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

                                TUESDAY,
                            SEPTEMBER 7, 1999

                The workshop took place in the Plaza Ballroom,
          DoubleTree Hotel, 1750 Rockville Pike, Rockville, MD
          20852 at 7:45 p.m., William Egan, Ph.D., Chair,
          presiding.

          PRESENT:

          WILLIAM EGAN, Ph.D., Chair
          ELWYN GRIFFITHS, Ph.D., D.Sc., Co-Chair
          REGINA RABINOVICH, M.D., Co-Chair
          LEONARD HAYFLICK, Ph.D., Speaker

          ANDREW LEWIS, Jr., M.D., Speaker
          PHILIP KRAUSE, M.D., Speaker

                                                                          2

                                 AGENDA

          Welcoming Remarks
            Regina Rabinovich, M.D. . . . . . . . . . . . .  3
              Institute of Allergy and Infectious Diseases, NIH
            Elwyn Griffiths, Ph.D., D.Sc. . . . . . . . . .  5
              WHO

          SESSION I - Evolution of Cell Substrates Used in the
                      Manufacture of Biologicals

          Review of workshop purpose and goals
            William Egan, Ph.D. . . . . . . . . . . . . . .  7
              CBER/FDA

          Evolution of cell substrates used for the production
          of human biologicals
            Leonard Hayflick, Ph.D. . . . . . . . . . . . . 11
              University of California, San Francisco

          Major issues associated with cell substrates
            Elwyn Griffiths, Ph.D., D.Sc. . . . . . . . . . 26

              WHO

          Developing an approach to evaluate the use of
          neoplastic cells as vaccine substrates
            Andrew Lewis, Jr., M.D. . . . . . . . . . . . . 58
              CBER/FDA

          Conceptual and experimental approaches to address
          product safety issues raised by novel cell substrates
            Philip Krause, M.D. . . . . . . . . . . . . . . 73
              CBER/FDA

                                                                          3

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

     2                                             (7:54 p.m.)

     3                DR. RABINOVICH:  Good evening.  I would

     4    like to ask those who would like to participate in

     5    this evening's session to please have a seat so we can

     6    get started.  My name is Regina Rabinovich.  I am here

     7    from the Institute of Allergy and Infectious Diseases

     8    from the NIH in the U.S., and on behalf of my co-

     9    sponsoring agencies and colleagues, the Center for

    10    Biologics Evaluation and Research, the International

    11    Association for Biologicals, the National Vaccine

    12    Program Office, which includes Marty Myers, who is

    13    supposed to be a co-chair for this session and is

    14    currently either grounded or wandering around Newark,

    15    New Jersey, and the World Health Organization, I

    16    welcome you to the meeting on Evolving Scientific and

    17    Regulatory Perspectives on Cell Substrates for Vaccine

    18    Development.

    19                I have a couple of housekeeping comments. 

    20    I won't bother to tell you where the bathrooms are. I

    21    am sure you can find those. There are two documents

    22    that I think would be very useful, if you haven't

    23    noticed them already, because they are key to the

    24    workshop.  The first is the document prepared by our

    25    colleagues from the Food and Drug Administration,

                                                                          4

     1    which is under your tab entitled, Draft CBER document.

     2    Because I think that the creation and comment upon

     3    this is central to the scope of this meeting, as will

     4    be elucidated further by Dr. William Egan.

     5                Because the sponsoring groups as well as

     6    the FDA particularly are so interested in the concepts

     7    within the document and the discussion that will take

     8    place, this meeting is being taped.  And for that

     9    reason, we must ask that speakers as well as those

    10    that are asking questions or getting answers speak

    11    into the microphones, and that we will work with the

    12    moderators to make sure that everyone that wants to

    13    ask a question, regardless of where they are in the

    14    room, have a microphone available to them because it

    15    is key that it be part of the written record.

    16                The steering committee would like to thank

    17    a couple of individuals that have worked very hard to

    18    make the meeting possible.  Two of our colleagues at

    19    FDA, Dr. Keith Peden and Dr. Phil Krause, and Dr. John

    20    Daugherty at NIAID.  

    21                In thinking about eight months ago about

    22    this workshop, the scope and what it should cover, we

    23    never in our wildest dreams considered that in

    24    September of 1999 we would have gone through a summer

    25    where there would be no question about how to spell

                                                                          5

     1    thimerosal, and that early information coming out of

     2    sort of a group analysis of all clinical trial data

     3    for the rotavirus vaccine would give us the first

     4    inkling to the potential connection to

     5    intussusception. Indeed, the issues regarding this

     6    topic, vaccine safety, which never makes sense to

     7    anyone, what we are talking about in terms of vaccine

     8    safety, are elucidated by the kinds of thoughts and

     9    strategies that will evolve from this meeting.  And if

    10    ever there was a connection between basic research and

    11    what we do with human subjects as an outgrowth of your

    12    deliberations, I think this is it.

    13                So on behalf of my colleagues at the

    14    National Institute of Allergy and Infectious Diseases,

    15    I thank you for your participation.  I would like to

    16    go ahead and introduce Dr. Elwyn Griffiths from the

    17    World Health Organization, who is my co-chair in my

    18    role as Dr. Marty Myers, and will introduce the first

    19    speaker.  Thank you.

    20                DR. GRIFFITHS:  Can you hear?  Oh, yes,

    21    okay.  (Speaking in Welsh.)  It is the only

    22    opportunity I have ever had to introduce a meeting

    23    such as this in Welsh.  I know there is one colleague

    24    of mine, Merfyn Williams, who used to be at FDA, and

    25    he is in the audience. So at least he understood that. 

                                                                          6

     1                It is really an honor and a pleasure for

     2    me to co-chair this session, the first session of this

     3    important meeting with Regina here.  Let me also say

     4    that the World Health Organization is very pleased

     5    indeed to be a co-sponsor of this workshop.  Because

     6    I think it considers the subject to be vitally

     7    important from a global perspective, and we take that

     8    view of course, for future development and production

     9    of vaccines in general.  

    10                I see this evening's activities really as

    11    a sort of an hors d'oeuvre, I guess you might say, or

    12    an appetizer to the entrees that come during the next

    13    two days or so.  So it is an overview of where we are

    14    going and where we have been.  I think perhaps we

    15    should, because of the time scale -- I think we should

    16    move forward with the program, taking particular pity

    17    on any colleagues who have just flown in from Europe,

    18    because they would be suffering from some jet lag or

    19    biological clock problem at the moment.  So I think we

    20    should move forward fairly quickly.

    21                So it is a pleasure then really to call

    22    upon Dr. Bill Egan, who is now the Acting Director of

    23    the Office of Vaccines at the Center for Biologics

    24    Research and Evaluation, CBER, who will actually give

    25    us a review of the works or purpose and the goals of

                                                                          7

     1    this.  So he will set the scene for where we are going

     2    to go during the next few days.  So, Bill?

     3                DR. EGAN:  Thank you, Elwyn.  Can I have

     4    the first slide, please?  I too would like to thank

     5    everybody who has been able to come to the meeting and

     6    everybody who helped in the organization of the

     7    meeting and all of the participants in the meeting. 

     8    This is a workshop and everyone will be working

     9    together to try and address some very interesting,

    10    some practical, and some very difficult questions.

    11                Every now and then I think we need to

    12    remind ourselves that viruses can propagate only in

    13    live cells, and this of course holds true for whole

    14    viral vaccines.  They can only be produced in cells. 

    15    The choice of suitable cell substrates for the

    16    manufacture of viral vaccines has over the years

    17    engendered considerable discussion.  The primary focus

    18    in these discussions on cell substrates have been

    19    safety, in particular the potential safety concerns

    20    from residual cellular DNA and from adventitious viral

    21    agents.

    22                As history has shown, the need for concern

    23    about cell substrate issues was real.  We have only to

    24    think back to the finding of SV40 in polio virus

    25    vaccines to realize the extent of the risk that any

                                                                          8

     1    cell substrate may pose, and there is still great need

     2    for concern.

     3                Early discussions on cell substrates

     4    allowed only for the use of "normal" cells, that is,

     5    cell populations that were derived from normal tissues

     6    and that had undergone no subculture.  And right now,

     7    I am referring primarily to the 1954 decision of the

     8    Armed Forces Epidemiological Board.  

     9                I can't read this -- it is hard to read

    10    from this perspective back here.  The use of only

    11    normal cells has prevailed for many years.  For

    12    example, in 1971, the regulations of the Public Health

    13    Service for the production of OPV stated that only

    14    primary monkey kidney tissue cultures may be used in

    15    the manufacture of polio virus vaccines.  There were

    16    similar regulations in place for the production of

    17    inactivated polio virus vaccine, live attenuated and

    18    inactivated measles vaccines, mumps vaccine, rubella

    19    vaccine, and the adenovirus vaccine.  

    20                This was taken from the regulations in

    21    1971, and it was in 1972 that the Federal Register

    22    notice was placed that contemplated the use of other

    23    cell lines other than these primary animal tissue

    24    cells for use in viral vaccines.

    25                Now our thinking and our practices with

                                                                          9

     1    regard to cell substrates has evolved over time.  At

     2    present, for example, in addition to primary animal

     3    cell culture, diploid cell lines, both of human and

     4    animal origin, are in routine use, as, for example,

     5    the mumps, rubella, IPV vaccines.  And in addition to

     6    being manufactured in the human diploid cell, live IPV

     7    is also produced in viro cells, a continuous cell

     8    line.

     9                We now wish to consider additional cell

    10    substrates or additional cell substrate uses.  In

    11    particular, we would like to consider the use of

    12    continuous cell lines for live attenuated viral

    13    vaccines, and the use of other neoplastic cell lines

    14    for both live attenuated and inactivated viral

    15    vaccines.  Indeed, the question that the Armed Forces

    16    Epidemiology Board met to consider 45 years ago,

    17    namely the use of tumor cells, specifically HeLa

    18    cells, as a cell substrate is a question that we are

    19    again raising.   

    20                The goals of the workshop are simple. 

    21    They are two.  The first is to identify the various

    22    concerns that are associated with using neoplastic

    23    cell lines, to catalog them as completely as possible. 

    24    The second is to determine approaches and to identify

    25    approaches to determining the level of the risk that

                                                                         10

     1    are associated with these concerns.  In the decisions

     2    that we will make with regard to the use of neoplastic

     3    cells as potential substrates for viral vaccines, we

     4    need to be guided by data; either data that already

     5    exists in the literature or the data that we need to

     6    generate.  We should not be guided by intuition or by

     7    opinion.  If I may quote Dr. Hilleman from a

     8    discussion about the use of tumor cell lines from a

     9    meeting that was held in 1968, he said, "If the debate

    10    has taken on an ecclesiastical or philosophical

    11    overtone, it should not be unexpected, since it is an

    12    area in which the definitive information for making a

    13    judgment is clearly missing.  In fact, I feel that the

    14    greatest problem of all may lie in ascertaining

    15    whether there is a problem in the first place."  

    16                The workshop in which we will be

    17    participating over the next four days is designed to

    18    see how far we have progressed and what we still need

    19    to do.  Thank you.

    20                DR. GRIFFITHS:  Thank you very much, Bill. 

    21    I think we have been given the task, then, for the

    22    next few days, that is, to identify these concerns and

    23    issues associated specifically with neoplastic cells

    24    for production of vaccines.  We are focusing on

    25    vaccines.  And to identify some approaches in

                                                                         11

     1    determining this risk associated with their use and

     2    really how to move forward on these issues.  I think

     3    those are going to be our main concerns over the next

     4    four days.  

     5                So we now move on to the second talk and

     6    we are really looking at historical perspective to a

     7    certain extent here, the evolution of cell substrates

     8    used for the production of human biologicals.  Because

     9    as Bill already mentioned, they have been in use for

    10    a long time and there has been a tremendous amount of

    11    discussion over the years. And who better to give us

    12    this talk and to take us through from the beginning of

    13    the evolution of these cell substrates than someone

    14    who has been involved right from the very beginning,

    15    and the name is synonymous really with diploid cells,

    16    Dr. Hayflick.  So would he come forward and give his

    17    talk on the evolution of cell substrates for the

    18    production of human biologicals.  Dr. Hayflick?

