than the classic tumor

    10    suppressor genes are emerging, and these are just a

    11    few, and they play a role in various kinds of

    12    processes, some for enzymes that guard against DNA

    13    damage such as methyl transferase, 06NGMT GSP pi. 

    14                Recently, for the P53-like gene where

    15    mutations of -- or P73 where mutations have not been

    16    found, very frequent to see hypermethylation of this

    17    gene in lymphomas.  So an increasing list of genes

    18    which can play a potentially important read in

    19    oncogenesis.

    20                Just to remind you, that in many of the

    21    instances where this has been defined, or in several,

    22    that gene might not have been recognized through

    23    classic mutational changes in the coding region for a

    24    role in tumors, because several of these genes such as

    25    P73, in some tumors even P16 like colon cancer, don't

                                                                         80

     1    seem to be altered through mutational change but have

     2    a high frequency of this promoter change.  Next slide,

     3    please.

     4                Just to remind you again how frequent this

     5    is, -- this is not to meant to be read -- this is a

     6    survey of major human tumors where Jim Harmon in our

     7    laboratory and others have been working out these

     8    hypermethylation events, and just to show you that

     9    virtually every kind of human neoplasm has multiple

    10    genes, not every tumor in every patient but, for

    11    example, in breast cancer, if you take the list of

    12    genes here shown to be hypermethylated and examine a

    13    series of breast cancers, you will find that every one

    14    of those breast cancers has one or more of these genes

    15    hypermethylated.  This now includes in about a quarter

    16    of them actually in the nonfamilial type of breast

    17    cancer the BRCA1 gene.  Next slide, please.

    18                Now in terms of what this might mean for

    19    therapy of cancers, the difference, of course, here

    20    from coding region mutations is that theoretically, if

    21    there are no base changes that inactivate the gene on

    22    a permanent basis, this is a potentially reversible

    23    situation, although indeed, if you look at it in

    24    culture and you look at it over time, for the most

    25    part it is a heritable event, the maintenance of this

                                                                         81

     1    kind of gene silencer. But it certainly has raised the

     2    question can reactivation of these hypermethylated

     3    genes serve as some sort of therapeutic target for

     4    cancer.

     5                This is not a new concept.  There has been

     6    a drug around for a long time -- next slide, please --

     7    Peter Jones and others introduced years ago when they

     8    found that drugs and congeners of 5 azacytidine can

     9    cause demethylation within the genome and reactive

    10    genes.  So this has been the drug that people have

    11    concentrated on.  

    12                It's only more recently that, I think,

    13    people are thinking about it in terms of specific gene

    14    reactivation for events in cancer, like John talked

    15    about.  Various other series of molecules, antisense

    16    again, so called DNA methyl transferase 1 and other

    17    molecules directed at the major mammalian DNA methyl

    18    transferase are being tried.

    19                It must be remembered that these other DNA

    20    methyl transferases that have recently been described

    21    are probably going to also play important roles in

    22    their separate genes with separate sequences, and so

    23    that's going to have to be taken into account.  Next

    24    slide.

    25                In our laboratory, it just shows you that

                                                                         82

     1    most of the genes where you see the CPG island

     2    methylation in culture, you can achieve at least

     3    partial reactivation of any or all of these genes

     4    through administration of the drug 5 deoxy

     5    azacytidine.  Next slide.

     6                And you can bring back functional events

     7    in the cells by reactivating these genes.  The classic

     8    example has been the loss of the mismatch repair gene

     9    hMLH1, which is probably the leading cause for the

    10    microsatellite instability phenotype in colon cancer. 

    11    Most of those colon cancers have this epigenetic

    12    change at the promoter of this gene.

    13                If you throw different mismatches at

    14    cultured cancer cells -- This is a Hela cell in Tom

    15    Kunkle's lab.  Actually, this is the 100 percent for

    16    each of the three different bases in a Hela cell which

    17    has effective repair.  

    18                Here's a hypermethylated colon cancer cell

    19    before azacytidine and, if you give 5 azacytidine to

    20    that cell, you can restore considerable mismatch

    21    function -- repair function to the cell by

    22    reactivating the mLH1 gene.  Next slide.

    23                In terms of manipulating the genome, I

    24    think what's become exciting in this area is to

    25    consider holistically the role of DNA methylation in

                                                                         83

     1    chromatin which is transcriptionally repressive, and

     2    this has been an exciting arena in the past year or

     3    two.

     4                Adrian Byrd's laboratory and Wolff's

     5    laboratory at the National Institutes of Health have

     6    made a big contribution to this recently when they

     7    showed that proteins such as mECP2 actually can bind

     8    preferentially to methylated cytosines in the genome

     9    and recruit proteins in a transcriptionally repressive

    10    complex which include a major player, histone de-

    11    acetylases, various histone de-acetylases.

    12                So what this does is suggest -- and

    13    there's been long an argument about the chicken or egg

    14    participation of methylation in the science -- that

    15    methylation can potentially through recruitment of

    16    proteins such as mECP2 actually see the repressive

    17    complexes around these methylated sites in the

    18    promoters of these genes.  Next slide, please.

    19                There are drugs that inhibit histone de-

    20    acetylase and will reactivate many genes.  This slide

    21    just shows you that in our hands for the

    22    hypermethylated genes in cancer cells and now in

    23    multiple others, that if you block histone de-

    24    acetylase alone with drugs like trichostatin at any

    25    doses -- and here is mLH1 in a colon cancer cell which

                                                                         84

     1    is hypermethylated, not expressed by rtPCR -- you

     2    can't reactivate, or we can't, using trichostatin

     3    alone.  However -- next slide -- if one combines a low

     4    amount of a drug like 5 deoxy azacytidine to achieve

     5    just a little bit of demethylation -- and here's the

     6    hMLH1 again.  

     7                So here's the azacytidine alone, and now

     8    you give trichostatin.  Within six hours of doing

     9    that, you can effectively reactivate the gene. We've

    10    seen at least four or five genes where this is the

    11    case.

    12                So this is suggesting to us the following

    13    type of scenario for work in a working construct, I

    14    think, that could prove fertile in this arena for

    15    understanding.  Just let me show you that this type of

    16    activation also brings the protein back to the cells.

    17                This is mLH1.  Here's an unmethylated

    18    colon cancer cell line, the green staining.  Here is

    19    a line that's methylated, and this is trichostatin

    20    alone.  Aza will bring back a few cells, and then the

    21    combination brings back in this short study a fraction

    22    of three or fourfold greater number of cells.  Next

    23    slide.

    24                In thinking about this, again in cartoon

    25    form is a working hypothesis, the following type of

                                                                         85

     1    scenario is one that is appealing to us.  As many

     2    people working in transcription machinery are showing,

     3    around the promoter of a gene generally there are a

     4    series of activating complexes, shown in the green

     5    here, and repressive complexes around the chromatin of

     6    this promoter that are simultaneously bound to the

     7    gene.

     8                The activator complexes, importantly,

     9    include coactivators, some of which, like CBP, pcaf,

    10    include acetylase activity.  The repressor complexes

    11    often include corepressors like syn 3 which are

    12    tethered to hdac, and the balance of these two,

    13    determined by incoming cell signals, would then

    14    determine the level of expression of that gene.  Next

    15    slide.

    16                What I think changes when you have the

    17    hypermethylation introduced into that promoter is that

    18    you change the nature of this repressive complex

    19    around the methylated CPG, shown here in the black, so

    20    that perhaps at least two things happen.

    21                One is that you tightly tether a complex

    22    that, through syn3 and probably other transcriptional

    23    repressive complexes which will behave in this way, to

    24    be shown, you tether hdac and deacetylase inhibition

    25    to that promoter region.

                                                                         86

     1                Our studies with the TSA would also

     2    suggest it's possible that you block or have less

     3    favorable access to a number of these coactivator

     4    complexes which have acetylase activity, and that

     5    leads to a balance of the acetylase activity and

     6    methylation around the gene that results in a rather

     7    tight transcriptional repression.

     8                What we may be doing when we begin to

     9    relieve this methylation with the drug in a little way

    10    is to allow return perhaps of these acetylases and

    11    coactivator complexes, and now the cell cares if you

    12    block the deacetylase, and it contributes to gene

    13    reactivation.  Next slide.  

    14                This needs to be worked out by showing who

    15    the players and the chromatin setup around these

    16    promoters really are at these genes in cells where

    17    they are methylated versus nonmethylated, which many

    18    groups obviously are now working on.

    19                So it has raised a possibility, which

    20    actually is getting some trials now in places like our

    21    institution and others, that by administration of

    22    several days of low dose 5 azacytadine to patients

    23    which may avoid some of the toxicity that's been seen

    24    with these kinds of compounds, much of which may not

    25    be due to actually effects on methylation but actually

                                                                         87

     1    binding of the drug to the DNA, that if you could do

     2    this in combination with drugs like phenylbutarate and

     3    others that are coming that inhibit histone

     4    deacetylase activity, one might get a favorable ratio

     5    for reactivating some of these key genes like P16 and

     6    others, which might have therapeutic benefit.  Next

     7    slide, please.  That may be my last.

     8                I think that is my last slide.  Thank you

     9    very much.

    10                (APPLAUSE.)

    11                DR. DOERFLER:  I would like to comment on

    12    the promoter models.  Now to my experience, the

    13    situation is slightly more complicated than you have

    14    depicted in some of these slides.

    15                For instance, the finding that some of the 

    16    promoters are rich in CPT dinucleotides would be

    17    completely unmethylated, I don't think, is so clearly

    18    true.  We have looked at many promoters, some of which

    19    are as you showed and are active or inactive.  Others

    20    are methylated in spurious nucleotides.  However, you

    21    find them only if you apply the genomic sequencing

    22    technique which detects all methyls in a promoter.

    23                Lastly, when one talks about the

    24    mechanism, of course, the one you proposed is very

    25    plausible.  On the other hand, I think there are

                                                                         88

     1    examples where a methide group inhibits the finding of

     2    a transcription factor.

     3                So there are several ways of how this

     4    scheme might work.

     5                DR. BAYLIN:  Well, I think Dr. Doerfler's

     6    comments -- I hope you could hear them -- are exactly

     7    right in the time frame.  So let me address the last

     8    one first.

     9                There are certainly genes which do not

    10    have CPG islands, most of them, in which occasionally

    11    a methylation event right in and around a

    12    transcription factor recognition site is important to

    13    access of that transcription factor to the gene, and

    14    can play a role in expression.  No question about

    15    that.

    16                Probably even within CPG islands, although

    17    density seems to be the most important determinant,

    18    there are also regions within the island of

    19    methylation that are important for where it occurs,

    20    but density is extremely important.

    21                I think he's again right that, if you do

    22    genomic sequencing around these promoters -- every

    23    dogma changes a bit -- do you find no methylation in

    24    virtually all of these CPG islands?  And no, you find

    25    methylation.  But what is often very much protective

                                                                         89

     1    is a region right in and around the transcription

     2    start site.

     3                Often the regions of methylation that you

     4    do find in normal cells in these islands is distal to

     5    that region right around the transcription start site,

     6    for the genes that we've sequenced at least, and this

     7    is true for Ecad here, and it's true for mLH1.  It may

     8    be that that's the regions in which their methylation

     9    is seeded into these areas during tumor progression

    10    that causes the increased density and begins to more

    11    and more affect the expression of that gene.  But

    12    you're right, it is more complex than the dogma would

    13    have indicated.

    14                CO-CHAIRMAN RUBIN:  Thank you.  I think

    15    we'll have to move to the next speaker, because

    16    otherwise we're not going to have a break.

    17                The next speaker is Dr. Walter Doerfler of

    18    the University of Cologne, who will speak on a new

    19    concept in viral oncogenesis.

    20                DR. DOERFLER:  I'd also like to thank the

    21    organizers for inviting me to this very interesting

    22    and constructive meeting.  Could I have the first

    23    slide, please.

    24                For actually not 50 years ago, but for

    25    more than 30 years we have used the adenovirus model

                                                                         90

     1    to look at problems of viral oncogenesis.  Here's a

     2    model of one of the adenoviruses.  We have been mainly

     3    using adenovirus type 12, and we've been following the

     4    model established in 1962 by Trenton Yabe and Taylor

     5    who showed that, when adenovirus type 12 in small

     6    doses is injected into newborn hamsters, actually

     7    about 70 percent of the animals that survive the

     8    injection develop tumors within 30 to 50 days.

     9                That model we have been following most of

    10    our studies.  The interesting pursuit mainly is that

    11    of viral DNA integration and its possible

    12    consequences, and that's what I'd like to discuss with

    13    you today.

    14                So here is my model and some of the

    15    concepts which at least for me are new and which we --

    16    I can't say we have proven these concepts, but we'd

    17    like to pursue them in future research.

    18                So definitely the adenovirus is

    19    integrating its DNA, and tumor cells are transformed

    20    cells.  There's no free viral DNA.  The site of

    21    insertion is nonspecific.  It's different in each

    22    tumor, and there are multiple copies inserted.

    23                The inserted DNA, much like any foreign

    24    DNA -- if you use lambda DNA, if you use plant cells

    25    and put transgenes in, the foreign DNA becomes de novo

                                                                         91

     1    methylated, an observation we made way back in '78.

     2                Recently, we have been able to show and

     3    represent some of the data today that as a consequence

     4    probably of this insertion, changes of DNA methylation

     5    occur at places distant, remote, from the site of

     6    insertion, although the immediate site of insertion

     7    can also be affected.

     8                Now changes in methylation, as the

     9    previous speaker just discussed with you, can lead to

    10    changes in transcription, and we'd like to pursue the

    11    idea that these changes might play a role in viral

    12    oncogenesis.

    13                Now from a totally different point of view

    14    -- and that project we've been pursuing only for 12

    15    years; I may not have time now to present all the data

    16    on that.  So I've asked for some minutes on Thursday

    17    night.  A much more frequent occurrence, a much more

    18    frequent encounter of an organism, this foreign DNA,

    19    is not necessarily the viral infection but it may be

    20    uptake of foreign DNA in the foods supply, and we've

    21    been studying that to some extent, and I may have a

    22    chance to present the data.

    23                Now here is again the concept.  Viral DNA

    24    or other foreign DNA inserts into the host genome. 

    25    This DNA is de novo methylated.  We'd like to think of

                                                                         92

     1    this de novo methylation as an ancient host defense

     2    mechanism which defends against the foreign genes.

     3                As a consequence of this insertion,

     4    changes in the patterns of DNA methylation occur at

     5    remote sites in cellular DNA.  Perhaps, depending on

     6    the site of foreign DNA insertion, this may also lead

     7    -- it's not been shown too stringently, at least not

     8    in our lab -- in the instances we have looked at,

     9    there's only indications, but there is evidence from

    10    other laboratories that alterations in methylation

    11    accompanied by alterations in chromatin structure, and

    12    these numerous changes can lead to alterations in

    13    transcription.  I will show you some evidence for

    14    that.

    15                Now let's look at the data.  When we

    16    infect cells, hela cells, with adenovirus Type 12 or

    17    when we -- Maybe we could dim the lights a little bit

    18    -- or when we look at transformed cells -- this is a

    19    transformed cell line, transformed by adenovirus Type

    20    12, and a stretched chromosome preparation --

    21    chromosomes have been subjected to low centrifugal

    22    force, and you can see the multiple copies of

    23    integrated viral DNA, or you could see them if we

    24    could dim the lights a little bit perhaps.  Thank you

    25    very much.

                                                                         93

     1                A very similar picture emerges when we

     2    look at the chromosomes in infected cells.  For a long

     3    time we're trying to show that also infected cells,

     4    productively infected cells at least early after

     5    infection do show some association, actually massive

     6    association, of viral DNA with the host chromosome and

     7    the pictures between infected cell upon stretching the

     8    chromosome and the transformed cells that we have

     9    definitely shown of this DNA integrated by cloning and

    10    sequencing junction fragments do look very similar.

    11                So we've pursued the possibility that at

    12    least transiently some of the DNA becomes also

    13    inserted and productively infect the cells.  As long

    14    as we have the lights down, this is a blow-up of the

    15    transformed cell.  

    16                You might imagine that there's multiple

    17    copies aligned in a string, pearl-like fashion, in the

    18    stretched DNA, in this case hamster DNA.  

    19                Now just a brief summary of some of the

    20    observations, summarizing many years of work.  At the

    21    chromosomal and nucleotide levels there is no

    22    specificity of insertion.  There may be rare

    23    exceptions.  The integration may occur preferentially

    24    at transcriptionally or otherwise may be replication

    25    active sites.

                                                                         94

     1                The consequences are, as I mentioned

     2    before, de novo methylation of the integrated foreign

     3    DNA, and secondly, changes in patterns of methylation

     4    in cellular DNA segments.  The integration is in

     5    general stable, but we have found revertants which

     6    have lost all or most of the viral genome, and are

     7    still oncogenic.  Actually, we reported that first in

     8    1982 in a paper with Ingrid Kuhlman, and in this case

     9    hamster tumors were induced by injecting adenovirus

    10    Type 12.

    11                The integrated state was demonstrated at

    12    that time by Southern blotting and also by cloning

    13    junction sites.  Upon cultivation of these cells,

    14    occasional loss of the viral DNA was observed,

    15    initially by Southern blotting in the old days, and

    16    these Southern blot negative cells, clonal lines, were

    17    still oncogenic just as much as the original cells

    18    from the tumor.

    19                Now when we used more recently, with Anna

    20    Pfeffer, PCR analysis, we found very similar to what

    21    Jim McDougall was just telling us, tiny bits of the

    22    adenovirus Type 12 DNA in several of these lines, none

    23    of the lefthand end but only snippets from the

    24    righthand end.

    25                Each clonal line seemed to have a

                                                                         95

     1    different persistent pattern of these small fragments. 

     2    When these cells were injected again, they were as

     3    oncogenic as the cells that carried the viral DNA.  So

     4    we had a discussion before on the hit and run

     5    mechanism.  It's a possibility, but of course, there

     6    are other ways of interpreting these data, but it's

     7    certainly interesting in this context.

     8                I will skip this slide, not to spend too

     9    much time, and return to the cell line T637, hamster

    10    cell line, and adenovirus Type 12 transformed.  It

    11    carries about 15 to 20 copies of viral DNA.  As you

    12    see on this fish preparation, there's a green spot

    13    that contains the adeno sequences.  The red regions

    14    are representing endogenous retroviral genomes. 

    15    That's about 900 copies per cell, often located on the

    16    short arms of the chromosome.  

    17                Now in the following slide I will use this

    18    cell line and others to look at changes in methylation

    19    patterns in these regions, which are abundant, and

    20    changes might be readily apparent.  

    21                In fact, when we do such a comparison,

    22    work done by Hilda Heller in our laboratory, and

    23    compare patterns of methylation in the IAP region of

    24    the BHK hamster cell -- that's the parent line for

    25    this transformed cell line that carries the adeno 12 -

                                                                         96

     1    - we see both for the hepa-2 and for the hh1 cleavage,

     2    both sensitive to methylation in CCCG or GCGC

     3    sequences, a very striking increase in DNA methylation

     4    as compared to the parent line.

     5                This increase persists when the TR3 line

     6    is investigated, which has lost all of the adeno 12

     7    copies.  This is both based on Southern blotting, but

     8    there may be still snippets left.  We haven't analyzed

     9    that extensively yet.  But it's important, I think, to

    10    our way of arguing that these changes in methylation

    11    persist.

    12                Hilda Heller has looked at another of

    13    other cellular regions, for instance, like the part of

    14    the major histocompatibility complex and others in

    15    these tumor cell lines and in other cell lines, and

    16    has found changes in some, not in others.

    17                So certainly, in these transformed tumor

    18    cells there are extensive changes in DNA methylation

    19    patterns.  Now this may not be so surprising, because

    20    we are talking about transformed cells with a

    21    transformed phenotype.  That most likely plays a role

    22    in the eliciting of changes in DNA methylation.

    23                We have considered other possibilities, in

    24    addition, like the function of early viral gene

    25    products which are expressed, but when we infect the

                                                                         97

     1    same cells, in the case of adeno 12 which also

     2    expresses early viral gene function under these

     3    conditions, we do not see these changes.  But we have

     4    not pursued this possibility, although it's not

     5    definitely ruled out.

     6                The third possibility we have investigated

     7    more extensively, namely, the notion that just the

     8    integration of foreign DNA nonviral and nonviral

     9    transforming could perhaps also elicit similar

    10    changes.

    11                So Hilda Heller and Kristina Kaemmer made

    12    a number of bhk hamster cell lines that were

    13    transgenic, not for adeno DNA but for bacteriophage

    14    lambda DNA.  In these panels you see -- You may not

    15    see it well in the back, but up front you can clearly

    16    see that these cell lines are all lambda DNA

    17    transgenic, as determined by FISH analysis.  This is

    18    a control, and again the lambda DNA is located in

    19    different positions and different clones, becomes de

    20    novo methylated, just like adeno DNA behaves like any

    21    foreign DNA in a cell line like bhk21 cells.

    22                Now we've now looked at these cells for

    23    changes in DNA methylation, and we've concentrated

    24    again on a subsegment of the IAP 1 region, which is

    25    shown here in full detail.  You see all the CG

                                                                         98

     1    dinucleotides.  These are the lollipops here.

     2                The HP sides are just these two sides.  So

     3    you see my concern when conclusions are drawn about

     4    DNA methylation using just restrictases, because with

     5    upper you just catch two out of 35, and with HP you

     6    just catch two out of 35, and with Hh, the dots, you

     7    catch maybe six out of 35, just a subpopulation of

     8    nucleotides -- dinucleotides that can be subject to

     9    changes in DNA methylation.

    10                So we first looked again at Hh and HP,

    11    cleaved DNA from these lambda transgenics.  Could I

    12    have the next slide, please?  Thank you.

    13                You see it mainly for the Hh.  For the HP

    14    it's less striking, although some changes are

    15    observed, but for the Hh it's very noticeable that

    16    even in sub-clones, different clones of lambda

    17    transgenic cell lines show these changes.

    18                I should mention that one of these cell

    19    lines has also now been subjected by Knut Mueller to

    20    an analysis on changes in transcription, by doing a

    21    differential hybridization of C-DNAs, and Knut can

    22    find quite considerable changes in the transcription

    23    level of these -- not only the total level but of

    24    cloned segments out of these cell lines.

    25                Now to convince ourselves of these

                                                                         99

     1    changes, and particularly the reviewers who were

     2    doubtful, we used the bisulfide treatment of the DNA. 

     3    In brief, the bisulfide converts a cytosine to a

     4    uracil, but a methyl C is resistant to this

     5    conversion.

     6                So then after complete conversion of the

     7    DNA, we PCR out the segment we are interested in -- in

     8    our case, the IEP1 region -- clone individual PCR

     9    products, and sequence up to several hundred of these

    10    clones.

    11                Whenever we find a C in a known sequence,

    12    it's a 5 methyl C after the treatment, and we see a U

    13    or a T, it's a C.  

    14                I just give you a quick summary of the

    15    data.  What you see here are the positions 1 to 34 in

    16    the segment I showed you on the map out of IAP.  Each

    17    of these numbers represents one CG dinucleotide, and

    18    in hundreds of clones we plot here the percentage of

    19    clones which are methyl C in a particular location.

    20                So let's first look at the bhk 21 cell

    21    line, which is sort of the base.  This has a certain

    22    pattern of methylation, as I showed you before in the

    23    Southern blot.  Of course, we didn't see any -- All

    24    this 34, you just saw the HP and Hh side.

    25                Now when we look at P637 cell line where

                                                                        100

     1    we find a blatant, a very obvious increase in DNA

     2    methylation by HP and Hh blotting -- cleavage, you

     3    find the same effect when we look at all the CGs and

     4    the percentage of those CGs that were methylated.

     5                Now the lambda lines are somewhere in

     6    between.  I've shown two examples here, and there are

     7    changes in some positions, not in others.  When we

     8    look at clonal sublines of CPHK21 cell line, we do not

     9    see these increases.  The possibility existed with

    10    respect to methylation IAP in the bhk population per

    11    se, but by this and another method we have applied, we

    12    did not see that.  So we don't pursue that any

    13    further.

    14                We conclude that after insertion of lambda

    15    DNA in these transgenic cell lines, we do see quite

    16    distinct changes in levels of methylation, and in one

    17    cell line investigated so far we also see changes in

    18    transcription, as done by differential hybridization

    19    and CDNAs.

    20                Now how might this change come about?  Of

    21    course, I don't know.  So I show you a cartoon, and

    22    one possibility we consider is that, due to the

    23    insertion of foreign DNA as an alteration or you might

    24    call it a destabilization of DNA surrounding here, and

    25    this may be -- since chromosomes have a unique

                                                                        101

     1    neighborhood in a nucleus, this may be somehow

     2    transmitted to other parts of the genome, and the

     3    parts affected naturally depend on where the foreign

     4    DNA inserts.

     5                This adeno is a recent immigrant of 30

     6    years ago.  The IAPs have been there for 5 million

     7    years, and the ancient genes like MHC can also be

     8    affected.  So this is a way of thinking about it.  Of

     9    course, no proof whatsoever, but I think the

    10    observation is quite clear, and we are now preparing

    11    other approaches to make -- obtain additional

    12    evidence.

    13                Particularly, we are thinking about using

    14    perhaps the primary human cell lines, because then we

    15    could have much better information about the genes in

    16    which the DNA methylation patterns are changing.

    17                Now lastly, and this I will just summarize

    18    very quickly, the more frequent encounter of an

    19    organism with DNA, at least to my judgment, is the

    20    daily uptake of food.  So we asked the question

    21    whether DNA that is orally administered to a mouse --

    22    we used M13 DNA or a green fluorescent protein cloned

    23    DNA or, more lately, we used leaf from soybeans and

    24    watched for one of the genes out of the light side,

    25    the ribulose biphosphate carboxylase, for its

                                                                        102

     1    persistence in the gut and places beyond.

     2                I will not have time to do just to the

     3    data, which were mainly obtained by Reiner Schubert

     4    and Gerte Holbeck.  The concept behind this, perhaps

     5    at first a strange experiment, is it's foreign DNA

     6    integrated into an established genome consistently or

     7    very frequently becomes de novo methylated, and I

     8    consider this a defense mechanism.

     9                If you postulate a defense mechanism, the

    10    next question is, well, where is the attack.  The

    11    attack very frequently, naturally, is in the

    12    gastrointestinal tract with a huge surface and a

    13    tremendous immune system.

    14                So we have asked the question, can

    15    intestinal depotea take up the DNA.  They seem to be

    16    able.  We find, first of all, the DNA persisting at

    17    certain times after feeding to ease the control of

    18    unfed animals, TE, EDTA buffer and then there is

    19    observation of M13 DNA one hours to eight hours after

    20    feeding, not later on, in the cecum or in the small

    21    intestine, the large intestine.  At about ten-to-

    22    hundredfold lower level we also find persistence in

    23    the blood of fragmented forms of DNA.  The largest

    24    fragments here are about 1700 out of 7,250 of the M13

    25    DNA, and about 800 in the blood.  So it's not the

                                                                        103

     1    total DNA that's persisting.

     2                When we go to sections -- again, you will

     3    have difficulty seeing that in the back.  If you want

     4    to see my printouts, I can show  them to you.  We have

     5    persistence in gut epithelia, in pia patches or in

     6    liver cells that are controls, and Reiner Schubert

     7    looked at hundreds of sections through the gut and

     8    through the liver and the spleen, and in some of these

     9    section he found -- by applying the FISH, he found DNA

    10    persisting over in the nuclei of these cells.

    11                Now FISH is nice, and PCR is nicer, but

    12    you always have nightmares, not only when you are in

    13    jet lag.  You wake up four o'clock in the morning

    14    thinking maybe this is the wrong interpretation.  

    15                So what Reiner Schubert and Doris Renz did

    16    was to reclone the DNA from the spleen of animals that

    17    have been fed for a week daily, and in 18 hours after

    18    the last feeding spleen DNA was prepared and clones

    19    were -- DNA was cloned into lambda-2 vector, and

    20    lambda plaques were investigated.

    21                Amongst several hundred million of

    22    plaques, about five to six were found positive for M13

    23    and for the mouse DNA.  Then what you see here are the

    24    coordinates of the M13 phage DNA.  I told you, it's

    25    one 7,250 nucleotide long molecule, and these are the

                                                                        104

     1    coordinates that we find persisting in individual

     2    clones.

     3                Two clones were of particular interest,

     4    because they had, as the nearest neighbor to the M13

     5    DNA, DNA with 70 and 80 percent homology to known

     6    mouse genes.  So we feel that perhaps the M13 DNA in

     7    rare instances has become integrated into mouse DNA in

     8    the neighborhood perhaps of pseudogenes.

     9                It did not escape our attention that in

    10    some clones we found 100 percent homology to known E.

    11    coli genes.  So if you have some fantasies, you can

    12    say, well, perhaps there's a steady stream of foreign

    13    DNA from the gut to the spleen, and then the uptake

    14    and perhaps also some of it integrated in rare

    15    instances in individual cells.

    16                I will not go into details now on feeding

    17    experiments where we have also looked at pregnant

    18    animals and transferred to the next generation, which

    19    can occur, and also found DNA in some cells in the

    20    fetus and in the newborn, perhaps also in an

    21    integrated state, but I will not have time to discuss

    22    that.

    23                Let me just say in closing, when Gerte

    24    Holbeck has investigated animals which have been fed

    25    soybean -- this is not naked DNA like we did in these

                                                                        105

     1    series of experiments which are a little bit way out,

     2    but we figured first let's try an extreme case.  If

     3    that DNA persists, then perhaps we can also expect

     4    natural feeding experiments to give results.

     5                Now when we look at the, as I said before,

     6    ribulose biphosphate carboxylase gene after feeding

     7    leafs of soybeans, and we can also find this DNA to

     8    persist actually in practically unfragmented form in

     9    the gut.  Of course, we don't know is it still in a

    10    cell or has it been liberated.  But in rare instances

    11    now in three cases, we could also see by PCR some of

    12    the DNA which has no homology to mouse, because mouse

    13    doesn't have such a gene, has no life cycle in spleen

    14    and in liver.

    15                So perhaps even by that route, the natural

    16    route, if you wish some of the foreign DNA, masses of

    17    which we ingest daily can persist in the gut.  I have

    18    no doubt about that, and can enter probably via

    19    columnar epithelia in the gut, pia patches, thoracic

    20    duct, white blood cells, spleen and places beyond.

    21                Whether it has any effect, any long range

    22    effects on the cells in the way I have been

    23    speculating in my first slide, of course, remains to

    24    be determined, and this will be a much harder task

    25    than looking for a tiny needle in a haystack, like in

                                                                        106

     1    a mouse.

     2                So in summary, we'll continue to pursue

     3    this concept that the insertion of foreign DNA may

     4    have much further effects, much more far reaching

     5    effects on an established mammalian genome than just

     6    a local perturbation, but this local perturbation may

     7    be transmitted to other parts of the genome and change

     8    transcriptional and certainly methylation patterns.

     9                Thank you very much for interest.

    10                (APPLAUSE.)

    11                CO-CHAIRMAN RUBIN:  Any questions?  

    12                DR. COFFIN:  You seem to reject -- Oh,

    13    John Coffin, sir.  You seem to reject hypotheses that

    14    relate to selection, say from a variety of expression

    15    of methylation in preexisting cell clones.  But at the

    16    same time you suggest that methylation is -- methylase

    17    is a guardian of the genome against integration

    18    expression of foreign genes.

    19                If that's the case, could not your data

    20    also be explained, at least so far as you presented

    21    it, by the idea that preexisting cells with lower

    22    amounts of methylation, the rare preexisting cells are

    23    much more sensitive to transformation events and,

    24    therefore, selected by the transformation experiments

    25    that you showed?

                                                                        107

     1                DR. DOERFLER:  Thank you very much for

     2    this question.  Of course, we are still considering

     3    this possibility, and it was one of the possibilities

     4    mentioned on my slide under number 4.

     5                Now I have two lines of thought which are

     6    at least unsupported -- can't rule it out.  One, when

     7    we look at many clones, subclones, of the bhk cell

     8    line that we use sort of as a total which could be

     9    heterogeneous, and look at -- I think Ralf Remos has

    10    looked at 70 or 80 different clones, again by HP and

    11    Hh cleavage.  Doesn't see any differences.

    12                Now that may not be good enough.  So we --

    13    In the CGG genomic sequencing analysis in the region

    14    that I discussed, IAP-1, Ralf also did the same type

    15    of genomic sequencing analysis, and again he found no

    16    striking differences --  I mean, there's always some

    17    variation, of course -- to the basic total cell line.

    18                So although when you have looked at 70

    19    clones or at five or ten clones, subclones, this is

    20    not very much, when you have to look at millions, it's

    21    difficult to rule it out completely.  But certainly,

    22    the data -- and we're continuing this sort of analysis

    23    -- do not support this notion.  It's difficult to rule

    24    it out completely.

    25                CO-CHAIRMAN RUBIN:  Are there anymore

                                                                        108

     1    questions or is everybody so eager to get to the

     2    break?  Okay, thank you very much, Dr. Doerfler.

     3                We're going to take a break now, and we'd

     4    like to be back by eleven o'clock to start again, for

     5    sure.

     6                (Whereupon, the foregoing matter went off

     7    the record at 10:33 a.m. and went back on the record

     8    at 11:01 a.m.)

     9                CO-CHAIRMAN MYERS:  If everybody could

    10    take their seats, we'll reconvene.  Probably the most

    11    difficult thing that has to occur in a meeting is

    12    ending the breaks.  