    19                DR. HAYFLICK:  Although the first cell

    20    culture is generally attributable to Ross Harrison in

    21    1907, it was not until 1949 that contrary to the then

    22    existing dogma, which held that the polio virus could

    23    replicate only in neural cells, that Enders, Weller

    24    and Robbins showed that the virus could replicate in

    25    cultured human fibroblasts.  This set the stage in the

                                                                         12

     1    late 1950's for the development by Salk and his

     2    colleagues of an inactivated polio vaccine prepared in

     3    primary monkey kidney cells.

     4                I should define primary cells, because

     5    neophytes are using it to mean something entirely

     6    different and are oblivious to the fact that its

     7    original meaning still has a legal FDA definition.  A

     8    primary culture is simply a population of cells

     9    derived from impact tissue that has not undergone any

    10    subcultivations. After a few years of use of primary

    11    monkey kidney cells, it became apparent that these

    12    cells contained many unwanted viruses, some of which

    13    were lethal to humans.  At about this time in 1961, we

    14    described the development of normal human diploid cell

    15    strains that were free of contaminating viruses, and

    16    we suggested that they may be useful for the

    17    preparation of human virus vaccines.  It took about 10

    18    years before our suggestion became generally accepted

    19    in the scientific community.

    20                Normal human diploid cell strains have

    21    several interesting properties.  If I can have the

    22    first slide?  The properties that we described are

    23    listed here.  Very briefly, point number one, if

    24    derived from human embryos, they undergo about 50

    25    population doublings.  Contrary to the belief that

                                                                         13

     1    this is a serious limitation, the limit is actually

     2    about 50 million metric tons after 50 doublings. 

     3    Human diploid cells undergo a number of population

     4    doublings inversely proportional to donor age.  This

     5    suggested to us that the finite replicative capacity

     6    of cultured normal cells is an expression of aging at

     7    the cell level.  This notion received considerable

     8    experimental support in subsequent years and is now

     9    the basis for the field of cell senescence or

    10    cytogerontology.  

    11                We also found, of course, that the cells

    12    derived from this tissue had the diploid karyotype and

    13    are incapable of replication in suspension culture. 

    14    The next point, number four, human cell strains will

    15    not produce tumors when inoculated into the hamster

    16    cheek pouch or even when we directly introduced them

    17    into terminal human cancer patients, something that

    18    was relatively easy to do in the 1960's.

    19                Human diploid cell strains can be

    20    cryogenically preserved.  When, for example, WI38,

    21    which was the first highly characterized human diploid

    22    cell strain which we developed in 1962, is preserved

    23    at a particular doubling level and then reconstituted,

    24    the number of doublings remaining is equivalent to 50

    25    minus the number of doublings spent prior to

                                                                         14

     1    preservation.  The cells have an extraordinary memory

     2    and remember at what doubling level they are

     3    preserved, even after 37 years of storage in liquid

     4    nitrogen.  WI38 has been cryogenically stored longer

     5    than any other normal human or animal cell population.

     6    We also reported that human diploid cell strains had

     7    the broadest virus spectrum of any cell population

     8    known, and even heretofore unknown common cold viruses

     9    were isolated with these cells at that time.  

    10                As a result of this characterization, we

    11    suggested that cell populations in culture could be

    12    classified into three groups: primary cultures, which

    13    I have already described: cell strains, which are

    14    populations that have a finite capacity to replicate

    15    and don't produce tumors when inoculated into

    16    experimental animals, have the karyology of the tissue

    17    of origin and are anchorage-dependent; and the third

    18    category, cell lines, which are populations consisting

    19    of immortal cells that may produce tumors when

    20    inoculated into lab animals, don't have the karyology

    21    of the tissue of origin, and are usually anchorage

    22    independent.  

    23                Our description of these three fundamental

    24    cell classes in which we define normal cell strains to

    25    be mortal overturned a dogma that existed from the

                                                                         15

     1    turn of the century.  The dogma was that all cells

     2    placed into culture have the capacity to replicate

     3    indefinitely.  And if they do die, you simply do not

     4    have the proper culture conditions.  We upset this 60-

     5    year-old dogma by proving that normal cells do have a

     6    finite capacity for replication and showed further

     7    that normal cells have an intracellular counting

     8    mechanism and that the replicometer is located in the

     9    nucleus.  Without our having proven that there exists

    10    mortal normal cells, then the field of cancer research

    11    rooted in the concept of immortalization of normal

    12    cells clearly could not have developed.  

    13                Our efforts to define these classes of

    14    cells were not universally accepted, and

    15    terminological chaos has reigned for the last 40

    16    years. A good recent example can be found in the very

    17    first paragraph of the major document that you all

    18    received in the package of materials for this

    19    workshop.  It is titled, "A Defined Risks Approach to

    20    the Regulatory Assessment of the Use of Neoplastic

    21    Cells as Substrates for Viral Vaccine Manufacture." 

    22    Reference is made to a 1954 U.S. Armed Forces

    23    Epidemiological Board recommendation that "normal

    24    cells, rather than cell lines established from human

    25    tumors be used for the development of adenovirus

                                                                         16

     1    vaccines."  By normal cells, this Board meant immortal

     2    cell lines as I have just defined them but derived

     3    from normal tissue.  The cells lines referred to by

     4    the Board are not normal by any criterion.  In fact,

     5    the normal human diploid number was not known in 1954,

     6    and no normal cell had yet to be described.

     7                This confusion led Maurice Hilleman to

     8    prepare a live adenovirus vaccine in the Henley

     9    intestine cell line, which was thought to have been

    10    derived from normal human tissue, but was in fact a

    11    grossly abnormal cell line later identified as HeLa. 

    12    The six human recipients of this vaccine fortunately

    13    did not suffer any ill effects after 25 years of

    14    follow-up.  It was not until 1961 that we showed how

    15    to make normal human diploid cell strains that normal

    16    cells became available.

    17                The advantages of these cells are shown on

    18    the next slide.  In 1961, we suggested that these

    19    normal diploid cells have definite advantages over

    20    primary cell populations.  I won't go into detail on

    21    each of these items, but simply give you some details

    22    on two of them, the first and the last actually.  In

    23    respect to latent viruses, primary adult monkey kidney

    24    cells, as we all know now, harbor quite a number of

    25    serologically distinct latent viruses, several of

                                                                         17

     1    which were demonstrably lethal for humans.  The B

     2    virus and the Marburg agent both were lethal for 23

     3    people after accidental percutaneous inoculation of

     4    handlers of monkeys and their primary kidney cultures. 

     5    SV40, as was mentioned earlier, known to produce

     6    tumors in rodents and with the unknown potential to do

     7    so with humans was also an early contaminant of

     8    primary monkey kidney cells.  SV40 was also capable of

     9    transforming normal human cell strains into cancer

    10    cell lines in vitro.  SV40 was in fact inadvertently

    11    administered to several million recipients of early

    12    lots of inactivated and attenuated polio virus

    13    vaccines.  Latent viruses have never been found in

    14    WI38.  To this day, no evidence exists for an

    15    endogenous human oncogenic agent, latent virus,

    16    transforming principle, slow virus or endogenous

    17    intact retro in any human diploid cell derived human

    18    virus vaccine.

    19                I won't discuss cost of cell procurement,

    20    available cells, cell storage.  These are all fairly

    21    obvious to most of us by now.  But the last point I

    22    think is worth emphasizing, and that is that the

    23    essential point that we tried to make was that a

    24    diploid cell strain was more attractive than a primary

    25    cell population in its ability to be thoroughly tested

                                                                         18

     1    before use.  In a word, standardization.  Thus, the

     2    concept of cell standardization was first introduced. 

     3    Once standardization is achieved, cryogenic storage

     4    permits the acceptable strain to be held indefinitely

     5    for subsequent use.  It is not possible, practical or

     6    economic for primary cells to be similarly handled. 

     7                The argument holds equally well now for

     8    cell lines that all of you now take as perfectly

     9    obvious. But it was not so obvious from 1962 to 1972. 

    10    In the late 1950's and early 1960's, cell lines were

    11    generally believed to be a forbidden candidate for the

    12    production of human biologicals because they shared

    13    properties with cancer cells.  Our suggestion that

    14    human diploid cells could provide a safe and superior

    15    substrate for the production of human virus vaccines

    16    was met with considerable resistance for the next

    17    decade.  The major reasons for this resistance were

    18    the belief that hypothetical cancer viruses might be

    19    present and that spontaneous transformation might

    20    occur resulting in the presence of human cancer cells. 

    21    Latent viruses were such a problem with primary monkey

    22    kidney cells that a worldwide moratorium on the

    23    licensing of all polio virus vaccines was called in

    24    1967 because of death and illnesses that occurred in

    25    monkey kidney workers and vaccine manufacturing

                                                                         19

     1    facilities in Germany and in Yugoslavia.  The

     2    responsible virus, of course, was the Marburg agent.

     3                The arguments against the use of human

     4    diploid cells for the preparation of human virus

     5    vaccines were used as the basis for the philosophy of

     6    the Division of Biologics standards, the precursor of

     7    what is today CBER, that "the devil you know is better

     8    than the devil you don't know."  

     9                In 1961, together with Dr. Stanley

    10    Plotkin, we prepared the first human biological

    11    produced in a human diploid cell.  It was an oral

    12    polio vaccine which we showed to be both safe and

    13    efficacious.  By 1963, 7,000 people received a similar

    14    vaccine produced in WI38 with no safety problems.  

    15    Many of you use the concept of the master working cell

    16    bank in the production of biologicals, but few know of

    17    its origins.  In 1963, I suggested the concept of the

    18    two-tiered system of master and working cell banks for

    19    WI38, although these terms were not used.  The terms

    20    I used were master and working cell seeds.  I based

    21    this reasoning and the terms on the identical way in

    22    which virus seeds were then utilized.  The first

    23    publication of these procedures was made at the

    24    landmark Opatia, Yugoslavia conference in 1963.

    25                By the 1980's, when heteroploid and mortal

                                                                         20

     1    cell lines like CHO lines were used for the

     2    manufacture of human biologicals, the procedures for

     3    characterizing and handling WI38 were lifted over for

     4    use with cell lines with very few changes even to this

     5    day.  In the mid-1960's, several WI38 users expressed

     6    difficulties growing the cells that were traceable to

     7    variations in the medium reagents used by various

     8    laboratories.  As a consequence of this, we developed

     9    a practical standardized cell culture powdered media

    10    in kilogram lots, that unlike wet media was easily

    11    transported worldwide and used by many labs

    12    simultaneously and for which only two variables

    13    existed, water and serum.  As a result of our

    14    development of powdered media, the WI38 growth problem

    15    was quickly resolved and the use of powdered media has

    16    now become commonplace throughout the world.

    17                In the years following our development of

    18    WI38 in 1962, as a matter of national pride other

    19    countries produced their own strains.  The Medical

    20    Research Council in the UK produced MRC5.  In 1970,

    21    the Japanese made TIG1 and the Chinese made 2BS, et

    22    cetera.  Today, most of the world's virus vaccines are

    23    made on WI38 or similar strains.  It has been used to

    24    provide more individual doses of a biological than any

    25    other cell substrate ever used. This slide following

                                                                         21

     1    gives you a summary of or a rough idea of the extent

     2    of the use of these populations.

     3                I am frankly astonished to observe that

     4    after 10 years of debate on the risks of moving from

     5    frequently contaminated primary monkey kidney cells to

     6    virus free normal human cells that the time required

     7    to make the final leap to abnormal cell lines for

     8    producing human biologicals occurred in the 1980's in

     9    a matter of weeks.  Today, the molecular mechanism for

    10    our finding that normal cells have a replicometer has

    11    been substantially described as a result of a

    12    fascinating story that time constraints prevent me

    13    from describing.  The explanation is briefly described

    14    on the next slide and consists, as most of you here

    15    know by now I am sure, of the shortening of telomeres

    16    in normal cells each time DNA replicates.  And for

    17    those cells that are immortal, they escape the

    18    inevitability of telomere shortening by switching on

    19    an enzyme called telomerase that adds on the

    20    nucleotides at each round of DNA replication, thus

    21    maintaining the telomere length constant.