    13                We're going to continue our morning

    14    session.  Our next speaker is Dr. James Cook, who is

    15    Professor of Medicine at the University of Illinois,

    16    who is going to talk about the role of nonspecific

    17    NK/macrophage cell host responses in assessing

    18    tumorigenicity.

    19                DR. COOK:  Well, Dr. Lewis asked me to

    20    talk about the terminus of tumorigenicity from the

    21    perspective of the recipient of the challenged host,

    22    what kinds of things determine the ability of cells to

    23    form tumors, what the limitations are of the

    24    tumorigenicity assays, and how the immune response may

    25    affect our perception of tumorigenicity, depending on

                                                                        109

     1    what's used as the animal.

     2                What I'll do is give an overview of the

     3    model system we've been studying.  The focus in these

     4    studies have been on DNA virus oncogenes.  Dr. van der

     5    Eb gave a nice summary this morning of how SV40-T and

     6    E1A of adenovirus immortalize and transform cells, and

     7    we're going to be looking at cells that are expressing

     8    E1A without worrying so much about whether they're

     9    immortalized, because most of these will be super

    10    transfected cells that are artificially created to

    11    express the adenovirus E1A oncogene, and to ask what

    12    effect that has on the tumorigenicity of the cells in

    13    various hosts.

    14                I'm going to focus on so called innate

    15    immune responses or natural killer cells, activated

    16    macrophages to a lesser extent, and Dr. Tevethia will

    17    talk more about specific immune responses in a

    18    following talk.

    19                The correlations that we'll discuss are

    20    between oncogene induced susceptibility of neoplastic

    21    cells to killer cell injury.  So this is sort of

    22    counterintuitive here in an oncogene that's supposed

    23    to be causing a cell to become immortalized and become

    24    virulent is actually inducing the susceptibility of

    25    the cell to immortalize it to destruction, and then

                                                                        110

     1    the tumorigenicity in various animals with a focus on

     2    the value of immunodeficient animals in assessing

     3    tumorigenicity, and then later on probably in the

     4    discussion session we'll talk about the

     5    generalizations of some of these data to broader terms

     6    for human cell evaluations.

     7                The points that I'll make are that viral

     8    oncogenes in certain contexts can sensitize neoplastic

     9    cells to the destruction by killer cells, can thus

    10    actually reduce or eliminate tumorigenicity if the

    11    host is immunocompetent, and that the results of

    12    tumorigenicity assays are highly dependent on the

    13    immunocompetence of the host.  

    14                So inducing a tumor is not the same.  I

    15    think we all know that, but tumorigenicity is a very

    16    relative phenomenon and can't be perceived as an

    17    absolute, and the level of tumorigenicity among

    18    different types of neoplastic cells can be widely

    19    variable.  So we can't assume that a single dose of a

    20    cell can always tell you the same thing.

    21                This all started with studies that we were

    22    doing looking at an explanation for the reason that

    23    adenovirus 2 or 5 transformed hamster cells were

    24    unable to form tumors in immunocompetent hamster,

    25    although they could form tumors in very young, two- to

                                                                        111

     1    three-day-old hamsters.  

     2                Through a long series of experiments, the

     3    upshot of that is the expression of this E1A

     4    immortalizing gene or oncogene of human adenovirus

     5    serotypes 2 and 5 appears to sensitize cells to host

     6    defenses that are absent in the immuno-immature animal

     7    or in animals that lack sufficient NK activity, and

     8    this is just a representation of the generality of

     9    this kind of observation.

    10                If E1A is transfected and caused to be

    11    expressed in early passage or low passage NIH 3T3

    12    cells in a rat cell line called RN 12 that actually is

    13    pre-immortalized with rats in a bhk 21 subclone or in

    14    a human fibrosarcoma cell, in every circumstance when

    15    these cells are tested for susceptibility to killing

    16    by the relevant natural killer cell population,

    17    they're much more sensitive.

    18                So it's not just the adenovirus

    19    transformed cell.  It appears to be perhaps many, if

    20    not almost any, type of cell in which E1A can be

    21    expressed in reasonable concentrations.

    22                Now just to tell you a summary of a lot of

    23    work about E1A, as I've just shown, E1A expression in

    24    cells can sensitize them to natural killers from many

    25    species of origin.  It also sensitizes to a wide

                                                                        112

     1    variety of injuries.  There's nothing unique about

     2    this NK cell injury.  In other words, it's not a

     3    specific killer cell receptor interaction with the

     4    cell surface.  It's something to do with injury per se

     5    of certain types, and those are injuries that tend to

     6    stimulate apoptosis or proapoptotic injuries.  

     7                From an immune perspective, natural killer

     8    cells, cytotoxic lymphocytes, activated macrophages or

     9    the dominant cytokine that macrophages produce -- that

    10    is a cytotoxic molecule tumor necrosis factor alpha --

    11    all can elicit this kind of apoptotic response in

    12    cells if they express E1A, and these are cells that

    13    are inherently resistant to these types of injuries.

    14                It's not unique to immune injuries, and

    15    the chemical injuries can do the same thing.  A

    16    topicide,  atopoisummaries 2 inhibitor, beauvericin,

    17    a calcium regulator, hydrogen peroxide and hygromycin

    18    or protein synthesis inhibitor all can trigger the

    19    same kind of response in E1A expressing cells. 

    20                So there's something inherently weak about

    21    E1A expressing cells in an Achilles heel kind of

    22    fashion that makes them sensitive to undergoing

    23    apoptosis when injured by exogenous agents.

    24                Now the immune mediated apoptosis per se

    25    is somewhat unusual from other types of E1A

                                                                        113

     1    sensitization in that it is P53 independent.  So it

     2    works equally well in cells that can't express P53,

     3    and it's also resistant to blockade by the E1B 19kD

     4    molecule, one of the small E1B proteins that Dr. van

     5    der Eb told you about that normally blocks E1A

     6    induction of apoptosis during the immoralization

     7    phenomenon.

     8                Now what I'll do is focus more on the

     9    animal model side of the story to show you something

    10    about how tumorigenicity of cells that have this

    11    phenotype are being sensitized to immune mediated

    12    injury can vary, depending on the type of host in

    13    which it's tested.

    14                So in this model bhk21 hamster cells were

    15    subcloned and then transfected in stably expressed E1A

    16    or E1A + E1B.  It turns that E1B expression is

    17    irrelevant for this model.  So I won't talk about it

    18    further.

    19                We tested the E1A expression for its

    20    ability to induce sensitivity to killing, as I've

    21    shown you, and then asked whether tumorigenicity in

    22    different types of animal hosts, depending on their

    23    cellular immune responses, varied.

    24                For example, in the bhk model we used

    25    adult hamsters that have, in fact, NK cell and NT cell

                                                                        114

     1    responses both, nude mice which are unusual in this

     2    respect in that they have defective T-cell responses

     3    which we all know about, but they also have something

     4    wrong with their NK cells when it comes to recognizing

     5    E1A oncoprotein expressing hamster or rat cells.  

     6                So the NK cells are defective in this

     7    regard, whereas they will kill other types of NK

     8    susceptible targets, and then nude rates which are

     9    more like we think of as an aphonic animal.  That is,

    10    they have defective NK cell responses but have

    11    perfectly healthy active NK cell responses -- I'm

    12    sorry, they have defective T-cell responses but active

    13    NK cell responses when it comes to killing E1A

    14    expressing cells.

    15                So the question was:  If we have E1A

    16    expressed in these bhk cells, we use these animals as

    17    the recipients, what happens in vitro and what happens

    18    in the in vivo tumor induction model?

    19                So here are the in vitro data, using

    20    hamster spleen natural killer cells as the source.  As

    21    I've shown you before, they can kill the bhk

    22    expressing cells perfectly well, as long as they

    23    express E1A.

    24                The nude mouse cells don't do very well. 

    25    They have this defect for killing hamster cells

                                                                        115

     1    expressing E1A, in contrast to the nude rats which

     2    kill them quite well unless the nude rates are

     3    previously depleted of NK cells with an antibody such

     4    as anti-AZ allow GM1.  So there is an NK recognition

     5    of E1A expressing bhk cells in these different types

     6    of animals.

     7                Now this is a busier slide looking at the

     8    results of tumor induction studies in these different

     9    animals, and what we're talking about are the NK

    10    resistant bhk 21 parental cells or the E1A expressing

    11    clone that was derived from that, that are inherently

    12    NK susceptible or resistant, and what happens when

    13    different animals are challenged, considering their NK

    14    phenotype when it comes to killing the E1A positive

    15    cell. 

    16                So hamster that have competent natural

    17    killer cells, that can kill the E1A expressing cells

    18    develop tumors with about 1,000 cells, and this has

    19    been true for over 30 years of testing of bhk 21

    20    cells, if you look back in the old literature.  It

    21    takes about 1,000 cells to form tumors that are 50

    22    percent endpoint in an adult hamster with bhk 21.

    23                If you ask what happens when those NK and

    24    T-cell competent animals are challenged with the E1A

    25    expressing cells, you virtually can't get tumors with

                                                                        116

     1    10 million cells.  So there is at least four orders of

     2    magnitude change in the ability of these cells to

     3    induce tumors in immunocompetent adult hamsters when

     4    the cells express E1A and become NK susceptible.

     5                If we ask what happens in the nude mice

     6    that lack both T-cell and NK cell defenses against the

     7    E1A expressing bhk cells, there's not a huge change,

     8    although there is some.  

     9                They go from again about 1,000 cells

    10    making a tumor in nude mice, a little less, to about

    11    10 to 80 times more cells being able to make tumors in

    12    nude mice, but certainly not the magnitude of increase

    13    of resistance of the animals against E1A expressing

    14    cells, as was seen in the normal hamster.

    15                In fact, nude mice have activated

    16    macrophages which can kill E1A expressing bhk 21

    17    cells, and so it may be that there are other defenses

    18    other than their Nk cells that can explain this

    19    smaller change in increased resistance to tumor

    20    development.  

    21                Then there are the nude rats that are

    22    highly NK competent against E1A expressing bhk cells. 

    23    It takes about 30,000 cells to make a tumor in a nude

    24    rate, and 10 million cells is insufficient to make any

    25    tumors in these animals.  

                                                                        117

     1                So again, there's a several orders of

     2    magnitude change in the resistance of nude rates

     3    against the E1A sensitized bhk 21 cells unless the

     4    nude rates are NK depleted in advance, in which case -

     5    - Now this was just a single challenge with 10 million

     6    cells, but in which case the animals become more

     7    susceptible to tumor development.  

     8                So it appears that E1A can reduce the

     9    tumorigenicity of these cells if the animals have a

    10    competent NK cell response and, obviously, there are

    11    other things going on in these animals as well that

    12    might lead to changes in tumor susceptibility.

    13                Now if we look at a completely different

    14    animal model with the same basic scenario -- that is,

    15    transfecting and expressing E1A and asking what

    16    happens to the susceptibility of the tumor cell in

    17    immunocompetent and immunodeficient animals.

    18                In this case, these are studies done with

    19    Jack Rudis in which the methylcholanthrene cell line

    20    MCA-102 was studied.  We tested them for

    21    susceptibility to killing, and also tested them across

    22    the range of animals, adult mice, nude mice, and in

    23    this case CD3 epsilon transgenic mice that are

    24    defective for both T and NK cell responses.

    25                The one difference here is that nude mouse

                                                                        118

     1    NK cells are competent to kill these E1A expressing

     2    mouse cells.  So we have a range of activities of

     3    intact NK and T, defective T but intact NK, and then

     4    defective both in these animals that basically lack

     5    the function of either of these types of killer cells.

     6                This just shows you that the nude mouse in

     7    a black 6 background can in fact kill E1A expressing

     8    methylcholanthrene sarcoma cells in contract to the

     9    E1A negative cells.   

    10                These are the survival curves of these

    11    three different kinds of animals challenged with the

    12    E1A expressing cells.  So this is the adult C57 black

    13    6 mouse that has both the ability to generate a T-cell

    14    response and a killer NK cell response.

    15                This is tumorigenicity induced by the

    16    methylcholanthrene cells.  So you can think of this as

    17    a survival curve over the 12 week time of tumor

    18    challenge, and this is the survival curve of the E1A

    19    expressing cells.  

    20                So there's basically very little

    21    detectable tumor induction even at 10 million cells in

    22    these animals.  So I think of this difference in the

    23    50 percent endpoints for tumor production after three

    24    months as reflecting the cumulative antineoplastic

    25    activities of these mice against the E1A expressing

                                                                        119

     1    sarcoma cells, realizing that some of those are immune

     2    mediated and some of those are likely to be other.

     3                If you do the same experiment in nude mice

     4    and follow them over time, you get a similar type of

     5    curve, although you'll notice that the tumor inducing

     6    capacity in these animals is lower.  

     7                It only takes about 100 cells to reach an

     8    endpoint in the nude mice, whereas it takes a little

     9    over 1,000 cells to reach an endpoint in black 6 mice. 

    10    So they are inherently more susceptible to the

    11    methylcholanthrene cells, in the first place.

    12                There still is some resistance that's

    13    present in the nude mice against the E1A expressing

    14    cells, but the amplitude of this difference at the end

    15    of the assay is smaller.

    16                Then if you look at the animals that lack

    17    any detectable T-cell or NK cell defenses because of

    18    the CD3 epsilon transgenic status, basically what you

    19    see is that the survival curves are not much

    20    different, and the simplest way that I think of these

    21    is that these mice don't really have any defenses, at

    22    least from the immune response point of view, against

    23    E1A expressing methylcholanthrene sarcoma cells.  

    24                So it probably suggests that these

    25    defenses are pretty important.  If it were other

                                                                        120

     1    nonimmune defenses, you would think of these mice as

     2    being able to mount them.   Here the difference in

     3    tumorigenicity is not significant.

     4                So what I'd like to conclude is that E1A

     5    can sensitize neoplastic cells to killer cells and

     6    markedly reduce the tumorigenicity of those neoplastic

     7    cells, again considering the fact that you test this

     8    in some kind of immunocompetent animal.

     9                The lack of tumor formation by one cell

    10    dose in one host at one time point may not indicate

    11    nontumorigenic phenotype.  So what I would do is go

    12    back to the previous slide and say that, if we picked

    13    a dose in here and said that we're just going to

    14    challenge with a single dose of maybe a million cells

    15    -- So here's a million cells at a given point in time

    16    -- if you looked at any point in time across this

    17    spectrum in the black 6 mice, you would find no tumor

    18    formation by the E1A+ methylcholanthrene sarcoma

    19    cells.

    20                If you looked at the nude mice, it would

    21    kind of depend on when in the follow-up period you

    22    picked.  If you looked at two weeks, you should see no

    23    tumor formation.  If you looked at four weeks, you

    24    would see tumor formation, and in the CD3 epsilon

    25    animals you would see tumor formation at two weeks.

                                                                        121

     1                So it depends on the animal.  It depends

     2    on the cell dose, and it depends on the time at which

     3    you look at these kinds of things as what result you

     4    might find.  

     5                So I think we can't just assume something

     6    is not tumorigenic based on a single dose, single

     7    time, single host assay, and that the tumorigenicity

     8    likely reflects a collection of neoplastic cell

     9    traits, not just a single trait in terms of

    10    susceptibility to one thing or another.

    11                In the context of the cumulative effects

    12    of the host defense, probably there are things such as

    13    natural killer cells, activated macrophages, host

    14    growth factors and a variety of other things that

    15    determine the outcomes of these experiments.  

    16                So I think it's important not to think too

    17    simplistically about tumor development in animals,

    18    irrespective of the type of neoplastic cell that's

    19    being tested.

    20                Thank you.

    21                (APPLAUSE.)

    22                DR. RUSSO:  Carlo Russo from Merck.

    23                You didn't mention anything about kera --

    24    the inhibitory -- that expressed by NK cells and what

    25    role this receptor may have in modulating the NK

                                                                        122

     1    activity, in view of the changes in MHC expression by

     2    the tumor.  

     3                DR. COOK:  So the question is:  Could

     4    oncoproteins alter class 1 expression on the cell

     5    surface of target cells?  

     6                It's known that high levels of class 1

     7    expression and inhibitor expression on target cells

     8    can actually lead to repressed NK cell activity, in

     9    contrast to CTL responses where high levels of class

    10    1 expression are necessary.  So actually, these are

    11    probably complementary host defenses.  

    12                What I can tell you is that class 1

    13    expression level appears to be unrelated to this

    14    sensitizing phenomenon in the E1A system.  So you can

    15    have cells that have normal levels of class 1 compared

    16    to normal fibroblasts for levels of class 1 that are

    17    very suppressed, and in both cases the cells are

    18    highly susceptible to NK killing.

    19                So class 1 as an inhibitor or a class 1-

    20    like molecules as an inhibitor for NK killing appears

    21    not to be a major phenomenon in this system.

    22                DR. RUSSO:  Do you think that in the

    23    experiment you should include the factors that may up-

    24    regulate MH expression in order to rule out the

    25    possibility of inhibition of NK cells, such as gamma

                                                                        123

     1    interferon?

     2                DR. COOK:  Well, you could treat -- Well,

     3    those experiments have been done.  You can treat the

     4    cells with interferon gamma.  Class 1 expression can

     5    go up.  It doesn't seem to change the cells'

     6    resistance, as long as they express E1A.

     7                Certainly, in other models that's not the

     8    case.  Dr. van der Eb?

     9                DR. VAN DER EB:  van der Eb, Leiden.

    10                If I understand it correctly, you talked

    11    about adeno 2 or 5 E1A expressing cells.  Right?

    12                DR. COOK:  Right.  Correct.

    13                DR. VAN DER EB:  Do you have any

    14    comparison with adeno 12 transformed cells which are

    15    oncogenic?

    16                DR. COOK:  We haven't done it in these

    17    exact models.  We've looked at adeno 12 transformed

    18    cells from viral transformation events in hamster and

    19    in mouse.  When you contrast adeno 2 or 5 transformed

    20    cells using virus to adeno 12 transformed cells, the

    21    adeno 12 transformed cells are much less NK

    22    susceptible, despite the fact -- and this kind of goes

    23    back to the last question -- despite the fact that

    24    they all express low levels of class 1.  

    25                So again, I think adenovirus violates

                                                                        124

     1    these models of natural killer cell recognition and

     2    killing in that adeno 2 and 5 appear to allow class 1

     3    to be expressed most of the time, and yet sensitized

     4    to natural killing which normally requires low levels

     5    of class 1.

     6                Adeno 12 represses class 1 and doesn't

     7    appear to sensitize to natural killing, despite the

     8    fact that the class 1 is reduced, and you would expect

     9    that to trigger a natural killer cell response.

    10                CO-CHAIRMAN MYERS:  One last question.

    11                DR. JOLICOEUR:  Jolicoeur from Montreal.

    12                I was wondering if you have other examples

    13    of the oncogene or viral oncogene which behave like

    14    that?

    15                DR. COOK:  I'm sorry?

    16                DR. JOLICOEUR:  If you have other examples

    17    of an oncogene which will behave as E1A here.

    18                DR. COOK:  A nonviral oncogene?

    19                DR. JOLICOEUR:  Nonviral or a viral, an

    20    autoviral.

    21                DR. COOK:  Some other example?  Well, in

    22    mouse systems SV40 T antigen can do something like

    23    this, or in rat where it can sensitize cells to

    24    killing by activated macrophages, and in some models

    25    natural killer cells.

                                                                        125

     1                There are examples of overexpressed MIC

     2    that can cause sensitivity to natural killer cell

     3    activity.  So I don't think E1A is unique in this

     4    regard, although it appears to be unique in the fact

     5    that it can do it in multiple different species.  

     6                SV-40, for example, if you compare

     7    hamsters and mice, SV-40 transformed mouse cells or

     8    sensitive SV-40 transformed hamster cells aren't, even

     9    though they express T antigen at equally high levels.

    10                So there may be some species specificity

    11    to other oncogenes' ability to convey these same kinds

    12    of traits to the targets.

    13                CO-CHAIRMAN MYERS:  Thank you very much. 

    14                Continuing on in the same theme, our next

    15    speaker is Dr. Tevethia from Penn State, who will talk

    16    about the role of CTL responses and their implications

    17    for tumorigenicity testing and the use of tumor cells

    18    as vaccine substrates.

    19                DR. TEVETHIA:  Thank you very much.  If I

    20    could have the first slide.  

    21                Everybody had a favorite virus and a

    22    favorite oncogene.  Mine is SV40, and the SV40 large

    23    T antigen is my favorite oncogene.  This has already

    24    been introduced.  So I can move on to the second

    25    slide.

                                                                        126

     1                The point here I want to make -- and I had

     2    to go back to some of the work we had done in the

     3    Sixties and Seventies to make this slide -- if you

     4    transform cells, either hamster cells or mouse cells,

     5    BALC mouse cells or C57 black 6 mouse cells, with the

     6    SV40 large T antigen, and try to transplant in nude

     7    mice, all of these are transplantable in nude mice. 

     8    But if you try to transplant in the adult animals, in

     9    hamsters they are immediately transplantable.  In 

    10    BALC they are rarely transplantable, but only after

    11    prolonged custody in vitro and passaging through the

    12    immunosuppressed host.

    13                In C57 black 6 mice, they are never

    14    transplantable.  They are only transplantable if you

    15    force them to lose MBC Class 21 antigen.  

    16                There's obviously a very simple conclusion

    17    to the role of immune response and the transplantation

    18    of these in vitro transformed cells.  Let me just give

    19    you a clue, that every time you transplant a cell line

    20    transformed by oncogene, there the oncogene acts as an

    21    antigen, exogenous antigen.

    22                It is processed quite differently as

    23    compared to the transgenic mouse.  If you express the

    24    same oncogene T antigen in transgenic mouse, we have

    25    60 or 70 examples of this.  I picked three of them,

                                                                        127

     1    expressed T antigen and its own promoter produces

     2    tumors in choroid plexus.

     3                The point I'm trying to make, no matter

     4    which promoter you pick, you can induce neoplasia in

     5    almost any tissue you like, and these tumors will

     6    continue to grow progressively until they kill the

     7    host at different times after birth.

     8                So the question really that I want to

     9    address is why is it hard to transplant the cell line

    10    without tumorigenic in nude mice into the adult

    11    immunocompetent mice, but the same tumors induced by

    12    the -- the tumors induced by the same oncogene in vivo

    13    as a transgene continue to grow progressively?

    14                I can tell you, the difference is where

    15    this oncogene becomes as a self-antigen and that is

    16    processed quite differently and the way it interacts

    17    with the immune response is quite different.

    18                Just to give you a just a little bit of

    19    introduction, and the only thing I want to point out,

    20    that the purified T antigen can induce the generation

    21    of cytotoxic T-cells and can induce in the

    22    participation of the tumor rejection.

    23                This is just a cartoon slide, just to

    24    indicate how the antigen processing works.  I'll go

    25    very, very briefly.  When the antigen comes from

                                                                        128

     1    outside, broken down into peptides, peptides are

     2    shunted to the tap into the endoplasmic reticulum

     3    where it is assembled with the molecules, and to

     4    microglobulin, is then transported back to the cell

     5    substrates where it is recognized by the cytotoxic T-

     6    cells.

     7                Just a cartoon slide to show the 3-D

     8    structure of the classical molecule with a peptide

     9    bound in the groove.  This is the immuno terminal. 

    10    This is the C terminal. What I'd like to point out,

    11    there are two residues in the peptide.  They are known

    12    as the anchor residues.  They are responsible for

    13    binding to the MIC molecule in a particular way.

    14                So these anchor residues will determine

    15    which peptide will bind to which MIC classical

    16    molecule.

    17                In SV40 large T antigen, there are four

    18    epitopes that we have defined, 206-215, 223-31, 404-

    19    411, 489-497.  They are restricted either by H2 DFP

    20    molecule or H21 molecule.  We have the cytotoxic T-

    21    cell clone for these -- especially for these epitopes,

    22    and these are the epitope designation 1, 2, 3, 4 and

    23    five.

    24                In order to work -- It is a complicated

    25    slide.  What I'd like to point out is we have cloned,

                                                                        129

     1    expressed these epitopes individually in the common

     2    vaccinia viruses in cooperation with John Yewdells and

     3    Jack Bennick, and either by themselves or behind the

     4    ES signal sequence of the glycoprotein E19.

     5                That allows these F clones to be delivered

     6    directly into the endoplasmic reticulum much more

     7    efficiently.

     8                Okay.  So what I'd like to describe is the

     9    two-transgenic system that inactivates the immune

    10    response, cytotoxic T-cell immune response by

    11    different mechanisms.

    12                One of them is a 501 transgenic mouse

    13    system.  It's driven -- T antigen driven by the late

    14    alpha amylase promoter and appears in salivary glands

    15    and osteoplaths.  It produces osteosarcoma about one

    16    year of age, and metastasized to lung, liver, and T-

    17    cells are positively selected.  Means that T-cells are

    18    present that are expressly for the large T antigen.

    19                SV11 tumor system is driven by its own

    20    promoter.  Tumors appear in choroid plexus.  T antigen

    21    most importantly appears in the thymus, expressed in

    22    thymus, as a result of which there is a negative

    23    selection in the T-cells, and these mice essentially

    24    can be considered as naive, because they don't select

    25    the T-cells.

                                                                        130

     1                So let me just describe the SV11 system. 

     2    This is a choroid plexus of the 100 day old normal

     3    mice, and this is histochemistry for T antigen. 

     4    Obviously, this is negative.  

     5                In the SV11 transgenic mice, by 40 days of

     6    age you barely see a few T antigen positive cells over

     7    here.  By 60 days you see a focus formation and

     8    corresponding expression of the T antigen in this

     9    focus.  

    10                By 80 days tumor grows, and you can see,

    11    it contains -- all cells contains the large T antigen,

    12    and by 100 days they are described by Terri Wendyke. 

    13    There's a big explosion in the tumor.  Means that it

    14    grows extremely large in a short period of time, and

    15    it will die at exactly 104 to 105 days after birth, a

    16    very predictable system.

    17                Now this is a very, very complicated

    18    slide.  The message I want to give is this, that if

    19    you immunize these mice, SV11s, either with a full T

    20    antigen vaccinia virus or with its epitopes, you can

    21    see they don't respond.  That's all you need to look

    22    at.

    23                The graph here shows they all respond to

    24    vaccinia virus vectors.  That means they are not

    25    immunosuppressed to general immunosuppression.  But

                                                                        131

     1    these are the normal mice, litter mates that don't

     2    have the transgene.  They respond to all of these

     3    epitopes.

     4                So what we have done is to -- carried out

     5    studies in which we could -- We have taken the

     6    transgenic mice.  We irradiated them briefly with low

     7    dose of the radiation 450r.  Then we reconstituted

     8    them with the normal T-cells from the normal animals.

     9                Then we can immunize them with either the

    10    full NT antigen or with immunogenes or we can leave

    11    them alone.  Then we look for the development of

    12    cytotoxic T-cells and look for the progression of the

    13    tumors.

    14                Okay.  If now you irradiate them, transfer

    15    the spleen cell, immunize them with a full NT antigen,

    16    you have the appearance of the cytotoxic T-cells only

    17    to epitope 4.  This is the most dominant epitope at

    18    this particular point.

    19                What is important is that, if you

    20    irradiate them first, as I said before, reconstitute

    21    them but don't immunize, they still develop high

    22    levels of cytotoxic T-cells to the dominant epitope 4. 

    23    Now this is important, because tumors now after

    24    radiation is starting to release the antigen, and this

    25    particular T antigen there is the one which has been

                                                                        132

     1    processed and inducing the development cytotoxic T-

     2    cells.

     3                By irradiation or reconstitution, you can

     4    start a program immune response against the tumor

     5    cells.  This is the tumor looks like.  I showed you

     6    what the tumor looks like at 40 days of age, very big

     7    tumor.  In this case, the tumor remains a very small

     8    size, hardly recognizable, but still you will see the

     9    T antigen positive cells.

    10                Now if you keep these animals long enough,

    11    until they die, and I'll show you when they die, that

    12    they will eventually die of the tumor, the tumor we

    13    grow.  This is the life span of these animals.  You

    14    can see the transgenic animals die, which are un-

    15    irradiated, by 107 days.

    16                Irradiation plus the spleen cell from the

    17    transgenic mice again, then they don't live very much

    18    longer; but if you irradiate and transfer normal

    19    spleen cells and don't immunize, they live over 320

    20    days.  But they eventually develop tumors and die.

    21                The second one that I want to discuss --

    22    So the previous example indicates that the T-cells are

    23    completely deleted in the thymus.  

    24                The second example where the T-cells are

    25    positive selected -- this is driven by amylate late

                                                                        133

     1    promoter, and it has all of the epitopes 1, 2, 3, 4

     2    and 5, and they come down with osteosarcoma before one

     3    year of age.

     4                This is what it looks like, the

     5    osteosarcoma impinging on the spinal cord.  This is

     6    the metastasizing to the lungs, and this is the

     7    metastasis to the liver.  You can see that all of the

     8    cells in the tumor have the T antigen by

     9    histochemistry.  So they have not lost the antigen.

    10                What I have done is to just simply

    11    summarize the immune responses to it.  What's

    12    interesting here is that, if you look at the four

    13    months of age, then all -- no matter how you immunize,

    14    they respond -- almost all -- they respond to almost

    15    all of the epitopes except 2 and 3, which is a lethal

    16    epitope.  They respond to vaccinia virus.  

    17                If you test the very same animals at 12

    18    months of age, then you can see they have lost the

    19    response to full NT antigen all to epitope 1, and also

    20    epitope 5.  They do respond on occasions to epitope 4.

    21                So we wanted to know what the mechanism

    22    may be, and to -- we want to differentiate between

    23    different possibilities, and one of the ones we zeroed

    24    in, that these T-cells which are already there are

    25    dying by clone deletion.

                                                                        134

     1                The way we have done that is to -- We have

     2    made the tetrameres.  The tetrameres are made by

     3    folding the class 1 molecules with the peptide and the

     4    beta 2 microglobulin.  Now these soluble class 1

     5    molecules have a signal for the biotin -- the

     6    biotinylated, and then with strep evident, you can see

     7    they will make  tetrameres in three of them and can

     8    bind to a single T-cell to different receptors.

     9                You simply to FAS analysis.  What one does

    10    is -- this is an example for it, and I'll just

    11    illustrate one of them.  We have used one of our CTL

    12    clone to epitope 1, for example.  They have the

    13    epitope 1 tetrameres, and with the antibodies, here

    14    are the CD 8.  With the tetrameres, you can see all of

    15    the CTL clones, they line up in the corner over here. 

    16    It means that -- So you can count -- you can actually

    17    count the T-cells that are especially for a particular

    18    epitope, and you can see that the -- This is a

    19    different tetramer.  It does not bind to the same CTL

    20    clone.

    21                So we have used this approach to identify

    22    the mechanism in the 501 mice, why these T-cells are

    23    being deleted.  If you look at the three-month-old

    24    mice 501, only 3.5 percent of the entire CDA positive

    25    population is directed to epitope 4.

                                                                        135

     1                You can see in the control mice over 14 to

     2    16 percent of the T-cells here are directed to RC

     3    epitope 4, once you immunize the animals with epitope

     4    4.  So they are much reduced in number, to begin with. 

     5    By the time 11 months comes around, you have only half

     6    a percent left of the epitope at specific T-cells.

     7                Now if you ask the question, do these

     8    animals have a tumor or do not have a tumor, and we

     9    assay the animals to determine if they have tumor or

    10    not, once they have tumors they have lost all of the

    11    T-cells, and you can see, once you have lost it, you

    12    cannot even expend them in vitro for lytic activity.

    13                What's interesting is that these are not

    14    anergic.  Even the .5 percent of the T-cells can be

    15    expended with full function.  

    16                Okay.  The last thing will take just a

    17    minute for me.  This is a study, not done in my

    18    laboratory, done by the laboratory of Judy Trevethia,

    19    and what we were interested in this study was the fact

    20    that she made the transgenic mice, which is the black

    21    6, that only have 1 through 128 -- actually 1 to 127

    22    amino acids of T antigen.  It had the RB binding site. 

    23    It had the intact J domain, and that's really about

    24    it.

    25                You can see -- What I want to emphasize

                                                                        136

     1    from our point of view here, that all of the epitopes

     2    are outside the region of this transgene.  That will

     3    be, in my opinion, might not be as good a news,

     4    because you cannot do anything immunologically, you

     5    know, to these animals.

     6                So these animals can come down with a SNR

     7    tumor.  These are the adeno carcinomas.  They are

     8    driven by the elastase promoter, and you can see this

     9    is the adeno carcinoma of the pancreas.  You can see

    10    a lot of duct formations.  You can see this is a

    11    normal pancreas with islets intact, metastasizes to

    12    the liver.  

    13                This is again liver metastasis.  What is

    14    interesting about this is that, if you take the

    15    newborn within four hours after birth and check the

    16    pancreas, you see a dysplastic pancreas at that

    17    particular point within four hours after birth.  As

    18    the normal pancreas of the four-hour-old, you know,

    19    newborn will look like this.

    20                So I guess I'll pass this one.  So I think

    21    my message is the following.  I think that

    22    transplantability may or may not give you the right

    23    answers, because there's a lot of variation, and

    24    beside the engine that's present in those tumor cells

    25    or transformed cells, then the exogenous engine

                                                                        137

     1    process differently as compared to the transgenic

     2    mice.

     3                These are some of the people that did the

     4    work.  These are the people we did work in

     5    collaboration, Bennick Yewdell, Barbara Knowles,

     6    Arnold Levine of Princeton.  Thanks very much.

     7                (APPLAUSE.)

     8                CO-CHAIRMAN MYERS:  We have time for one

     9    or two questions.  Thank you.

    10                Our last paper this morning is by Frank

    11    Sistare and was on transgenic animals that might be

    12    useful in identifying unsuspected oncogenic factors in

    13    tumor cell substrates, from CDER.

    14                DR. SISTARE:  I want to thank the

    15    organizers for this opportunity to speak.  May I have

    16    the first slide?

    17                What I want to do is, first of all, spend

    18    some time on how our center -- I'm from the Center for

    19    Drug Evaluation and Research -- got involved in sort

    20    of changing, as we heard this morning, the gold

    21    standard, standard two-year bioassay, for ascertaining

    22    the carcinogenic potential of pharmaceuticals.

    23                This was an initiative that was an

    24    outgrowth of an ICH process that started a few years

    25    ago.  A guidance was developed, again with the premise

                                                                        138

     1    that there was a general dissatisfaction with the

     2    standard two-year bioassay, and are there alternatives

     3    for assessing pharmaceutical carcinogenicity.

     4                So a consortium was formed as an outgrowth

     5    of that by the International Life Sciences Institute. 

     6    This consortium has over 50 labs worldwide.  There are

     7    28 pharmaceutical companies.  There's members from the

     8    United States government regulatory agencies, Japanese

     9    and European regulatory agencies as well, that are

    10    part of that.

    11                Why we're focusing on alternatives -- Like

    12    I said, there are often ambiguities and

    13    dissatisfaction with the conventional "gold standard"

    14    two-year rodent bioassay.  Saccharin, as an example,

    15    comes to mind as the strain or species specific event

    16    which is deemed irrelevant to humans, and there are a

    17    host of others.

    18                The feeling is that transgenics will carry

    19    target genes that are involved in human and rodent

    20    cancers.  During the prime course of these transgenic

    21    assays, there are much lower backgrounds as opposed to

    22    the backgrounds associated with some of the two-year

    23    bioassays.  

    24                It's a six-month assay which is a

    25    tremendous savings in time in terms of making a

                                                                        139

     1    decision on whether a pharmaceutical is going to be

     2    developed or not, and there's tremendous cost savings

     3    just in the conduct of the assay.  In often cases, the

     4    endpoints are very simple.

     5                The ILSI initiative has named a number of

     6    specific models.  As you've heard this morning, there

     7    are a host of transgenic models that could be

     8    explored, but several were brought into the limelight.

     9                There were the initiation promotion

    10    models, which is not a transgenic model, which Dr.

    11    Pitot reviewed for you this morning.  There's a

    12    newborn rodent assay, which again is not a transgenic

    13    model, but I'm going to spend some time talking about

    14    that, because that is one model system that the ICH

    15    consortium -- I'm sorry, the ILSI consortium has

    16    decided to focus on.

    17                Then several transgenic rodent models,

    18    though not named by ICH, are emerging from this ILSI

    19    initiative:  The two knockout models, the P53 knockout

    20    mouse heterozygous, and the XPA homozygous knockout

    21    mouse, the TJC mouse and the TJ RAS H2 mouse.

    22                So what I want to do for you now is

    23    describe these test models, give a brief overview of

    24    some of the pragmatic protocol design issues that are

    25    going to impact on whether or not some of the models

                                                                        140

     1    might be practical for assessment of vaccines.

     2                I want to review for you some of the test

     3    compound results.  Again, these are going to be based

     4    on pharmaceuticals, and then give you some mechanistic

     5    speculations that are based on some of the data that

     6    we're seeing now.

     7                First of all, the neonatal mouse model: 

     8    This model has been around for probably the longest

     9    time of the models that the ILSI consortium is being

    10    focused on.  A number of compounds have been tested.

    11                The ILSI consortium has formed a standard

    12    protocol for assessing these.  So some of these other

    13    compounds that have been tested have been used have

    14    been used in different schedules and different

    15    strains.

    16                The protocol is -- One nice virtue of it

    17    is the protocol, there's only two dosing periods, one

    18    on day eight and one on day 15 of the mouse.  One-

    19    third of the total dose of the compound is given

    20    initially and then two-thirds seven days later.  The

    21    test agent can be given either IG or PO by gavage.

    22                The doses that are decided on are based on

    23    the 28 day dose range finding study, and you simply

    24    dose them for that period of time and feed them, take

    25    care of them for a year, and then one year in necropsy

                                                                        141

     1    and do some histopathology, and then assess the

     2    outcome.

     3                The general concepts that are forming are

     4    that this model will pick up genotoxic carcinogens. 

     5    Nongenotoxic or indirect active carcinogens appear

     6    negative and, specifically, the genotoxic carcinogens

     7    that seem to be picking up are mutagens.

     8                There's some question about whether it can

     9    pick up deletionogens, and some of those questions are

    10    being addressed presently.  Conceptually, the model is

    11    -- It's like an initiation or mutation just prior to

    12    periods of normal high DNA replication.  So it's like

    13    an induced initiation of that early on, and then the

    14    normal promotion processes.

    15                The P53 alluded to earlier -- mouse model,

    16    P53 tumor suppressive gene is the most frequently

    17    mutated gene in cancers.   The human Li Fraumeni

    18    syndrome has a mutated P53.  Those patients have

    19    greater cancer risk.

    20                There's a conservation of P53 across

    21    species, and the function of the P53 gene product

    22    involved in cell cycle regulation, involved in

    23    trafficking toward apoptosis, and also involved in DNA

    24    repair, makes it an attractive product in terms of a

    25    target for mutation that might knock out a lot of

                                                                        142

     1    these functions.

     2                The assay is a six-month assay with

     3    continuous dosing.  There is -- It's a hemizygous

     4    model.  So there's one functional allele in all cells. 

     5    What's been found is that induced tumors, chemically

     6    induced tumors -- the tumors are associated with a

     7    loss of the wild type P53 allele.

     8                One pragmatic concern is that one thing

     9    we're learning is that, when there are subcutaneous

    10    injections, when that's the route by which the drug

    11    may want to be delivered, tumors are sometimes

    12    associated with injection site.  So the control

    13    animals will often show tumors at the injection site. 

    14    So that can be a problem.

    15                The TJC mouse model:  The mouse zetaglobin

    16    promoter linked to v Harvey RAS gene, and assay 40

    17    terminus.  This is a micro injected mouse model. 

    18    There were thought to be three to ten copies in the

    19    initial founder mice that were generated here, but it

    20    looks like these animals have wanted to go on an

    21    amplification of transgene, and in our own we've shown

    22    that there's about 40 copies presently now of this

    23    transgene.

    24                The rationale for the use of v Harvey RAS

    25    is that human cancers are associated -- 25 percent of

                                                                        143

     1    human cancers are associated with a mutated RAS gene,

     2    and conceptually all cells have this mutation.  All

     3    cells in this mouse were preinitiated, and what this

     4    model is waiting for is a strong promotion event.  

     5    That becomes the event in the induction of tumors.

     6                It's been found that tumors that result

     7    are associated with a loss of methylation, and all

     8    tumors that have been examined to date for expression

     9    do express the RAS -- mutated RAS gene.  So it is

    10    expression dependent.

    11                In terms of practical aspects of its

    12    utility, it is primarily a skin paint model.  So the

    13    test compound is painted onto the skin.  Approximately

    14    200 microliters is applied to the skin.  As a positive

    15    control for bolestra, PMA is applied three times a

    16    week.

    17                Issues surround how the drug is going to

    18    be delivered.  You have a very aqueous, soluble drug. 

    19    That could be a problem.  It has to be soluble in a

    20    solvent that can be delivered to the animal's skin and

    21    will evaporate and deliver the dose.

    22                There is an alternative route that is

    23    being explored.  Oral gavage with

    24    dimethylvinylchloride has been shown as a positive

    25    control to result in four stomach tumors.  So again

                                                                        144

     1    it's almost like a local effect, and also some rare

     2    leukemic tumors.  Leukemias have been seen with, for

     3    example, benzine administration.

     4                Oddly enough, one example of systemic

     5    cyclosporin A given by gavage has resulted in topical

     6    skin tumors, but that's the only example that I know

     7    of where a systemic drug has resulted in a skin tumor. 

     8                The TgrasH2 transgenic mouse model:  This

     9    model is reported to have five to six copies of the

    10    human normal ras gene with its own promoter and its

    11    own enhancer region.  There are no mutations in the

    12    coding region of this transgene.

    13                What's been found in transgenic mice is

    14    that the 221 protein seems to be up regulated.   It's

    15    about two to threefold higher expression in cells, and

    16    that when tumors are looked at -- when one examines

    17    tumors from -- chemically induced tumors, that there

    18    are a lot of somatic point mutations that are found,

    19    found very frequently in these tumors.  This is also

    20    a six-month assay.

    21                Some of the data:  This is some of the

    22    data that preceded the whole ILSI initiative, and this

    23    is the kind of thing we're looking for.  Can these

    24    models pick up known human carcinogens?  When you look

    25    at the ones that have been tested, yes, they all can

                                                                        145

     1    pick up the genotoxic in carcinogens.  

     2                We look, for example, at P53.  It doesn't

     3    pick up cyclosporin A, Tcdd or diethylstilbestrol;

     4    whereas, Tgac and TgrasH2.  So Tgac seems to pick up

     5    both genotoxic and nongenotoxic.  So it picks up the

     6    promoters and the complete carcinogens; whereas, P53

     7    is limited to the genotoxic.

     8                TgrasH2 so far seems to be able to pick up

     9    both categories, although you'll see there are some

    10    other examples where they can't.  

    11                If you look only at compounds that are

    12    associated with rodent tumors, the same sort of

    13    pattern falls out.  Tjc can pick up genotoxic and

    14    nongenotoxics.  For the most part there are a few

    15    deficiencies here that you'll see.  Ethyl acolate, for

    16    example, Tjc did not pick up.  

    17                P53 again, for the most part, picks up the

    18    genotoxic.  Glysodol is an example where both P53 and

    19    Tjc did not result in a positive finding, but this was

    20    multi-site, two-species, both male and female

    21    carcinogen, conventionally treated bioassay.   The

    22    reason for these negative findings are being explored

    23    presently in Dr. Tennant's lab.

    24                Finally, you want models that do not react

    25    to everything that's given to them.  So you want to

                                                                        146

     1    check the question of specificity.  Do they not react

     2    to noncarcinogens?  

     3                Again, for the most part, for Tgac except

     4    for resorcinol here, and rotenone, the specificity is

     5    good.  P53 specificity is good, and rasH2 specificity

     6    is good.  But again, there is a lot more work being

     7    done presently with these things.

     8                The other question is not just things that

     9    are four-cell negative, meaning negative in both male

    10    and female rats and mice into your bioassay, or things

    11    that are positive in male and female rats and mice to

    12    your bioassay.  What about those things that get us

    13    into regulatory dilemmas where it's positive in the

    14    mouse but it's not in the rat, or it's positive in the

    15    males but not in the females, and what do you do with

    16    that kind of data?

    17                Some of the compounds that gave us these

    18    sort of ambiguous findings where there were some mouse

    19    only findings or male only findings in the mouse and

    20    rat were put into Tgac and P53.  For the most part, 

    21    these things are not viewed as like "overly"

    22    sensitive.

    23                There is some level of selectivity, in a

    24    sense, that again these things were negative in the

    25    rat and positive in the mouse, but they came out

                                                                        147

     1    negative in Tgac.  It wasn't sure what was going to

     2    happen here.

     3                In terms of our charge today is to ask

     4    questions about -- You know, when we're talking about

     5    vaccines, we ask questions about DNA -- when these

     6    transgenic models are exposed to DNA.  I don't know

     7    the answer to that question, how these things will

     8    respond to exogenous DNA, but one abstract that I was

     9    able to locate, Gina Clarke at Genentech asked the

    10    question about growth factors, mouse abnormal growth

    11    factor or recombinant human IGF-1. 

    12                It was administered subcutaneously for six

    13    months to both P53 and Tgac, and it came out negative. 

    14    Again, like I say, Tgac is primarily a skin model. 

    15    This was a subcutaneous injection.  This was an

    16    example where they did see some subcutaneous injection

    17    site tumors, but there were no increases in the ones

    18    with EGF or IGF-1.

    19                The XPA deficient transgenic mouse has not

    20    received near as much testing as the other models, but

    21    the thinking here is that there is deficient repair

    22    capabilities, and it's expected that this model will

    23    be able to pick up genotoxic carcinogens.

    24                For the most part, the few chemicals that

    25    it has been exposed to, it seems to be bearing out. 

                                                                        148

     1    A question of whether it can respond to nongenotox is

     2    wide open, and what about genotoxes don't rely on --

     3    repair.  We don't know the answer to that yet.

     4                There are promising prospects sort of off

     5    in the wings, if you want to think of it that way. 

     6    There is a sort of TgrasH2 equivalent rat model. 

     7    Again, if you want to ask this question about across

     8    species, there's another very interesting model, a K6

     9    promoter, ontheondecarboxylase transgenic mouse made

    10    by Tom O'Brien.

    11                This mouse behaves like a pre-promoted

    12    model.  Apparently, all the polyamines being

    13    synthesized by ODC have the skin that occurred to the

    14    sites as a state of promotion.  What they're waiting

    15    for is an initiation of that.  They've shown with some

    16    chemicals that a preinitiator will activate this

    17    model.  Again, it's a skin paint model.  So it's sort

    18    of like the slip side of the coin of the TgAC mouse.

    19                There are also some very creative kind of

    20    things.  I just put an example from Dr. Inoue's group

    21    in Japan where they're taking TgrasH2 mouse and then

    22    giving it IP urethane, to accelerate the whole thing. 

    23    So you've got an initiator here.  Then you add your

    24    test compound, and they can get an answer in three

    25    weeks as opposed to waiting six months.

                                                                        149

     1                So there's some efforts like that to

     2    accelerate even further from the six month assay on 

     3    forward.

     4                So in conclusion, these short term models

     5    to define transgene carcinogens are promising.  There

     6    are strengths and limitations that are being

     7    elucidated, and mechanistic understanding of these

     8    models is expanding.

     9                The P53 and the neonatal mouse identifies

    10    genotoxic carcinogens but really not nongenotoxic

    11    carcinogens.  TgAC identifies both.  TgrasH2

    12    identifies genotoxic and the nongenotoxic so far. 

    13    Looks promising.  There's further evaluation going on 

    14    there, and with XPA we don't have a lot of test

    15    results yet to go with that.

    16                I'll entertain any questions.  Thanks.

    17                (APPLAUSE.)

    18                CO-CHAIRMAN MYERS:  -- cell substrates

    19    been tested in any of these models?

    20                DR. SISTARE:  I am unaware of anything

    21    like that having been done.  It certainly is something

    22    that could be done, and the beauty of these things is

    23    it can be done pretty rapidly, because they are six-

    24    month assays.  The cost is not prohibitive.

    25                The animals are $150-$200 a piece.  Some

                                                                        150

     1    of these transgenic animals, $150 a piece, but you

     2    only need like 15 animals per group.   So these things

     3    are doable, but to my knowledge they haven't been done

     4    yet.

     5                DR. ONIONS:  David Onions, Glasgow.  I

     6    enjoyed the talk very much.  I just wanted to ask a

     7    question in terms of how the endpoint is read.  Mike

     8    has very kindly allowed me to show some data later,

     9    and one of the points that we find is that by looking

    10    at the actual kinetics of the tumor development, that

    11    actually gives you much finer data than looking at a

    12    single time endpoint.  But by looking at the kinetics,

    13    you can actually discriminate between very low

    14    carcinogenic events in multiple transgenic animals.

    15                I was wondering how the readouts are done

    16    in your particular case.

    17                DR. SISTARE:  In the TgAC, for example,

    18    you can look at both the numbers of animals developing

    19    papillomas and also the number of papillomas per

    20    animal as a function of time, and that data is

    21    analyzed in that way.  But, you know, how we are

    22    ultimately going to decide whether something develops

    23    a tumor faster than another thing, is a more potent

    24    carcinogen,  that's something that has to be done

    25    later.

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     1                With P53, you don't know until you dissect

     2    the animal at the end.  So there's really not a time

     3    component that goes in there, but certainly, they will

     4    look at numbers of tumors per animal, number of sites,

     5    you know, is it a multi-site or is it a single site,

     6    that kind of thing?  Is it only the males, only the

     7    females?  So all these kind of things.

     8                DR. ONIONS:  I just have one very quick

     9    comment about the P53 heterozygotes.  We've used a

    10    slight different line, which is the Edinburgh knockout

    11    mouse, but its function is the same.

    12                If you use the heterozygote, what is quite

    13    surprising with some carcinogenic insults -- for

    14    instance, a retroviral insertion -- you would expect

    15    the normal allele to be the primary target of

    16    insertion in tumor development, and it's not.

    17                It actually is our other genes, but

    18    actually universally the heterozygote P53 normal

    19    allele is rearranged.  So it's, I think, very

    20    important to be very careful about assuming that that

    21    is always the primary target and that you need to be

    22    very careful about that assumption.

    23                DR. SISTARE:  Yes.  No, I agree with that. 

    24    There has been some data that's shown that there is

    25    some rearrangement in that locus, and also that, you

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     1    know, one has to keep in mind that the P53 is sort of

     2    -- having only one allele, having sort of a gene

     3    dosage effect there, that the loss of that function of

     4    that protein is going to have these other cells in a

     5    state of readiness for whatever.  They're going to be

     6    less prone to apoptosis -- less capable of apoptosing;

     7    so other injuries, yes.  I agree with that.

     8                DR. LEWIS:  Andrew Lewis, CBER.  Has there

     9    been any attempt to establish tissue culture cells

    10    from these various models and to see whether you can

    11    replicate the findings with genotoxic versus

    12    nongenotoxic carcinogens in these tissue culture

    13    systems?

    14                DR. SISTARE:  Well, there are P53

    15    deficient cells, as we know, which kind of looks a lot

    16    like the P53 model, Li Fraumeni cell lines, for

    17    example.  But from these particular cells, the TgAC,

    18    for example -- The thing about TgAC is that it appears

    19    that the target cell, at least for skin tumorigenesis,

    20    is a follicular cell, a cell at the base of a hair

    21    follicle.  So it's not just any keratinocyte there.

    22                There have been attempts, none of which I

    23    know have been successful.  Now there have been some

    24    tumors -- tumor lines based on papillomas, but those

    25    aren't as useful.  TgrasH2, none to my knowledge.

                                                                        153

     1                DR. LEWIS:  And how frequent are these

     2    tumors that appear at the wound site in these P53

     3    mice?  

     4                DR. SISTARE:  In P53s with the

     5    subcutaneous injection sites, it's not an

     6    insignificant frequency. It's something that we've got

     7    to reckon with.  We've got to work out things like is

     8    it pH related, what is it, that kind of thing.

     9                With TgAC, if there's a wound, a full

    10    length wound, you will get a papilloma every time

    11    there's a wound.  So there are concerns about

    12    fighting, wounding, that kind of thing.  You usually

    13    have to guard against those kinds of things.

    14                CO-CHAIRMAN MYERS:  After lunch Dr.  Fried

    15    and the morning speakers are going to try and put this

    16    all -- lead us in a discussion to put this all into

    17    perspective, and we'll reconvene at 1:20 promptly.

    18                There's one announcement.

    19                DR. PEDEN:  Yes.  For the late breaking

    20    session on Thursday night, there's about an hour for

    21    a number of presentations.  So if you want to present

    22    data that will appear in the publication that comes

    23    out of this meeting, come and see me, and we'll

    24    decide,  if we have too many, which gets presented.

    25                So I'm Keith Peden, and I'll be around

                                                                        154

     1    here.  Thanks.

     2                CO-CHAIRMAN MYERS:  Thank you.

     3                (Whereupon, the foregoing matter went off

     4    the record at 12:01 p.m.)

     5                            - - -

     6

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     1              A F T E R N O O N   S E S S I O N

     2                                        Time:  1:24 p.m.

     3                DR. FRIED:  Welcome to the first panel-

     4    audience discussion on mechanisms of neoplastic

     5    development and neoplastic cell tumorigenicity:

     6    implications for cell substrate development.

     7                What we're going to have first is two

     8    short talks which are relevant to the information we

     9    will discuss, and the first one will be given by David

    10    Onions, and it's the use of transgenics to detect rare

    11    oncogene events.

    12                DR. ONIONS:  Thank you very much, Mike. 

    13    It's kind of you.  

    14                What I'd like to just talk to you about is

    15    some work that I've been doing with my colleagues, Jim

    16    Neal and June Cameron at Glasgow and relates to using

    17    transgenic mice with transgenes targeted to the

    18    lymphoid compartment using the CD2 locus control

    19    region, and also controlling the functional expression

    20    of these genes using a estrogen receptor so you can

    21    control the actual target into the nucleus with

    22    tamoxifen.  So you can either have an inactive or an

    23    active transgene.

    24                Next slide, please.  Now this is a

    25    survival curve showing 100 percent survival here and

                                                                        156

     1    survival in days along this axis here.  What you're

     2    looking at at the green line is the survival of

     3    transgenic mice carrying the human CD2 myc transgene.

     4                As you can see, what happens is there's a

     5    background rate of about 13 percent of lymphomas in

     6    these mice over a period of about a year, and that's

     7    been replicated by other people.

     8                On the second curve here is a quite

     9    different mouse.  This is the P53 knockout mouse.  In

    10    this case it's the homozygous knockout mouse.  It's

    11    the Edinburgh strain of mice, and again there is tumor

    12    development, again predominantly lymphomas, over about

    13    a year period.  In fact, they're all dead by a year

    14    from lymphoma.

    15                So that's the starting point.  But what

    16    becomes interesting is when you make genetic crosses

    17    of these mice.  So if you look at this curve here,

    18    this is a cross now between this mouse and this mouse,

    19    and you see that there is a very rapid acceleration in

    20    tumor development.  This shows at least two genes are

    21    collaborating in the development of lymphoma.  

    22                What's more interesting, I think, from the

    23    point of view of this discussion here is that you can

    24    even push this system further.  Notice these mice are

    25    all dead by 90 days.  But if you superinfect these

                                                                        157

     1    mice with murine leukemia virus, you can actually get

     2    insertional mutagenesis that results in further

     3    acceleration of tumor development.

     4                By cloning the sites where that virus is

     5    integrated, you can identify new genes, as we've done

     6    -- in this case, new progression genes.  But the

     7    functionally important point I want to make here, I

     8    think, from the issues we've been discussing about the

     9    oncogenicity of residual DNA is that you can

    10    discriminate even in mice that have this very, very

    11    short life span by doing kinetic studies and looking

    12    at the pattern of tumor development over time.

    13                You can actually pick up very rare

    14    oncogenic events or very weak oncogenic events that

    15    you would certainly miss, even if you put these

    16    insults into mice that carry the myc gene.  So I think

    17    that's the first point I would like to make.

    18                This might be a very useful model system,

    19    for instance, for looking at certain insertional

    20    vectors like AAV that have got the mutated -- or have

    21    got lacorep gene or certain retroviral vectors, as

    22    well as perhaps residual DNA.

    23                Could I have the next slide, please.  The

    24    second point I wanted to make is that not all

    25    oncogenes collaborate.  This just shows genes that we

                                                                        158

     1    have identified as collaborating genes.  

     2                In this case this is a collaborating we

     3    identify with myc which we just published called

     4    CBFA1, and CBFA was identified by this retroviral

     5    tagging technique.

     6                When we make transgenics with that and

     7    then superinfect murine leukemia virus, we can ask

     8    what genes are activated, and we find, not

     9    surprisingly, that myc is activated; because we know

    10    that myc interacts with CBFA1.  But you can also find

    11    that other genes like pim and pal are activated, but

    12    they tend to be exclusive.  So that we know now that

    13    pim and pal lethal form the same complementation

    14    group.

    15                Could I have the next slide?  So in fact,

    16    we can begin to bring a pattern of progression

    17    starting in this case with myc, and then we can put

    18    pim and pal which we now put in the same

    19    complementation group, and we've identified further

    20    progression genes.

    21                So you can begin to build up a pattern of

    22    tumor -- of genes necessary for tumor progression.  

    23                The final point I wanted to make in

    24    relevance to the kind of use of these models in a

    25    functional sense to screen out of the carcinogens or

                                                                        159

     1    the oncogenic effect of residual DNA is this

     2    conjecture slide.  This is not data slide.  This is a

     3    conjecture slide, and it's a simplification.

     4                For instance, we heard from Dr. van der Eb

     5    today about the complexity of transformation

     6    associated with DNA viruses.  They cram a lot of

     7    functions into a small space.  But predominantly E1A

     8    has a myc-like function.

     9                So that if you were to test the

    10    oncogenicity of E1A regions in a myc transgenic model,

    11    it's unlikely that you would detect an acceleration of

    12    tumor development.

    13                On the other hand, if you were to take

    14    another mouse like the CBFA mouse, which we know

    15    collaborates with myc, you would probably find that

    16    this wouldn't collaborate, and you would detect

    17    deceleration.

    18                So my final point is that when picking

    19    these models, it will have to be models plural,

    20    because I think you need to use a range of different

    21    mice with different oncogenes to cover the

    22    complementation groups.

    23                (APPLAUSE.)

    24                DR. FRIED:  Are there any questions or

    25    comments?

                                                                        160

     1                (AUDIENCE PARTICIPANT)

     2                DR. ONIONS:  Yes, we do.  I didn't

     3    actually show that one, but those were critical

     4    experiments for us.  If you take --

     5                DR. FRIED;  Is your microphone on?

     6                DR. ONIONS:  Well, the question was, if

     7    you take the myc transgenic mice and superinfect those

     8    with murine leukemia virus, do you see acceleration. 

     9    The answer is, yes, you do.  We published this in

    10    Oncogene a few years ago.

    11                In fact, we used those mice to actually

    12    identify the new oncogenes like CBFA1 as a new

    13    oncogene.

    14                AUDIENCE PARTICIPANT:  I would like to add

    15    a note of complexity to this scheme.  I would like to

    16    add that not only with a single oncogene you can

    17    detect a complementing -- depending on the strain of

    18    mouse, the MULV you are using, you will get a

    19    different complementation.

    20                For example, with myc we have detected

    21    over 60 percent of notch activation, while you don't

    22    detect that in your system.

    23                DR. ONIONS:  That's right.  That's

    24    correct, yes.  That's absolutely true.  I think that

    25    really just highlights one of the issues here, that

                                                                        161

     1    all animal models are extremely complex.

     2                In instance, the LTRs and the retrovirus

     3    used will bias which particular genes are activated

     4    and so on.  So that's absolutely correct.  Again, I

     5    think, reinforces that, if you are to use these model

     6    systems, that you need to use a variety of model

     7    systems and not rely on one.

     8                DR. FRIED:  Does E1A and myc collaborate

     9    in vitro?

    10                DR. ONIONS:  Not as far as I'm aware.  I

    11    think that goes back to early experiments of Helmut

    12    Lang who did these experiments and showed that

    13    predominantly E1A had a myc-like function.  So that it

    14    didn't seem to collaborate.

    15                Of course, if you put in E1B, then you've

    16    got other functions there.

    17                DR. FRIED:  Thank you very much.  Is Jim

    18    Cook here?  Okay, I think we'll go on.  Jim was going

    19    to give us a short talk about the generalities about

    20    tumor genecosities, but I'm sure we could take that

    21    later if he shows up.

    22                From the talks we have heard, we know that

    23    to generate a fully neoplastic or tumor state is not

    24    simple, and it requires multiple events and multiple

    25    stages.  You know, we need sort of activation of

                                                                        162

     1    mitogenic signals.  This is usually through oncogenes

     2    and the oncogenes -- we might need more than one.  We

     3    might need cooperation.

     4                We also probably need inactivation of

     5    tumor suppressor genes, at least in the P53 and the RB

     6    pathways.  We have to overcome senescence.  We have to

     7    make sure, if the tumor is going to grow in the

     8    animal, that the division is greater than the death

     9    rate of the apoptosis.  

    10                This probably changes at the cell surface. 

    11    You need vascularization for the tumor and, of course,

    12    we've heard a lot about overcoming immune

    13    surveillance.

    14                It's quite clear that the formation of

    15    transformed cells or immortalized cells in vitro are

    16    not necessarily the same as forming a tumor in vivo. 

    17                So the first panel-audience discussion

    18    should probably highlight features that the regulatory

    19    agencies should consider when assessing neoplastic

    20    cell substrates for vaccine development.  

    21                I would ask the audience to actually

    22    participate as much as possible, because I'm sure that

    23    everybody here could answer all the questions that you

    24    may have questions.  So it will be a blend of both the

    25    panel and the audience.  

                                                                        163

     1                Also, if you want to ask people questions

     2    that you've thought of later after they gave their

     3    talk, this is the time.  

     4                So I think one of the first questions we

     5    should ask is should we use neoplastic cells for

     6    vaccine substrates, and what are the positive features

     7    of neoplastic cells?  Does anybody want to start on

     8    that?

     9                I mean, we have heard that we can grow

    10    them to large numbers, and we can get their

    11    reproducibility, always having the same one, freezing

    12    them down.  We certainly might need certain types of

    13    neoplastic cells for virus growth.  For high titers 

    14    we might need to get specific cell types.  

    15                Also, more recently, growth of disabled

    16    virus has been done in sort of P93 cells, which Alex

    17    van der Eb highlighted in his talk, which actually

    18    contain some adenovirus genes, and they're sort of, I

    19    guess you would say, immortal but not tumor producing. 

    20    They don't produce tumors at all, Alex?

    21                DR. VAN DER EB:  They produce tumors now,

    22    I think, but initially -- That has been tested many

    23    years ago, I think.  They were not tumorigenic in the

    24    nude mouse system, but they became it later.

    25                DR. FRIED:  But when you say they produced

                                                                        164

     1    tumors, was this on passage or do you need a lot of

     2    cells?

     3                DR. VAN DER EB:  I think it was on

     4    passage.  This happened in 1974-75 or something like

     5    that, and so it's a long time ago, and there is not

     6    much written down, not by me.

     7                DR. FRIED:  Because that's mentioned in

     8    the document, the CBER document, but this type of cell

     9    is really used, and people are injecting things,

    10    because most of the adenovectors which people are

    11    using for gene therapy are grown in 293 or equivalent

    12    cells, because they complement the E1A gene and allow

    13    you to get disabled virus, which then is produced and

    14    will not replicate.

    15                On the other hand, we know Onyx is using

    16    a virus for sort of gene therapy to selectively kill

    17    tumor cells which do not have P53, and they also grow

    18    their virus in those cells because they have E1B, and

    19    that's a complementation.

    20                So could you think of any other cases

    21    where we would need neoplastic cells?

    22                DR. PITOT:  Well, being very naive in

    23    this, I just have one question.  Since I think already

    24    on the market or at least in some trials a product of

    25    neoplastic cells that is basically a product of

                                                                        165

     1    myeloma cells have been used already in the treatment

     2    of neoplasia.  So it seems that question already, in

     3    a sense, has been answered in that it's been done.

     4                That's quite different from the sort of

     5    things you've been talking about up here.  But perhaps

     6    if one is talking about a protein that's going to be

     7    produced, I personally wouldn't see any problem with

     8    the neoplastic.

     9                So I think where it may get into a

    10    difficulty is in the DNA vaccine production.

    11                DR. FRIED:  Right.  Well, in the case of

    12    the hybridomas you're purifying a protein.  In the

    13    case of growing the viruses that you want live, you're 

    14    actually just -- You don't purify it to the same

    15    degree as you would purify the antibody molecules, and

    16    then you would inject in virus; because you want it to

    17    get into -- infect a lot of cells.

    18                So it's slightly different, I think the

    19    less critical.

    20                DR. SHEETS:  Might  I respond to your

    21    question about other reasons we might want to use

    22    neoplastic cells?

    23                My name is Becky Sheets, and I work in

    24    vaccines at CBER.

    25                A couple of other things that we have

                                                                        166

     1    considered in terms of why we are thinking about the

     2    acceptability of these sorts of cells is (1) you

     3    mentioned adenovirus as a recombinant vector, but also

     4    traditional HIV vaccine -- traditional approaches to

     5    HIV vaccines have not been tried in preventive

     6    settings, and by traditional approach I mean live

     7    attenuated and inactivated.  Part of that is because

     8    HIV only really grows well in human tumors, in T-cell

     9    lines.  

    10                Another basis for using these sorts of

    11    cell substrates for vaccine production is because they

    12    can be grown in -- They can be adapted to growth in

    13    serum free medium.  Therefore, you're taking away the

    14    risk of an animal derived substance that you would be

    15    adding extraneously to the vaccine.  So you're

    16    removing some of the risk for BSE or for some of the

    17    other bovine viruses.

    18                So there are additional reasons for

    19    considering neoplastic cells for vaccine production.

    20                DR. FRIED:  Right.  Also, I guess, people

    21    are going to use, say, adenovirus vectors to bring in

    22    other antigens, maybe HIV-type antigens.

    23                DR. SHEETS:  We have been approached about

    24    that.  Some of the adenovirus vectors are replication

    25    competent and can be grown in other things, but those

                                                                        167

     1    that are replication defective would need to be grown

     2    in a cell line such as 293.  Correct.

     3                DR. FRIED:  I think that's a strong point. 

     4    Also, I guess certain viruses that one would want,

     5    higher titers might be achieved in neoplastic cells

     6    than some of the cells that they're growing; because

     7    you can get them from different tissues where might

     8    need tissue specificity for the virus to really grow

     9    well.

    10                Okay.  So that's some of the positive

    11    features.  Then the question is what are some of the

    12    negative features for the substrate?