    22                The remaining question, as I just

    23    described, in respect to telomere shortening was the

    24    discovery of this enzyme called telomerase, an enzyme

    25    that contains an RNA template and a catalytic moiety,

                                                                         22

     1    among other components.  This is the only enzyme that

     2    contains a reverse transcriptase and is part of normal

     3    cell biology.  90 percent of cancer cells express the

     4    enzyme.  Only normal stem cell populations do so.

     5                As for the use of abnormal and mortal cell

     6    lines for the production of human vaccines, that has

     7    already been done.  The Vero cell line derived from

     8    the kidney of an African green monkey kidney has been

     9    licensed in France, Belgium, the United States and

    10    Thailand for the production of both live and

    11    attenuated polio and rabies vaccines.  There are now

    12    more than 20 million vaccinees.  The Chinese have made

    13    a Japanese encephalitis vaccine in the abnormal cell

    14    line, BHK21.  Recombinant vaccines have been licensed

    15    for hepatitis B grown in a CHO cell line, and phase

    16    III trials are currently underway for an HIV vaccine

    17    in these same cells.  

    18                There are other immortal cell lines that

    19    bear consideration as substrates for the preparation

    20    of human virus vaccines. About two years ago, I

    21    initiated a culture of fibroblasts from a skin biopsy

    22    that I took from my knee.  This culture was the first

    23    to be immortalized by transpection with human

    24    telomerase reverse transcriptase.  Three other human

    25    cell strains were similarly treated and these results

                                                                         23

     1    were published in Science in January of 1998.  These

     2    immortalized normal cell populations have now

     3    undergone well over 400 population doublings.  This

     4    opens up enormous opportunities, not only to

     5    immortalize WI38, but many other normal cell types for

     6    applications like -- for exploitation actually as

     7    vaccine substrates among many other potential uses.

     8                Another category of immortal cell lines

     9    that merits consideration are those transformed by

    10    agents other than viruses.  SUSM1 is a cell line

    11    derived from normal human lung that we transformed

    12    with a chemical carcinogen.  KMSD6 is also an immortal

    13    cell line derived from normal human fibroblasts and

    14    transformed by exposure to cobalt 60 gamma radiation. 

    15    I might add that contrary to the belief of some, early

    16    passage WI38 does exist in ample quantities, and

    17    anyone wishing to obtain these cells should feel free

    18    to contact me.  

    19                Thus, there exists several methods for

    20    transforming well-characterized normal human and

    21    animal cell populations to immortal cell lines using

    22    at least three proven methods.  What should not be

    23    overlooked, however, in our zeal to condemn any

    24    immortal abnormal cell line from consideration as a

    25    human virus vaccine substrate is the fact that

                                                                         24

     1    downstream processing has reached such a level of

     2    efficiency that the preparation of a human virus

     3    vaccine containing only the purified moieties capable

     4    of eliciting an immune response is possible now. 

     5    Affinity technology in particular is clearly capable

     6    of meeting this exacting goal.  Nevertheless, as we

     7    all know, economic considerations will surely guide

     8    most of these decisions.  Thank you very much.

     9                DR. GRIFFITHS:  Thank you very much

    10    indeed. We do have time for one or two questions if

    11    people want to start the ball rolling and ask Dr.

    12    Hayflick any questions.  I don't see any immediate --

    13    oh okay.  Sorry, I can't see your name from here. Can

    14    you introduce yourself?  Is there a microphone?

    15                PARTICIPANT:  I am Jim Cook from Chicago. 

    16    What all types of animals have WI38's been inoculated

    17    into for tumorigenicity testing besides the hamster

    18    cheek pouch?

    19                DR. HAYFLICK:  Oh my. 

    20                PARTICIPANT:  I mean, have they been put

    21    into newts.  Have they been put into other kinds of

    22    immunocompromised animals?  

    23                DR. HAYFLICK:  All immune compromised lab

    24    animals -- mice, rats -- 

    25                PARTICIPANT:  What about things like the

                                                                         25

     1    odd observation that Balb C3T3's when hooked onto

     2    plastic disks will make tumors in newts?  Has anything

     3    unusual like that been done with WI38's?

     4                DR. HAYFLICK:  Yes.  And to the best of my

     5    knowledge, there has been no tumor formation.  There

     6    have been occasional reports of nodules where the

     7    cells will replicate to a point where they reach a

     8    macroscopic size.  But usually what happens is that

     9    they fail to grow beyond that state.  

    10                DR. GRIFFITHS:  Johannes?

    11                PARTICIPANT:  Johannes Loewer.  You

    12    mentioned that the human diploid cell lines have a

    13    very broad host range for many different viruses.  Is

    14    it known whether neoplastic cells have even a broader

    15    host range or is it more or less similar to diploid

    16    cells?

    17                DR. HAYFLICK:  You are talking about cell

    18    -- immortal cell lines?

    19                PARTICIPANT:  Yes, immortalized cell lines

    20    or neoplastic cell lines.

    21                DR. HAYFLICK:  From human material?

    22                PARTICIPANT:  Yes.

    23                DR. HAYFLICK:  To the best of my

    24    knowledge, the human diploid cell strains have a far

    25    broader virus range than the immortal cell

                                                                         26

     1    populations.  Most or many of the rhinoviruses were

     2    first discovered on WI38 and later on MRC5.  But the

     3    fact that they could ultimately be adapted to growth

     4    in immortal cell populations was also revealed. 

     5    However, I think it is still fair to say that normal

     6    human diploid cells are the most sensitive cell

     7    population to most of the viruses that are now well

     8    characterized.  

     9                DR. GRIFFITHS:  Any other points that we

    10    should take?  Because we are right on time, so we are

    11    really doing quite well this evening.  If not, then

    12    thank you very much indeed.

    13                DR. HAYFLICK:  Sure.  

    14                DR. RABINOVICH:  Our next speaker is Dr.

    15    Elwyn Griffiths from the World Health Organization,

    16    who has also worked in this field for -- I won't even

    17    venture to guess how many years.  He is going to talk

    18    about major issues associated with cell substrates.

    19                DR. GRIFFITHS:  Thank you very much.  I

    20    must say I was very impressed when I got this badge

    21    actually from the organizers. I mean, I don't know if

    22    they get a prize for the number of flags that is on

    23    it.  It is very impressive.  I mean, I was only asked

    24    to do this particular talk and stand in just a couple

    25    of weeks ago. I think it should have been Dr.

                                                                         27

     1    Petricciani who would have done this and done it far

     2    better than I am sure.  But in standing in, perhaps I

     3    should have stood in for the patron committee or

     4    something just to have another badge on here.

     5                Anyway, for this evening to a certain

     6    extent this talk really is something of a trailer to

     7    the more detailed discussion we are going to have over

     8    the next few days, and really an extension of the

     9    points already touched upon by Dr. Hayflick.  

    10                The way I thought I would like to do it is

    11    to take a different stance in the beginning.  Before

    12    dealing with the major issues which really revolve

    13    around these different cell substrates, to start with

    14    a reminder.  And I think I would like this reminder to

    15    be borne in mind throughout the meeting that what we

    16    are dealing with has great benefits for mankind.  I

    17    mean, the benefits of the vaccines produced on cell

    18    substrates.  There has been enormous success in cell

    19    substrates for producing vaccines.  These particular

    20    vaccines have had a tremendous influence on human

    21    health.

    22                I would just want to pick up on polio, for

    23    example, because that is something which the WHO

    24    clearly has a -- and everybody else, I guess -- has a

    25    major interest in.  The last case of polio in the

                                                                         28

     1    whole of the Americas, that is PAHO, was in 1991.  In

     2    the Western Pacific region, it was in March of 1997. 

     3    The world is moving towards global eradication of

     4    polio in roughly about -- it is expected in 2000.  I

     5    am not sure if 2000 is realistic -- 2000, 2002 or 2003

     6    or so.  You can see the difference from -- the top one

     7    is 1988 really to 1998.  There are patches left in

     8    1998, and this is gradually getting smaller and

     9    smaller.

    10                So essentially, global eradication of

    11    polio, which will have enormous benefits for mankind,

    12    has been achieved primarily through live polio

    13    vaccines.  These vaccines are produced on a number of

    14    cell lines -- primary monkey kidney cells, diploid

    15    cells, and also in vero cells.  I think it is true to

    16    say on a global perspective that the majority of the

    17    producers are producing on primary monkey kidney

    18    cells.

    19                Now having said that, I just wanted to

    20    have that borne in mind.  When we deal with the

    21    issues, we really do have some major benefits from

    22    these products. So it really is how much of a

    23    risk/benefit equation which we have to deal with here

    24    and not forget the actual benefits by focusing too

    25    much on the issues.  That isn't to say the issue is

                                                                         29

     1    not important.

     2                The major issues associated with the use

     3    of cell substrates really relate to safety. I mean,

     4    that is our main problem here.  This is something

     5    which has been really extensively debated over the

     6    last 40 years or so, with the acceptance of cells for

     7    production really being a hot topic as they come up

     8    from time to time -- primary cells, diploid cells and

     9    continuous cell lines, as Dr. Hayflick already

    10    mentioned.  The perceived risks being attributed to

    11    contaminants coming from the cells.  The main ones of

    12    interest really being viral contamination, DNA, and

    13    transforming proteins.

    14                The whole of these issues have been

    15    discussed -- I wouldn't say ad nauseam, but they have

    16    been extensively discussed over -- what is it, from

    17    1967 -- over 30 years or so. And what is interesting

    18    is when you look back over this, this is a selection

    19    of meetings.  I am sure there are many, many more

    20    meetings.  You see, I am only just taking this from

    21    1967.  It is the same players essentially or the same

    22    cast as we have for this meeting.  We have the FDA. 

    23    The IABS features strongly in this, the WHO, the NIH. 

    24    So they are all there really right from the very

    25    beginning.

                                                                         30

     1                The main point here is that some of the

     2    issues -- I mean, each meeting will have a special hot

     3    topic, if you like.  In 1967, it was diploid cells. 

     4    Then in 1978, that meeting was organized essentially

     5    to see whether we could move forward with the use of

     6    continuous cell lines, primarily because namalva cells

     7    have already been produced by the Wellcome Institute

     8    for producing interferon.  And I suppose the 1978 Lake

     9    Placid conference gave us a cautious yes to the use of

    10    continuous cell lines for the production of

    11    biologicals.  And I am broadening this now not just

    12    for vaccines, but for the production of biologicals.

    13                But there was still a lot of "yes, but" at

    14    that meeting. I wasn't there, and I am sure that John

    15    and other people who would have been there would say

    16    the same thing.  It seems that it was, yes, we can go

    17    forward, but, but.  And certainly not in the USA, I

    18    guess.  That was the sort of general feeling at that

    19    time.  It was really the meeting in 1986, the WHO

    20    meeting, a study group in Geneva, which really allowed

    21    the field to move forward in so far as use of

    22    continuous cell lines were concerned for the

    23    production of biologicals. 

    24                At that time, there was an agreement from

    25    this particular study group that continuous cell lines

                                                                         31

     1    could be used for the production of biologicals

     2    provided that the level of residual DNA or residual

     3    host cell DNA was kept below 100 picograms of DNA per

     4    dose. That is the origin of the magic figure which has

     5    been used for the last 10 years or so.  And this

     6    really allowed -- this decision really allowed the

     7    field to move forward in the expansion of the

     8    biotechnology industry to produce recombinant DNA

     9    biologicals in continuous cell lines.  This has been

    10    the situation for the last 10 years or so or more now. 

    11                At the last meeting of the WHO, which was

    12    1996, a WHO and NIABS consultation at Merieux

    13    Fondation in Annecy, that is the time where the field

    14    was reviewed, if you like.   Where are we after 10

    15    years or so of using this figure of 100 picograms? 