    13                I guess the ones are that they contain

    14    activated oncogenes, and the question is whether these

    15    oncogenes could somehow make it over to transfer

    16    through the vaccination procedure.

    17                Has anybody any feelings on that?  That

    18    would be -- The other thing is, if there were cell

    19    contamination, would cells of a neoplastic cell be

    20    transferred, and could that cause a tumor?  I guess

    21    one could very carefully get rid of most of the cells

    22    in the preparation, but how many cells do we need for

    23    tumors when we transfer it into the vaccination?

    24                AUDIENCE PARTICIPANT:  I have to express

    25    some bewilderment about some of these issues, simply

                                                                        168

     1    because, as I mentioned last night, the vero cell,

     2    which under many conditions is neoplastic, has been

     3    licensed for the production of IPV and OPV in the

     4    United States, Thailand, Belgium and France, and these

     5    issues apparently have been resolved, at least to the

     6    extent that they apply to vero cells.

     7                Is there something that I'm missing that

     8    applies to cells other than vero that are immortal in

     9    neoplastic?

    10                DR. FRIED:  Well, I think in the document

    11    it was considered that -- Bob?

    12                AUDIENCE PARTICIPANT:  Just to clarify, in

    13    the United States at least vero cells have not been

    14    licensed for an unpurified vaccine, for instance like

    15    OPV, to my knowledge.  IPV can be produced in vero

    16    cells.

    17                Of course, vero cells being used for OPV

    18    may be a special case, because whatever you're giving

    19    is being presented orally rather than parentally.  So

    20    I think that these still are issues, even with vero

    21    cells, that the world is struggling with.

    22                AUDIENCE PARTICIPANT:  Well, we have

    23    enough representatives here from FDA to tell us

    24    whether IPV and OPV are licensed in this country,

    25    produced in vero cells.  Does anyone have a definitive

                                                                        169

     1    answer?  OPV is not?  Yours is definitive?  

     2                Okay.  So IPV is, which implies strongly

     3    that vaccines that are administered orally and

     4    produced in continuously propagable abnormal

     5    neoplastic heteroploid cell populations should be

     6    acceptable.  

     7                AUDIENCE PARTICIPANT:  I would like  to

     8    add that OPV prepared in vero cells is also pre-trial. 

     9                AUDIENCE PARTICIPANT:  I was going back to

    10    try and look at the basis of our understanding of

    11    whether DNA -- residual DNA is a risk.  I thought

    12    there were some interesting talks today that perhaps

    13    highlighted some of the issues.

    14                One of the issues that wasn't stressed,

    15    though, was in fact the nature of the DNA that we see

    16    in Crucip and Ames, which are the primary danger, it

    17    seems to me, because blood products are purified.

    18                Then most of the DNA is pretty badly

    19    sheared, and it's sheared below a size that would be

    20    normally associated with a functional gene.  Now

    21    that's an average size, and we have to be careful

    22    because there's clearly a distribution in size.

    23                It seems to me that you're left with two

    24    primary worries -- well, three worries perhaps.  One

    25    is of promoter insertion, because there's still some

                                                                        170

     1    of these size of around about a promoter.  

     2                The second level of concern would be, I

     3    think, the still intriguing data on the MTR-like

     4    sequences in herpes viruses and whether there are

     5    other insertion elements like that, and they truly

     6    operate.

     7                I suppose the third concern I would have 

     8    really relates to adventitious agents we don't know

     9    about, like say a good example would be bovine polyoma 

    10    virus which we didn't really know a lot about until

    11    recently.  So you have an unknown polyoma virus in a

    12    cell line, and as we know, as we've heard today, you

    13    can get a lot of transforming functions in a small

    14    domain.

    15                So I think those are the three areas which

    16    seem to me to be perhaps residual concern in residual

    17    DNA.

    18                My only comment would be that you can talk

    19    about it a lot, but the only way of actually testing

    20    that, apart from large scale epidemiological studies,

    21    are the kinds of models that, I think, that Frank

    22    Sistare and myself mentioned.

    23                DR. FRIED:  But, David, I mean, none of

    24    those are really neoplastic cell specific.

    25                AUDIENCE PARTICIPANT:  No, they're not.

                                                                        171

     1                DR. FRIED:  They're the same for both.  I

     2    mean it's just DNA.  I was thinking more of, you know,

     3    if you start with a cell that has an activated

     4    oncogene or something, could that get over.  

     5                I think, since we need multiple genes for

     6    neoplasia, the chance of getting two or three cellular

     7    genes to meet and get in is very rare.

     8                AUDIENCE PARTICIPANT:  I think I was

     9    working around the conservative approach to exactly

    10    that same conclusion.  In other words, I think you can

    11    enumerate a number of issues, but those seem to me so

    12    remote at the moment that I would not, without further

    13    experimentation -- I would go in your direction, yes.

    14                DR. FRIED:  Right.  But I think one should

    15    consider the viruses.  Now the viruses put -- They get

    16    a lot for their money, because they have either

    17    overlapping reading frames, so one piece of DNA, in

    18    the case of polyoma viruses, three genes and SV40 is

    19    two genes, or they have common things that are hooked

    20    up to each other, are very closely linked.

    21                So we don't need different parts of the

    22    genome together.  I think that's something one might

    23    think about.

    24                DR. BERKOWER:  I'm from CBER.  Ira

    25    Berkower is my name.  

                                                                        172

     1                I'd like to make two points and ask for

     2    comments on these points.  The first one is:  I see

     3    vaccines, particularly anything that is an assembled

     4    antigen, as being entirely different from other

     5    products that might -- biomedical products that might

     6    be made by recombinant DNA methods and expressed in

     7    even in the same cells.

     8                For example, monoclonal antibodies made in

     9    myeloma cells -- myeloma cells are -- In hybridoma

    10    cells myeloma cells are highly malignant, but the

    11    antibodies can be highly purified.  Steps like protein

    12    A affinity columns and various other chromatographic

    13    steps can highly purify away adventitious agents,

    14    harmful DNA sequences and so on, and really should be

    15    considered, in my view, completely different from

    16    vaccines.

    17                Vaccine purification, particularly if it's

    18    a particulate type of antigen, would tend to select

    19    for things that are the size of viruses.  They would

    20    tend not to be inactivated by any harsh chemicals or

    21    denaturing agents.  They don't -- They may not have a

    22    specific affinity step.

    23                So -- and even some of the purification

    24    steps might tend to concentrate viruses into your

    25    fractions rather than remove them.  

                                                                        173

     1                So I think it's a mistake to reason from

     2    other products that might be made in tumor lines to

     3    vaccines.  Vaccines, I think, are quite distinct in

     4    that regard.  In many cases, they either can't be

     5    purified away from harmful agents or they can't be

     6    inactivated to the same extent as would ordinarily be

     7    done for other products produced in the same cells.

     8                So arguing that the same cells are used

     9    would not apply to vaccines.  That's my first point.

    10                DR. FRIED:  Well, but on that point, the

    11    differences between neoplastic and immortalized cells

    12    -- I mean diploid cells -- there's not much difference

    13    there.

    14                DR. BERKOWER:  That's true, but I think

    15    one of the arguments that these things are safe is

    16    that they've been used to make other products.  That

    17    argument, I think, just doesn't hold water.

    18                DR. FRIED:  And your second point?

    19                DR. BERKOWER:  The second point was: 

    20    Seems to me that, something I asked about before, that

    21    there ought to be a notion of sort of the malignant

    22    potential of some of these lines that are being

    23    considered, that some lines are what I would call

    24    highly malignant and probably shouldn't be used to

    25    produce vaccines, even though viruses can grow in

                                                                        174

     1    those cells.

     2                The cell itself would intrinsically  have

     3    a higher risk, whereas other cells would be much

     4    closer to normal somehow with a lower, what I would

     5    call, malignant potential, and might -- Perhaps we

     6    could identify these cells and, because there are

     7    continuous lines, characterize them as well as

     8    possible and actually show somehow that they were less

     9    malignant and, therefore, a more suitable substrate

    10    for producing vaccines, say by virus growing on those

    11    cells.

    12                I could imagine, for example, classifying

    13    cells based on some kind of oncogene inventory, and I

    14    wonder if that would be possible.

    15                DR. FRIED:  Does the panel have any

    16    comments on that?

    17                DR. COOK:  I think that gets back to the

    18    same point Mike Fried just made, which is that until

    19    you have some way to assess the relevance of what

    20    highly malignant is and whether the things that convey

    21    those traits to the neoplastic cells that you're using

    22    for the vaccine substrate -- whether those genes could

    23    be reasonably expected to be transferred across in

    24    some kind of residual DNA.

    25                It's sort of a nightmare and a worry, but

                                                                        175

     1    there's not much science to it.  I mean, the question

     2    is how can you say that a cell that varies by as much

     3    as a millionfold compared to another cell is any

     4    greater risk, other than the fact that it feels bad?

     5                If nothing is reasonably expected to be

     6    transferred and you have four or five oncogenes in

     7    that cell, what's the likelihood that those genes are

     8    going to be transferred across?  I mean, think of a

     9    simple experiment like a cell transformed by a pair of

    10    oncogenes, that you're required to get that cell to be

    11    reasonably immortalized and transformed in a

    12    conventional in vitro assay.

    13                Shear up that DNA and try to transfect it

    14    into another cell, and see if that DNA itself rather

    15    than the purified genes like we all do in plasma

    16    preparations could actually create a focus in anything

    17    you could imagine.  I think the answer is probably

    18    not.  It's unlikely that that would be an issue.

    19                Now how you test that is another question,

    20    and even more fundamental to your issue is what's

    21    highly oncogenic mean?   It's relative to how you

    22    assess the cells, how you quantitate those sorts of

    23    things, which cells you test, what kind of host you

    24    put them in, and you could probably define it to meet

    25    whatever criteria you wanted to.

                                                                        176

     1                I think it's a real dilemma and it

     2    probably requires some experiments to come up with how

     3    you're going to define these issues.  What do the

     4    terms mean, and how are you going to use these to set

     5    the guidelines that people would have to do this awful

     6    job will do?

     7                DR. BERKOWER:  I mean, one example, which

     8    is not directly related -- It seemed obvious to me

     9    that, if you were going to use, for example, EBV

    10    transformed B cells as a substrate, at least pick the

    11    nonproducer lines.  Okay?  That's obvious -- to grow

    12    the virus in.

    13                DR. COOK:  I think that's a different

    14    issue, and that is you're at risk of transmitting an

    15    infectious agent that can amplify itself.  That's

    16    different than saying you have a neoplastic cell free

    17    of all known adventitious agents that contains either

    18    a known or an unknown gene that regulates the behavior

    19    of that cell.

    20                I think transmitting infectious agents

    21    should be at a different level of concern, because

    22    then if that could survive the purification process,

    23    that's a big problem.

    24                DR. FRIED;  I mean, if it's a really

    25    highly tumorigenic cell, and we're not quite sure what

                                                                        177

     1    that means, then I think one has to be careful in

     2    whether cells are contaminating the vaccine.  If it's

     3    four or five or six oncogenes together that are making

     4    it so powerful, the chance of getting them all over in

     5    the vaccine is very remote, because you need six

     6    pieces.

     7                AUDIENCE PARTICIPANT:  I find myself in

     8    the reluctant position of trying to argue against what

     9    you just said, because I actually don't believe that

    10    oncogenic -- DNA from the lines we've been talking

    11    about is a risk.  But I think I'm not sure that I

    12    agree with the reasoning that you've just adduced.

    13                I do think there's a difference between

    14    cell lines.  I think the very fact that we use vero

    15    cells usually at a passage level below the level at

    16    which they're tumorigenic in animal models is a

    17    testimony to that.

    18                I think there clearly is a difference.  We

    19    know that, as we pass these cells, that different

    20    genetic changes are occurring, and we now know that --

    21    it's a kind of paradigm of tumor biology that multiple

    22    genetic changes are required.

    23                So if you're going to pick a line, why

    24    don't you pick one earlier on where there's likely

    25    only to be one or two changes and not one where

                                                                        178

     1    there's seven?

     2                I think your argument, Mike, was that it's

     3    so unlikely to transfer four oncogenes into a single

     4    cell, but surely all our transfection data in gene

     5    therapy suggests entirely the opposite.

     6                If you make a cocktail of a cytokine gene

     7    and a couple of other genes and inject them in, you

     8    can find generally the cells that take that out have

     9    all of those genes in.  It's rather like a cell

    10    transfection.

    11                DR. FRIED:  But you're putting in the

    12    purified genes, not the whole cell.

    13                AUDIENCE PARTICIPANT:  Oh, sure.  I accept

    14    that.  That's why I still think that this is a highly,

    15    highly improbable effect.

    16                DR. FRIED:  I was only commenting to him,

    17    saying that there may be multiple genes, you know.  If

    18    there are four or five, it's going to be worse.

    19                AUDIENCE PARTICIPANT:  But this is -- I

    20    would like to say I very much agree with you, that the

    21    idea is to take a cell that is barely immortalized

    22    rather than the cell that is soundly full of all kinds

    23    of mutations and could potentially transfer more than

    24    one at a time, like you just said.

    25                DR. FRIED:  I don't think anybody is

                                                                        179

     1    disagreeing with you, that you take it earliest and

     2    safest.  I mean, the question is what are the risks as

     3    you passage it later?

     4                AUDIENCE PARTICIPANT:  Well, okay.  To sum

     5    up my question then, would there be some convenient

     6    quantitative way to assess, oh, this one is earlier,

     7    that one is later?  

     8                DR. FRIED:  Does the panel have any

     9    comments?  Anyone?

    10                DR. COOK:  So you're talking about two

    11    different cell lines.

    12                AUDIENCE PARTICIPANT:  Yes.

    13                DR. COOK:  Trying to assess the relative--

    14                AUDIENCE PARTICIPANT:  Which one would be

    15    better for vaccine --

    16                DR. COOK:  -- that cell line?

    17                AUDIENCE PARTICIPANT:  -- for use as a

    18    cell substrate, as we're trying to decide today.

    19                DR. COOK:  Well, you can create artificial

    20    models in which you can do that, like the animal

    21    models I described this morning, and at some point I

    22    guess I can present these data.  But you could say,

    23    okay, we're going to decide that host A is the host in

    24    which this is going to be defined.

    25                Somebody is going to have to make that

                                                                        180

     1    decision, because you can always come up with a weaker

     2    host, what I call a walking test tube.  You know,

     3    you've got an animal, but it lacks any kind of defense

     4    against a tumor, and you're going to be able to change

     5    the tumorigenicity of that cell, depending on what you

     6    test it in.

     7                So somebody is going to have to decide,

     8    well, what is the animal in which we test this.  The

     9    data I showed this morning was that, if you have a

    10    cell line that can be easily rejected by a normal

    11    mouse or a nude mouse at a given concentration, it can

    12    be highly tumorigenic in a CD3 epsilon transgenic

    13    mouse.

    14                So you've got to define the animal in

    15    which this is going to be done, and then you can say

    16    how can we test the cells.  Well, there are

    17    quantitative ways to compare cell line A with cell

    18    line B, once you settle on that in vivo model.

    19                Then you have to accept the fact that

    20    you're making an arbitrary decision about, okay, if

    21    this one has a millionfold greater ability to make

    22    tumors at this inoculum over this period of time or

    23    over a range of inocula, then does that meet the

    24    definition of a worse cell line versus a better cell

    25    line.

                                                                        181

     1                AUDIENCE PARTICIPANT:  Yes, but I'd prefer

     2    a molecular diagnosis, if you see what I mean.

     3                DR. COOK:  A molecular probe that will

     4    tell you what's the worst cell?

     5                AUDIENCE PARTICIPANT:  No.  Multiple

     6    probes to try and define, you know, which ones have

     7    accumulated more activated oncogenes and which ones

     8    are running with fewer or almost none.

     9                AUDIENCE PARTICIPANT:  To respond to this,

    10    I would suggest that there's at least the possibility

    11    that focus on the malignancy of the cell is the wrong

    12    problem to be concerned with, that what we're really

    13    interested in is the ability of the information in

    14    that cell to be transferred, not the state of the

    15    information in the cell.

    16                For example, primary tissues  from

    17    monkeys, for example, can have pathogenic organisms in

    18    there that have nothing to do with transformation and

    19    would not score in a test for neoplasia, but would be

    20    certainly something we would want to avoid.

    21                I mean, the assumption in the question is

    22    that there's a correlation between the presence of

    23    information of a neoplastic nature and its ability to

    24    be transferred from one cell to another.  And it

    25    doesn't seem to me that we have any evidence to

                                                                        182

     1    suggest that that correlation holds.

     2                Now, certainly, if you have a scenario in

     3    which you have two cells that are completely identical

     4    in terms of their ability to donate information, you

     5    want the one with the less damage or accumulated

     6    changes in it.  But that's an assumption that I don't

     7    think there's any basis to make.

     8                In fact, I would question whether there's

     9    any reason to believe that there's any correlation at

    10    all between neoplastic potential and ability to donate

    11    information in a vaccine problem.

    12                DR. FRIED:  That was one of the topics I

    13    wanted to bring up, is whether the mechanism by which

    14    the cells become neoplastic could contribute to their

    15    relative risk as a vaccine substrate.  

    16                You know, certainly, if you knock out

    17    tumor suppressive genes, that's a loss of a gene. 

    18    That might, you know, make the cell more tumorigenic,

    19    but you're not going to really transfer that unless

    20    you're transferring a dominant negative P53.  If it's

    21    just mutated and gone, it's gone, and it's not a

    22    positive situation.

    23                I don't know about methylation, how people

    24    feel, whether that is something that's going to change

    25    that.  Walter?

                                                                        183

     1                DR. DOERFLER:  Well, you know, with

     2    methylation you have to be very cautious in

     3    interpreting into biological problems, because we --

     4    To be quite honest, I don't think we understand in

     5    full detail the implication.

     6                So from my judgment at this point, I would

     7    say, well, methylation may be -- the pattern across a

     8    certain stretch of the genome may be just a model on

     9    which chromatin structure is built, and in some

    10    instances this relates to expression of genes,

    11    permanent silencing of genes, and then how does that

    12    relate to the tumorigenesis?

    13                Well, one model I've got up in my mind can

    14    pursue, that by changing a pattern like Dr. Baylin,

    15    for instance, gave you several examples where in tumor

    16    cells the patterns are changed, and early on we saw

    17    viral transformed cells at a very different pattern

    18    into different segments.  

    19                So, certainly, things change, but to say

    20    it is an increase or a decrease, I wouldn't be able to

    21    do that; because we have looked at a fair number of

    22    human tumors in a number of mainly lymphomas and

    23    Hodgkin in Cologne, and looked at different -- used

    24    different probes.

    25                For some of the probes there was an

                                                                        184

     1    increase; for other ones there was a decrease, and for

     2    third ones there was no change at all.  

     3                So if I should translate that into

     4    biologic significance, my feeling at this point is

     5    perhaps chromatin structures are changed segment-wise

     6    in different parts of the genome in a tumor cell as

     7    compared to normal cell.  That's almost a sort of a

     8    given.  

     9                That's a blatant thing that chromatin

    10    structure changes, but I always thought changes in

    11    methylation patterns would be an interesting indicator

    12    for such change, and then we could, once we have the

    13    technology, try to delve deeper and see what do these

    14    changes mean in terms of altered chromatin structure

    15    and altered methylation pattern -- and altered

    16    transcription patterns.

    17                So when we saw these changes in lambda DNA

    18    transgenic cells, of course, we got very excited that

    19    this might be a more general phenomenon somehow

    20    relating to tumorigenesis.  I don't know how Dr.

    21    Baylin would feel about these proposals.

    22                DR. BAYLIN:  Well, again, if the question

    23    becomes more the one of transfer, which I think is the

    24    issue, it's hard to quite envision.  The biggest

    25    danger -- If you're using any virus, I think, as a

                                                                        185

     1    vehicle for inserting or carrying something, the virus

     2    itself is the biggest threat.

     3                I think there's going to be a lot more

     4    learned, and some of these viral proteins, in addition

     5    to binding up and taking away RB function and P53

     6    function, I think, can cause probably -- It's going to

     7    emerge that they probably can cause methylation

     8    changes in trans by binding up or changing parts of

     9    what is the methylation machinery itself, the ability

    10    to target the methyl transferases and all this.

    11                This is very little studied, but I think

    12    most viruses can set up methylation changes, and part

    13    of it may be that.  It's harder to envision with

    14    antigens and proteins things where the background of

    15    the cell itself will influence what you could

    16    transfer.  That would be a danger, I think.  That

    17    would be my view.

    18                 DR. DOERFLER:  Perhaps I could still make

    19    -- cite one example, because in general it's -- One

    20    uses the sort of the correlation or inverse

    21    correlation between gene activity and levels of

    22    methylation very frequently and loosely.

    23                So over the years I have learned the hard

    24    way that one has to be sort of very cautious, because

    25    it may be only certain segments of a promoter, and

                                                                        186

     1    unfortunately one cannot give a blueprint to say,

     2    well, it has to be 35 or 38 base pairs upstream from

     3    such and such a site.

     4                It doesn't seem to be the case.  It's more

     5    complicated.  Then an extreme case, which has taught

     6    me a lot, is again a virus, frog virus 3, which was

     7    initially worked on by Dawn Willis and Allan Graniff

     8    in Memphis.

     9                This is a DNA virus which replicates both

    10    in fish cells, frogs probably as well, and also in

    11    mammalian cells like BHK.  When you look at this DNA

    12    with any methylation sensitive restrictase, it seems

    13    to be totally methylated.

    14                Several groups, including our own, have

    15    used genomic sequencing techniques to look at all the

    16    CGs, and again this is a completely methylated genome. 

    17    And yet, a late expression is going on, and with

    18    genomes that are totally methylated in all CGs.

    19                So Mark Monitz in our lab a number of

    20    years ago did an experiment in which he used one of

    21    these late promoters which are active, fully

    22    methylated state in a viral infection, and hooked it

    23    up to an indicator gene like Luciferase and

    24    transfected it in the fully methylated stage.  Yes, 

    25    it was active.

                                                                        187

     1                However, when he just did a Hapa 2

     2    methylation, a CCGT which -- there were about eight

     3    sides -- it dropped its activity.  So here's an

     4    example which would support the notion that perhaps a

     5    certain pattern has to be present for an active or an

     6    inactive promoter, and this frog virus, for reasons I

     7    don't think we understand, prefers a completely

     8    methylated promoter to attract proteins possibly that

     9    facilitate transcription.  

    10                So one has to look in each promoter and

    11    each biologic system separately what this methylation

    12    means.  So my suggestion is it's a modulator of DNA

    13    protein interactions and that we have to be working

    14    very hard to find out for each promoter what is the

    15    important pattern for the active or inactive state or

    16    for building a chromatin structure that is consistent

    17    with biologic activity.

    18                DR. FRIED:  Thank you.  I want to get back

    19    to -- DNA would be the things that we're really afraid

    20    of, if there are some oncogenes in a cell.  So I don't

    21    know how efficient DNA transfer is.  

    22                I mean, are there people who do DND

    23    vaccines?  Could they say there's a sort of naked DNA

    24    go over or do you have to shield it somehow?  What's

    25    the efficiency if you put it in the microgram, how

                                                                        188

     1    many cells get it?  Does anybody know those answers or

     2    can say anything?

     3                AUDIENCE PARTICIPANT:  Ron Desrosiers

     4    might make a comment, because of the SIV experiments,

     5    which I think are probably the pertinent ones where

     6    you get an actual readout of virus production from

     7    putting in intact genomes.

     8                We've done some of the experiments to him

     9    using SIV, and you need to put in very large amounts

    10    of DNA relatively still to get a functional expression

    11    of a replication combinant virus.  

    12                So you're still talking in tens of

    13    micrograms into an animal before you get out a

    14    functional virus.  So it does imply that the uptake of

    15    DNA to result in a functional end expression is

    16    actually extraordinarily rare, and we're talking in

    17    micrograms of DNA.

    18                I think the SIV experiments are the same.

    19                AUDIENCE PARTICIPANT:  The question was

    20    what was the nature of the DNA.  This is linearalized

    21    proviral -- intact proviral DNA going in.  We have

    22    actually shown that if you actually take a retrovirus

    23    and you split it into gag and pol and env constructs,

    24    just as in a packaging line, put those in, you can

    25    also get expression in vivo and get particle

                                                                        189

     1    production.

     2                DR. PURCELL:  Yes.  My name is Damian

     3    Purcell from Australia.  I was just going to make a

     4    comment of some work that -- on SIV that I'll present

     5    on Thursday night regarding infection similar to Ruth

     6    Ruprecht and de Rossier's experiments with injected

     7    the naked DNA as plasmid, circular plasmid, in small

     8    numbers of animals.  

     9                We found that we can very efficiently

    10    initiate infection.  When we put this DNA onto, for

    11    example, gold beads, we get down below ten nanograms.

    12                DR. FRIED:  You're putting it on beads? 

    13    If you just --

    14                DR. PURCELL;  On beads as, yes, gene gum,

    15    and we haven't titrated it, but putting it on the

    16    method of transfer, the naked DNA can under some

    17    circumstances such as gene gum inoculation be

    18    extremely efficient.

    19                DR. FRIED:  But would the naked DNA be

    20    efficient?  I mean, if some was -- there was some DNA

    21    that got out of a cell, what's the chance of that

    22    getting over?

    23                DR. PURCELL:  Yes.  We can't really

    24    comment on that, because we didn't titrate the DNA,

    25    but in our experiments with naked DNA injection, we

                                                                        190

     1    just followed a 300 microgram amount, which --

     2                DR. FRIED:  When you say it was efficient

     3    with the beads, what kind of numbers are we talking

     4    about?

     5                DR. PURCELL:  In terms of numbers of

     6    animals?

     7                DR. FRIED;  No, in terms of amount of DNA

     8    in successful infection, how could you dilute it down

     9    to?

    10                DR. PURCELL:  We've only gone as low as

    11    ten nanograms, but maybe one additional piece of

    12    information is that included among the DNAs are DNAs

    13    where we've made deletions in the promoter region of

    14    SIV in the U3, so that the expected expression might

    15    be lower than wild type and still, even with ten

    16    nanograms delivered by gene gum, there's efficient

    17    initiation of infection under those circumstances.

    18                DR. FRIED:  Thank you.

    19                DR. PETRICCIANI: John Petricciani. 

    20    There's a large body of data actually in the

    21    literature that goes back a couple of decades.  One of

    22    the experiments -- these are in vitro experiments on

    23    DNA transfer.  One of the experiments that was

    24    mentioned earlier today was the transformation of 3T3

    25    by human tumor cell DNA.

                                                                        191

     1                In that particular set of experiments, the

     2    amount of DNA required to get any positive readout was

     3    20 micrograms, and it required also carrier DNA in

     4    addition to the human tumor cell DNA.  The bottom line 

     5    is that in the studies of which I'm aware in vitro,

     6    let alone in vivo, it's extraordinarily difficult to

     7    get a positive uptake in expression of DNA, whether

     8    it's cell transformation or other gene expression.

     9                DR. FRIED:  I assume that, if somebody

    10    aware of these things, they would be nucleasing up

    11    their preparations before they -- I mean, they would

    12    be using -- trying to break down the DNA as small as

    13    possible.  

    14                AUDIENCE PARTICIPANT:  Well, it's a

    15    question of inactivation, reduction through one or

    16    another mechanism.  Right.

    17                DR. SHIVER:  John Shiver from Merck.  Our

    18    experience is that plasmid DNA is not efficiently

    19    taken up by direct injection into most tissues into

    20    animals, whether they're non-even primates or mice,

    21    and that it generally requires some degree of

    22    amplification of whatever response you're looking for

    23    in order to detect it, such as an immune response to

    24    a transgene encoded by the plasmid or actually a virus

    25    virion developing capable of replication, as perhaps

                                                                        192

     1    in the SIV or FIV model.

     2                Looking for direct gene expressions

     3    systemically is generally not successful.  Also, the

     4    uptake of the DNA is highly dependent upon the state

     5    of the DNA.  It really needs to be compacted

     6    supercoiled to really -- to work even as well as it

     7    does, generally, with naked DNA injection.

     8                DR. FRIED:  So your feeling, if there was

     9    oncogenes inside of a DNA and some of that was taken

    10    up by recipient cell, it would be very inefficient to

    11    get that expressed?

    12                DR. SHIVER:  Should be very inefficient,

    13    especially in the linearized fragments and form that

    14    you would expect that DNA to be in.  

    15                DR. DOERFLER:  Just in response to this

    16    interesting comment, in addition to feeding DNA with 

    17    all that we're injecting DNA, mainly the green

    18    fluorescent plasmid construct, fewest cytomegalovirus

    19    promoter or SV40 promoter, recently also the ras

    20    sarcoma virus promoter but we haven't done many

    21    experiments on that yet, and we've also mainly failed

    22    to find expression except locally in the muscle where

    23    we injected it into.

    24                It could be traced by FISH and PCR in

    25    various organs for quite a long time, and although

                                                                        193

     1    these data are repeated only a number of times and not

     2    for years, we've also seen it in testing the contents. 

     3                So this may be an interesting observation

     4    for people around here.  It might be excreted probably

     5    via the bile route.  I'm not sure how it works.  But

     6    definitely we can find some of this DNA in the

     7    intestinal contents.

     8                DR. SHIVER:  We would agree with that

     9    experience overall.  The DNA that is residual and

    10    remains tends to be extrachromosomally located -- you

    11    know, with some persistence time, but generally gene

    12    expression only near the site of injection.

    13                DR. DOERFLER:  Right.  Right.  So you do

    14    find expression at site of injection?  We do, too.

    15                DR. FRIED:  So, I mean, I think the

    16    general feeling is, if there was DNA, cellular DNA

    17    with cellular oncogenes, that would be very

    18    inefficiently transferred, and I think anybody

    19    preparing a vaccine would take great precautions in

    20    trying to destroy the DNA or make it as small as

    21    possible.  Is that what the panel would think?

    22                So the way the cell -- the neoplastic cell

    23    was formed, we don't really expect most of these

    24    properties, methylation or loss of tumor suppressor

    25    genes or viral oncogenes to be transferred over in a

                                                                        194

     1    vaccine preparation.  

     2                Is there anybody who disagrees with that?

     3                AUDIENCE PARTICIPANT:  I don't disagree,

     4    but maybe I could ask you to clarify a bit the

     5    statement that you made a moment ago, that you'd like

     6    to see the DNA made as small as possible in order to

     7    avoid these kinds of problems.  And of course, from a

     8    regulatory perspective, somebody comes in with a

     9    product and they're doing a particular purification

    10    scheme on it and it makes the DNA a certain size, and

    11    so we can always say can you make it smaller.  But the

    12    question is how small then is small enough?

    13                What if somebody comes in with a product,

    14    and they haven't made the DNA smaller?  Does that mean

    15    we should be worried about it?  Likewise then, the

    16    question of if you have one cell line which is more

    17    tumorigenic than another, again the question is, if we

    18    truly think that one which is less tumorigenic is

    19    safer, then that should be something that one can put

    20    some numbers on.  Then one has to, obviously, come

    21    with some kind of a threshold of sort of trying to

    22    define this.

    23                So I know it's thorny and difficult when

    24    trying to put numbers on this kind of thing, but I

    25    think the kind of information that the FDA needs to

                                                                        195

     1    make these decisions requires some more definition.

     2                DR. FRIED:  I think you probably have to

     3    spike your preparations with, you know, some really

     4    efficient either phage DNA or something or some of

     5    these really nice, you know, circular small polyoma

     6    SV40, which are probably efficient in assays, and then

     7    you know, at different stages of the preparation see

     8    how much and where it goes.

     9                AUDIENCE PARTICIPANT:  That certainly

    10    sounds like a reasonable suggestion, if in fact the

    11    recommendation then is that one has to presume that

    12    such DNA is there, in the first place, and that one

    13    does have to get it to a size that's small enough to

    14    prevent these kinds of outcomes.

    15                DR. FRIED:  I mean, I was going to get to

    16    it.  But the only -- Is there a comment in the back? 

    17    The only thing that I was not including is possibly

    18    gene amplification, which people don't look at.

    19                I would certainly look at cells, because

    20    those are cells that usually amplify oncogenes.  They

    21    also amplify more than one oncogene.  More and more

    22    information is coming out that oncogenes get together. 

    23                There are large circles that we could see

    24    satellogically, but there's also generated small

    25    circles of DNA.  So there you're sort of amplifying

                                                                        196

     1    up, and I certainly -- If one was using neoplastic

     2    cells -- I was going to get to this later -- you would

     3    certainly look for -- you know, cytologically, for

     4    double minutes or HSRs, and I wouldn't stay away from

     5    those, and I certainly would look for polydispersed

     6    circles which have been reported in cells, small

     7    circular DNA.

     8                We don't know where that comes from, but

     9    certainly, a lot of the gene amplifications has to do

    10    with oncogenes and myc.  I think all -- If anybody is

    11    using cells, they should certainly do EM analysis for

    12    circular DNA and for HSRs and double minutes.  

    13                 DR. KAPPES:  John Kappes from UAB.  I'd

    14    like to suggest an alternative mechanism for genetic

    15    transfer, at least where the vaccine would be

    16    retroviral derived or relying on retroviruses for

    17    cancer vaccination, for example, if our hopes of gene

    18    therapy ever come to fruition.

    19                Data I will present tomorrow could suggest

    20    transfer of that information via the messenger RNA. 

    21    That is, instead of relying on DNA transfer, with HIV

    22    based vectors I have evidence that the poly-A signal

    23    could help in some way I don't understand yet mediate

    24    -- transfer that information.

    25                That is, if the oncogene message was

                                                                        197

     1    packaged and that polyadenylation somehow modulated

     2    recombination with the transfer vector, and if that

     3    was integrated, that might be an alternative concern

     4    to take into account for transfer that type of genetic

     5    information.

     6                DR. NICHOLS:  Warren Nichols, Merck. 

     7    There's certainly been a lot of data published over

     8    several years now about taking tumorigenic DNA from

     9    cells that are tumorigenic and injecting very large

    10    quantities into a variety of laboratory animals and

    11    newborn rats, newborn hamsters, nude mice,

    12    antithymocyte treated rats and mice, and when giving

    13    up to 100 micrograms of these, things like HeLa cells,

    14    CHO cells, the DNA from HeLa cells, CHO cells, human

    15    bladder tumor cells, cloned activated onc genes, there

    16    has been no evidence of any tumor formation in any of

    17    them.

    18                Just as a comment on distribution, this

    19    isn't a comment on distribution of cellular DNA, but

    20    in looking at a plasmid from DNA vaccines, when

    21    injecting 1011 plasmids into a muscle and then looking

    22    at various time periods after that, we can see a great

    23    many plasmids still in the muscle six weeks or 12

    24    weeks later.  But in the other tissues, in blood and

    25    brain and spleen and a variety of tissues, 13 that we

                                                                        198

     1    looked at, by six weeks there's never any present.

     2                If we look just to try to see where it has

     3    been distributed, in day one and day two we can find

     4    it in most organs.  By day seven, it spontaneously has

     5    all gone.  When we use purification methods to

     6    separate plasmid from a genomic DNA even at two days

     7    or three days, it all disappears.

     8                We use in looking at this PCR methods that

     9    are sensitive to .005 to .01 femptograms.  It's about

    10    one plasmid in 150,000 nuclei per microgram of DNA. 

    11    So that it certainly does get around, but it certainly

    12    does disappear quite quickly.  None of it has ever

    13    been demonstrated to be integrated in that kind of

    14    system.

    15                I do think that the injections with the

    16    DNA derived from tumor cells in huge quantities

    17    compared to what is present in any vaccine is very

    18    important.

    19                DR. FRIED:  Could you repeat the question?

    20                AUDIENCE PARTICIPANT:  Were those done in

    21    normal animals?  

    22                DR. FRIED:  Were which done in normal

    23    animals?  

    24                AUDIENCE PARTICIPANT:  Injected from the

    25    DNA.

                                                                        199

     1                DR. NICHOLS:  The injection of the

     2    plasmid?  The question is were the injections of DNA

     3    done in normal animals?  The injections of plasmid

     4    into animals and then following and looking -- that

     5    was in normal animals.

     6                The injection of cellular DNA that's in

     7    the literature is injecting into newborn hamsters,

     8    newborn rats, newborn nude mice and rats and mice,

     9    newborn rats and mice that had antithymocytes here.

    10                AUDIENCE PARTICIPANT:  Dr. Nichols, excuse

    11    me.  Have you published that data?

    12                DR. NICHOLS:  Which data?

    13                AUDIENCE PARTICIPANT:  The ones you're

    14    just referring to?

    15                DR. NICHOLS:  The data about the plasmid

    16    injections, that is published; and there are two other

    17    papers that are in preparation right now, but the

    18    first one is published.  The other data from the

    19    standpoint of the cellular DNA being injected, that's

    20    all from literature that exists now, not from us but

    21    from other -- many other people.

    22                DR. FRIED:  Thank you.  

    23                DR. MINOR:  Philip Minor from NIBSC. 

    24    Warren, how do you square your lack of detecting any

    25    integration with Dr. Doerfler's comments this morning

                                                                        200

     1    about integrating M13 after you feed it to mice?  It

     2    does seem to me, there's a little bit of ambiguity

     3    here about what's really going on.

     4                DR. NICHOLS:  Well, let's say that there

     5    are always other systems you can look at, but up to

     6    now with that sensitivity we haven't seen anything

     7    that has remained after separation techniques.

     8                We were talking about the possibility with

     9    Dr. Doerfler of having a ligase in the process being

    10    able to hook up things that make it look like

    11    integration that isn't really integration, and he has

    12    told us that there are very rare events, you know. 

    13    There are just two different observations, and it's

    14    hard to explain what --

    15                DR. FRIED:  But one is cellular DNA, and

    16    Walter, yours was just adenovirus.  It wasn't -- I

    17    mean, Walter didn't do the comparable experiment.

    18                DR. DOERFLER:  Well, if I just can comment

    19    on that, the DNAs we have been using were mostly DFH

    20    M13 DNA double stranded form, either linear or

    21    circular.  Most of the experiments were done with ECO

    22    1 linearized DNA.

    23                I don't think there is so much of a

    24    discrepancy between these observations, because we

    25    looked for -- In most experiments, we looked

                                                                        201

     1    relatively, you know, early after application.  So the

     2    recloning was done over 18 hours after feeding, from

     3    the last feeding, from the spleen.  So we can see it

     4    in the nucleus and perhaps chromosomally associated or

     5    even linked to cellular DNA.

     6                This was a recloning experiment and, I

     7    think, for the definite this is M13 DNA.  Whether or

     8    not it's linked to mouse DNA, well, it was suggested

     9    it could be.  But, of course, there are other

    10    possibilities, how we might find such a linkage.

    11                That was the latest we could do.  That was

    12    18 hours after the last feeding.  If we do it later,

    13    we have great difficulties finding it.  So we

    14    considered that a rare event that only -- persistence

    15    only for a certain time, although experiments we

    16    didn't discuss in feeding pregnant animals, there may

    17    be a longer persistence in the offspring.

    18                DR. MINOR:  It wasn't the question of

    19    persistence so much as the question of actually any

    20    integration at all, you see.  I think what Warren was

    21    implying to me was that there is no integration of the

    22    plasmid DNA that he injects into the mice; whereas, as

    23    I understood it from your feeding experiments, there

    24    was evidence of such expression.

    25                DR. DOERFLER:  Well, you know, we have two

                                                                        202

     1    lines of evidence.  One is the recloning, and the

     2    second is data I didn't show, chromosomal association

     3    in embryonic cells after feeding pregnant animals in

     4    rare cases.  So one really has to look hard for it.

     5                DR. FRIED:  How long after did you look?

     6                DR. DOERFLER:  Well, in the directly fed

     7    animals, it was -- The animals had received a dose of

     8    50 micrograms day before one week, and then 18 hours

     9    after the last feeding the animal was sacrificed,

    10    spleen extubated, and the DNA was cut and cloned.

    11                For the offspring of pregnant animals, we

    12    looked in fetuses and in embryos.

    13                DR. MINOR:  I've got a related sort of

    14    technical question, too, I think.  As Warren said, I

    15    mean, it depends very much on what system you're

    16    looking at.  I wondered, most of the carcinogenicities

    17    or assays that we've been hearing about today are

    18    really being chemical carcinogenicity type approaches.

    19                I wondered how appropriate those would be

    20    to something like a DNA where you're talking about a

    21    particular gene going in.  I mean, to what extent is

    22    this system actually appropriate for what you're

    23    looking for, if you're looking for DNA oncogenicity.

    24                DR. FRIED:  Well, that gets to the next

    25    sort of topic.  How do we test for oncogenicity. 

                                                                        203

     1    Maybe Jim will want to give his talk now.

     2                AUDIENCE PARTICIPANT:  Can I ask a

     3    question about one of the earlier comments?

     4                Did you use any transgenic mice in that

     5    DNA study, because it's for the very reason that was

     6    just mentioned.  If an activated oncogene was

     7    involved, it wouldn't cause, you know, global changes. 

     8    It might cause changes that would just be measured in

     9    an animal that only needed a promoter or only needed

    10    a knocking out of -- that was heterozygous and only

    11    needed a knocking out of one copy of RB or something

    12    like that.

    13                The other comment:  I also see a paradox

    14    here in saying that there's no real risk of this DNA

    15    being taken up.  When you consider that  almost

    16    everyone of these vaccines is injected right into the

    17    tissue, which is the preferred site for DNA gene

    18    therapy, which is the preferred site for DNA vaccines,

    19    which is the best way to get expression of

    20    extracellular just naked DNA -- the best place you

    21    could go is right into those same muscles.

    22                I'm a little surprised.  I'm actually very

    23    surprised to hear that you think that just injecting

    24    DNA there won't be taken up.  In fact, the fact is DNA

    25    at that site will be taken up.  That's why Dr.

                                                                        204

     1    Doerfler's results are what they are, and they will be

     2    expressed, and they will persist for quite a long

     3    time, surprisingly long.

     4                I don't know about you.  I was very

     5    surprised to see how long DNA injected directly into

     6    a muscle will be expressed there.  Whether that

     7    oncogene being expressed at that site will cause

     8    cancer in a mouse, whether it even could cause cancer

     9    in a mouse, is maybe the next question.  But I think

    10    you couldn't do much more to get the DNA expressed

    11    than to inject it into a muscle the way it's being

    12    done.

    13                I mean you could.  You could damage the

    14    muscle.  You could inject lidocaine and various other

    15    things, but it would be hard to maintain that -- I'd

    16    like to interpret your result, which is that if you

    17    don't add enough DNA, you don't get it; and if you add

    18    too much, kill the cells that would have responded.

    19                AUDIENCE PARTICIPANT:  Let me just say

    20    there's a big difference between being taken up and

    21    being integrated.  The DNA is certainly taken up by

    22    the cells, and it is expressed by the cells.

    23                What we're saying is there's no evidence

    24    that it's integrated into the genome of the recipient

    25    cells that can be determined in our studies up to this

                                                                        205

     1    time.

     2                DR. FRIED:  So it's taken up, but it's not

     3    necessarily integrated.  Is that -- So there's no long

     4    term -- The long term persistence is

     5    extrachromosomally?

     6                AUDIENCE PARTICIPANT:  Yes, that's

     7    correct.  At least with the sensitivity that we can

     8    get of the .005 to .01 femptograms, it all is taken

     9    away by the purification procedures that we do, and

    10    nothing remains.

    11                AUDIENCE PARTICIPANT:  And also somewhat

    12    related to this, I heard you say that you hope the DNA

    13    is reduced to its smallest possible size.  I believe

    14    it's true, the statement that not a single vaccine is

    15    treated with DNAase and that any considerations of

    16    reducing DNA to its smallest size have simply been

    17    missed by people who make vaccines.

    18                DR. RUUD:  Rupert Rudd from Solfi.  We are

    19    using DNAs or our vaccine, and in general here I want

    20    to state that we are now looking at cells, viruses

    21    that are being expressed and can be transferred into

    22    vaccines.

    23                I think it's also fair to say what you

    24    define as vaccine -- I mean, if your final product is

    25    like a monoclonal antibody, we have no problems here. 

                                                                        206

     1    If you have a purified inactive phage, it's a purer

     2    product which is also a protein, but it is from virus

     3    that has been grown on certain cells.  Then you may

     4    have a problem.

     5                I think this dispairment should be

     6    broadened and more defined in what we call vaccines

     7    and what kind of purpose it is, and list a series of

     8    the amounts that are necessary for that particular

     9    line.

    10                DR. FRIED:  Walter?

    11                DR. DOERFLER:  Just one other comment to

    12    the ongoing discussion on persistence and in what

    13    form.  Now when we look by sectioning different organs

    14    and fish after feeding DNA, then we invariably see the

    15    DNA inside the nucleus.  That doesn't mean it's

    16    integrated.

    17                Now the recloning and also chromosomal

    18    association would indicate to me that perhaps very,

    19    very rarely it can be integrated.  So the limit Warren

    20    has given us are perfectly agreeable with me, because

    21    it's a very, very rare event, if it occurs at all. But

    22    it's frequently in the nucleus.

    23                So there's no question about its uptake

    24    and, once it's in the nucleus, of course, one doesn't

    25    know what might happen.

                                                                        207

     1                In the embryo we've also seen it in the

     2    nucleus, usually in more than one cell, cell type

     3    justice, which might indicate that the cells still

     4    divide after receiving some of the DNA, and then this

     5    pattern emerges after what we consider transplacental

     6    uptake.

     7                DR. FRIED:  Do you think you have to have

     8    cells dividing for the DNA to become integrated and,

     9    when they're injected into certain tissues, that the

    10    nondivided cells that DNA just persists and then goes

    11    away?

    12                DR. DOERFLER:  Possibly.

    13                AUDIENCE PARTICIPANT:  I want to follow

    14    up.  Jim McDougall presented some data that I was not

    15    aware of, and that is that fairly small pieces of the

    16    herpes virus genome seemed to be associated with this

    17    hit and run mechanism.  

    18                I didn't count all the base pairs in those

    19    stem loop structures he had.  It looked like to me

    20    somewhere between maybe 25 and 75, but I think the

    21    sequences you had that was about 500 base pairs, and

    22    I don't believe you looked at any smaller than that.

    23                The question is whether you could chop

    24    away at those things and still show that that stem

    25    loop was associated with that type of event.  But I

                                                                        208

     1    think the question that really comes to my mind is to

     2    whether those stem loop type structures are present in

     3    mammalian cell DNA, and if they are associated with

     4    hit and run transmission of herpes viruses and, if

     5    similar stem loop structures are present in mammalian

     6    DNA, is that something that we would have to worry

     7    about from a regulatory perspective?

     8                DR. McDOUGALL:  Well, I think it's not

     9    difficult to find stem loop structures in almost any

    10    source that you go and look at.  Their presence is

    11    certainly there.

    12                Did we cut them down any further?  No. 

    13    The size that I showed was the -- That was the

    14    structure that we could see in a piece of DNA that

    15    actually was bigger than that, but that was the

    16    consistent structure that was always present and was

    17    capable of transforming cells.  But again, you know,

    18    I continually come back to what I know for sure.

    19                That is, you have to use large amounts of

    20    DNA to get this sort of transfection to work, and I

    21    cannot believe that in any vaccine that's produced

    22    there would ever be residual DNA at that sort of

    23    level.

    24                Now it's true that, you know, what you may

    25    be looking at is a single event, and so if you

                                                                        209

     1    vaccinate 10 million people, you're likely to find one

     2    somewhere out on the end of the bell curve that gets

     3    a reaction to that.  But that's a price that you may

     4    have to pay in this situation.

     5                The other -- While I'm on, the other thing

     6    that I do feel is that a decision whether or not to go

     7    ahead with this sort of -- this type of cell line

     8    that's been immortalized or transformed by a DNA

     9    virus, by papilloma or SV40 or adenovirus or whatever

    10    -- I still think that that sort of decision should

    11    only be made on the question of what residual DNA

    12    might not be in the vaccine, and not on the basis of

    13    whether a cell is partially moving toward neoplasia or

    14    completely moving toward it; because I think the truth

    15    is that, once you make a cell genetically unstable, it

    16    is going to accumulate more changes, whatever you do.

    17                So the one thing I keep coming back to is

    18    how much DNA is going to be in one dose of a vaccine,

    19    and I cannot see that that will ever carry enough

    20    material to actually be damaging.

    21                DR. HOEKENOF: I'm Ray Hoekenof of

    22    Intergene in Holland.  I collaborate with Alex von

    23    Raab who is in the forum.

    24                I'd like to add one thing to the

    25    discussion whether there is good cells or bad cells. 

                                                                        210

     1    I think there was a comment earlier this afternoon

     2    that there is no evidence of any correlation between

     3    the neoplastic potential of a cell and its potential

     4    to transfer that neoplastic potential to the vaccine.

     5                I think there is one example that was not

     6    mentioned yet of the potential of transfer of such

     7    potential, which is the mechanism of homologous

     8    recombination.

     9                I think the 293 cells which are generally

    10    used for gene therapy vectors but have also been

    11    mentioned for production of vaccines provide a clear

    12    risk that you can generate or can transfer elements

    13    from the cell line to the vaccine simply by homologous

    14    recombination.  In 293 cells the 5 prime ITRS is

    15    present.  The encapsulation signal is present. 

    16                So, for instance, if you would produce

    17    adenoviral batches on that cell line, you can very

    18    quickly generate recombinant adenoviruses  that have

    19    taken up parts of the 293 cell line by homologous

    20    recombination, a phenomenon known as RCA.

    21                I think there will be a paper presented

    22    later this week by Fritz Fallaux from van der Eb's lab

    23    to present cell lines that eliminate that risk, and I

    24    think homologous recombination between a neoplastic

    25    cell line and a vaccine is something that should be

                                                                        211

     1    kept in mind as a risk factor for transfer of

     2    neoplastic characteristics.

     3                DR. FRIED:  So you would think all cell

     4    lines that had transformed by viruses -- I mean, but

     5    293 doesn't have -- It only has a couple of genes

     6    there.  Is that --

     7                DR. HOEKENOF:  That is correct, but --

     8                DR. FRIED:  So you think when they come in

     9    with the E1A minus vectors, they're going to recombine

    10    with the endogenous genes?

    11                DR. HOEKENOF:  Oh, in 293 that is not

    12    something that you expect, but it is a fact.  You very

    13    quickly start to generate revertant adenoviruses which

    14    result from homologous recombination between the cell

    15    line and the vector.

    16                So the point I'm trying to make is, if you

    17    want to produce a vaccine on a cell line, make sure

    18    that there is no homology present between your DNA on

    19    the vector and the cell line, and maybe van der Eb can

    20    comment on that.

    21                DR. VAN DER EB:  I agree with that. 

    22    There's little I have to add.

    23                DR. RUSSO:  Carlo Russo from Merck.  Are

    24    you referring to recombination with viral genes, not

    25    with cellular genes?

                                                                        212

     1                DR. HOEKENOF:  Well, in this case I'm

     2    referring to recombination with viral genes, of

     3    course, but any homologous recombination can lead to

     4    uptake of cellular DNA into your vector.  So I think

     5    you should avoid that.

     6                DR. FRIED:  But again let's not drift

     7    away.  We're asking in neoplastic cells, is it any

     8    different than, say, diploid cells, and are these

     9    phenomena that are specific to neoplastic as opposed

    10    to diploid cells?

    11                DR. RUSSO:  I think what we need to

    12    clarify is this concept of malignancy, because it

    13    seems to the FDA, at least to a couple of their

    14    representatives here have this concept that there are

    15    different degrees of malignancy, and perhaps the cell

    16    substrate should be identified, high malignancy, low

    17    malignancy, medium malignancy.

    18                I think we should have a consensus here

    19    whether this is considered to be true or

    20    independently, as somebody suggested, the only concern

    21    should be on the final.  What is the residual DNA that

    22    we're going to have in our vaccine, independently from

    23    which cell line the DNA came.  

    24                DR. FRIED:  Do people on the panel have

    25    anything to say on what's malignant, how we would

                                                                        213

     1    measure that?

     2                DR. COOK:  Well, we've covered this in

     3    bits already, it seems like, in three parts.  One is:

     4    How do you measure this?  And we've already talked

     5    about quantitating the malignancy of the neoplastic

     6    potential or the tumorigenicity of the cell.

     7                The second is, once you have one cell line

     8    of a given kind of tumorigenic potential, what's the

     9    likelihood that something from that cell line could be

    10    transferred through residual DNA in such a way that it

    11    would be expressed functionally to lead to that in a

    12    recipient of some sort?

    13                I think those are probably quite different

    14    questions.  You can measure the first, once you define

    15    the parameters, and we can talk more about that if you

    16    want to at some point.  But the second issue, I think,

    17    requires experiments like were referred to that are

    18    published in literature that I don't know about where

    19    you put in large amounts of tumor cell DNA into

    20    immunodeficient recipients, and you find no evidence

    21    of pathology, whether it's tumor development or some

    22    other kind of illness in those animals.

    23                If you can't prove that transfer of that

    24    information, even though it's all sheared and chopped

    25    up, is going to do anything to the recipient host,

                                                                        214

     1    it's kind of hard to translate the information from

     2    the virulence of the cell, if you will, to the risk of

     3    that cell being used to convey something, if there's

     4    no evidence that it's conveyed and if it's unlikely

     5    that you're going to get the right combination of

     6    genes put together after the sheared DNA is carried

     7    over into the recipient.

     8                I mean, that's where the question is.  How

     9    do you test that?  You can test the first, but does it

    10    have any relevance for the second; that is, the

    11    transfer phenomenon, and what happens in the recipient

    12    -- human vaccine recipient?

    13                DR. FRIED:  Is it worth testing it in the

    14    transgenic models?  I mean, which would be the best

    15    that people who presented on the transgenes?

    16                AUDIENCE PARTICIPANT:  Well, I think the

    17    question that we sort of have to get after is whether

    18    it's worth pursuing and how far or how hard we should

    19    pursue the need to ask the second question.

    20                We can define the first and, in fact, as

    21    you know, the issue of whether cells that have very

    22    low TPD50 values on the order of one to a few hundred

    23    cells compared to cells that have very high TPD50

    24    values of, say, in the order of 105 or 106 -- the

    25    question is whether we should be more worried and

                                                                        215

     1    pursue with some -- oh, I can't think of the word I

     2    want to use right now, but with some eagerness to look

     3    at that question with cells that have that.

     4                Now in order to make that decision, the

     5    first thing you have to have is data that says there

     6    is a cell that this particular substrate of this cell

     7    that's being proposed for this particular substrate

     8    falls into that category of cells that are highly

     9    neoplastic or highly tumorigenic, if you will.

    10                Neoplastic in this situation, I think, is

    11    not quite the right word, because you can't -- I don't

    12    know how you measure neoplastic.  You can measure

    13    tumorigenicity, but the other thing is a more nebulous

    14    term when it's a tissue culture cell.

    15                So if you have a situation in which you

    16    have a very aggressive or a highly tumorigenic cell

    17    line, should we in fact be concerned about pursuing

    18    whether the components of that cell represent more of

    19    a risk, and then we should in fact worry about how to

    20    measure whether those risks are transferable or not,

    21    I think, is the heart of our question.

    22                We can measure the one, but we're not

    23    quite sure we know how to measure the other, and when

    24    you look at the models that are available for that,

    25    there aren't very many of them, especially the tissue

                                                                        216

     1    culture model.        

     2                Now I don't know, with regard to looking

     3    at DNA from cells that are highly aggressive or highly

     4    tumorigenic versus cells that are weakly tumorigenic,

     5    whether those types of studies have actually been

     6    done.  

     7                Dr. Nichols is talking about a lot of

     8    data.  Unfortunately, I'm not familiar with that data. 

     9    So I don't really know whether he's looked at cells

    10    that have very low TPD50 values, comparing it with

    11    cells that have very high TPD50 values in his models

    12    or not.  But I think that's one of the things that we

    13    could think of doing, if we need to get at that kind

    14    of data.

    15                So I think the question to the panel is

    16    whether we in fact need to get at that kind of data,

    17    and if so, you know, is there any collective wisdom

    18    among you all that says how we should go about doing

    19    it?

    20                DR. SISTARE:  Two questions, I think.  One

    21    is, if we're concerned that the cell is going to be

    22    surviving some processing and the cell is going to

    23    remain intact in the vaccine, then we got to approach

    24    it a certain way.  There, the question of this

    25    neoplastic potential in the cell is a very real one.

                                                                        217

     1                If, on the other hand, our concern is not

     2    the cell -- we don't think the cell is going to

     3    survive the processing of the vaccine, and it's not

     4    going to be carried over, and what we're concerned

     5    about is the DNA  itself, that's a different question,

     6    and I don't know that it's been addressed yet.

     7                It strikes me that some of the experiments

     8    that Dr. Doerfler referred to and Dr. Onions referred

     9    to were newborn hamsters, I believe it was, and

    10    newborn young animals.  They were injected or gavaged

    11    or whatever.  

    12                I don't know how the stuff was given, but

    13    they were given DNA in some form, and these animals

    14    came down with tumors, if I understood your statement

    15    correctly.  It wasn't a primary part of your

    16    presentation, but you referred to it, some old data.

    17                In a sense, the newborn mouse model, the

    18    neonatal mouse model that we use for testing

    19    pharmaceuticals is that same model.  It's a single hit

    20    or double hit kind of a model with a long latency, a

    21    lifetime -- you know, just a one exposure, wait a

    22    lifetime and then see if a tumor develops.  That might

    23    be the kind of model to ask the kind of question, 

    24    neoplastic DNA versus primary cell DNA.

    25                Now another thing that Dr. McDougall

                                                                        218

     1    brought up -- and it's another reason why this is a

     2    very interesting experiment -- If you remember

     3    correctly, he indicated that these small pieces of

     4    palindromic DNA seemed to be doing the work, seemed to

     5    be causing the problems, and it was the more mature

     6    cell line the tumor actually lost these pieces of DNA.

     7                So one might guess that the early phase of

     8    the tumor might be the more pathogenic, the DNA from

     9    that, as opposed to the later, the more mature, more

    10    "tumorigenic" DNA. 

    11                So you don't know how the experiment is

    12    going to turn out.  If this palindromic DNA is really

    13    the important stuff, it's going to go in and not

    14    express -- if expression is not the most important

    15    thing, but disruption of normal expression of the cell

    16    is the key thing, then these are all kinds of

    17    questions that could be addressed, and there are

    18    models that we could do this with.

    19                I don't know if the data is out there

    20    already for these kinds of things.  I don't know.  An

    21    we keep talking about efficiency of transfer and

    22    looking for expression.  That's not what we're trying

    23    to do here, I don't think, is ask the question about

    24    necessarily whether oncogenes are going to be

    25    transferred.

                                                                        219

     1                I think the question we have to ask for is

     2    the rare event.  If there is transfer, and we know

     3    it's going to be very efficient, when that transfer

     4    occurs is it going to integrate into a part of the

     5    cellular genome that's critical to the function of

     6    that cell?  And if it's only one cell, and it gives it

     7    some selective advantage, then it's a concern.

     8                So it can be a rare event that we have to

     9    concern ourselves with.

    10                DR. FRIED:  Any other comments?  

    11                AUDIENCE PARTICIPANT:  Given what you all

    12    know as experts on neoplastic cells, suppose you have

    13    a cell which is neoplastic for which you don't know

    14    the mechanism by which it became immortal.  What is

    15    the relative pretest probability as compared with

    16    other types of cells that these cells will contain an

    17    abortitious agent, either infectious or latent, known

    18    or unknown?  

    19                We'd sort of just be interested in, in the

    20    sense of the panel, whether it is reasonable or

    21    unreasonable to be more worried about the possibility

    22    of abortitious agents being in these kinds of

    23    neoplastic cells.

    24                DR.  FRIED:  I guess, if you passage

    25    things more, they may be there, and it depends on what

                                                                        220

     1    species we're looking at.  I mean, I think Harry Rubin

     2    said this morning something about if they came from

     3    chicken, it probably wouldn't infect humans.  Then I

     4    think John Coffin said maybe it would.

     5                So I don't know.  I mean, I guess agents -

     6    - I mean, there's just the normal tests looking for

     7    viruses and nuclease resistant DNA in particles and

     8    PCRing up, as mentioned in the document.  I don't know

     9    if the panel has any other ideas.

    10                AUDIENCE PARTICIPANT:  I was actually

    11    going to address a different issue which was mentioned

    12    earlier and has been mentioned a couple of times so

    13    far, which has to do with the damage due to

    14    integration of DNA in disruption or activation of

    15    genes.      

    16                In the very best models that we have for

    17    that, which is infection of chickens or mice with

    18    certain retroviruses, where very reproducibly every

    19    single infected animal will come down with a tumor, if

    20    you actually look in cases where the tumor is being

    21    caused by insertional activation and not expression of

    22    preexisting oncogenes, if you actually count the

    23    numbers of cells that are infected as compared to the

    24    ones that actually become transformed in this way, the

    25    ratio is probably a million to 10 million to one or

                                                                        221

     1    something like that.

     2                These events are extremely rare at the

     3    cellular level.  The introduction of foreign DNA has

     4    a very, very low probability of doing any harm to a

     5    cell in terms of induction of malignancy.  So we have

     6    to keep that in mind.  We can't think that

     7    introduction of DNA is per se a very damaging event to

     8    a cell.

     9                You have to do it a lot of times.  You got

    10    to do it a lot of times in an infected animal in order

    11    to get a tumor to appear.

    12                DR. FRIED:  You think it's preselecting a

    13    cell already is halfway there?

    14                AUDIENCE PARTICIPANT:  Well, there might

    15    be some of that.  There's certainly a multi-step event

    16    to it, and it's certainly true that only certain cells

    17    in the body are primed for this.  

    18                It may not be because they're abnormal. 

    19    It may just have to do with something about the

    20    differentiation, say, of that particular cell, but you

    21    can infect many cells with avian leukosis virus, for

    22    example, in a young chick, but you will virtually

    23    always get a B cell tumor that arises in the bird.  So

    24    presumably, because there are some particular features

    25    about those cells that prime them, not necessarily

                                                                        222

     1    mutagenically, but in a differentiation specific way

     2    for the kinds of events that can then lead to

     3    oncogenesis.

     4                DR. FRIED:  The same with the P53 mice. 

     5    I mean, they all get tumors by 200 days, but they

     6    don't get tumors on day one or day ten.  Other events

     7    have to happen, and there's also a story of BRCA-1.

     8                There's a woman in Scotland in a little

     9    village where everybody knows everybody, and she was

    10    actually a knockout.  She had had both BRCA-1 genes

    11    knocked out, and she didn't get a breast tumor until

    12    39.  So again, other events have to happen.

    13                AUDIENCE PARTICIPANT:  The rarity of

    14    these, I think, is a very important thing to keep in

    15    mind.

    16                DR. TEVETHIA:  I'd like to get back to the

    17    issue of the degree of tumorigenicity.  I think it's

    18    a very important point for the cell line you're going

    19    to consider making in vaccines.

    20                Seems to me that we seem to be using this

    21    word rather more loosely, and I think the question

    22    really becomes that, if you look at any of the DNA

    23    virus transformed cells, they are transformed, but

    24    they don't metastasize.

    25                Yet you express a gene in transgenic mice

                                                                        223

     1    as metastasizing.  So, obviously, we're looking at a

     2    lot of artificial situations when you transplant a

     3    cell line into a tumor.  The cell may grow rapidly. 

     4    It may look like a tumor.

     5                One really has to define actually what the

     6    tumor looks like, at the blood supply, how soon the

     7    blood supply is established, and other factors that

     8    are produced.

     9                So I think just because the cell line 107

    10    cells end up producing a nodule doesn't necessarily 

    11    mean that is a very high cell line to make vaccine in.

    12                DR. FRIED:  Okay.  Is there anything more?

    13                DR. McDOUGALL:  I'd just like to make one

    14    observation on that observation.  That is that human

    15    papilloma viruses actually do create a tumor that's

    16    capable of metastasizing, and this is an event that's

    17    just initiated by a DNA virus infection.

    18                So it's not -- Although in vitro and the

    19    sort of lab experiments we do it's true that these

    20    cells don't metastasize, in vivo they certainly do.

    21                AUDIENCE PARTICIPANT:  Yes.  I have some

    22    unpublished data on the ability of a lot of these

    23    cells metastasize, and it's, in fact, quite striking.

    24                If you look at SV40 transformed hamster

    25    cells, it's the size of the tumor that depends on

                                                                        224

     1    whether the cells metastasize or not.  They won't --

     2    You can't begin to see lung metastasis in animals with

     3    SV40 transformed cell tumors, hamster cells, until the

     4    tumor is probably 30-40 millimeters in diameter.  Then

     5    the lungs are studded with metastasis.

     6                We found the same thing for BHK 21 cells. 

     7    Now the interesting thing is that adeno-12 transformed

     8    cells, which have the reputation of being highly

     9    tumorigenic -- we've never seen a lung metastasis in

    10    animals, even though the tumor is half the size of the

    11    animal.

    12                So there are differences there.  We've not

    13    looked -- made the same observations in mouse models,

    14    but the idea that DNA virus transformed cells,

    15    especially SV40 and some of these types of viruses,

    16    won't produce metastasis, I think, is based on the

    17    fact that you really haven't looked very hard, and you

    18    have to wait until the animal gets around to

    19    development of lesions.  They're probably microscopic,

    20    and you can't see them.

    21                DR. COOK:  I would just agree.  I think

    22    that the way these experiments are done is usually to

    23    inject a large number of cells that causes tumors in

    24    a fairly short period of time, and it creates an

    25    environment, like Tev was saying, where you develop

                                                                        225

     1    localized tumors that don't look like the metastasize.

     2                If you put in smaller numbers of cells and

     3    wait a very long period of time, like Dr. Lewis'

     4    experiments, we've seen the same thing in the BHK

     5    model in newborn nude rats inoculated with small

     6    numbers of adeno-5 E1A expressing cells that I

     7    wouldn't h ave intuitively thought would make anything

     8    once they sat around in this nude mouse to the time

     9    that they were an adult.  But they can have lungs full

    10    of metastases if you let them sit for a very long

    11    time, and the cells are put in under the cover of

    12    immunodeficiency.

    13                So again, this relative question about how

    14    tumorigenic a cell line is is interesting, but whether

    15    it has anything to do with the risk of that cell, I

    16    think, is another question.

    17                DR. FRIED:  Yes?

    18                AUDIENCE PARTICIPANT:  If I may suggest, 

    19    you still haven't addressed that question.  That's

    20    what we want you to address.

    21                DR. COOK:  Well, I want to go back to your

    22    comment, and that is, if we wanted to do number 2,

    23    which is to ask about the transfer of DNA and how

    24    could that be tested, because we're all talking about

    25    religion here -- I mean, there should be an

                                                                        226

     1    experiment.