    16    Incidentally, that figure of 100 picograms was used as

    17    the guidance for all the national regulatory

    18    authorities. The FDA went a little bit further and

    19    went down to 50 at one time, less than 50 picograms. 

    20    Industry was pushing toward the limited detection. 

    21    And it is interesting to see the European

    22    Pharmacopoeia has a limit for recombinant hepatitis B

    23    vaccine produced in continuous cell lines of 20

    24    picograms per dose, which is very low and very

    25    stringent.  This meeting in 1996 was to review that,

                                                                         32

     1    and things have changed a little since then and I will

     2    come to that later on.

     3                The present meeting, of course, really

     4    focuses on cell substrate for vaccine production, not

     5    for biologicals in general. I think there is this

     6    distinction which I would like to make later on. We

     7    are now moving to potential use of overtly neoplastic

     8    cells for vaccine production, and I think this is a

     9    very timely meeting.

    10                The sort of landmarks -- and I am really

    11    going over a little bit the ground which has already

    12    been covered by Dr. Hayflick.  These were sort of, I

    13    think, the sort of landmarks as you might say.  The

    14    gradual overcoming of the hurdles -- the use of

    15    primary cells for vaccine production in polio really

    16    exemplifies that in the 1950's.  In the 1970's,

    17    diploid cells were accepted for vaccine production,

    18    but not without problems as we have already heard. 

    19    Then in the 1980's, after the WHO meeting, continuous

    20    cell lines then became acceptable for vaccine

    21    production -- namalva cells for interferon, vero cells

    22    and so on -- hybridomas for monoclonal antibodies

    23    before then of course, and recombinant DNA

    24    derivatives.

    25                Now the progress during this period of the

                                                                         33

     1    1960's to the 1990's has been really on a number of

     2    fronts.  I think it is fair to say -- I can say this

     3    because the WHO isn't really a regulatory agency. But

     4    I think one can now say that regulatory decisions are

     5    made more on a scientific basis and not on opinions as

     6    such.  And the progress that has been made in

     7    scientific knowledge really over this period is

     8    enormous.  We have to remember that.  I just put a few

     9    up here to flag them -- new diagnostic procedures,

    10    let's say, such as PCR; better understanding of

    11    molecular mechanisms of pathogenicity; and new

    12    concepts, the concept of validation has been

    13    introduced.  

    14                So we are now in a position -- it is

    15    perhaps unfair to say that the regulatory decisions

    16    can be made more on a scientific basis.  We have got

    17    more scientific basis to make the decisions.  I think

    18    that is perhaps the main point to make there.  We can

    19    make decisions, better informed decisions, and I think

    20    this is an important part of the gradual move to

    21    acceptance of different cell substrates.

    22                Part of this process as well has been co-

    23    defining, if you look -- perhaps co-define is too

    24    strict.  Having regulations and guidelines and

    25    recommendations for production and quality control of

                                                                         34

     1    these products using cell substrates has also been a

     2    major development since that time, both on a national

     3    basis, on a regional basis, such as in the European

     4    Union and the European Commission, and the WHO, of

     5    course, on a global perspective. And I think what we

     6    are doing is really learning how to manage the risks

     7    to take advantage of the benefits.  And I think that

     8    is the important -- what we want to do to take the

     9    field forward.

    10                Very briefly then I will go through just

    11    to remind ourselves and really to reiterate what Dr.

    12    Hayflick has said.  The primary cells -- I mean, I

    13    will just mention the disadvantages here in the sense

    14    that many people tend to use them because they tend to

    15    be easy to prepare and so on.  But they really do have

    16    this major disadvantage of microbial contamination and

    17    especially of viruses, of course.  

    18                Can we go to diploid, the next one, and

    19    just go through these very quickly.  Again, and it has

    20    already been eluded to, the great advantage of using

    21    diploid cells is that they can be well characterized

    22    and standardized. And so production then becomes based

    23    on well characterized cell banks, the master cell bank

    24    and a working cell bank.  And this is true also for

    25    the continuous cell line.  

                                                                         35

     1                But they all have their advantages and

     2    disadvantages.  I just want to focus on the

     3    advantages.  An infinite life span for the continuous

     4    cell lines, so you can keep them going forever I guess

     5    really.  And again, production on well characterized

     6    and standardized cells for an indefinite time basis

     7    really.  

     8                Now when you look at the disadvantages

     9    amongst these cell lines, the primary cells had the

    10    problem with the contamination.  But the main

    11    disadvantage of the continuous cell line is that many

    12    do express endogenous viruses and there has always

    13    been this concern over tumorigenic potential, should

    14    we say, associated with cellular DNA.  

    15                The main three risks then with these

    16    different cell lines for producing biologicals are

    17    contaminating viruses, and we must include here, the

    18    TSEs, the transmissible spongiform encephalitis

    19    agents, whatever that may be in the end, residual host

    20    cell DNA, and growth-promoting proteins. And as I

    21    said, the trick really is how to manage these.  And by

    22    developing procedures, we can actually move forward

    23    onto guidelines and so on.

    24                In fact, there are three principles

    25    essentially really to production of biologicals.  And

                                                                         36

     1    these are really the basis for moving forward on all

     2    these cell lines.  Control of the starting material,

     3    such as the baseline data, characterization of the

     4    host cell, and so on, if you are able to do that

     5    depending on what sort of cell line you're using. 

     6    Then you control the manufacturing process itself,

     7    this is for adventitious agents and so on, and also

     8    control of the final product.  And that includes tests

     9    performed during development.  Because not all tests

    10    will be carried out on a routine lot-to-lot basis. 

    11    There will be some testing on development.  And then

    12    there is a subset of those tests, which will be

    13    carried out on a routine basis. These are the lot-to-

    14    lot releases.

    15                Now historically, assuring the quality and

    16    safety of what you might call traditional biologicals

    17    has been what I call disaster-led.  And you can make

    18    a nice list of all the problems we have had over the

    19    years and you find that control procedures are really

    20    established or altered following some major mishap,

    21    let's say.  For example, Creutzfeldt Jakob disease

    22    from the bottom there in recipients of pituitary-

    23    derived growth hormone.  I mean, that meant that we

    24    weren't now going to use the pituitary-derived growth

    25    hormone and move over to recombinant. So it is these

                                                                         37

     1    sorts of issues which really trigger the traditional.

     2                But if you look at the situation which I

     3    call applied to biotechnology products -- I mean novel

     4    biotechnology products.  I mean, it is all

     5    biotechnology I guess really, but I am thinking of

     6    recombinant DNA, monoclonal antibodies, cell cultures

     7    and so on.  The approach has been somewhat different. 

     8    Guidelines have been established early-on in the

     9    development of the new field to try and pre-empt any

    10    disaster.  I mean, the idea here is that we think of

    11    what the problems might be and try to make sure that

    12    we don't land ourselves in these sort of situations. 

    13    And developing guidelines and points to consider,

    14    whatever you might want to call them -- and the

    15    different countries and different agencies call them

    16    different things -- I think these have been

    17    instrumental in establishing safety and quality of

    18    these newer type of biologicals, the biotech products

    19    if you like.  Because they are by far the best

    20    characterized and best purified biological medicines

    21    in clinical use.  

    22                You see during this period in the 1970's

    23    and 1980's, guidelines on production and control or

    24    you might call them points to consider have been

    25    derived by many agencies. The main ones being the CBER

                                                                         38

     1    FDA points to consider, Japan has guidelines, the

     2    European CPMP biotech working group, those are the

     3    European Commission guidelines, and WHO. Now what is

     4    interesting is that the majority of these guidelines

     5    do have the benefit of mutual consultation during

     6    their development.  If you look at them in some

     7    detail, you find that essentially they all say the

     8    same thing, but they are actually viewing it from a

     9    slightly different perspective with slightly different

    10    words.  The ICH guidelines, for example, on cell

    11    substrates, that actually deals with more of the

    12    characterization of the cell lines and so on. So we

    13    find that these different type of guidelines do all

    14    have the same thrust, but they take it slightly in

    15    different perspectives. But they all say essentially

    16    the same thing. 

    17                Now as far as WHO is concerned, the first

    18    requirements for cell cultures for production of

    19    biologicals was the 1959 requirements for primary

    20    monkey kidney cell production of polio vaccine.  Now

    21    all the other major agencies -- FDA and in Europe and

    22    the individual national states -- would also have

    23    their requirements as well.  But WHO is the embodiment

    24    of a global situation. And using the primary cells,

    25    monkey kidney cells, for polio vaccine really did lead

                                                                         39

     1    to some confidence in developing other primary cell

     2    lines for producing a range of vaccines.  And these

     3    are still in use.  We mustn't forget that.  The

     4    primary cell is still a major source -- for example,

     5    the chick embryo cells for measles vaccine and mumps

     6    vaccine as well. So the development of these at this

     7    point was a sort of major move forward.

     8                Now I am not going to try and sort of

     9    compare and contrast the different guidelines, because

    10    I don't think that is particularly helpful.  But what

    11    I will try and do is just to scan briefly through the

    12    guidelines and points to consider which have been

    13    developed in relation to the cell cultures.  These,

    14    again, reflect considerable discussion. I mean, I have

    15    just put the WHO ones. The first one there was the one

    16    for oral OPV, and these are updating. As time goes on,

    17    there is always a need for updating because some

    18    technology changes and things move forward.  

    19                I have already mentioned the meeting of

    20    the study group in 1986.  It was published in 1987,

    21    which allowed the field to move forward, and that was

    22    the acceptability of cell substrate for production of

    23    biologicals. And then in 1987 as well, there was the

    24    continuous cell line for the production of

    25    biologicals.  The FDA, the ICH, the CPMP also have

                                                                         40

     1    their guidance documents as well specifically on cell

     2    substrates.

     3                Now I just want to say something about the

     4    situation of 1998 as far as the WHO cell substrate

     5    document is concerned.  This document actually

     6    reflects considerable international consultation. I

     7    think this is probably the latest one or major

     8    international one. It was published last year, but the

     9    actual meeting -- there was a meeting in 1996 with

    10    WHO, IBS, and we had the benefit of input from the

    11    ICH, which went into great detail about how we should

    12    control the quality of the biologicals produced on

    13    primary, diploid and continuous cell lines. It covers

    14    the whole three for production of viral vaccines and

    15    the biologicals.  It is not restricted to viral

    16    vaccines. And it is interesting that this document is

    17    very clear in that it encourages the move away from

    18    primary cells to cell substrates which can be

    19    generated from well characterized cell lines.  As we

    20    have already mentioned -- Dr. Hayflick mentioned this

    21    idea of the well characterized cell line goes back a

    22    long time, probably to the meeting -- I think there

    23    was a meeting in Zagreb where this was developed of

    24    having the master cell bank and so on.  

    25                Now in this particular document, there has

                                                                         41

     1    been a major change in certain parts of the way we

     2    handle the control of some of these cell lines. There

     3    is, for example, for well characterized diploid cells,

     4    a relaxation of the karyology and tumorigenicity

     5    testing and so on. But I just want to touch on one

     6    particular point which is important for the next part

     7    of the issues. And that is related to the DNA and the

     8    viruses.  The main risks -- in this document, we still

     9    see that the main risks still are for the continuous

    10    cell lines residual DNA and maybe growth promoting

    11    proteins, and I will come to that in a moment, and for

    12    all cell lines, contaminating viruses.

    13                Now as far as the DNA is concerned, there

    14    has been a major departure in the previous

    15    recommendation regarding residual DNA.  And this is

    16    really due to a reevaluation of the situation and new

    17    data over this decade from 1986.  But also new

    18    technologies have appeared on the scene -- gene

    19    therapy and DNA vaccines, which actually are involving

    20    injection or administration of in the case of DNA

    21    vaccines quite large quantities of DNA. So one has to

    22    be consistent here.  Either DNA is really a big

    23    problem or it isn't.  Or is it the type of DNA?  And

    24    this is the issue.