     2                It seems like to put together your

     3    question with Mike's suggestion earlier, has anybody

     4    done a simple experiment like take DNA, like polyoma

     5    DNA that's infectious, run it through a vaccine

     6    production schedule of some sort, and find out whether

     7    that DNA which is -- I mean, you need a positive

     8    control for these experiments.

     9                We could all create a lot of experiments

    10    that would result in no tumors and say that the

    11    residual DNA in this vaccine prep didn't do anything

    12    bad.  What about some DNA that we know can cause

    13    neoplasia, run it through a vaccine preparation, put

    14    it into a animal that's known to be sensitive?

    15                If those animals don't get tumors when

    16    inoculated with spiked preparations that have been

    17    processed, then I think the idea of working backwards

    18    from a positive control to what's likely to be a

    19    negative experiment might be more logical.

    20                AUDIENCE PARTICIPANT:  Well, I think that

    21    the question -- One of the questions that you're

    22    asking is, in fact, something that we thought about a

    23    little bit.  That is the possibility of using -- to

    24    get around this whole issue of the problem of DNA is

    25    to use the same concept that was applied to the

                                                                        227

     1    business of viral clearance.

     2                That is the concept of DNA clearance.  In

     3    other words, you would spike DNA with DNA containing

     4    defined or known viral oncogenes, if you will, and

     5    then show that the manufacturing process actually

     6    clears that DNA to a certain -- with a certain

     7    efficiency, and then using those numbers, you can then

     8    make a prediction as to what else would be removed

     9    during the same procedure.

    10                So that's one way that you can think about

    11    possibly getting around this business of testing.  The

    12    other thing that comes to mind when you're thinking

    13    about risks associated with elements that are

    14    components of vaccines is the fact, if you -- and

    15    we're talking about tumorigenesis -- but the fact, if

    16    you worry about what is in the tumor cell and the

    17    possibility that the tumor cell might contain an

    18    adventitious agent or, in face, is induced by an

    19    adventitious agent -- and I think one of your

    20    examples, that's a ras sarcoma cell -- you infect ras

    21    sarcoma cells in chickens, and the chickens get ras

    22    sarcomas, but they also get ras sarcoma virus.

    23                So the fact that a cell line makes a tumor

    24    could be obscuring -- The tumor itself could be caused

    25    by some sort of oncogenic agents, especially if you're

                                                                        228

     1    dealing with an unknown cell line.

     2                The tumor could be obscuring the presence

     3    of an oncogenic agent.  The oncogenic agent could be

     4    there whether you get a tumor or not, simply because

     5    the animal model you're using is not sensitive to

     6    that.  

     7                So if you wanted to go out and begin to

     8    poke at the issue of how to assess the transfer of a

     9    neoplastic event or some type of infectious event,

    10    then you need to compare the tumorigenicity of the

    11    cell line with the possible tumorigenicity of the X

    12    rac and things like that.

    13                So there are models that -- things that

    14    you need to do, but I think the question is how can

    15    you do those quantitatively and at what point in time

    16    do you need to begin to define where you go in that

    17    direction; because this is not a small amount of work.

    18                DR. FRIED:  Okay, thank you very much.  I

    19    want to thank everybody here.  I think we've overrun

    20    our break.  I feel I should summarize, but maybe we'll

    21    leave it to the last session.

    22                I think we're much more amenable that DNA

    23    is not as big a hazard as we might have thought in the

    24    beginning.

    25                (Whereupon, the foregoing matter went off

                                                                        229

     1    the record at 3:00 p.m. and went back on the record at

     2    3:19 p.m.)

     3                DR. ROSENBERG:  If everyone could take

     4    their seats, we would like to get started.  

     5                We're going to be shifting gears only

     6    slightly, I think, in this afternoon's session, and

     7    then continuing in the same vein tomorrow morning to

     8    a discussion that will highlight for you issues that

     9    relate to retroviruses and ways in which retroviruses

    10    are able to alter particularly the growth of cells and

    11    ways in which retroviruses can change during their

    12    replication in cells.

    13                I want to very briefly introduce this by

    14    showing a couple of slides, and these will just

    15    highlight for you -- I think if we could have the

    16    lights down, please -- some of the aspects that will

    17    be coming up in the talks this afternoon and this

    18    morning.

    19                Certainly, you're all aware that

    20    retroviruses, unlike some of the agents that have been

    21    discussed already, have as a natural part of their

    22    life cycle the ability or an obligate need to

    23    integrate into the DNA of the host.  As a consequence

    24    of this, we appreciate that there are a number of

    25    things that can occur.

                                                                        230

     1                These are illustrated here.  Here we see

     2    an insertion and an activation of an oncogene, leading

     3    eventually to the development of a mammary tumor in

     4    this mouse.

     5                Other things can happen as well.  While we

     6    may be focusing on these events, I think it's

     7    important to remember, these are not the only

     8    consequences.  Two other inactivations, one an

     9    activation rather seen here, results in the hairless

    10    phenotype in this mouse, and an insertion here results

    11    in up regulation of amylase, which has been suggested

    12    at least by some, including the people who made this

    13    figure, perhaps to explain our great love for

    14    starches, including some that were just consumed in

    15    the hall.  

    16                So there's a diverse set of things that

    17    can happen when retroviruses integrate.  To illustrate

    18    this in perhaps a more mechanistic fashion, because of

    19    the cleverness, or at least to me the cleverness of

    20    these agents, a number of ways that the integrated

    21    virus, shown here, can impact on genes nearby, it's

    22    illustrated.

    23                Here we have a circumstance where we have

    24    expression of the neighboring gene under the influence

    25    of promoters here.  

                                                                        231

     1                Here we have a circumstance where strong

     2    enhancers contained within viral control elements are

     3    again affecting the expression of a neighboring gene,

     4    and we certainly appreciate, although the slide

     5    illustrates that this gene looks very close, that this

     6    can occur over reasonably long distance.

     7                Here we have another circumstance where

     8    viral control elements identical to these but located

     9    at a different position in the viral genome again are

    10    affecting expression of the gene.

    11                So these insertions don't have to be

    12    completely specific with respect to cellular genes in

    13    order to impact upon them.  In addition, in

    14    circumstances where replication occurs, not only can

    15    there be effects on gene expression.  Here we have the

    16    integrated provirus, just as in the earlier slide, and

    17    a cellular gene.

    18                In this case we have transcription, as I

    19    showed before, mediated from the virus into the

    20    cellular gene, creating a hybrid viral cellular

    21    transcript, and subsequent, although all the steps are

    22    not diagramed here, packaging of this transcript into

    23    a virion, subsequent replication, and in this case

    24    incorporation of part of the cellular sequence into

    25    the virus, then having the capacity to be transmitted

                                                                        232

     1    to cells subsequently if this structure is contained

     2    within a cell with a replicating virus.

     3                 Clearly, this is a very low frequency

     4    event, but not one that occurs at such a low frequency

     5    that we haven't been presented with enough examples of

     6    these types of viruses to employ many retrovirologists

     7    over very long periods of time.

     8                Finally, another event that we will hear

     9    about today involves the ability of retroviruses to

    10    recombine, and this slide illustrates recombinations

    11    that go on in the generation of a particular set of

    12    pathogenic murine leukemia viruses.  However, this

    13    slide could apply to other situations with other types

    14    of retroviruses as well.

    15                Here we have acquisition of sequences in

    16    this region from endogenous viruses, and -- hopefully

    17    the colors show up, although I can't see them from

    18    here -- changes also affecting the LTRs of this virus. 

    19                As a consequence of these, the virus

    20    tropism changes, and confers to this virus a highly

    21    tumorigenic phenotype.  Other kinds of recombinations

    22    can occur as well and, again, alter both host range

    23    and tissue tropism.

    24                This is an extremely important way in

    25    which viruses can infect new hosts or new cells, again

                                                                        233

     1    leading to diverse consequences.

     2                So I think, as we discuss all of these and

     3    present model systems in which these are well

     4    characterize, we'll hopefully set the stage for the

     5    discussion that will follow these retroviral sessions.

     6                With nothing further, I'd like to turn

     7    this over to Sandy Ruscetti, who will present the

     8    first talk. 

     9                DR. RUSCETTI:   I'm going to move down

    10    here, because I understand you can't see your slides

    11    otherwise.

    12                Thank you, Naomi.  As Naomi mentioned,

    13    when one uses neoplastic cells as substrates for

    14    vaccine development, one can inadvertently get viral-

    15    viral or viral-cellular interactions that could have

    16    unknown biological consequences.  

    17                So the organizers here asked me today to

    18    talk about a model system in the mouse, and that's the

    19    generation of MCF retroviruses as a model for

    20    understanding how viral-viral and viral-cellular

    21    interactions can result in the generation of new

    22    viruses that can have pathological consequences.  

    23                Now mouse cells are just loaded with

    24    endogenous retroviruses of various subtypes.  Some

    25    strains have ecotropic MuLVs in small copy numbers

                                                                        234

     1    and, when these are expressed, they are involved in

     2    the development of leukemia in these mice.  

     3                The AKR mouse is an example of such a

     4    mouse, but all strains of mice contain many copies of

     5    xenotropic, polytropic and modified polytropic MuLVs. 

     6    Now these viruses really don't cause any problems to

     7    the mouse or the mouse cells, but when they are

     8    expressed with ecotropic virus or if ecotropic virus

     9    is injected into the mice, recombination can occur

    10    with these endogenous sequences, particularly the

    11    polytropic sequences, and this can result in the

    12    development of a pathogenic virus.

    13                These viruses are called MCF viruses, and

    14    what I'm going to do today is to give you a background

    15    about MCF viruses in the mice, how they're generated

    16    and what their characteristics are, and give you a

    17    little food for thought about whether we should worry

    18    about these in using mouse cells as substrates for

    19    vaccine development.

    20                Now these are just some properties about

    21    MCF viruses.  MCF viruses are recombinant viruses

    22    between an ecotropic virus and endogenous sequences

    23    present in mouse DNA.

    24                These viruses contain ons genes.  So they

    25    are derived from polytropic or modified polytropic

                                                                        235

     1    MuLV sequences, and they contain oftentimes LTR

     2    sequences that are also derived from the endogenous 

     3    sequences in the mouse or certainly modified so that

     4    they can be expressed at high levels in certain

     5    tissues.

     6                Now one of the properties of MCF viruses

     7    that gave it its name is that they can induce unique

     8    changes, and they were called foci, but not

     9    transformed foci, in mink lung fibroblasts.  But other

    10    studies have shown that not all MCF viruses have this

    11    property.

    12                One of the interesting things about MCF

    13    viruses, and another reason why we're interested in

    14    them today, is that compared with ecotropic MuLVs,

    15    these viruses have an extended host range which allows

    16    them to replicate both in mouse cells and non-mouse

    17    cells.  This is because MCF viruses use a different

    18    receptor than ecotropic viruses to enter cells.  

    19                Now when one looks at the host range of

    20    MCF viruses compared to other classes of murine

    21    leukemia viruses, one can see that it has a host range

    22    that's a combination of them.  Ecotropic MuLVs are

    23    mouse-tropic.  They can only infect mouse cells and

    24    not non-mouse cells.  

    25                Xenotropic MuLVs cannot infect mouse

                                                                        236

     1    cells, but they are very efficient in infecting non-

     2    mouse cells, including human cells.  Polytropic or MCF

     3    viruses can infect both mouse and non-mouse cells.

     4                Now it's still unclear how infectious MCF

     5    viruses are for human cells, and I thought it was

     6    worth bringing that up at this conference.  Working 

     7    with the prototype MCF virus, AKR MCF 247, some

     8    studies have shown that certain human cell lines can

     9    be efficiently infected with this virus.  

    10                Other studies show that they can't, and I

    11    think, importantly, four different primary human cell

    12    cultures could not be infected with AKR MCF virus.  So

    13    it's unclear just how infectious MCF viruses are for

    14    human cells, and they are certainly less infectious

    15    than the xenotropic or even amphatropic MuLVs.

    16                The prototype model for the generation of

    17    MCF viruses is the AKR mouse.  Now these mice develop

    18    thymic lymphomas at a high frequency when they are

    19    about six to 12 months of age.  

    20                It was shown early on that these mice

    21    contain in their genome an endogenous ecotropic MuLV. 

    22    It's called AKV or several copies of AKV, from which

    23    infectious ecotropic replicating MuLV is produced at

    24    birth.

    25                Now it's always been a mystery why these

                                                                        237

     1    animals who produce lots of this virus do not get

     2    leukemia for six to 12 months, especially since early

     3    studies indicated that AKV was certainly important for

     4    the generation of this leukemia; because you could

     5    take AKV and express it in a low leukemic strain, and 

     6    they would become a high leukemic strain.  

     7                Well, in 1977 Jan Hartley discovered a new

     8    virus from these tissues of these diseased mice that

     9    provided an answer.  What she and her colleagues were

    10    able to do were isolate this new class of MCF viruses

    11    from the pre-leukemic and the leukemic thymuses. 

    12                Further studies showed that the MCF

    13    viruses were recombinants between the AKV and

    14    endogenous MuLV sequences, and replication of both the

    15    AKV ecotropic and the MCF viruses in these thymuses

    16    were what were responsible for the development of the

    17    leukemia that occurred when the animals were six to 12

    18    months of age.

    19                Now how are these MCF viruses generated? 

    20    Studies have shown that there are three endogenous

    21    MuLVs that are involved in the generation of AKR MCF

    22    viruses:  AKV, which is the ecotropic virus I just

    23    mentioned; VXV1, which is an endogenous xenotropic

    24    MuLV; and various copies of polytropic and modified

    25    polytropic MuLV sequences.

                                                                        238

     1                The generation of MCF virus in the AKR

     2    mouse is thought to involve several different steps. 

     3    First of all, the AKV is thought to recombine with the

     4    om genes of one of these endogenous polytropic MuLVs,

     5    generating a new virus which looks basically like AKV,

     6    but it has new om gene sequences that are derived from

     7    the polytropic virus.

     8                Then the second even occurs.  This

     9    recombinant recombines with the endogenous xenotropic

    10    virus to generate a new LTR sequence.

    11                Now it's not clear which of these events

    12    occur first, but both of them apparently need to

    13    occur.  Finally, alterations appear to occur in the

    14    LTR sequences that the viruses acquired from the

    15    xenotropic MuLV, which often involve the duplication

    16    of the enhancer sequence, obviously making this now a

    17    pathogenic virus in the AKR mouse.

    18                Now the AKR mouse is not the only mouse in

    19    which MCF viruses are generated.  You can generate MCF

    20    viruses in low leukemic strains of mice if you inject

    21    them with ecotropic MuLV.  Two good examples of that

    22    are Frend and Moloney MuLV induced leukemia.

    23                If you take newborn mice and inject them

    24    with Frend MuLV -- this is an ecotropic MuLV -- you

    25    get the generation of MCF viruses, and these animals

                                                                        239

     1    develop erythroleukemia several weeks later.

     2                If you take Moloney MuLV and inject those

     3    into newborn mice, you also get the generation of MCF

     4    viruses, but these animals now develop thymic

     5    lymphoma.  A lot of studies have been done with these

     6    two systems.  

     7                It's a little bit easier to do studies

     8    with the input virus, because one can inject that into

     9    the mice when it's not just relying on AKV sequences

    10    in the AKR mouse, and it was shown -- several things

    11    that I think are important.

    12                The type of disease induced is determined

    13    by the input LTR.  So if you have  a Frend MuLV LTR,

    14    you'll get erythroleukemia.  If you have a Moloney,

    15    you'll get thymic lymphoma.  This was determined by

    16    making chimeras between the two viruses.

    17                It's thought that perhaps the MuLV --

    18    Frend MuLV LTR may replicate better in erythroid

    19    target cells, and the Moloney in T-cells, and that may

    20    be why you get that particular type of tissue tropism. 

    21    But also, too -- Actually, Leonard Evans who is here

    22    in the audience -- he showed that Frend and Moloney

    23    ecotropic viruses actually recombine with different

    24    endogenous polytropic om gene sequences to generate

    25    MCF viruses.

                                                                        240

     1                So the particular sequences that they

     2    recombine with may determine the type of disease

     3    that's induced.

     4                Another thing:  Adult mice are resistant

     5    to leukemia development by injecting these viruses. 

     6    You need to inject newborn mice, and it's thought that

     7    the reason you need to do this is because adult mice

     8    may either have fewer target cells for the virus or

     9    may have an efficient immune response that allows you

    10    to clear either the input virus or the generated MCF

    11    virus.

    12                We showed a number of years ago with Frend

    13    MCF that you could treat adult mice to increase the

    14    target cells or decrease the immune response, and they

    15    would be susceptible as adults.

    16                Finally, there are strains of mice that

    17    are resistant to the development of leukemia induced

    18    by these MuLVs, even if you inject them into newborn

    19    mice, and these strains of mice do not replicate MCF

    20    viruses; because they contain genes that suggest they

    21    are MCF genes which prevent the replication of the MCF

    22    viruses.

    23                This makes the case even stronger that

    24    efficient generation and replication of MCF viruses in

    25    these mice are responsible and are necessary for the

                                                                        241

     1    development of the leukemias that follow.  

     2                Now the generation of MCF viruses after

     3    injection of Frend or Moloney is a little less

     4    complicated than in AKR mice.  Just showing this same

     5    slide with AKR, with this being the ecotropic MuLVs.

     6                The ecotropic MuLV does recombine with

     7    endogenous polytropic MuLV sequences to generate a new

     8    om gene.  Again, particular polytropic sequences they

     9    recombine with are different, but usually the viruses

    10    do not need to acquire a new LTR.

    11                The ecotropic Frend and Moloney LTR seems

    12    to work fairly well to allow this virus to replicate

    13    in the target cell.  Now sometimes there have been

    14    observed some changes in the enhancer region of the

    15    LTR, and these may be to allow the particular MCF to

    16    replicate to high levels in the target tissue.

    17                One point I wanted to make was that both

    18    in AKR mice and in MuLV infected mice, one may

    19    generate lots of different types of MCF viruses, and

    20    maybe only a certain subset of those MCF viruses are

    21    actually pathogenic, ones that have acquired all the

    22    right changes to cause disease.

    23                Now these studies have all been done where

    24    MCF viruses are generated in situ in the mouth.  Now

    25    what happens if you take the MCF virus from the

                                                                        242

     1    diseased tissue, purify it away from the ecotropic

     2    virus, and inject it back into mice?  Nothing really

     3    happens.

     4                That's because MCF viruses need to be

     5    pseudotyped with ecotropic MuLV in order to cause

     6    disease when you take them out and try to put them

     7    back in.  For example, AKR MCF virus will accelerate

     8    leukemia in AKR mice, but it will not cause leukemia

     9    in the low leukemic NIH mouse unless these mice

    10    express an AKV ecotropic virus as a transgene.

    11                Okay.  Frend MCF virus will not cause

    12    leukemia in mice unless it's pseudotyped with

    13    ecotropic MuLV.  Also I put this in here, because we

    14    had showed a number of years ago that, if we infected

    15    a packaging cell line inside two lines with Frend MCF

    16    virus, we could get a virus preparation that would

    17    cause leukemia, erythroleukemia in NIH Swiss mice.

    18                So this suggested that the ecotropic MuLV

    19    may not have to replicate in the mouse once it gets

    20    the MCF there, and we actually showed that we did not

    21    regenerate an infectious ecotropic MuLV from these

    22    studies.

    23                Now it's thought that pseudotyping may

    24    prevent the inactivation of MCF viruses by maybe serum

    25    factors complement or it may allow the virus to bypass

                                                                        243

     1    an effective immune response that might be against the

     2    MCF virus.

     3                So how to MCF viruses cause leukemia? 

     4    It's not completely clear, but there have been several

     5    hypotheses, and it may be actually a combination of

     6    several of these.

     7                It's possible that the MCF viral envelope

     8    proteins may interact with cytokine receptors on

     9    hematopoietic cells and alter their growth and

    10    differentiation.  Also integration of MCF viral genome

    11    into the host DNA may lead to activation of certain

    12    proto oncogenes whose inappropriate expression in

    13    hematopoietic cells may also lead to the alterations

    14    in their growth and differentiation.

    15                Also, since MCF viruses use a different

    16    receptor than ecotropic viruses, and since both

    17    viruses are present in the diseased animal, that would

    18    allow two MuLVs to get into the same cell and increase

    19    the changes of an activation event due to viral

    20    integration.

    21                So the data with studies of MCF viruses in

    22    mice indicates that you can generate in mice

    23    recombinant MCF viruses, and that these are really

    24    crucial for the development of leukemia in these mice. 

    25                But the question that's more relevant for

                                                                        244

     1    this  conference is can these viruses be generated in

     2    vitro, and if they can, is that a problem when using

     3    mouse cells as substrates for the development of

     4    vaccines?

     5                Also, even if these could be generated,

     6    could they cause a biological effect if they could

     7    actually replicate in humans?  I think this will be

     8    perhaps addressed by some of the other talks in this

     9    session, and I think certainly will be a topic of

    10    discussion when we have our panel tomorrow, late

    11    tomorrow morning.

    12                So if you have any questions in general

    13    about MCF viruses and how they are generated and how

    14    they are used as a model for viral-viral interaction,

    15    I'll be happy to answer those for you.

    16                (APPLAUSE.)

    17                DR. EVANS:  I'm Leonard Evans from Rocky

    18    Mountain Labs.

    19                I wanted to clarify about the

    20    recombination with distinct sequences.  Those studies

    21    were done where the viruses were initially selected on

    22    mink cells.

    23                We found subsequently that the viruses --

    24    many more viruses that could be found on mouse cells

    25    than on mink cells, largely because they were

                                                                        245

     1    serotyped.  When we analyzed the larger population of

     2    viruses, we found there was a significant overlap. 

     3    Frend does not specifically recombine with something

     4    different than Moloney.

     5                There are differences in the preponderance

     6    of the viruses, but not in the identify of those

     7    viruses.

     8                The second point I wanted to make is that

     9    we can inject Frend MCF into NFS mice, and they do

    10    develop erythroleukemia.  

    11                DR. RUSCETTI:  In  the absence of eco?

    12                DR. EVANS:  Yes.  Markedly cloned and

    13    transfected.

    14                DR. RUSCETTI:  Well, I know one of the

    15    questions that always arises:  When you purify an MCF

    16    virus out of a diseased tissue, are you purifying the

    17    pathogenic one or not?  

    18                Possibly, some of the reasons why you

    19    might not be able to induce disease with a given MCF

    20    virus is because you haven't actually cloned the

    21    pathogenic MCF virus.  But I know our studies with our

    22    MCF virus, we were never able by itself to, but I

    23    agree, that doesn't mean that you always have to have

    24    that.  That's generally a problem, though, with MCF

    25    viruses replicating once you take them out of the

                                                                        246

     1    animal.

     2                DR. EVANS:  Yes.  It's also been shown

     3    that there's a tremendous range of infectivities for

     4    different heterologous cell lines like mink rhesus

     5    mouse.  It can be vary over several orders of

     6    magnitude in terms of the ratio, the relative

     7    infectivity, and there's such a heterogeneity of

     8    polytropic viruses that to generalize that they do not

     9    infect human cells is probably a leap, you know.

    10                They don't readily affect them.  Maybe

    11    someone --

    12                DR. RUSCETTI:  All right.  Now I wasn't

    13    trying to say that.

    14                DR. EVANS:  No, no, I know.

    15                DR. RUSCETTI:  I just think that people

    16    might have an impression that they are very infectious

    17    for human cells, and that may not be the case.

    18                DR. EVANS:  Some of them might be.

    19                DR. LINIAL:  Maxine Linial, Hutchinson

    20    Cancer Center.

    21                So there are a whole bunch of other

    22    endogenous retro elements in mouse cells like DL-30s

    23    which, at least in one defective virus, have been

    24    shown to be recombined, and there are mouse endogenous

    25    viruses.d

                                                                        247

     1                So has anyone looked in replication

     2    competent MCFs, maybe not the ones that grow out but

     3    in the pool, to see if they have acquired other

     4    endogenous retroviral sequences?

     5                DR. RUSCETTI:  Not that I'm aware of.  I

     6    don't know.  Somebody else might be able to address

     7    that, because I'm really not working in that field.

     8                DR. ONIONS:  David Onions.  Hi, Sandra.

     9                I didn't quite understand your last point

    10    when you were suggesting that the -- the possibility

    11    that the pseudotyping with ecotropic envelope was

    12    necessary to evade the immune response or perhaps to

    13    avoid a complement factor.

    14                I would assume, as in other pseudotyping

    15    situations, the envelopes are chimeric with both

    16    ecotropic and MCF type sequences.  So wouldn't that

    17    make them susceptible to complement, and wouldn't it

    18    also make them susceptible to the immune response?

    19                DR. RUSCETTI:  Oh, no.  It would just be

    20    the MCF genome that's packaged in an ecotropic coat

    21    would be able to get past those inactivating factors

    22    in order to infect the cells that it needed to infect

    23    to cause disease.

    24                DR. ONIONS:  So you think that

    25    pseudotyping is absolute, as in there's a complete

                                                                        248

     1    ecotropic coat there?

     2                DR. RUSCETTI:  Right.  Well, as Pug

     3    mentioned, that may not be absolute, but certainly it

     4    appears to be the common event that occurs, that you

     5    do get pseudotyping, but it's actually -- It's a

     6    complete ecotropic coat.  It's not a chimera between

     7    the eco and the MCF.

     8                DR. ROSENBERG:  Thank you.  The next talk

     9    will be by Clive Patience on MLV packaging systems.

    10                DR. PATIENCE:  Okay.  Firstly,  I'd like

    11    to thank the organizers for inviting me to be here to

    12    present some data.  If I could have the first slide

    13    and the lights down a bit, it would be great.

    14                The work I'll be presenting was primarily

    15    performed in Robin Weiss's laboratory in cancer

    16    research in London.  However, recently -- it does not

    17    account for my jetlag, which I don't have -- I've

    18    moved to BioTransplant, a xenotransplantation company

    19    in Boston.

    20                Now when I was invited I made the

    21    assumption that I'd be talking to a diverse audience,

    22    ranging from those which know very little about

    23    retroviruses up to those which are astral heights of

    24    editors of retroviral textbooks that should remain

    25    nameless.  

                                                                        249

     1                So what I'll try and do is give you a

     2    story which I hope everyone will be able to follow,

     3    and it's going to deal with the murine leukemia based

     4    virus packaging systems, gene therapy models, and

     5    basically estimation of potential recombination.

     6                Okay.  For the uninitiated, this is the

     7    basic system, which I shall try and get in focus for

     8    you somewhat.  I think that's as good as we're going

     9    to do.  Basically, there's three components.  

    10                You have the packaging cell line at the

    11    top here.  The function of the packaging cell line is

    12    to create the virus.  There's generally three

    13    components, the gag and pol expression plasmid which

    14    make the body of the virus, and the envelope which

    15    makes the outer covering of the virus which determines

    16    what sort of cells you will ultimately infect.

    17                Into that virus, rather than packaging the

    18    normal virus genome, you put in your therapeutic

    19    vector.  You make your virus, and that virus then is

    20    used to infect your target recipient cells.  Where the

    21    RNAs converts to DNA, integrate it and your gene is

    22    expressed.

    23                A little bit more detail on  the actual

    24    structure of a packaging cell line:  Basically, we use

    25    the immortalized cell line HT1080 for the particular

                                                                        250

     1    cell line I'm going to describe.

     2                We compared this human cell line in

     3    comparison to a 3T3 based murine based system.  It's

     4    called FLY.  It's got nothing to do with little

     5    insects.  It's actually the initials of the

     6    discoverers, or I should say the inventors.

     7                Okay.  Firstly, you put in your gag pol

     8    expression plasmid, which basically produced the core

     9    of the virus.  You then cotransfect in your envelope

    10    plasmid.  So you can then make a complete virus

    11    particle with all the machinery required for

    12    retroviral integration.

    13                You then insert your vector of choice, and

    14    basically that can be a therapeutic vector or in the

    15    laboratory a reporter vector.  The particular plasmids

    16    which I'm going to discuss here, you can see, is

    17    diverged somewhat away from its wild type parent. 

    18                It has a Moloney murine leukemic virus, 5

    19    prime LTR, as well as Moloney gag and pol.  It then

    20    has basically a gap through here after the stop code

    21    on drug selection.  So basically you get a read-

    22    through here.

    23                About five percent of ribosomes will read

    24    through this stop code and go on to transcribe your

    25    drug resistance gene.  So if you select the drug

                                                                        251

     1    resistance, you should, and indeed you do, select for

     2    very high expressors of the gag/pol proteins and,

     3    therefore, high levels of virus.

     4                Top down then we have a polydentylation

     5    signal which I believe was SB40, if I remember right. 

     6    The envelope comes out very similar, 5 prime

     7    retroviral LTR, no packaging signal.  This region here

     8    determines whether the RNA construct will actually be

     9    packaged into virus particles.  The envelope is

    10    expressed up here, and again SV40, and your reporter

    11    vector -- the important part here is the presence of

    12    a packaging signal.  So it should be efficiently

    13    packaged into virus particles.

    14                Okay.  We were trying to get some sort of

    15    feeling for the degree of recombination that could

    16    occur, and our logic was that it would be greatly

    17    increased if there was any generation of replication

    18    competent retroviruses in the packaging system.

    19                Now the recombination could either come

    20    from the -- The viruses -- sorry -- could either come

    21    from a combination of DNA present in the packaging

    22    cell lines, and you may have noticed that all of the

    23    plasmid constructs have quite a number of regions

    24    deleted or mutated.  Very elaborate cell lines have

    25    been produced now over many years.

                                                                        252

     1                The alternative is that the recombination

     2    events could actually occur during reverse

     3    transcription of the RNA in the virus particle.  For

     4    that to occur you need expression of the unwanted RNAs

     5    in the packaging cell line, and that in turn will be

     6    enhanced.  This is significant similarity between the

     7    vector and the endogenous sequences.

     8                Some bad news to you people.  About one

     9    percent of your genome is retroviral.  Yes, we're

    10    derived from apes, but we're also derived from

    11    retroviruses.

    12                Basically, so about one percent of your

    13    genome is retroviral.  It probably represents the

    14    ancient remains of past retrovirus infections where

    15    germ line cells have become infected.  Over the

    16    millions of years, most of these copies of the various

    17    viruses, which range number from one up to thousand,

    18    have become deleted and mutated.  So they can no

    19    longer encode for infectious virus or, in most cases,

    20    even proteins.

    21                Of particular interest or those which have

    22    received particular attention are the HERV-H or RTVL-H

    23    family of viruses.  There's approximately a thousand

    24    copies of these in our genome, about 200 full length,

    25    about 800 deleted versions, very highly transcribed in

                                                                        253

     1    general terms.

     2                HERB-K, another family, has been the focus

     3    of a lot of attention, primarily because it has pretty

     4    well conserved open reading frames.  It still has the

     5    ability to encode envelope proteins, gag proteins, pol

     6    proteins, and there's been a lot of effort, people

     7    trying to grow this guy and correlate it with disease,

     8    etcetera, with not too much success compared to the

     9    amount of effort which has been put in.

    10                So this is the basic scenario we were

    11    looking at.  We were looking at the packaging cell

    12    line, which was human.  Normal situation is that you

    13    would like for your therapeutic transcript to be

    14    packaged into the virus.  We were looking to try and

    15    determine whether the human endogenous retroviral

    16    transcripts could also be co-packaged and get into

    17    your virus particle.

    18                So the first scenario or the first

    19    requirement is that you have HERV expression in your

    20    cell lines.  Now we compared two cell lines, the old

    21    and inverted comers M-12 and murine 3T3 based system

    22    and the human based system which we've developed in

    23    the laboratory.

    24                This is basically an RT PCR.  For those of

    25    you who are not familiar with PCR, basically a bright

                                                                        254

     1    band means a positive.  A lack of band is a negative. 

     2    Therapeutic --  so that what we were looking at was

     3    actually reported back to LacZ, and you can see there

     4    it's expressed in both the human and the murine cell

     5    line.

     6                We performed a degenerate PCR approach,

     7    basically using PCR primers designed to conserve

     8    regions in the virus and try and pull out as much as

     9    we possibly could and identify as many different

    10    sequences as we could.

    11                In the human cell line we managed to

    12    identify sequences which showed similarity to the B

    13    and B-type retroviruses as well as the RTDL-H family,

    14    which I have mentioned earlier.  

    15                In the murine cell line we again managed

    16    to pull out the D type sequences and also another

    17    retroviral sequences called BL-30 which is an

    18    endogenous retroelement of mice which has been shown

    19    to be packaged in murine cell lines,  not by myself

    20    but by Damian Purcell and others maybe -- and others.

    21                We basic scenario we then went to do was

    22    we had confirmed RNA expression in the cells.  We then

    23    wanted to try and identify whether this got as far as

    24    the virus particles.  And if it gets into particles,

    25    it may be available for recombination events.

                                                                        255

     1                So we took these from the various

     2    packaging cell lines and spun it through a sucrose

     3    density gradient to purify and concentrate the

     4    particles with respect to their density.  What we

     5    could then do was analyze the various fractions where

     6    I've put up the enhanced RT assays or PERT assays to

     7    identify the fractions with the highest virus content,

     8    and then also examine exactly those fractions for the

     9    presence of unwanted retroviral transcripts.

    10                This is a typical section across a

    11    gradient where you see banding of virus particles at

    12    a density appropriate for mature retroviruses.

    13                Okay.  If we look at the old system, to

    14    start off with, the RT assay here identifies a

    15    concentration of particles, at one point 174

    16    approximately grams per mil, which is appropriate for

    17    virus particles.  We see, as expected, the reporter

    18    transcript as it possesses a packaging signal co-

    19    purifying or co-concentrating in the same fraction.

    20                What we also see is significant levels of

    21    the endogenous retroviral element packaging in the

    22    particles.  

    23                When we looked at the human system, again

    24    we get a RT fraction, positive fraction, here with

    25    high levels of beta galactoside as reporter vector. 

                                                                        256

     1    However, in comparison to the murine system we don't

     2    detect any packaging of endogenous sequences, RTVL-H

     3    family, BLD type sequences or use in pan-retroviral

     4    sequences.

     5                We do see a faint band or found a faint

     6    band when we were looking with C-type primers, and

     7    I'll come back to the identity of what this product

     8    was a little bit later.

     9                So this phase basically identified RNA

    10    expression in the cells, and we had in some cases

    11    shown that some of the endogenous retroviral sequences

    12    could be packaged in particles.

    13                The next thing we really had to do was try

    14    and put the numbers on this.  Really, a plus and a

    15    minus wasn't very informative.  So the methodology

    16    which we undertook was some in vitro transcription

    17    whereby we cloned some of the PCR products' retroviral

    18    sequences, cleaved before your 5 prime end,

    19    effectively your ATG, if it were there, and

    20    synthesized RNA from the transcripts.

    21                We could then use this RNA to actually

    22    determine accurately the sensitivity of our RT PCRs,

    23    and that would allow us to eventually estimate the

    24    concentration of RNA in the virus particles.  

    25                Shown in this slide here is the

                                                                        257

     1    sensitivities that we found for our different PCRs,

     2    and it was really -- We were glad we did this, because

     3    we got a huge range of sensitivities down from

     4    approximately 1 femptogram reaction way up to in the

     5    500+ femptograms a quad in the RT PCR to get a

     6    positive signal.

     7                For the reporter vector, which was the

     8    least sensitive, it didn't appear to be due to

     9    primers.  We varied primers quite considerably and

    10    didn't really see any improvement in the

    11    sensitivities.  It was probably due to the actual

    12    sequence that you're trying to amplify any reactions.

    13                So we then basically determined the

    14    sensitivities of the various PCRs, whether it be

    15    through reporter or for the different families of

    16    endogenous sequences.

    17                What we then went on to do was to perform

    18    dilutions of the peak fractions.  Basically, once we

    19    had the sensitivities, we could combine these with the

    20    dilution series of the fractions to determine the RNA

    21    concentration.

    22                If we look at the murine system at the top

    23    here, the beta galactosidase, twofold dilutions,

    24    looked to be in very similar concentration to the VL-

    25    30 endogenous retroviral elements.

                                                                        258

     1                In the human based system we saw

     2    significantly more beta galactosidase present in the

     3    fraction, which is reflected in a higher titer of

     4    virus, and relatively little C-type product.

     5                So basically, that allowed us to estimate

     6    the ratio of the desired therapeutic or recorder

     7    transcript in comparison to the unwanted endogenous

     8    retroviral transcripts in the cells. 

     9                The peak fraction of the murine based

    10    system contained 6.3 times 103 femptograms and nearly

    11    900 femptograms of the endogenous retroviral sequence. 

    12    If you do the mathematics, convert these to

    13    molarities, that meant, basically speaking, for every

    14    seven virus particles which contain the LacZ genome,

    15    which you want, you have one virus particle which

    16    contains the endogenous retroviral sequence.

    17                Obviously, that means for every

    18    integration, every cell that you treat, one in seven

    19    is going to be the wrong molecule being integrated

    20    into the genome.  In fact, it's slightly more

    21    complicated as the retro-particle actually carries two

    22    copies of viral RNA, but the mathematics is still

    23    basically the same.

    24                When we looked at the human FLY packaging

    25    system, you see we saw much, as I had mentioned

                                                                        259

     1    earlier, higher level of reporter vector and

     2    basically, looking at the endogenous retroviral

     3    sequences, these were much cleaner than the seven to

     4    one which we saw with the murine system.

     5                For the C type product which we saw, we

     6    had calculated that about one in 19,000 virus

     7    particles would contain this retroviral sequence.  We

     8    went on to identify what this was.

     9                We cloned and sequenced it and, actually,

    10    it turned out to be on sequencing identical to the

    11    gag/pol expression plasmid which we were using to

    12    actually create the particles.  So we inserted that as

    13    representing basically the packaging of random

    14    cellular transcript.  There's no real way of getting

    15    around this.  You will get random RNAs incorporated

    16    into viral particles at a certain, albeit low, degree.

    17                What I'd like to -- Oh, sorry, I should

    18    state that, since performing this data, we've gone on

    19    to try and address the potential problem with this

    20    approach, whereby we're revising our sequences to

    21    known retroviral sequence.

    22                What we've done is to actually perform a

    23    random PCR on CDNAs made from the virus particles, to

    24    try and identify unknown viral families which may be

    25    being packaged and other viral sequences.  

                                                                        260

     1                I don't have any slides, but a summary of

     2    the results would be that in the murine based system

     3    the predominant sequence which we identified was VL-

     4    30, as you might expect, because we could even detect

     5    it with this system.

     6                In the human based system, again we

     7    identified the gag/pol expression plasmid and a whole

     8    range of various, apparently random, in many cases

     9    unidentified RNA transcripts, very few of which had

    10    open reading frames, and most of which were quite

    11    small in size.  So the significance of that data I'd

    12    have to come back to in perhaps a year's time or

    13    whenever.

    14                Regards another potential risk of

    15    retroviral gene therapy, we tried to address or what

    16    we wanted to address was whether, once you had treated

    17    a cell, if the endogenous retroviruses in that cell

    18    were expressing gag proteins, whether they could pick

    19    up the RNA transcripts from your integrated

    20    therapeutic vector, package them, and potentially

    21    immobilize them and bounce them around the genome.  

    22                So this would be a disadvantage due to

    23    insertional mutagenesis, and there are some mechanisms

    24    which we had talked about earlier.

    25                In our bodies there are very few cells

                                                                        261

     1    which express significant levels of retroviral

     2    particles.  One major exception is the placenta which

     3    is absolutely heaving with BD type virus particles

     4    butting from the few cyncitial trophoblast layer into

     5    the cytotrophoblast which forms basically the barrier

     6    between the mother and the fetus.

     7                There are all of these particles as yet to

     8    be determined.  It's a lot of research going into

     9    that.  So the cell line which we analyzed to try and

    10    assess this case was one of the few cell lines where

    11    we do get lots of virus production, HERV-K based BD

    12    type virus.

    13                What we did was to look for the -- There's

    14    a Gh cell line for those that would like the

    15    information -- again, performed sucrose gradients on

    16    the virus particles.  We could see indeed endogenous

    17    retrovirus particles being produced of the B and D

    18    type sequence, which were banding at densities

    19    appropriate for BD type viruses.

    20                Interestingly, in those cells we could

    21    also detect the packaging of RTVL-H sequences, another

    22    endogenous family of sequences.  The significance of

    23    that we're really not sure, and haven't taken this

    24    work any further at the moment.  But the reassuring

    25    point was the murine leukemia based virus vectors

                                                                        262

     1    which we had introduced into these cells did not

     2    appear to be packaged into these particles. 

     3    Absolutely blank.  So that was somewhat reassuring.

     4                So in conclusion, I think we have to, and

     5    will always have to, put up with the problem that gene

     6    therapy packaging cell lines, whether they be derived

     7    from human or murine systems, dog, you name it, will

     8    express endogenous retroviral sequences which can

     9    potentially interact with your gene therapy vectors.

    10                The FLY line, human baseline, and probably

    11    other packaging cell lines seem to produce virus

    12    particles which are significantly less contaminated

    13    with endogenous retroviral sequences and do murine

    14    cells.

    15                As a result, these are probably less

    16    likely to produce replication competent recombinant

    17    retroviruses which could be a problem in such

    18    therapies and, encouragingly, again human endogenous

    19    retroviral gag proteins, core proteins, only interact

    20    weakly, if at all, with the NOV based viral RNA

    21    transcripts.

    22                Once again, thank you for the invitation

    23    to speak, and I'll welcome any questions.

    24                (APPLAUSE.)

    25                DR. COFFIN:  It's an easy one.  I'm John

                                                                        263

     1    Coffin from Tufts.  It's also been reported that MLV

     2    based vectors produced on certain packaging cell lines

     3    ex situ contain very high levels of an MLV related

     4    RNA, from Jim Cunningham's lab some years ago, for

     5    example.  Did you see any of those -- any of that? 

     6    Did you look for that?

     7                DR. PATIENCE:  We didn't look, actually. 

     8    It should be done, actually.

     9                DR. KAPPES:  John Kappes from UAB.  You

    10    said you identified that the gag/pol transcript was

    11    derived from the plasmid you transfected.  Through

    12    your sequencing, were you able to gain any information

    13    that helped you understand how it recombined?

    14                DR. PATIENCE:  I don't think it was a

    15    recombination at all.  I think it's basically a

    16    nonspecific packaging of RNA transcripts, which you do

    17    see in retroviruses.  You can pick up viral sequences. 

    18    I don't think it was recombination there.

    19                DR. KAPPES:  If you looked in infected

    20    cells for evidence of recombination -- Have you looked

    21    in infected cells for evidence of recombination?

    22                DR. PATIENCE:  Oh, yes, sure.  We've

    23    looked for both the transfer of the gag/pol construct

    24    and rescue of -- Basically, we targeted cells with

    25    envelope only, introduced our high titer virus, and

                                                                        264

     1    looked for rescue of a reporter vector by the

     2    envelope, which we had already put in, and the

     3    gag/pol.

     4                We could detect very, very low levels of

     5    recombinant viruses -- very, very low levels.

     6                DR. PURCELL:  Damian Purcell.  With the

     7    endogenous viruses that you've detected packaged, the

     8    human elements, have you been able to establish

     9    transmission and expression for those endogenous

    10    elements?

    11                DR. PATIENCE:  We  haven't seen any co-

    12    packaging and, therefore, we've been unable to detect

    13    any transmission.  We did, unfortunately, do the

    14    experiment back to front where we infected cells and

    15    then looked for human transcripts, and couldn't see

    16    any, which I guess again is even more reassuring, but

    17    nothing is getting across.

    18                DR. ROSENBERG:  Thank you.  The next talk

    19    is by Leonard Evans on, again, mixed retrovirus

    20    infection.

    21                DR. EVANS:  Okay.  What I want to talk

    22    about today are mixed retrovirus infections between

    23    two different types of viruses that have already been

    24    discussed.  That's the polytropic viruses and

    25    ecotropic viruses, and these are experiments that

                                                                        265

     1    we've done in mice.

     2                I guess the take-home message from this is

     3    that it's really going to be difficult to try to

     4    anticipate what the effects of a mixed retrovirus

     5    infection might bring.

     6                The initial experiments I'm going to --

     7    How this actually started out was not look at mixed

     8    retrovirus infections.  What I wanted to look at was

     9    recombination, and I wanted to look at the

    10    recombination of a polytropic virus when it was

    11    inoculated into a mouse and see whether or not it

    12    underwent further recombination.

    13                The reason we wanted to do this is because

    14    we saw that the population in the individual mouse of

    15    recombinant viruses that came up was plural.  There

    16    were a lot of different types of viruses, and we

    17    wanted to know whether this was a sequential situation

    18    or whether or not they were independent events.

    19                Now the virus that we chose is a virus

    20    called Fr98, and Fr98 was constructed in John Portiss'

    21    lab or Rocky Mountain Labs, and it's constructed by

    22    taking the envelope of the polytropic isolate and

    23    sticking it into a backbone of Frend murine leukemia

    24    virus, and in that way it was advantageous for this,

    25    because we knew that there were no other alterations

                                                                        266

     1    in the genome other than the envelope and part of the

     2    integrase gene.

     3                The other reason we chose Fr98 is that it

     4    replicates very well in mouse genes.  So we thought

     5    that -- I mean, if we're looking for recombination, we

     6    definitely have to have something that's replicating.

     7                The other had to do with the antigenic

     8    properties of this virus.  That's shown -- I'll

     9    explain that on the basis of this slide.

    10                Okay.  This slide describes the monoclonal

    11    antibodies that we used in these studies.  There's

    12    four monoclonal antibodies here.  The first three of

    13    these react specifically with polytropic viruses, and

    14    the last one reacts with the Frend ecotropic virus,

    15    which is the ecotropic virus we used in these studies.

    16                The first two antibodies here, hybridoma

    17    7 and 516, define two antigenic subclasses in these

    18    viruses.  Almost every virus that we ever looked at,

    19    including our isolates and other people's isolates --

    20    virtually all of them react with one of the two of

    21    these antibodies.

    22                They're mutually exclusive, and they map

    23    to the same position on the Su proteins and alternate

    24    amino acids.  We've also looked at endogenous

    25    sequences, many of them, not all of them, and all of

                                                                        267

     1    the endogenous sequences, including the modified and

     2    the regular polytropic viruses, correspond to either

     3    this one or this one in terms of the amino acid.

     4                Now what was unique about Fr98 is that it

     5    was a virus that didn't react with either one of these

     6    antibodies, although it did react with 514 which

     7    reacts with all polytropic MuLVs.

     8                S for our purposes, it was really a nice

     9    virus, because any emerging viruses we thought that

    10    would arise by recombination would either react with

    11    hybridoma 7 or 516.  So we would be able to watch

    12    this.

    13                Now another property of Fr98 is that it's

    14    a neuropathogenic virus.  It causes a neurological

    15    disease in IRW mice, but at the doses we were going to

    16    use we didn't think we would probably see a disease

    17    for about 30 days.  We weren't using IRW mice.  We

    18    were using NFS mice.

    19                When we inoculated Fr98 into NFS mice, we

    20    were able to actually follow the disease or follow the

    21    mice for the generation of new polytropic viruses that

    22    react with hybridoma 7 or 516 for about three or four

    23    months, because it turned out that these mice didn't

    24    get neurological disease with Fr98.  They developed

    25    erythroleukemia with a latency somewhat longer than

                                                                        268

     1    you see with the ecotropic virus.

     2                During the course of that time, we didn't

     3    see anything that suggested any emerging viruses with

     4    hybridoma 7 or 516.  Everything was reactive with the

     5    514, which is what the Fr98 is reactive with.

     6                It occurred to us that maybe what was

     7    happening is, because we're putting in a polytropic

     8    virus, even if we generated new polytropic viruses,

     9    because of the vicinity that we're apt to be

    10    generating this in and it would be a high level of

    11    polytropic infection, that there some interference so

    12    that we would never see the virus, because it was

    13    never, ever able to spread.

    14                Then it further occurred to us that, if

    15    this actually was happening, then maybe if we

    16    inoculated an ecotropic virus with the polytropic

    17    virus, that we could suppress the development -- the

    18    de novo development of new polytropic viruses.

    19                So we co-inoculated Fr98 with Frend MuLV,

    20    and that's the next slide.  Okay.  Now I expected

    21    there might be -- You know, I hoped there might be a

    22    little suppression.  What I didn't expect was the

    23    complete turnoff of any de novo polytropic viruses.

    24                Normally, when you inoculate Frend MuLV,

    25    you get -- It's predominantly hybridoma 7, but there's

                                                                        269

     1    some -- two or three percent that are -- of the

     2    polytropic viruses that are reactive with 516.  

     3                We got about 60,000 here on the average of

     4    12 days, and we got absolutely nothing, no hybridoma

     5    7 and no 516.  We have -- In fact, in any co-

     6    inoculation experiments, we have never seen any of the

     7    normal -- any hybridoma 7 or 516 polytropic viruses

     8    coming out.

     9                We did, however, see a huge amount of --

    10    This is the sera.  We saw a huge amount of virus that

    11    corresponded to Fr98 that's reactive to 514 only. 

    12    This is nearly 107, but there was a second observation

    13    that was really striking, and that's showing on the

    14    next slide.

    15                We found that the mice developed -- The

    16    reason those experiments only went for 12 days is

    17    because that 12 days after, the mice are gone.  We

    18    found out that, when you inoculated Fr98 with fMuLV,

    19    we got a very rapid development of neurological

    20    disease, very similar to what's seen in IRW mouse

    21    except for the onset.  At this dose in IRW mouse, they

    22    would normally come up about here.

    23                The mice appear really normal for about

    24    ten days, and when they're suckling, there didn't seem

    25    to be any problem.  Then from 11 to 15 days these mice

                                                                        270

     1    get sick.  They become ataxic.  They fall over.  They

     2    have tremors, and then they die.

     3                It's so rapid that, I mean, if -- The

     4    range here appears to be about a week, but in fact,

     5    when the first mouse in a litter develops symptoms,

     6    usually within a few hours all of the mice will

     7    develop those symptoms, and within 12 hours they will

     8    all be dead.

     9                Okay.  John Portiss looked at the brains

    10    of these mice.  He could find no spongiform

    11    encephalopathy at all.  There's no hemorrhagic

    12    differences.  John is a pathologist at our lab.  So --

    13    which is very similar to what you see in IRW mice.  In

    14    fact, a higher levels of virus inoculum -- and this is

    15    with Fr98 alone in IRW mice -- you can actually

    16    shorten the time of incubation here to come very close

    17    to this curve.

    18                Well, we wanted to know what was going on

    19    in these mice.  So we had a hint from the fact that

    20    the viremia was 107 at 12 days.  So we decided to

    21    follow the virus load in these mice, in mice that were

    22    inoculated with Fr98 alone, with MuLV or with the

    23    combination.

    24                So we looked at both ecotropic, compared

    25    the ecotropic with the co-inoculation, and the

                                                                        271

     1    polytropic virus load and the co-inoculation.  Next

     2    slide shows those experiments.

     3                This is the ecotropic virus.  This is off

     4    course here a little bit, but basically with ecotropic

     5    virus we could see no differences in the -- This is a

     6    serum.  This is the spleen, and this is the brain,

     7    which are the tissues we looked at in these mice.

     8                We could see no difference in the

     9    ecotropic virus replication in these mice.  It was the

    10    same, whether or not it was inoculated by itself or

    11    whether or not it was inoculated in combination with

    12    the polytropic virus.

    13                Completely different situation, though,

    14    with the polytropic virus.  If I can get the next

    15    slide.  Okay.  Here in the serum there is about a two-

    16    to-four hundredfold difference in the level of viremia

    17    between the co-inoculated mouse in the red and the

    18    mouse inoculated with simply Fr98.  It's even more in

    19    the spleen in terms of infectious centers.

    20                These are cell free viruses.  These are

    21    infectious centers.  A little less pronounced in the

    22    brain, but still a ten-to-twenty-fold difference in

    23    the level of infection in the brain.

    24                So it appeared that the viruses were,

    25    obviously, interacting in these mice, and one of the

                                                                        272

     1    things a person might suspect would be pseudotyping

     2    between the viruses, even though they were inoculated

     3    as separate -- well, as a mixture of stock, but they

     4    were inoculated as separate viruses.

     5                So could I have the next slide?  We looked

     6    at pseudotyping in these mice.  There's a couple of

     7    things I've got to point out about this, a couple of

     8    striking things.

     9                One is -- that I'll come back to in a

    10    second -- the fact that the polytropic virus appeared

    11    to be pseudotyped completely, almost through the whole

    12    course of infection, and pseudotyped by the ecotropic

    13    virus.  These assays are done by comparing the titers

    14    on 3T3 cells versus the titers of the virus on 3T3

    15    cells that are infected with fMuLV.

    16                So that if it's pseudotyped because of

    17    viral interference, it won't score on the fMuLV

    18    infected 3T3s, but it will score on the 3T3s that are

    19    not infected, because both eco and poly will go into

    20    those cells.

    21                The first striking thing here is the

    22    pattern of pseudotyping in the brain.  The pattern of

    23    pseudotyping changed abruptly right before the onset

    24    of disease, and this is one of the few instances where

    25    we see polytropic viruses that are not pseudotyped by

                                                                        273

     1    ectotropic virus, and the simplest explanation of

     2    this, which we're currently trying to resolve, is that

     3    the polytropic virus has spread to a new population of

     4    cells, and either that population is not coinfected

     5    with ecotropic virus or that population of cells has

     6    a different property in that it does not promote

     7    pseudotyping of the polytropic virus by the ecotropic

     8    virus.

     9                The other puzzling thing here is that --

    10    I can now extend these curves clear back to here for

    11    all this -- is the level of pseudotyping of the

    12    polytropic virus by the ecotropic virus -- the level

    13    of pseudotyping is nearly 100 percent here, and at

    14    this point in tim the level of infection is very, very

    15    low.  We're getting around 1000 titers.

    16                So the very first viruses that we're

    17    detecting in these mice are, in fact, pseudotyped. 

    18    Well, the only way you can get pseudotyping that I

    19    know of is to have co-infected cells.  So this

    20    suggested then that we actually have a very small

    21    population of cells that are infected by both viruses,

    22    which is kind of remarkable, considering they both use

    23    different receptors.

    24                It occurred to me that maybe this was

    25    because of the route of inoculation, because we were

                                                                        274

     1    mixing the viruses and sticking it in a compartment,

     2    actually in the peritoneum of the mouse.  So we

     3    thought that possibly it might be that.

     4                So what we did was to inoculate the

     5    viruses subcu. as different stalks of viruses.  So

     6    Fr98 is inoculated in one spot subcutaneously, and

     7    then like in the right haunch and the left shoulder,

     8    the ecotropic virus was inoculated.

     9                When we did that, we found the same thing,

    10    complete -- almost complete pseudotyping by the time

    11    we could get a good measure of the virus.  

    12                Well, that suggested to me that it might

    13    be a mobile population of cells that was actually

    14    being infected by these viruses.  So -- and that at

    15    some point in time a person should be able to find

    16    cells that were not pseudotyped, so that you could

    17    find cells that were not co-infected.

    18                So we did a -- God, more experiments than

    19    I want to know to look at this at very early times. 

    20    That's shown on the last slide.

    21                We could, in fact, find a time during the

    22    infection where the pseudotyping was minimal.  This

    23    computer moved this curve.  So it's supposed to be

    24    like this.  But basically, at about a tenth of a

    25    percent of there cells infected we could find free

                                                                        275

     1    polytropic viruses.  

     2                After that there was a significant level

     3    of pseudotyping.  So it looked to us then that there

     4    must be a very small percentage of circulating cells

     5    that are the initially infected cells by both of these

     6    viruses.  I guess one of the messages here that this

     7    implies is that just because the virus's dose and

     8    infection is a quite low dose, it does not preclude

     9    fairly immediate interaction of the viruses.

    10                I suppose I should say something about the

    11    possibility of this interactions in humans, not these

    12    interactions but the possibility of interactions of

    13    different types of viruses.

    14                There have been some examples of co-

    15    infection in different types of viruses in humans.  I

    16    mean, it's well documented, HtLV and HIV, but there is

    17    no good documentation, even though there was a lot of

    18    suggestion earlier, that HtLV may accelerate HIV.  

    19                To my knowledge, looking at larger cohorts

    20    there, there was no evidence of that, and also SiV and

    21    StLV infections in an experimental situation didn't

    22    show any synergism.  But definitely, the tools for a

    23    mixed retrovirus infection exists.  

    24                I mean, we talked a little bit yesterday

    25    about cells -- or viruses that could infect humans. 

                                                                        276

     1    There's a lot of viruses from a lot of different

     2    sources that can infect heterologous cells, and in

     3    fact, that may be the rule instead of the exception.

     4                With the murine system, the only ones that

     5    have never been, to my knowledge, demonstrated to

     6    infect human cells are the ecotropic viruses.  So we

     7    know there's -- from the bovine, from the feline

     8    system, obviously from the higher primates, there's

     9    all kinds of viruses that can affect human cells.  So

    10    that's the one prerequisite for setting up such a

    11    mixed retrovirus infection.

    12                The other prerequisite is for these

    13    viruses to interact.  Now they're somewhat restricted

    14    in the interaction in terms of packaging, but even

    15    that is not a foolproof situation.  C-type viruses

    16    appear to package C-type genomes more easily, but

    17    there is still -- Moloney has been demonstrated to

    18    package the genome of HIV and also the unspliced -- or

    19    the unspliced and the spliced messengers.  

    20                So it may not be a specific packaging, but

    21    it definitely was found within the virion, although it

    22    wasn't shown to be passed, and it wasn't tried in

    23    those particular experiments.

    24                In terms of utilizing glycoproteins and

    25    incorporating surface proteins in different

                                                                        277

     1    retroviruses, they're very promiscuous.  Again, you

     2    have to search for places where it doesn't happen.  It

     3    doesn't happen very easily with HIV and MuLV, because

     4    -- presumably because of the long cytoplasmid material

     5    of HIV, although SIV will incorporate into -- which

     6    also has a long cytoplasmid tail, will incorporate

     7    into MuLV particles.

     8                You also have to be concerned about

     9    heterologous pseudotyping between different kinds of

    10    viruses.  It's been shown that fowl plague virus,

    11    which is an influenza virus, can pseudotype MuLV. 

    12    Sendai virus, both proteins in Sendai virus can

    13    pseudotype MuLVs.  E. bola virus can, but that's

    14    probably a moot point.

    15                So I think that the potential for a mixed

    16    retrovirus infection is definitely there.  Whether or

    17    not it can be easily avoided is a question.  Thank

    18    you.

    19                (APPLAUSE.)

    20                DR. ROSENBERG:  Are there questions?  

    21    Thank you very much.

    22                The next talk is by Damian Purcell.

    23                DR. PURCELL:  Can I have my first slide? 

    24    So we've been introduced to gene therapy and the

    25    complications of co-packaging between multiple RNAs,

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     1    two RNAs into the retroviral particle.

     2                What I want to talk to you about today is

     3    some studies analyzing some of the early gene therapy

     4    vectors and packaging cell lines that would -- using

     5    what are now fairly unsophisticated reagents and the

     6    vectors based on Moloney murine leukemia virus and the

     7    N2 vector containing the packaging signal of Moloney

     8    and the helper constructs based on the amphotropic

     9    MuLV 4070A; and they have deletions in the packaging

    10    signal, but these constructs have regions of hemology

    11    where two recombination events can regenerate

    12    replication competent retrovirus.

    13                So the emergence of these replication

    14    competent retroviruses was observed, and the safety of

    15    -- potential risks from those replication competent

    16    retroviruses in primates has been studied.  I'll just

    17    review quickly the studies that have been published.

    18                There are essentially three of them.  The

    19    first one by Coronetta, et al. in 1990 examined a

    20    replication competent retrovirus coming from sax

    21    retroviral vector producer clone, which is essentially

    22    the same as the one on the previous slide.

    23                So in this study there was an intravenous

    24    infusion 7 by 107 focus forming units of this

    25    replication competent retrovirus or RCR, and though

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     1    that entity wasn't really well characterized

     2    molecularly, presumably it had an ecotropic Moloney

     3    MuLV gag/pol with the ampitrophic envelope.

     4                Four rhesus macaques were infused

     5    intravenously, three normals and one immunosuppressed

     6    with prednisolone and cyclosporin, and in these

     7    studies the virus was cleared in 15 minutes with,

     8    obviously, no detectable pathology after 15 year

     9    follow-up.

    10                So even within this study it was

    11    recognized that in these four animals infused with

    12    replication competent retrovirus produced in a murine

    13    cell line that probably a xeno antigen effect led to

    14    virolysis by complement where the enzyme alpha 13

    15    galactosol transferase adds the alpha gel residue onto

    16    the glycoproteins of the virus particles, and

    17    antibodies that are raised in humans, petrophile

    18    antibodies, recognize this activate complement and

    19    lead to a lysis of these murine cell derived viruses

    20    very quickly.

    21                So two ways to avoid this is, one, to

    22    reduce the virus in human or primate cells where you

    23    don't have the enzyme, and you also acquire passively

    24    into the envelope of the viral particles molecules

    25    that protect against the activation of complement such

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     1    as CD46, CD55 and CD59.

     2                The other way is to introduce the

     3    replication competent retrovirus in vitro into

     4    autologous cells and then implant the infected cells. 

     5    This is what was done in this study where 6 by 107

     6    fibroblasts from a rhesus macaque were infected with

     7    the recombinant retrovirus, both subcu. and IP, and

     8    also for good measure there was 3 by 108 focus forming

     9    units of the prototypic amphotropic virus MuLV 4070A

    10    injected IP into a single macaque.  

    11                This animal was immunosuppressed.  The

    12    animal was viremic for two days, and virus was

    13    recovered from PBMC for several weeks.  PCR could

    14    detect the presence of the amphotropic MuLV envelope

    15    up to 200 days, but there was no detectable pathology

    16    after five years of follow-up.

    17                In the second study, also published by

    18    Coronetta et al. in '91, the following year, they took

    19    the examination of the safety of these replication

    20    competent retroviruses derived from the mouse and

    21    murine system in primates one step further where they

    22    examined the transplantation of replication competent

    23    virus infected autologous bone marrow cells, in this

    24    case four cynamologous macaques.

    25                So in this protocol 1 by 106 focus forming

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     1    units of the replication competent retrovirus, again

     2    from a sax derived vector but carrying the ADA gene

     3    this time, were used to infect bone marrow, and they

     4    used supernate directly from these producer cell

     5    clones.

     6                The animals for the purpose of bone marrow

     7    transplantation received very strong immunosuppression

     8    with 1000 rads of total body irradiation, gamma rays,

     9    immediately prior to transplantation.  On follow-up

    10    there was no detectable viremia at early or late time

    11    points, no detection of the amphotropic envelope by

    12    PCR for up to three years, after long term follow-up

    13    no detectable pathology out to seven years.

    14                So on the basis of those studies, the

    15    replication competent retroviruses are probably

    16    thought of as being fairly innocuous in primates.  So

    17    a large study was done in the lab of Art Niehise at

    18    the NIH examining the transduction of autologous bone

    19    marrow in a group of ten rhesus macaques.

    20                In this protocol, using the vector

    21    packaging system on the first slide, they had a new

    22    twist to increase the titer of virus introduced into

    23    the bone marrow cells, and that was an amplification

    24    technique that they termed the ping-pong amplification

    25    where they basically bounce the vector between

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     1    ecotropic and amphotropic packaging cell lines in a

     2    co-culture system.

     3                This increased the titer of the N2 neo-

     4    vector carrying neomycin marker gene in the study, and

     5    also produced low levels of replication competent

     6    retrovirus, which they knew existed at around -- up to

     7    103 focus forming units, although the number does

     8    bounce a bit between the papers.

     9                So if we describe the largest number, 106

    10    focus forming units of the replication competent

    11    retrovirus, this was infected into 108 CD34 selected

    12    bone marrow cells in vitro in a system that cultured

    13    the bone marrow cells with the vector for 80 hours,

    14    and then these were infused into ten rhesus macaques.

    15                There was immunosuppression supplied prior

    16    to harvesting or just after harvesting the bone

    17    marrow, 5 FU for nine days, and then one and two days

    18    prior to transplantation there were two doses of 500

    19    rad of total body gamma irradiation, so total again of

    20    1000 rads of irradiation.

    21                So the surprise was that chronic

    22    retroviremia appeared in three animals, and these

    23    animals were noted not to have antibody against the

    24    GP70 or P30 capsid proteins of replicating competent

    25    retrovirus.

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     1                These three animals went on to develop T-

     2    cell lymphomas after 200 days, and these lymphomas

     3    were, obviously, rapidly fatal.

     4                So just to summarize the animals there and

     5    the data that I've just described to you, we had an

     6    opportunity to follow up in particularly two of these

     7    animals the types of viruses that were administered

     8    through this protocol.  

     9                One animal in particular had very well

    10    preserved samples, this 15445, the other 88049, had

    11    suffered a degradation post mortem, but we were able

    12    to use these samples from animals to see what had

    13    happened -- what had generated the problem here.

    14                So the first thing we looked at was the

    15    replication competence of these viruses in diploid

    16    rhesus cells.  We wanted to avoid selecting particular

    17    murine viruses by culture on rodent cells, and we

    18    could find that readily infect this rhesus lung

    19    fibroblast cell line with -- directly with plasma or

    20    with virus pelleted out of plasma.  

    21                What was the surprise when we took that

    22    virus harvested out of the rhesus cells, along with

    23    virus similarly cultured in rhesus cells, and that

    24    virus being the 4070A amphotropic retrovirus -- so the

    25    prototypic amphotropic retrovirus -- normalizing

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     1    inoculant, reinfected that back into rhesus lung

     2    fibroblasts, and the virus that was present in monkeys

     3    had a much greater capacity for replication in the

     4    rhesus cells compared to the prototypic amphotropic

     5    retrovirus.

     6                So when these viruses with the same

     7    inoculant were applied to Morsoduni fibroblasts which

     8    are very permissive for the growth of murine leukemia

     9    viruses, this growth differential was not evident.

    10                So summarizing the properties in terms of

    11    infectivity of these viruses in various cell types,

    12    reverse transcriptase activity was directly detectable

    13    in the plasma of these animals.  As I've said, the

    14    rhesus lung fibroblasts were regularly infected,

    15    bronchus fibroblasts.  Human helo cells were also

    16    infectable, and rodent fibroblasts and Morsoduni

    17    cells.

    18                What were not infectable after many

    19    attempts were rhesus PDMC, which we had activated with

    20    THA, and this is a bit of a pity, because the PDMC are

    21    obviously the easiest cells to examine from animals or

    22    patients for replication competent retroviruses, but 

    23    may probably provide a poor source of deriving virus

    24    back.

    25                So we next undertook a molecular

                                                                        285

     1    characterization of the types of virus present and

     2    transmitted and expressed in the host animal.  We

     3    chose to us an RT PCR protocol and focusing on two

     4    areas of the virus, the envelope region which, we have 

     5    already heard, determines tropism of the virus, and

     6    also the LTR, particularly the U-3 region containing

     7    the promoter and the enhancer sequences, because these

     8    also determine the tropism of the murine viruses, as

     9    we've heard.