    25                If you look at the 1986 summary, I guess

                                                                         42

     1    really, the cause of concern for that time was

     2    transmission of oncogenes. And the 1986 consultation

     3    provided this way forward by saying that if you can

     4    get your DNA down to less than 100 picograms per dose,

     5    then there is no realistic risk, and this is what

     6    everybody went forward on.  The perceived problem was

     7    really not DNA post-op, but the specific sequences of

     8    DNA which may be encoding oncogenes and the possible

     9    insertion of endogenous or host cell protooncogenes or

    10    inactivation of suppressor genes by the DNA.  The DNA

    11    itself was largely ignored, I think, and that is were

    12    the move forward is on DNA vaccines. We can go forward

    13    there because although there is a risk, the acceptance

    14    of this minimum risk is something which has been

    15    actively discussed and debated for the last two or

    16    three years.

    17                So the figure now is that we now think --

    18    by we, the current opinion.  I think this is

    19    everybody.  As I say, everybody has partaken into

    20    these general discussions here.  They are based on

    21    current state of knowledge.  The suggestion is that

    22    DNA from continuous cell lines can be considered --

    23    the DNA itself now -- as really a cellular contaminant

    24    rather than itself as a significant risk factor

    25    requiring removal to extremely low levels which needed

                                                                         43

     1    validation and testing of each lot and so on.  And the

     2    relaxation now is that we think that a figure of 10

     3    nanograms of residual DNA from a continuous cell line

     4    will apply.  And this applies primarily, of course, to

     5    purified recombinant DNA biologicals.  

     6                This is something which I want to touch on

     7    here. What we are talking about is removal of DNA to

     8    reasonably low levels.  But the guidelines do make the

     9    point that residual DNA from continuous cell line may

    10    pose a higher risk if it might include infectious

    11    retroviral or provirion sequences, and then we may

    12    well still have to go down to lower levels.  Now this

    13    is something I am sure that is going to come up in

    14    this meeting.  What is the actual level which is

    15    applicable to vaccines.  Can you actually go to that

    16    lower level.

    17                I don't want to say much about the growth

    18    of multi-proteins.  These are sort of more or less

    19    dismissed, I suppose, in the WHO guidelines. Growth

    20    factors that are produced by cells, of course, the

    21    risk is very limited.  Yes, they do affect cell

    22    growth.  It is usually transient and reversible.  They

    23    don't replicate and many are inactivated in vivo.  So

    24    the general thrust of the argument there was that they

    25    are not such a problem as we originally thought.  

                                                                         44

     1                Now that might be different today, but the

     2    viruses remain the major and real concern for all cell

     3    types.  The test for viral contamination really is a

     4    major part of cell bank characterization.  In the

     5    guidelines, in the WHO ones, it covers the primary

     6    cells here.  Now, here we are taking cells directly

     7    from animals.  Sometimes in the wild animals for polio

     8    -- from monkeys for polio. And quite clearly now, we

     9    have to move into a situation if you are going to use

    10    animals that they must be healthy animals subject to

    11    veterinary and laboratory monitoring, very closed

    12    colonies wherever possible, not from the wild if

    13    possible.  And animals, if they do come in from the

    14    wild, then they really have to be quarantined and

    15    appropriately monitored in the time before you

    16    actually take the cells for preparation of vaccines. 

    17    And serological screening of donor animals for

    18    relevant pathogens.  

    19                Now you might say what is a relevant

    20    pathogen.  Well that really is changing as time goes

    21    on, of course.  The problem with doing this for a

    22    primary cell is that you have to do it for every

    23    vaccine batch.  I mean, it is not the case of doing it

    24    once.  You need to test the cells vigorously.  It is

    25    an awful lot of work.  And of course you can only test

                                                                         45

     1    for a certain number of known viruses.  You can have

     2    a sort of catch-all, but there are mishaps. As we

     3    know, SV40 was missed, and this is an issue that comes

     4    back to haunt us as time goes on.

     5                With a cell bank, we can actually do this

     6    testing, extensive testing for exogenous and

     7    endogenous agents, once only.  You can do it and then

     8    you can make sure that this is the master cell and

     9    then you move on to the working cell bank.  The

    10    further testing really is on the working cell bank or

    11    production cells really to detecting common

    12    adventitious agents, and that could be sort of a

    13    catch-all.  It is not such a major issue.

    14                It is interesting that the working group,

    15    the WHO study group, in 1986 valued the well

    16    characterized cell banks so much that it recommended

    17    the establishment of a well characterized cell line

    18    that would be available to national control

    19    authorities and manufacturers of biologicals globally.

    20    And, in fact, the WHO master cell bank for vero cells

    21    was established following the recommendation by this

    22    group.  It comes from African green monkeys and it was

    23    selected -- the vero cell was apparently selected

    24    because of the immediate prospect for improving the

    25    quality and quantity of several vaccines.  Now it has

                                                                         46

     1    been used but perhaps not used as widely as was

     2    thought at the time.  

     3                Now the strategy for preventing viral

     4    contamination then is basically what all the documents

     5    go through.  Tests on starting materials -- not just

     6    the cells.  We have to remember that some of the

     7    agents will come in from the reagents -- the serum and

     8    so on.  We have to be very aware of excipients as

     9    well.  

    10                Evaluation of production processes.  That

    11    is the ability to remove or inactivate potential viral

    12    contaminants. That is your validation essentially. 

    13    This is the concept where validation has come in.  And

    14    then tests on final product or appropriate

    15    intermediates.  And essentially what people think is

    16    that it is not a single approach.  One approach isn't

    17    sufficient.  It is the combination of approaches which

    18    gives you this degree of security.

    19                Now if you look at the WHO guideline, and

    20    I think this is true of most of the points to consider

    21    in the guidelines, they are really focusing on biotech

    22    products, that is, highly purified recombinant DNA

    23    products essentially.  Now if you look down at the

    24    bottom, the purified biotechnology product -- you can

    25    have a very robust virus clearance system that you can

                                                                         47

     1    check for validation of virus clearance downstream and

     2    so on.  So even if you are -- I mean, I don't think

     3    there are any biotech products produced or recombinant

     4    DNA products produced in primary cells, but even if

     5    you were doing it that way, you could be sure that --

     6    you could actually be sure that everything was going

     7    to be cleared up by the system. When you come to virus

     8    vaccines, you have much less purification, and the

     9    possibility of virus/virus interaction during

    10    production needs to be borne in mind, and this is an

    11    issue which will come up later in the meeting.

    12                So virus vaccines, though, are a different

    13    ballpark really from the general biologicals, which

    14    everybody -- you know, most of these guidelines are

    15    focusing on clearance of DNA and clearance of viruses,

    16    looking at the characterization of cells.  When you

    17    move to virus vaccines, there is less you can do.

    18                Now we mustn't think, of course, that we

    19    know everything at this stage.  I mentioned that we

    20    have had enormous progress in the last 30 years or so. 

    21    We must be prepared for surprises.  Two recent events

    22    I think have raised awareness of the challenge there

    23    is in dealing with viral contamination of cell lines

    24    and vaccines which are used in their preparations and

    25    the consequences.  One was the detection of very low

                                                                         48

     1    levels of reverse transcriptase in chicken cell-

     2    derived vaccines using this newly developed assay --

     3    we call it PERT assay.  This is from -- like measles

     4    vaccine is an example and mumps is another example. 

     5    All the vaccines produced in eggs will contain very,

     6    very low levels of RTAs.  And this, of course, when it

     7    first was discovered raised a lot of alarm bells. 

     8    People were thinking, yes, there must be retroviruses

     9    in here and so on. And this really led to quite a lot

    10    of activity trying to show where this activity was

    11    coming from.  Is it real retrovirus or what do we have

    12    there?  What is the problem?  Or do we just have some

    13    non-specific activity which really looks like reverse

    14    transcriptase?  And much work -- and WHO was very much

    15    interested in this, of course, because if national

    16    control authorities of one country ban a vaccine

    17    because it is considered to be potentially unsafe,

    18    this has ripples throughout the whole of the vaccine

    19    community. I mean, what is not safe for one set of

    20    kids in one country must be unsafe for other kids of

    21    course.

    22                So there was a lot of work to try and make

    23    sure that this activity -- where did it come from and

    24    so on.  And manufacturers and the national control

    25    authorities were very active here.  It has now been

                                                                         49

     1    shown to be particle associated, but extensive studies

     2    have shown no transmission or productive infection,

     3    and that of course is good news.  But as we will hear

     4    later on in the meeting, what about the potential for

     5    interaction between these particles and some of the

     6    viral vaccines -- the actual virus which is used for

     7    producing the vaccine during growth.  We need to be

     8    sure -- I am thinking of pseudotypes and so on here. 

     9    This is an issue which we need to address in this

    10    meeting.

    11                The other surprise was the detection of

    12    SV40 genome in rare human tumors.  This is something

    13    which has come back to haunt us after 30 years or so. 

    14    I am sure you all know that SV40 was a contaminant of

    15    some of the early batches of primary rhesus monkey

    16    kidney cells used to produce polio vaccines.  This is

    17    no surprise.  During the 1950's, these were actually

    18    used in a large number of people -- in the millions. 

    19    There was follow-up with that to see whether they

    20    actually caused any problems, and nothing much

    21    materialized. And then suddenly about three or four

    22    years ago, the SV40 sequences were picked up in

    23    various rare human tumors.  That raised the issue of

    24    was the vaccine -- was the polio vaccine made in

    25    primary kidney cells actually still transmitting SV40

                                                                         50

     1    or SV40 sequences.  Because right in the beginning

     2    when SV40 was discovered, measures were introduced

     3    very quickly by national regulatory authorities to

     4    exclude SV40 from polio vaccines. For example, I am

     5    thinking here of the WHO.  The 1959 requirements had

     6    to be modified and they were updated and the

     7    regulations in all the major national authorities were

     8    also changed to exclude SV40.  And for more than 30

     9    years then, polio vaccine made in primary monkey

    10    kidney cells have been shown to be free of SV40.  Now

    11    by shown to be free, it was shown to be free by

    12    technology of the 1960's, I guess.  I mean, it is old

    13    technology.  And the question which everybody was

    14    rather anxious about was were we actually missing

    15    something here?  And then the application of new

    16    highly sensitive PCR techniques for detecting SV40

    17    genome was then introduced to see whether batches of

    18    vaccines were actually carrying any SV40 sequences. 

    19    This is work carried out by CBER and also by NIBSC,

    20    and we are looking back on historical samples as being

    21    quite clear that the methods introduced in the 1960's

    22    were effectively excluding SV40.

    23                Now if I can sort of come back to where I

    24    started from in the beginning in relation to polio,

    25    the polio eradication is going to be with us in about

                                                                         51

     1    two or three years, and this is really an end-game and

     2    how do we move forward here.  Once polio has been

     3    eradicated, transmission of wild polio would be

     4    stopped.  And the global certification of polio

     5    eradication will come in around 2005, I guess.  But

     6    there will be continued use of the OPV expected for

     7    maybe 5 or 10 years. It is unclear.  The end-game here

     8    is unclear really and how we move forward.  But WHO is

     9    looking towards cessation of immunization with OPV

    10    around 2005 or 2010.  USA will be moving to IPV in

    11    2000 -- in January of the year 2000. So the question

    12    is do we need to do anything about the vaccine, the

    13    OPV.  And we need now, because -- I think now as we

    14    move to eradication, I think we do need to be

    15    absolutely sure of the vaccine and introduce an

    16    additional level of security.  This is provided by

    17    ensuring that SV40 sequences are absent from polio

    18    virus seed.  Because during the actual survey of the

    19    vaccines, one seed I think from a manufacture was

    20    found to be positive.  The seed was positive.  There

    21    was no actual live SV40 there.  There were some SV40

    22    sequences there.  It is considered that it is much

    23    better to have the seed free of SV40.  And we are now

    24    actually updating the original requirements -- this

    25    will come up this year -- making sure that all seed

                                                                         52

     1    will be tested for absence of SV40 sequences.  The

     2    primary monkey kidney cells for production will now

     3    have to come, I think, from closed, intensely

     4    monitored colonies of animals and not from the wild. 