    10                So we extracted the RNA directly from the

    11    tumor tissue, made poly-A RNA from it, synthesized

    12    CDNA.  Then we chose, after a lot of sequence

    13    alignments, sequence primers that would amplify as

    14    many of the murine leukemia viruses that we could pull

    15    out of the database and stuff into an alignment.

    16                So we're essentially going for full length

    17    envelope sequences here, and we were successful using

    18    different samples of RNA from the rhesus tumor and

    19    amplifying full length envelope CDNAs, and we

    20    amplified no bands from the rhesus lung cell line.

    21                I guess what is important to point out in

    22    this slide is that what amplifies is mostly the full

    23    length envelope, not other shorter products that might

    24    indicate the transduction with cellular components, as

    25    we might find after passage of the virus in the rhesus

                                                                        286

     1    lung fibroblast cell line.

     2                So we sequenced -- or we cloned those PCR

     3    products and sequenced the clones that we derived. 

     4    Essentially, three types of envelope structures were

     5    observed, the amphotropic envelope which was directly

     6    the same as that derived from the packaging cell

     7    construction 4070A with clear recombination back into

     8    Moloney right here around the R protein region of

     9    Moloney.

    10                There was a variance within this type of

    11    envelope that had deleted six base pairs in a key

    12    region determining the tropism that's in the VRB or

    13    variable receptor binding domain of the envelope

    14    protein, and this led to an amino acid sequence change

    15    within that crucial amino acid loop structure.

    16                What was a surprise in this animal was to

    17    find a new player, an MCFY envelope where the bulk of

    18    the envelope sequence had come from the Frend MCF

    19    strain with sequence towards the junction with the

    20    4070A amphotropic sequence, resembling a cluster

    21    alignment with xenotropic envelopes.

    22                So this was in one animal.  When we looked

    23    in the other two where we had less good starting

    24    material to do an RNA analysis, looking instead at

    25    DNA, it was clear that all of the animals, all three,

                                                                        287

     1    had the amphotropic envelope sequence present, but

     2    only the one animal had the either MCF sequence with

     3    primers within the MCF region only or the

     4    recombination between the amphotropic and MCF

     5    sequence.  None of these animals amplified with

     6    xenotropic primers.

     7                The recombinant amphotropic MCF virus

     8    could be traced back to the producer cell clone and

     9    its emergence appearing between the 22nd and 25th

    10    week.  So it seems that its derivation was from the

    11    packaging cell line.

    12                So next we examined the LTR structures

    13    within the various players here.  So firstly, I draw

    14    your attention to this line here, which is

    15    amplification across the U-3 region of the LTR.  So

    16    we're looking at the structure of the core enhancer

    17    and the promoter region.

    18                The amphotropic virus has a single core

    19    enhancer, giving rise to this size fragment here.  The

    20    A2 producer cell line has predominantly the Moloney

    21    LTR which drives the expression of vector and also the

    22    recombinant retroviruses from the previous slide, and

    23    Moloney has a duplication of the core enhancer, and we

    24    see a larger size PCR product.

    25                Also evident was another faint band which

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     1    was further duplication of the enhancer element being

     2    expressed within RNA in the producer clone.

     3                In the two rhesus samples that we

     4    examined, the predominant forms of LTR expressing RNA

     5    were those that had  the three copies of core enhancer

     6    and four copies of core enhancer was also evident in

     7    these cells.

     8                We infect the same virus into the Mosduny

     9    cell type, the indicator, it more closely resembles

    10    the expression seen in the A2 producer.  So this

    11    greater addition of enhancer sequences seems to lead

    12    to preferential expression in rhesus.

    13                So just to give a cartoon version, this is

    14    the amphotropic type U3 region.  This is what Moloney

    15    has.  These forms here seem to be expressed in the

    16    tumors.

    17                So knowing the structures of the LTRs, we

    18    did PCR analyses that would match the envelope types

    19    to the LTRs and, as we expected, the three envelope

    20    types characterized earlier, each had the LTRs that I

    21    described, each having duplications of the core

    22    enhancer.  But when we used a specific primer for the

    23    MCF type LTR, the U-3 region, we asked whether this

    24    was also being expressed at very low levels in this

    25    specific PCR for the MCF endogenous virus itself, we

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     1    indeed found expression of this in the tumors.

     2                So I just wanted to point out that these

     3    viruses here, the MCF viruses, we couldn't detect

     4    infection in either the Mosduny indicator cell type or

     5    the diploid rhesus lung, but these do grow in mixed 

     6    cell lines.

     7                So if we look at the genetic structures

     8    that we pulled out, so that the main player is the

     9    recombination between the vector and helper virus.  So

    10    it's integrated into the DNA and does express RNA in

    11    the lymphoma.

    12                Also, we find recombinations between

    13    endogenous retroviruses in two types.  We see the MCF

    14    endogenous envelope represented in this main

    15    replication competent entity, but also we see just the

    16    expression of the endogenous retroviral elements

    17    themselves as RNA.

    18                As Clive Patience mentioned earlier, we

    19    also looked for VL30, and we found lots of VL30 DNA

    20    present in the rhesus tumors, but no RNA expression

    21    from these endogenous elements.  We also looked for

    22    the co-packaging of cellular RNA species or DNAs,

    23    which is not that easy to do, and we didn't find

    24    anything.

    25                So if we want to summarize the factors

                                                                        290

     1    that might account for the pathogenesis of the

     2    replication competent retroviruses in three of the ten

     3    monkeys in this study, first we would have to question

     4    the use of mixed cell populations or the co-culture

     5    technique that might have fostered additional rounds

     6    of replication to generate novel replication competent

     7    entities.

     8                The high level of post-immunosuppression

     9    is something that's also being pointed to, although

    10    this has been debated quite a bit, whether that was

    11    particularly high or more significant than previous

    12    studies where this didn't seem to contribute to the

    13    emergence of pathogenesis.

    14                Certainly, the lack of response or lack of

    15    antibody response to the presence of the replication

    16    competent retroviruses in those three animals is

    17    another thing that seems to indicate a problem for

    18    that particular animal, and probably also associated

    19    with the emergence of the chronic retroviremia.  But

    20    the adaption of the replication competent viruses is

    21    also, we think, a very important feature.

    22                So the genetic factors, particularly, the

    23    recombination events that arise and are associated

    24    with elements from within the packaging cell lines,

    25    particularly the expansion of the core enhancer

                                                                        291

     1    elements in the U3 region of the LTR, the modification

     2    of the BRB domain is a consistent feature between the

     3    animals, and we're unsure of the relevance of this,

     4    but probably enhances the infectivity to rhesus cells,

     5    and also the recombination with endogenous MCF is also

     6    an interesting thing, but doesn't correlate with each

     7    of the tumors in all of the animals.

     8                Epigenetic factors, the alpha gel, so the 

     9    lack of addition of the 1-3 galactose on the

    10    carbohydrates when virus is derived out of primate

    11    cells in this final study compared to the first one,

    12    clearly makes the difference here.  So the viruses

    13    that emerge out of the autologous cells are resistant

    14    to complement lysis.

    15                There was a very multiplicity of

    16    integrations after the prolonged in vitro infection, 

    17    the 80 hours of exposure of the bone marrow cells to

    18    the vector and RCR mix.

    19                So the implications are to select

    20    packaging cell types with the fewest endogenous viral

    21    RNAs, that the properties of the replication competent

    22    virus derived that emerge that combine the endogenous

    23    elements may not be totally predictable, are certainly

    24    not the same as prototypic viruses that go up to make

    25    the elements placed into the packaging cell lines, and

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     1    this has implications when selecting the indicator

     2    cell lines for detecting the presence of RCRs in males

     3    that have implications of prime transfer across

     4    species.

     5                Certainly, the immune compromising or

     6    immunosuppression of recipients for gene therapy

     7    protocols would place them at greater susceptibility

     8    to spreading infection with RCRs.  

     9                Now because the organizers have put

    10    implications of this work for viral vaccines, I've

    11    included this slide here to try raising some of these

    12    points.  

    13                Endogenous retroviral elements that are

    14    present in many neoplastic cell lines, and

    15    particularly the murine cell lines, as we've heard

    16    earlier in this session, might receive help function

    17    from the expression of functional viral proteins which

    18    may be a component of a vaccine producing a functional

    19    viral protein that might interact with elements, be

    20    they RNA or protein, in the cell line.

    21                The expression from viral genes may lead

    22    to repair of replication competent retrovirus, and

    23    that really is the really nasty element that is the

    24    most obvious thing to guard against, the greatest

    25    danger, if you like.  

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     1                RCR might have novel cell tropism, an

     2    enhanced infectivity in vitro compared to prototypic

     3    retroviruses due to modifications in the envelope and

     4    LTR.  Live attenuated retroviral vaccines may

     5    recombine with endogenous elements during production

     6    in cells expressing these endogenous retroviral

     7    elements, and mixed cell populations may facilitate

     8    rounds of replication that foster the emergence of a

     9    replication competent retrovirus.

    10                So whether this argues maybe that some

    11    cell lines may be better because there's definitely a

    12    clonal population, that's another way of interpreting

    13    this.

    14                I just wanted to thank my co-workers who

    15    helped with this work while at the lab of molecular

    16    microbiology at NIH, Norton Martin's lab, Elio Vannen

    17    from Gene Therapy, Inc., who did another similar study

    18    also published, and Art Niehise who is now at St.

    19    Jude's.  Thank you.

    20                (APPLAUSE.)

    21                AUDIENCE PARTICIPANT:  Have you looked at

    22    the integration sites of those lymphomas?

    23                DR. PURCELL:  That was -- Some studies

    24    were done with that, but it was appreciated that in

    25    the lymphoma there were up to 35 integrants from the

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     1    replicating competent retroviruses, and it was

     2    considered to be at that point too difficult, too many

     3    integrations.  So that's clearly the undercurrent of

     4    the pathogenesis, the insertional mutagenesis, from

     5    having so many integrations into the genome.

     6                DR. COFFIN:  John Coffin from Tufts.  Your

     7    talk and several before emphasized a very important

     8    but not often completely appreciated point about

     9    retroviruses, and that is that their really very large

    10    genetic flexibility allows them to respond to all

    11    kinds of different selection pressures in all kinds of

    12    remarkable and surprising ways.

    13                So when one is thinking about handling

    14    systems that have retroviruses in them, one should

    15    think not only about the genetic variation, the

    16    mechanism of genetic variation that you undergo, but

    17    also and probably much more importantly the kinds of

    18    selective pressures you're putting on these viruses to

    19    enhance their growth.

    20                For example, in the AKR mouse it's really

    21    remarkable that every single animal in lockstep

    22    virtually undergoes the sorts of changes that we saw

    23    in slides earlier, and the same kind of remarkable

    24    changes that you see here, and there are many, many

    25    other examples of these kind of things in

                                                                        295

     1    retrovirology.

     2                So I think we always have to be very

     3    careful in our thinking about how this goes, not only

     4    for understanding molecular mechanisms but also for

     5    understanding selective pressures that we're putting

     6    on the viruses and the cells to produce them.

     7                DR. PURCELL:  I couldn't agree more.  I

     8    mean, also an interesting component of this things is

     9    when you put these viruses together in vitro where you

    10    don't have immune selections and you're greatly

    11    enhancing the start of the virus to recombine and make

    12    a whole host of new entities, do we immune suppress

    13    the animal?  That gives it a new environment in which

    14    to make changes and get going.  

    15                DR. ROSENBERG:  The last question, because

    16    we are running and want people to stay and hear our

    17    final speaker.

    18                AUDIENCE PARTICIPANT:  I'll speak quickly. 

    19    Two very short questions.

    20                Damian, do you know if anyone actually

    21    measured the levels of natural antibody in the primate

    22    recipients of the various viruses?

    23                Secondly, have the complement inhibitors

    24    ever been directly shown to protect viruses produced

    25    from potentially sensitive cell lines?

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     1                DR. PURCELL:  There are studies published,

     2    I believe, that look at the lysis of murine derived

     3    retroviruses by human complement, and ascribe that

     4    activity.  But I'm not sure that it's been conducted

     5    in molecular rigor.

     6                AUDIENCE PARTICIPANT:  hose studies

     7    certainly do implicate alpha 1-3 gal as the culprit

     8    molecule, but I don't think anyone has ever shown that

     9    the complement inhibitors work directly on the virus

    10    particles by accidental packaging of the inhibitors.

    11                DR. PURCELL:  Yes, I have to agree with

    12    that.  I mean, my slide there was more deduction of

    13    the data rather than a good study to explain those

    14    results.  So, yes, more work needed.

    15                DR. ROSENBERG:  Our final talk this

    16    afternoon is by Paul Jolicoeur on defective

    17    retroviruses.

    18                DR. JOLICOEUR:  Yes.  I should thank the

    19    organizer for giving me the opportunity to talk.  I'm

    20    going to talk mainly about pathogenesis of defective

    21    viruses, as I was asked to talk about.  The first

    22    slide, please.

    23                What are defective retroviruses?  This is

    24    illustrated here.  They are a shorter genome and, in

    25    general, they have deleted or mutated genes. 

                                                                        297

     1    Therefore, they cannot replicate by themselves, and

     2    they do need a nondefective, which we call helper

     3    retrovirus for full replication.  

     4                Some of them, however, are not a gene in

     5    general, which I think capture many of the oncogenes,

     6    and we'll see a little bit later what they are.  These

     7    defective viruses can be expressed normally, once

     8    integrated, and we count helper proteins.  This is

     9    important for pathogenesis of some of them; and if

    10    expressed, they can also be rescued by other

    11    nondefective viral proteins, even those encoded in

    12    other defective viruses.

    13                In other words, in helper free stock you

    14    can generate one-cycle replication, and you heard

    15    about that earlier, in the packaging system where two

    16    defective viruses complement, in a way, each other.

    17                Why are there concern about defective

    18    retroviruses in vaccine preparation?  Again, you heard

    19    about it from the previous speaker, that we do have

    20    several copies of endogenous defective retroviruses

    21    which are either nondefective or nondefective

    22    retroviral sequence, including in humans where most of

    23    them, if not all of them, are defective.

    24                These endogenous sequences participate in

    25    recombination events, give rise to novel viral

                                                                        298

     1    entities, novel viruses, again as you heard from

     2    previous speakers.  And several strains of defective

     3    retroviruses have been shown to be pathogenic.

     4                There are broadly two classes of defective

     5    viruses, the one which essentially encode only viral

     6    genes such as this one, and the best known examples

     7    are the Frend virus and the duplan strain virus or the

     8    MAIDS virus on which I will talk just a little bit

     9    more; and obviously, the one which encodes V onc,

    10    which have captured a nonviral cellular gene, and from

    11    which we have learned most about oncogene and from

    12    which the first oncogenes were derived from and labels

    13    on the FBJ, the RAS Harvey and Kersten and so on, and

    14    where you do have a V onc. which has been captured in

    15    the middle of the defective viruses.

    16                The SFFV containing only viral sequences

    17    is defective in the sense that it has gag and pol --

    18    several gag and pol mutations.  It has a complete and

    19    open reading frame, but its end sequences are a form

    20    of two types of sequences, nonecotropic and ecotropic

    21    recombinant, plus an insertion, a one base pairs

    22    insertion here, giving rise to this new protein which

    23    is able now to recognize a structure which normally

    24    none of the parents recognize, that is, the CD EPO

    25    receptor.

                                                                        299

     1                The GP55 which is encoded by this Frend

     2    virus would stimulate the EPO receptor and give rise

     3    to this massive proliferation and erythroleukemia. 

     4    This is an example of a virus which is defective and

     5    is able to participate in tumor formation very easily.

     6                I have listed here some of the

     7    characteristics of defective retroviruses.  We are

     8    going to go through some of them, and I will

     9    illustrate many of them in a model which we call the

    10    MAIDS model.

    11                First, defective viruses can be generated

    12    in a lifetime of an animal, and this has been the

    13    hallmark of the V onc. defective viruses which all

    14    occur in one animal and were isolated in this animal, 

    15    although very rare genetic events are required to

    16    generate in general these viruses, sometimes two or

    17    three recombinations of them.  

    18                Defective and recombination retroviruses 

    19    appear to be generated more easily in vivo than in

    20    vitro.  Defective viruses or pathogenic viral genome

    21    can emerge from nonpathogenic viral sequences.

    22                As I said previously, two defective

    23    retroviruses can complement each other.  Individual

    24    retroviral sequence expressed in absence of

    25    replication in defective systems, such as the

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     1    packaging system, can be pathogenic or, in the context

     2    of transgenic where you express only one gene.

     3                In general, those viral genes interfere

     4    with -- or participate in cell signaling.  We also

     5    know that defective viral sequence such as VL30 can

     6    enhance V onc. activity or such as gag in gag oncogene

     7    fusion protein.  It has been shown, for example, with

     8    a gag Abelson fusion protein where gag is important

     9    for myristylation and location in the membrane.

    10                Finally, defective viral genomes are as

    11    effective as nondefective viral genomes as insertional

    12    mutant, and we'll see some example of that.

    13                Just to show a cartoon here, how can rare

    14    events -- very rare events, in fact, get -- occur in

    15    a live form of an animal as a capture of an oncogene

    16    where it has been alluded before by Naomi.  First, it

    17    has to -- This is a model which has been postulated by

    18    many labs, including Mike Bishop, and has been

    19    reviewed here.  

    20                You have to have -- It is postulated that

    21    there is an insertion in front of a C onc., that you

    22    do have deletion which by itself is rare.  First event

    23    is rare.  Second event, this deletion is relatively

    24    rare, and you do have a transcript.  

    25                You make a packaging sequence which has

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     1    been conserved here so that you can package this with

     2    an infection of a new cell where you do have

     3    transcription and a jump, which is also a relatively

     4    rare event, where you do have a jump to capture this

     5    sequence and put it between two LTR in chicken and

     6    mice, mainly in these two species.  It has been well

     7    documented that you do see that in the lifetime of a

     8    single animal.

     9                I will not go through that, because many

    10    people have talked about the packaging system where

    11    two defective genomes finally can be produced in a

    12    one-cycle event.

    13                Let me focus now on an animal model which

    14    is called MAIDS, which has been induced by defective

    15    viruses and where we can see the plasticity of what

    16    these viruses are.

    17                MAIDS stands for murine AIDS, essentially

    18    a lymphoproliferative disorders with splenomegaly and

    19    lymphadenopathy where you do have severe immune

    20    dysfunction of TNB cell lymphocyte. 

    21                There is massive lymphoproliferation in 

    22    these mice.  You can have as much as two or three

    23    grams of lymph node tissue, due to this massive

    24    proliferation.  It's basically caused by defective

    25    viruses which has a single open reading frame in the

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     1    gag region with severe deletion in pol MRV  regions.

     2                So essentially it's only a viral gene, and

     3    one gene -- one viral gene has been kept open.  The

     4    origin of this virus is interesting.  It arose after

     5    successive passage of cell-free extract for mix-free

     6    induced thymomas in C67 blacks.  

     7                The crude stock themselves contain several

     8    substrains of MuLV initially, and it is likely to

     9    originate from endogenous proviruses.  In fact, some

    10    of the parents have been partially identified.

    11                One is a family of endogenous MAIDS

    12    related virus, MRV, identified in the mouse genome --

    13    In fact, there are five members of that -- by Neil

    14    Kaplan, where the gag is intact and where a deletion

    15    of pol MRV is present, very much like the MAIDS virus.

    16                The gag region is homologous to the MAIDS

    17    virus and harbors some additional mutation, however,

    18    and the LTR is this thing.  The MRV is a xenotropic

    19    LTR, while the maze virus is an ecotropic LTR.

    20                There is another family of expressed

    21    genome, EDV, where the gag is homologous to that of

    22    the MAIDS virus if you allow a frame shifting. 

    23    Therefore, it appears MAIDS is a recombinant virus,

    24    probably of at least three distinct endogenous

    25    retroviral sequences, which again occur during passage

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     1    of irradiated tissues.

     2                It encodes a single protein, which is a

     3    gag protein, which is not cleaved in matrix or capsid

     4    or nucleocapsid proteins.  It is not cleaved.  This is

     5    a peculiarity of this gag protein.

     6                It is myristylated and well produced in

     7    cells, and our hints that this system could be

     8    interesting to study the pathogenesis came when we, in

     9    fact, used a packaging system to produce helper free

    10    stocks of these defective viruses and to show that, in

    11    fact, these helper free stocks, which are replicating

    12    incompetent except for one cycle, would cause disease 

    13    to a high frequency -- this is a Type 2 virus here -- 

    14    in fact, at 100 percent as compared to a replication

    15    competent helper virus.

    16                This is remarkable, because when you think

    17    about it, when you showed this prep in IP, it gets

    18    into the peritoneum, and the very first cell it's

    19    going to see and infect will be the last one, and

    20    these cells will cause the pathology you just saw.

    21                We are talking here of very low titer.  In

    22    the range of 103, 104 will give rise to this disease. 

    23    So 1000 cells to 10,000 cells in the body at maximum,

    24    if we don't account for the loss of infectivity going

    25    through the peritoneum, are responsible for the

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     1    disease.

     2                What's happened when you do that and look

     3    in the spleen, for example, or lymph node, you do very

     4    early see little clones of proliferation which are

     5    independent.  So a little polyclonal proliferation in

     6    distinct nodes and lymph nodes which eventually, as

     7    the time goes on, will fuse and get massive polyclonal

     8    proliferations.

     9                We have been able, through different

    10    techniques -- I'm showing only one here; this is

    11    mainly a typing by immunocytochemistry to show that

    12    these cells are B cells.  This is a B220 staining as

    13    opposed to the control, different controls in lymph

    14    node or a spread of cells here.  This is a positive

    15    cell, because you see the blastic-like inside two

    16    positive cells, and the noninfected, more mature B

    17    cells which are smaller.  So that you have the

    18    features of blastic, and they are B220 low.  

    19                Another interesting feature of this growth

    20    is that it's thought in many mice as polyclonal.  This

    21    is a blot where we look at integration site, on the

    22    junction of integration site, and you can see that

    23    different nodes in the same animals -- in this animal,

    24    for example, or this animal -- you do have a

    25    polyclonal or different -- not polyclonal but distinct

                                                                        305

     1    integration.  But in some animals the same clone has

     2    taken over the whole animal here.

     3                That is to say, a single clone here, a

     4    single cell really, took over and appears in these

     5    lymph nodes.

     6                Looking at that, we hypothesize that in

     7    fact not only the gag was providing a growth advantage

     8    to these cells -- that is quite obvious, because you

     9    can see it polyclonally and very quickly -- but also

    10    the genome itself was an insertional mutagent.

    11                We test this hypothesis by cloning some of

    12    these integration events and asking whether these loci

    13    were occupying a certain percentage of the tumor, and

    14    we did find at least two loci like that which we call

    15    Dis-2 where we did find common integration.  

    16                We have not yet identified the genes

    17    involved here, but the Dis-1 -- and you can see here

    18    the rearrangement and different tumor or lymph node

    19    enlargements, if you wish, in the band of the Dis-2 or

    20    Dis-1 locus here.

    21                The other locus that we have identified is

    22    the PU.1 locus which is a transcription factor which

    23    is specific for B cells in macrophage.  You can see in

    24    a certain percentage of tumors that the integration

    25    occurs at the same place where there were initially

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     1    identified in erythroleukemia and the Frend's systems.

     2                Therefore, this defective virus

     3    participates in growth of these cells, not only by

     4    providing the gag protein but also by serving as an

     5    insertional mutagent, and this is an example of a

     6    defective virus genome which can serve as an

     7    insertional mutagent.

     8                Now we did the structure function of that,

     9    and again this illustrates a point.  When we do point

    10    mutation in this protein very early at the subcode

    11    down here within a few amino acid of the beginning of

    12    gag, disease capacity of that virus is lost.

    13                If you do other point mutation along the

    14    protein or the sequence, you do see that this viral

    15    genome is pathogenic, but at the same time we found

    16    recombination in these viruses.

    17                So, in fact, these are nonmutant, because

    18    they are revertant, and you will see what kind of

    19    version we found.  So the only two viruses we could

    20    use where we found loss of capacity to induce disease

    21    was this early mutant stop coder and a myristylation

    22    negative mutant, as you can see here.

    23                Surprisingly, despite the fact that we had

    24    a single point mutation in the middle of this

    25    sequence, the type of reversion we got was not a

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     1    reversion of that mutation, but in fact was a

     2    recombinant.

     3                As you can see here, this is the virus we

     4    put in with the capacity to encode now a very short

     5    protein as opposed to P63 the encoder protein of P21. 

     6    This is in fibroblasts where we produce a stock of

     7    virus.

     8                In every mouse inoculated with this virus

     9    where we looked in every lymph node, what we found is

    10    a recombinant having the molecular where it closed to

    11    PR60, and you can see they act differently in each

    12    other.

    13                So again this illustrates the point with

    14    virology that John was coming at a few minutes ago,

    15    that those are very plastic and even, surprisingly,

    16    with a point mutation like that, what you achieve is

    17    you achieve a highest recombination event presumably

    18    with other endogenous sequence, and we know this was

    19    not -- we know it was not generated in vitro.

    20                The myristylation negative mutants tell us

    21    something about that.  First, it tells us that

    22    myristylation itself is required.  So, therefore,

    23    attachment to the membrane is important for the

    24    pathogenicity of that virus, and this suggests that

    25    PR60 molecule is the only protein required for disease

                                                                        308

     1    induction.

     2                Two, it suggests that the processing of

     3    that protein or that antigen, if you wish, by B cell,

     4    which are APC, is not required for this effect,

     5    although some people could argue about it; and PR60 is

     6    used as a docking side.

     7                The way we see it now is that you do have

     8    this protein here, and presumably, by interacting with

     9    some protein, you do send two messages.  One is

    10    proliferation, as illustrated by the very quick,

    11    within a day or two -- very quick proliferation of B

    12    cell polyclonal and, two, by reprogramming of these B

    13    cells the sense that B cells which proliferate will

    14    not induce total anergy of T-cells, as we do find in

    15    these mice.

    16                So, therefore, there is something special

    17    about these cells which make them energize T-cells

    18    which are not themselves infected, and there are some

    19    motif, especially in the P12 domain of this protein,

    20    which is a very proline rich protein as opposed to

    21    either P12 protein of other MuLV, and where you do

    22    create pyridivous H-3 finding site, and as well some

    23    H-2 phosphotyrosine binding sites, as you will find in

    24    signaling molecule.

    25                So to search for a putative factor binding

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     1    to PR60 gag, we use the yeast-two hybrid.  As a bait

     2    we use the PR60 in the LXA system, and the very first

     3    clone we got was an interesting clone.  It was the

     4    only small clone containing the SH-2 - SH-3 domain of

     5    C-ABL.

     6                We did some coimmunoprecipitation and

     7    other studies that I don't have time to go through

     8    here, showing that this interaction was -- could be

     9    seen in vitro and in cells infected with the virus

    10    itself, as you can see here with an anti-IPA both, for

    11    example, and coming back with anti-CA.

    12                Therefore, we do see this molecule, PR60

    13    gag, as a docking molecule where protein or a

    14    signaling molecule such as C-ABL will dock to, or

    15    other putative molecule to send a single proliferation

    16    and reprogramming of these B cells.

    17                So I will conclude with that, to state

    18    again that this is probably a docking site for C-ABL

    19    through the SH-3 domain, and the translocation of C-

    20    ABL to the plasma membrane in this system may release

    21    its block on the cell cycle progression and may give

    22    it access to novel target substrates for tyrosine

    23    phosphorylation.

    24                The way we see the pathogenesis of this

    25    virus is summarized here in a cartoon where the

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     1    defective viruses, whether with a helper or without a

     2    helper, as we have shown, can infect a B cell which

     3    will start proliferating locally in the germinal

     4    center -- I forgot to mention that these cells are

     5    located -- these B cells are located in the germinal

     6    centers -- which eventually will progress, and at the

     7    same time influence other cells, either to progress or

     8    attract them.

     9                I should mention that the infected cells

    10    in the huge lymph node sometimes represent only five

    11    percent of the cells of the node, a little bit like

    12    you will find with the Reed Stanford cells where the

    13    majority of the node is composed of normal cells. 

    14    This is also the case here in most of the time, which

    15    is by itself an interesting biological feature of the

    16    system.

    17                Consecutive to the growth of these cells,

    18    you do find in the body a defect in non-infected

    19    cells, which are themselves T-cells where you do find

    20    total anergy of all the B bearer T-cells, as well as

    21    defect of D cells.  Obviously, the infected B cells

    22    are themselves un-normal as well.

    23                So again, I'm just putting the same slide

    24    you saw before, which illustrates some of the

    25    characteristics and, in fact, you find many of them in

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     1    this system of the MAIDS virus.  

     2                I'm not going to go through all of them

     3    here, but again to point toward one important aspect

     4    is that defective pathogenic viral genomes can emerge

     5    from nonpathogenic viral sequences, and that this

     6    viral gene products are involved in cell signaling and

     7    are able to interfere with the cells.

     8                I will stop there, because time is running

     9    short, and I will thank my collaborator who has worked

    10    on this system.  Thank you.

    11                (APPLAUSE.)

    12                DR. HENEINE:  Walid Heneine, CDC.  

    13                My question is about set of data you

    14    mentioned on the insertional mutagenesis with this

    15    virus, and has relevance to what we've been discussing

    16    earlier about transfer of DNA.

    17                If I get it right, you said that one

    18    mechanism also involved -- of the disease involves

    19    insertional mutagenesis, despite the use of very small

    20    virus inoculum.  Right?

    21                DR. JOLICOEUR:  Yes.

    22                DR. HENEINE:  And you mentioned -- What is

    23    the number you mentioned about those viruses that you

    24    inoculate with -- the number?

    25                DR. JOLICOEUR:  Number of virus particle? 

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     1    Infectious virus particle are in general 103, 104.  

     2                I should stress here that the insertional

     3    mutagenesis is not an essential component of the

     4    disease.  In other words, you do have disease when you

     5    do have a polyclonal growth of many clones.  

     6                The fact that some of these clones emerge

     7    and take over is not a feature -- is not a necessarily

     8    feature of the disease.  In other words, to anergize

     9    T-cell, you don't need this novel event, if you wish,

    10    but it does occur presumably because there are so many

    11    cells being in proliferation that some of them are at

    12    an advantage over others.

    13                DR. HENEINE:  So it has no causal

    14    relationship, you're saying?

    15                DR. JOLICOEUR:  Not for the disease

    16    itself, no.

    17                DR. HENEINE:  Not for the disease.  

    18                DR. JOLICOEUR:  No, but it occurs.

    19                DR. HENEINE:  Thank you.

    20                AUDIENCE PARTICIPANT:  I was very

    21    interested in your apparently pathology of B cells

    22    from the peritoneum into the germinal centers of the

    23    lymph nodes and ultimately the systemic tolerance of

    24    anergise in the T-cells.

    25                Did you look at all in the bone marrow to

                                                                        313

     1    see if any of the B cells eventually resided, as I

     2    say, into the marrow?

     3                DR. JOLICOEUR:  If the B cell, the

     4    infected B cell?

     5                AUDIENCE PARTICIPANT:  Yes.

     6                DR. JOLICOEUR:  We take over the whole

     7    body.  We know that initially it's not a B cell from

     8    the bone marrow which is infected.  It's a B cell

     9    which is localized at the site of inoculation.  We

    10    have done time course.

    11                If you inoculate IP the very first cells

    12    are within the mediastinal lymph node.  If you

    13    inoculate in the foot pad, it's in the right or left,

    14    whatever you inoculate, lymph node and so on.

    15                So the target cell is localized at the

    16    draining lymph node.

    17                AUDIENCE PARTICIPANT:  Right.  But do you

    18    know if any of the B cells from the germinal center in

    19    the lymph nodes eventually get back to the marrow

    20    where they could have --

    21                DR. JOLICOEUR:  They do, yes.  They take

    22    over the whole body.  Oh, yes, all over, including --

    23    and quite infiltrating.  Some of them infiltrate the

    24    liver, infiltrate the brain and so on.  Some get quite

    25    aggressive.

                                                                        314

     1                DR. ROSENBERG:  Thank you.  

     2                I'd like to thank all of our speakers --

     3    Oh, one more?  Okay.

     4                AUDIENCE PARTICIPANT:  What is  there --

     5    I'm Carlo Russo from Merck.

     6                What is the receptor on the B cells?

     7                DR. JOLICOEUR:  Oh, this is the eco-

     8    receptor. We use -- In each case the helper has been

     9    the Moloney helper or the ecotropic helper. So,

    10    therefore, the cell that would get infected is

    11    infected through the eco-receptor.

    12                Other group have used the amphotropic

    13    receptor for entrance, and they get the same disease.

    14                AUDIENCE PARTICIPANT:  And the mechanism

    15    of T-cell anergy, you think, is mediated by what?

    16                DR. JOLICOEUR:  This is a big mystery. 

    17    One theory has been that it is a super antigen effect,

    18    but I think this has been quite ruled out by our group

    19    and other groups now.  

    20                It's totally mysterious.  It's mysterious

    21    whether you need the contact with B cells to get this

    22    or whether it's a cytokine type of release from the B

    23    cells.

    24                DR. ROSENBERG:  If there are no more

    25    questions, we'll adjourn, I think, until eight o'clock

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     1    tomorrow morning when there will be more presenters.

     2                (Whereupon, the foregoing matter went off

     3    the record at 5 :30 p.m.)

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