     5                As time goes on, of course, new viruses

     6    are discovered and new problems arise.  The foamy

     7    virus has been identified as one that we should be

     8    really sure is absent from these vaccines.  So

     9    updating is something which we are moving forward

    10    towards in this end-game for on the polio. 

    11                What is the next step then?  What are the

    12    challenges for the future?  The use of neoplastic

    13    cells or novel cell lines for vaccine production I

    14    think is really a challenge.  And I think one could

    15    say as with any new technology, new sets of safety

    16    issues are generated for consideration. Not just by

    17    regulatory agents but by industry and regulatory

    18    agencies together.  I mean, everybody is on a learning

    19    curve here.  It is not one or the other dictating. 

    20    Everybody is together here.  We need to consider

    21    again, I think, some of the issues of residual DNA. 

    22    Is it oncogenic?  What is the issue there?  Is there

    23    an infectious DNA in relation to what sorts of cell

    24    line you've got and what is in the cell? It is really

    25    timely to review and assess the risks in light of a

                                                                         53

     1    much better understanding of the molecular mechanisms

     2    of geo-originicity and of viral/viral interaction.  As

     3    I mentioned, I think we will touch on that during the

     4    meeting.

     5                What I think we have to do is to come to

     6    some realistic and scientifically sound decisions

     7    concerning the use of these substrates.  Now, a point

     8    which I from sort of a WHO and a global perspective

     9    think is very important -- I think science and

    10    commerce in biologicals -- I mean, vaccines are made

    11    in one country and travel the world.  They are not

    12    necessarily just used in one country.  It really is an

    13    international situation.  And so too are the public

    14    health questions which the use of these products

    15    raise. So if there is an issue in relation to a

    16    problem in one country, it is a global issue.  And the

    17    international dimension of the discussion of these

    18    issues is vital.  And I am glad that we do have an

    19    international group here, and I think it is important

    20    that WHO is part of this and we are very pleased to be

    21    co-sponsoring this meeting.  Thank you very much.

    22                DR. RABINOVICH:  Are there any questions

    23    specifically for Dr. Griffiths?  Dr. Rubin?

    24                PARTICIPANT:  I was not quite clear on

    25    what you were saying about the presence of

                                                                         54

     1    retroviruses in chick embryo cell cultures.  Whether

     2    you thought that in fact it was present or not.  And

     3    the reason I am asking is we had a lot of experience

     4    with chicken leukosis viruses in chick embryo cells

     5    beginning back in 1960.  And the thing about them is

     6    they are not easy to detect because they don't produce

     7    any pathogenic effect and they have to be detected in

     8    indirect means.  The indirect means in those days when

     9    they were first found by us, at least, was

    10    interference with RAS sarcoma virus preventing.  And

    11    then much later on came -- at least 10 years later

    12    came reverse transcriptase.  The thing about these

    13    leukemia viruses or leukosis viruses, these

    14    retroviruses, other than the really pathogenic ones,

    15    is that they are highly species specific for one

    16    thing.  So chicken viruses in general, with some

    17    exceptions, will not infect other species. So if we go

    18    up the scale, I think, to the mouse leukemia viruses,

    19    I think it is generally true of them also that they

    20    are not cytopathic in culture. They can't be detected. 

    21    And probably they are of no concern to other species

    22    unless they are very closely related.  The concern I

    23    would have of vaccines made in higher species, monkeys

    24    or humans, is that probably there are -- or not

    25    probably, there may be some stealth viruses like these

                                                                         55

     1    that don't produce any obvious effect and that we

     2    don't even know about their presence to even detect

     3    them in animals.  Let's say the chicken virus requires

     4    a flock of chickens that is not infected with them

     5    that has to be infected at a very early age and you

     6    have to practically wait for half a lifetime of the

     7    animal to produce any statistical effect among them.

     8    So I wonder if there is any concern about these points

     9    with the retroviruses.

    10                DR. GRIFFITHS:  Can I just clarify the

    11    point about the ALV, the avian leukosis virus.

    12                PARTICIPANT:  Yes.

    13                DR. GRIFFITHS:  The vaccine production

    14    stipulates that the eggs are free from the -- the

    15    chickens are free from the avian leukosis virus.

    16                PARTICIPANT:  Yes.

    17                DR. GRIFFITHS:  So the reverse

    18    transcriptase activity wasn't -- it wasn't expected. 

    19    There are very low levels of RTAs.  I mean, it has

    20    been tested by the ordinary methodologies -- the sort

    21    of standard methods.  It was only when you went down

    22    to these very low levels. And the origin is now known. 

    23    Where it comes from is endogenous viruses, retros. 

    24    And they do form particles, but as I said they are

    25    non-infectious particles.  But on a global basis,

                                                                         56

     1    yellow fever for example -- the yellow fever vaccine 

     2    -- this is some sort of a risk/benefit which the WHO

     3    has to sort of wrestle with which a national authority

     4    has the benefit of not necessarily -- like the USA,

     5    for example, doesn't have to.  It can make a decision

     6    right away.  But, for example, WHO requirements --

     7    they will be changing, but they have allowed

     8    production in ALV positive eggs because you cannot get

     9    ALV-free flocks in many -- in places where you are

    10    actually making yellow fever vaccine, and it was

    11    considered that the lack of yellow fever vaccine was

    12    much more of a risk than the presence of the ALV. 

    13    Nevertheless, Western Europe and USA making yellow

    14    fever vaccine is all in ALV-free -- from eggs from

    15    ALV-free chickens.  And that will now be introduced

    16    into WHO requirements as well, I think, in the future

    17    because these countries which were producing in ALV-

    18    positives can now actually get the eggs free.

    19                PARTICIPANT:  Okay.

    20                DR. RABINOVICH:  One last question?  

    21                PARTICIPANT:  I was really going to

    22    comment on actually the last comment from Dr. Rubin. 

    23    And reiterate your point that the sequence that appear

    24    to be responsible for the RTA activity appear to be

    25    the AEV gene and not the ALV gene.

                                                                         57

     1                DR. GRIFFITHS:  That is right.  Yes.

     2                PARTICIPANT:  But actually both the AEV

     3    and ALV genomes can actually undergo recombination. 

     4    One of the subgroups of ALV is actually based on the

     5    AEV envelope gene sequence.  But I really wanted to

     6    challenge the assumption that we should not be

     7    concerned about the species specificity of the

     8    retroviruses.  In fact, you can induce tumors quite

     9    readily in certain species quite diverse from any

    10    species with RAS sarcoma virus that contains the ALV

    11    envelope gene in subgroups like Subgroup D.  So it is,

    12    I don't think, a correct assumption to state that

    13    viruses like the avian retroviruses are not of

    14    concern.  They could be.

    15                DR. GRIFFITHS:  I think David's point --

    16    did you introduce yourself David? That is David Onions

    17    for the record.  

    18                DR. RABINOVICH:  Thank you.

    19                DR. GRIFFITHS:  But I think the point made

    20    is very important that although we assume that some of

    21    these things are innocuous, I think it is much better

    22    if you can get rid of them to have them out of the

    23    way, and I think this is the point really.  Because

    24    there are a lot of interactions going on and there is

    25    a lot of new information coming through, which I am

                                                                         58

     1    sure will be discussed over the next few days.

     2                DR. RABINOVICH:  Thank you, Dr. Griffiths. 

     3    I would like to introduce our next speaker, who every

     4    single presenter and panel chair at this conference

     5    knows well because he has been contacted individually

     6    by him.  It is Dr. Andrew Lewis from the Center of

     7    Biologics and Review at the FDA.

     8                DR. LEWIS:  Thank you, Regina. I think

     9    pestering is a better word perhaps.

    10                DR. RABINOVICH:  Persistence, leadership.

    11                DR. LEWIS:  We have had a lot of

    12    conversations with everybody.  As you've heard from

    13    Dr. Hayflick and Dr. Griffiths, until the end of the

    14    1980's, the use of all types of neoplastic cells for

    15    the production of biologicals was controversial.  With

    16    the development of the defined 100 picogram limits of

    17    residual DNA, the development of the concept of viral

    18    clearance, and the World Health Organization's

    19    acceptance in 1987 of interferons and monoclonal

    20    antibodies that were produced in tumor cells, the

    21    issues regarding the use of neoplastic cells for the

    22    production of purified biological products were

    23    resolved.  However, the use of neoplastic cells as

    24    substrates for live virus vaccines continue to be

    25    controversial.

                                                                         59

     1                A number of factors are motivating the

     2    need to reconsider using neoplastic cells for vaccine

     3    development, and I have listed these factors on the

     4    next slide.  These factors include the development of

     5    the whole virus or traditional vaccines to HIV,

     6    bioengineering approaches to viral attenuation to

     7    vaccine development, the rapid development of vaccines

     8    to emerging viruses such as the H5N1 and the H9N2

     9    influenza viruses, significant progress in

    10    understanding carcinogenesis and in detecting

    11    adventitious agents, and finally the very successful

    12    experience with the highly purified biologicals that

    13    were derived from neoplastic cells.

    14                Now conflicting with the need to use

    15    neoplastic cells as vaccine substrates are the

    16    regulatory concerns over the possible risks associated

    17    with vaccine manufacturers in these cell types.  I

    18    think it is no secret that issues regarding vaccine

    19    safety receive a very high level of public attention. 

    20    It is also no secret that public confidence in vaccine

    21    safety is a critical component for the success of

    22    immunization programs that are important to the public

    23    health.  

    24                The scientific and regulatory challenge is

    25    to develop the concepts and the technologies that can

                                                                         60

     1    be used to assess the risks perceived to be associated

     2    with neoplastic cell substrates in a manner which

     3    sustains public confidence in vaccine safety.  To

     4    achieve this goal, we at CBER believe that it is

     5    essential that an approach be developed which can be

     6    used to objectively and critically assess using state

     7    of the art technology those issues associated with the

     8    use of neoplastic cells as substrates for vaccine

     9    manufacture.  

    10                The purpose of my talk tonight is to

    11    outline an approach that CBER is proposing to

    12    establish an objective and systematic means of

    13    managing the regulatory concerns that are associated

    14    with the development of vaccines in neoplastic cells. 

    15    To begin the discussion of this approach, I would like

    16    to define exactly what we mean by the term neoplastic

    17    cells.

    18                For my talk, the term neoplastic cells

    19    refers to immortalized cells that are derived from

    20    either tumors or from transformation of instant tissue

    21    culture, and these cells can either be tumorigenic or

    22    non-tumorigenic when injected into animals.  

    23                The approach that CBER has followed to

    24    consider neoplastic cells as possible vaccine

    25    substrates is based on the approach worked out in the

                                                                         61

     1    1980's to evaluate the use of continuous cell lines to

     2    manufacture biologicals.  Now as shown in the next

     3    slide, this approach consisted of five components. 

     4    These components included identifying the issues,

     5    developing theoretical and experimental models to

     6    evaluate each issue, validating the models for issue-

     7    associated risk evaluation, developing criteria to

     8    evaluate the levels of risk, and finally discussing

     9    these issues and the approaches in public meetings,

    10    which we are doing over the next several days.  

    11                The process of developing the CBER

    12    approach began with organizing and presenting a

    13    discussion of the use of neoplastic cells as vaccine

    14    substrates before CBER's Vaccines and Related

    15    Biological Products Advisory Committee in November of

    16    1998.  Following this meeting, CBER drafted a proposal

    17    which evolved from the Advisory Committee

    18    presentations.  This proposal represents the first

    19    attempt in CBER to formally address the regulatory

    20    issues associated with the use of neoplastic cells as

    21    substrates for vaccine development.  

    22                Perhaps I should also point out that the

    23    draft proposal which has been distributed in your

    24    meeting package, as Dr. Rabinovich mentioned in the

    25    introductory remarks, is not -- and I will emphasis

                                                                         62

     1    the word not -- an official, approved FDA document

     2    offering guidance on the use of neoplastic cell

     3    substrates.  The issues and concerns that were

     4    presented to the Advisory Committee were developed in

     5    greater detail in this CBER draft proposal, and these

     6    issues and concerns were use to develop the agenda for

     7    this meeting. And I am going to review them for you in

     8    the next series of slides.

     9                Now in this series of slides, I have

    10    attempted to organize the issues in a common format. 

    11    The title of the slide represents the issue of

    12    concern.  The first bullet represents the cell

    13    substrates that are involved.  The second bullet

    14    illustrates the issues that are responsible for the

    15    concern.  And the third bullet is a very brief summary

    16    of data that documents why the concern or why the

    17    issue is in fact a concern.  

    18                Now concern 1 represents tumor cell

    19    contamination.  With regard to the presence of tumor

    20    cells as contaminants, at first glance this concern

    21    appears to be trivial, as viable cell substrates are

    22    almost always or always removed from viral vaccines. 

    23    However, if cells from human tumors are used as a

    24    manufacturing substrate, the issue becomes a bit more

    25    complex.  As noted in bullet 3, the inoculation of

                                                                         63

     1    human tumor cells into humans has in fact produced

     2    tumor allografts on some occasions.  Thus, for

     3    regulatory purposes, not only will it be necessary to

     4    remove or eliminate the cell substrate, it will

     5    probably be necessary to document by validated

     6    procedures the efficiency with which the manufacturing

     7    process removes tumor cells from the final product.

     8                The second concern is a concern with

     9    adventitious agent contamination.  All cell

    10    substrates, as Dr. Griffiths has pointed out, are

    11    subject to contamination with adventitious agents. 

    12    Such agents might infect vaccine recipients, and the

    13    ample history of substrate contamination with viruses

    14    and with other agents has provided reason enough for

    15    this concern.  Without saying much more about this,

    16    Dr. Phil Minor is going to review this topic in

    17    considerable detail during the Friday meeting.

    18                Concern 3 is the concern with cell

    19    substrate DNA contamination.  Most vaccines consisting

    20    of whole varions contain some level of residual DNA

    21    from the cell substrate in which the vaccine was

    22    manufactured.  There has been considerable discussion

    23    over the years as to the possibility that residual DNA

    24    could be the source of activated oncogenes or other

    25    manifestations of a neoplastic process and might

                                                                         64

     1    result in the transfer of neoplastic activity to

     2    vaccine recipients.  Even though some live virus

     3    vaccines contain microgram quantities of DNA from

     4    their diploid cell substrates, there is no evidence

     5    that any adverse event has resulted in an exposure to

     6    residual cell DNA.  While diploid cell DNA is thought

     7    to be free of all the accoutrements of neoplasia,

     8    immortalized cells and tumor cells contain various

     9    combinations of genetic alterations that contribute to

    10    their neoplastic state.  For this reason, we believe

    11    it is necessary to revisit the possible risk

    12    associated with the residual DNA from neoplastic cell

    13    substrates.  

    14                Now in addition, tumor cells can carry

    15    part of or the entire genome of oncogenic viruses. 

    16    Furthermore, the need to produce or develop retrovirus

    17    vectors and retrovirus vaccines means, as Dr.

    18    Griffiths has pointed out, that retrovirus proviruses

    19    can also be present in any substrate DNA that remains

    20    in retrovirus vaccines that are manufactured in

    21    neoplastic cells.  These possibilities generate the

    22    concerns produced by the presence of residual cell DNA

    23    in viral vaccines manufactured in neoplastic cells and

    24    are the reasons why this topic is covered in the

    25    session on Thursday.

                                                                         65

     1                Concern 4 represents cell protein

     2    contamination.  Tumor cells produce a variety of

     3    cytokines and lymphokines and other biologically

     4    active proteins.  Some of these proteins have the

     5    capacity to produce local and systemic reactions.  In

     6    addition, random selection of cells from the human

     7    population as well as alterations that occur with some

     8    frequency in the genomes of neoplastic cells might

     9    result in the presence of abnormal PRP proteins, and

    10    whether such proteins might be able to produce

    11    spongiform encephalopathy so far as I am aware is not

    12    really known, but the theoretical possibility that

    13    such events can occur means that we should be

    14    addressing these concerns and discussing them at this

    15    meeting.  

    16                Concern 5 is the concern with viral/viral

    17    and viral/cellular interactions. In cells infected

    18    with more than one virus, a variety of interactions

    19    are known to occur between the replicating viral

    20    genomes.  These interactions include genetic

    21    recombination, genome reassortments, pseudotyping or

    22    sequestering of the genomes of one of the viruses

    23    within the capsid of another virus, and finally the

    24    parasitism of the genome of large viruses by the

    25    entire genomes of the smaller viruses.  If vaccines

                                                                         66

     1    are manufactured in cells containing adventitious

     2    agents, each of these processes could be associated

     3    with the transfer of non-viral components to vaccine

     4    recipients or their non-vaccine viral components. 

     5    Evaluating the possibility that these events could

     6    occur and developing methods to search for novel virus

     7    derivatives and establishing baselines or threshold

     8    levels considered to be necessary to document the

     9    absence of such novel agents will occupy two sessions

    10    of the workshop.

    11                The final and sixth concern is the concern

    12    with genomic instability.  In contrast to diploid

    13    cells, immortalized cells and especially tumor cells

    14    evolve by changing the configuration and expression of

    15    their genomes.  The question, and therefore the

    16    concern, raised by this characteristic of neoplastic

    17    cells is whether such changes could over time mobilize

    18    occult viruses or proviruses or result in the

    19    production of biologically active proteins resulting

    20    in adverse events.  Although we are not aware of any

    21    evidence that such events have occurred, the ability

    22    of tumor cells to evolve over time is well established

    23    and we suggest that the consequences of neoplastic

    24    evolution for the safety of vaccines manufactured in

    25    neoplastic cells needs to be considered.

                                                                         67

     1                Now once the issues and concerns

     2    associated with the possible use of neoplastic cells

     3    as vaccine substrates were identified, it was

     4    necessary to consider an approach that would allow the

     5    possible risk posed by these issues to be assessed. 

     6    As issues of risk assessment are best evaluated using

     7    quantitative data and as current technology provides

     8    the opportunity to measure most biological events, we

     9    are attempting to develop a systematic, quantitative

    10    approach to assessing the concerns associated with

    11    neoplastic cell substrates using what we have

    12    designated as a defined risk approach algorithm.  This

    13    algorithm is presented in the next slide.

    14                The basic features of this algorithm

    15    include assessing the levels of risk posed by each

    16    issue quantitatively, establishing the probability of

    17    a worst case scenario for each issue, using the data

    18    to evaluate the risk individually and cumulatively,

    19    and then using the data to assess the relative risk of

    20    the product.  In the next talk, Dr. Phil Krause is

    21    going to discuss risk factors and the use of

    22    experimental data and the application of this

    23    algorithm to specific concerns.  

    24                I would like to end my talk with sort of

    25    a short story.  In June of 1998, I was visiting with

                                                                         68

     1    two friends from medical school.  Both of these

     2    friends are practicing physicians and have been in the

     3    business of delivering health care for many years. 

     4    During one of the many conversations about

     5    professional issues that always come up when we get

     6    together, I asked my friends if they would consider

     7    giving vaccines produced in cancer cells to their

     8    family and their patients.  There was a slight pause

     9    in the conversation and then one of them answered with

    10    a seven-word remark that was both an expression of

    11    confidence as well as a reminder of responsibility. 

    12    When this remark is committed to paper, it also turns

    13    out that it can be somewhat poetic as well as

    14    prophetic.  And since it has been following me around

    15    for the past 14 months, I thought it would be

    16    appropriate to share it with you as my last slide.

    17                In response to the question about using

    18    vaccines made in cancer cells, my friend simply

    19    replied, "If the FDA says they are okay."  That

    20    question is being addressed at this meeting.  The

    21    question that is actually being addressed at this

    22    meeting is how to find out if they are okay.  

    23    Beginning with the controversy over the distribution

    24    of non-defective adeno SV40 hybrids that were

    25    developed in my laboratory in the late 1960's, with

                                                                         69

     1    the Oscillimar (Phonetic) Conference on recombinant

     2    DNA in 1975, and with xenoscience transplantation

     3    issues since I have been with CBER, I have observed

     4    that when technological advances make it necessary to

     5    address precedent setting issues, it is essential to

     6    first bring together knowledgeable scientists and

     7    interested individuals from other disciplines to

     8    discuss the issues and the data that are available for

     9    informed decision making.  Following these initial

    10    instructions, consultations, collaborations and

    11    cooperation among the interested parties, we have

    12    usually been able to resolve many or most of these

    13    issues.  

    14                Technological advances are once again

    15    asking us to address a precedent setting issue in the

    16    field of vaccinology. With this meeting on cell

    17    substrates, we are in the process of reviewing and

    18    evaluating the data that are available for informed

    19    decision making.  And I would like to conclude by

    20    saying that those of us at CBER who have been working

    21    on cell substrate issues believe that through

    22    consultation, collaboration and cooperation with our

    23    colleagues in academia, industry and the regulatory

    24    community that it should be possible to develop an

    25    effective approach to evaluate and manage the use of

                                                                         70

     1    neoplastic cell substrates for vaccine manufacture. 

     2    Thank you.

     3                DR. RABINOVICH:  Are there any questions

     4    specifically for Dr. Lewis?  Please identify yourself.

     5                PARTICIPANT:  Dr. Tevethia from Penn

     6    State. In the study that you cited where the

     7    allografts were accepted in the patients, you didn't

     8    mention, did they progress to the tumor stage or did

     9    they regress or disappear?

    10                DR. LEWIS:  I am sorry?

    11                PARTICIPANT:  Did they progress?

    12                DR. LEWIS:  Yes, they did.  These studies

    13    -- if you look at that literature, Tev, it goes back

    14    to the late 1940's and early 1950's most of it, and

    15    the cell line that was particularly problematic was

    16    called hep-3.  And most of these studies were done in

    17    patients with terminal cancer.  And so it is very hard

    18    to assess the implications of that for a more modern

    19    problem.  But preceding that information, there were

    20    several reported cases in the literature, and also

    21    cited by Dr. Luci Gross in his book "Oncogenic

    22    Viruses".  So reviews -- this is where most of his

    23    information came from.  But there were several cases

    24    in the literature in which surgeons had actually

    25    grafted themselves with a tumor cell from patients who

                                                                         71

     1    they were operating on in the 1920's and 1930's.  So

     2    it is an unusual phenomenon, but it is not unheard of. 

     3    And I think when we are talking about vaccines that

     4    are going to go into possibly millions of people, we

     5    have to consider the breadth of the experience that

     6    the population would be subjected to.

     7                PARTICIPANT:  Dr. Fried from ICRF.  How

     8    many cells were injected to get these nodules?

     9                DR. LEWIS:  Mike, a lot of those things

    10    were done not with cells but with explants.  That was

    11    before the days when trypsin was used to break up

    12    cells.  So they would just take a little piece of the

    13    tumor with a trocar and stick it in.  The tumors were

    14    generally several mm, but it was not -- that is just

    15    a rough estimate because this is a fragment.  Of

    16    course with the surgical -- when the surgeons

    17    inoculated themselves, there is no way to know.

    18                DR. HAYFLICK:  Actually, many of those

    19    studies were done by Chester Savin.

    20                DR. LEWIS:  Yes, and Alice Moore.

    21                DR. HAYFLICK:  And Alice Moore.  And they

    22    were done with prisoner volunteers at the state

    23    penitentiary in Ohio.  So they were not terminal

    24    cancer patients.

    25                DR. LEWIS:  Well, yes.  I went back and

                                                                         72

     1    looked at that data, Dr. Hayflick, and the prisoners

     2    uniformly rejected the tumors.

     3                DR. HAYFLICK:  Correct.

     4                DR. LEWIS:  Yes.

     5                DR. HAYFLICK:  Correct.  And I think that

     6    is important to know.

     7                DR. LEWIS:  Yes.

     8                DR. HAYFLICK:  However, when we inoculated

     9    HeLa into terminal human cancer patients, the HeLa

    10    cells did indeed grow to the point that they had to be

    11    extirpated.

    12                DR. LEWIS:  Yes.

    13                DR. HAYFLICK:  I also should point out

    14    that hep-3 later turned out to be HeLa.  

    15                DR. RABINOVICH:  Thank you very much, Dr.

    16    Lewis.  Last but not least, Dr. Phil Krause has the

    17    unenviable position of being between you and bed

    18    before tomorrow morning. So I welcome him and his

    19    presentation of setting the stage for conceptual and

    20    experimental approaches to address product safety

    21    issues raised by novel cell substrates.  And he

    22    becomes our guinea pig for testing of the computer

    23    system.  

    24                While he is doing that, let me remind all

    25    speakers that the manuscripts are to go to our

                                                                         73

     1    colleagues at IQ Solutions, and that there is, I

     2    believe, still space at the late breaker session for

     3    tomorrow evening which was thought up by Dr. Lewis,

     4    but we welcome comments, arguments and particularly

     5    data that people may want to share at the late breaker

     6    session.

     7                DR. KRAUSE:  What I want to talk to you

     8    about tonight is the defined risks approach that Andy

     9    just alluded to.  And the idea is that we would like

    10    to develop a quantitative approach that would enable

    11    us to use laboratory data to assess risks associated

    12    with cell substrates.  The change in precedent against

    13    the use of neoplastic cells as vaccine substrates

    14    needs to be based on objective scientific data that

    15    can be used to evaluate levels of risk.

    16                This approach is an example of science-

    17    based regulation in which FDA attempts to gather the

    18    relevant scientific data before making a decision. 

    19    And as Andy pointed out or as Dr. Lewis pointed out,

    20    this could be implemented by assessing the level of

    21    risk posed by each issue quantitatively, establishing

    22    the probability of a worst case scenario for each

    23    issue, using data to evaluate the risks individually

    24    and cumulatively, and using the data to assess the

    25    relative risk of the product.  So I am going to go

                                                                         74

     1    through these four points in this talk.

     2                The first of which is to quantitatively

     3    assess the level of risk posed by each issue.  I am

     4    also going to go through a simple example of how the

     5    defined risks approach can be applied to an issue.  In

     6    order to quantitatively assess the level of risk posed

     7    by each issue, it is necessary to first assess what

     8    are the issues associated with the plausible risk to

     9    vaccine recipients, and Andy Lewis went through some

    10    of these, evaluate the need for and availability of

    11    quantitative data to assess the risks in a regulatory

    12    setting, to evaluate factors that could increase or

    13    decrease this possible risk, and to consider

    14    development of assays that could be used in evaluating

    15    specific products and the general issue.  

    16                As it happens, these are four of the major

    17    points which are going to come up in a recurrent way

    18    in the panel discussions later on during this meeting. 

    19    So much of the discussion in this meeting is going to

    20    focus on this first part of the defined risks approach

    21    algorithm.  

    22                Now I am going to briefly discuss how this

    23    algorithm could be applied to the issue of infectious

    24    residual DNA in biological products.  And I don't mean

    25    to prejudice the discussions that will occur later in

                                                                         75

     1    the meeting, but I think it would be useful to go

     2    through these steps as we have considered them so far

     3    for this issue to illustrate the kind of analysis that

     4    we think will be valuable for all of the issues to be

     5    discussed at the meeting.

     6                You have already heard some of this from

     7    Dr. Hayflick and from Dr. Griffiths, but according to

     8    the recently revised WHO requirements for the use of

     9    animal cells as in vitro substrates for the production

    10    of biologics, the amount of cellular DNA in biological

    11    products should be limited to 10 nanograms per dose. 

    12    This is an increase from a previously established

    13    limit of 100 picograms per dose.  This limit is meant

    14    to apply to continuous cell lines but not to products

    15    given orally or to products derived from microbial,

    16    diploid or primary cell culture systems.  The 10

    17    nanogram figure was derived by considering data and

    18    theoretical calculations regarding the tumorigenicity

    19    of injected DNA.  

    20                While CBER is attentive to WHO guidelines,

    21    CBER evaluates products on a case-by-case basis in

    22    determining appropriate limits for cell substrate DNA. 

    23    Moreover, it should be noted that for live viral

    24    vaccines and other less purified products, it may not

    25    be possible to limit the total DNA to 10 nanograms. 

                                                                         76

     1    Thus the question is raised, what data would be

     2    required to provide assurances that this or any

     3    specific limit would be appropriate for vaccines

     4    produced in novel cell substrates, including

     5    neoplastic cells?  

     6                The first question is whether there is a

     7    plausible risk or not.  Viral genomic DNA is

     8    infectious when injected into animals.  Moreover,

     9    tumor cells and primary cells may contain virus

    10    genomes and may harbor latent viruses.  And DNA from

    11    cells used to produce retroviral or DNA-viral vaccines

    12    may contain viral genomes as a by-product of vaccine

    13    production.  Thus, based on the data, it would appear

    14    that under some circumstances there could be an

    15    infectious risk from residual DNA.

    16                Several factors may influence an

    17    assessment of the tumorigenicity or infectivity risk

    18    associated with residual DNA.  These include the total

    19    quantity of DNA in the vaccine, the number of doses to

    20    be given, the size of the DNA, sequence-related

    21    properties of the DNA, for example whether it encodes

    22    a virus, the number of copies of potentially

    23    infectious sequences per cell, and the state of the

    24    DNA, which I take to mean such factors as whether it

    25    is chromatin-associated, whether it is integrated into

                                                                         77

     1    a cellular genome, whether it is linearized or

     2    circular, et cetera.  For many of these kinds of

     3    considerations raised on the slide, although it is

     4    likely that they have an effect on the ultimate

     5    infectivity or tumorigenicity of the DNA, these

     6    studies have not in general been performed in a

     7    quantitative fashion that would enable us to apply a

     8    quantitative risk assessment model.

     9                This slide presents the quantitative data

    10    that we are aware of regarding the potential

    11    infectivity of cloned or purified viral genomic DNA. 

    12    Information on tumorigenicity is also presented based

    13    on the assumption that viral genomic DNA is at least

    14    as infectious as it is tumorigenic.  In these

    15    experiments, cloned or purified genomic DNA was

    16    injected directly into various animal models.  On the

    17    right-hand side of the table, I have calculated the

    18    theoretical risk associated with a product that

    19    contains one microgram per dose of cellular DNA that

    20    contains a single genome per cell.  This calculation

    21    accounts for the dilution of the viral genome and the

    22    cellular genome, and assumes that the viral genomic

    23    DNA is as infectious or tumorigenic when incorporated

    24    in cell substrate DNA as it is when it is linearized

    25    and injected directly.  

                                                                         78

     1                The estimated risk of infection associated

     2    with the theoretical product would thus range from as

     3    high as 1 in 8,000 for polyoma virus DNA to 1 in tens

     4    of millions for other DNAs.  An improved understanding

     5    of the relative infectivity of different types of DNAs

     6    might assist in developing tests to ensure that

     7    unacceptable quantities of infectious DNAs are not in

     8    biological products produced in novel cell substrates. 

     9                Another key point for discussion at the

    10    meeting will be the development of appropriate assays

    11    or methods to better evaluate these risks.  It should

    12    be pointed out that direct assessment of the

    13    tumorigenicity and infectivity of residual DNA

    14    associated with a particular product may be

    15    impractical.  This is because in order to achieve

    16    meaningful safety margins, very large quantities of

    17    DNA would need to be purified and tested in multiple

    18    animal models for infectivity and tumorigenicity.  The

    19    example on this slide, for example, if a dose of a

    20    product contained one microgram of residual cellular

    21    DNA, assessment of a million doses in a single type of

    22    assay would require testing of an entire gram of DNA. 

    23    This is why I believe it is important to take a more

    24    general approach and understand the potential

    25    underlying infectivity and tumorigenicity of different

                                                                         79

     1    types of DNA.

     2                Based on the data that I have presented,

     3    one might conclude that additional work could be done

     4    to achieve a more complete understanding of this

     5    issue.  This might include studies of how to detect

     6    latent genomes, including for unknown viruses, how to

     7    quantitatively assess the effect of various factors on

     8    DNA infectivity, and improved assessment of DNA

     9    infectivity for different types of viruses.

    10                Now it may be helpful to think about this

    11    issue in the context of a hypothetical example.

    12    Suppose an unpurified, live, attenuated viral vaccine

    13    is grown in a cell line that contains 50 copies per

    14    cell of a latent virus.  This is the approximate

    15    number of copies of HPV DNA that are in HeLa cells. 

    16    If there were 10 nanograms of residual DNA per dose,

    17    which is the current WHO recommendation, and if two

    18    doses were recommended per child, as for instance is

    19    the case with MMR vaccine, and the infectivity of

    20    viral DNA in the vaccine were comparable to that of a

    21    purified polyoma virus DNA, we can calculate the

    22    theoretical infectivity risk from the DNA in this

    23    product, which I proceeded to do on this slide. 

    24    Multiplying these numbers together with the

    25    infectivity of polyoma virus DNA yields a theoretical

                                                                         80

     1    infectivity risk of 1.2 infections per 10,000

     2    children.  For a vaccine that is universally

     3    administered to the U.S. birth cohort of 4 million

     4    children, this would represent about 500 infections

     5    per year, clearly an unacceptable rate.  Of course,

     6    this assumes that the DNA is as infectious as polyoma

     7    virus DNA.  It assumes that this DNA is as infectious

     8    as residual DNA in a vaccine as it is when it is

     9    cloned and purified.  And it doesn't account for the

    10    potential effect of DNA fractionation or other

    11    measures that might reduce its infectivity.  Based on

    12    the limited data I showed you, retrovirus DNA may be

    13    about 1000 to 10,000-fold less infectious than polyoma

    14    virus DNA.  Thus, this calculation is very dependent

    15    on a clear understanding of the infectivity of the

    16    viral DNA and a clear understanding of what type of

    17    viral genomes a neoplastic cell could harbor.

    18                The example I just went through could be

    19    thought of as a worst case scenario. And in fact, the

    20    second element of the defined risk approach algorithm

    21    is to establish the probability of a worst case

    22    scenario for each issue.  Thinking of these kinds of

    23    issues in the context of their worst case is justified

    24    by the need for public confidence in vaccines and

    25    their safety, which requires that we take a very

                                                                         81

     1    conservative approach.  This includes identification

     2    and discussion of all of the issues, determining which

     3    can be dismissed, which are relevant, and which are

     4    amenable to a resolution with additional data.

     5                The third element of the defined risk

     6    approach algorithm is to use data to evaluate the

     7    risks individually and cumulatively.  In this meeting,

     8    we will be individually considering a broad variety of

     9    potential risks posed by neoplastic cell substrates. 

    10    Each of the meeting sessions is devoted to discussion

    11    of these potential risks.  The meeting starts tomorrow

    12    morning with the consideration of mechanisms of

    13    oncogenicity because this is a final common pathway

    14    for many of the potential concerns with neoplastic

    15    cells.  We then consider virus/virus and virus/cell

    16    interactions, residual DNA, and adventitious agents. 

    17    We then turn to a discussion of how cell substrates

    18    might be designed or chosen to minimize these types of

    19    concerns. 

    20                In lieu of a poster session, there will be

    21    a session on miscellaneous topics on Thursday evening

    22    to permit participants to present additional relevant

    23    data.  The final panel discussion on Friday afternoon

    24    will then summarize the results of the meeting. It is

    25    our hope that a comprehensive discussion of these

                                                                         82

     1    individual potential risks will help CBER also

     2    consider them cumulatively.

     3                The fourth element of the defined risks

     4    approach algorithm is to use the data to assess the

     5    relative risk of the product.  Potential risks must be

     6    placed in the context of the potential benefits of the

     7    product.  This is something which may need to be

     8    considered by CBER on a product-by-product basis. 

     9    These risks also must be placed in the context of

    10    risks already found acceptable and unacceptable by

    11    society, and this can be a changing target.  For

    12    example, society has been willing to accept the one in

    13    a few million or so risks of a vaccine associated with

    14    paralytic polio for many years, but more recently it

    15    appears less willing to do so.  

    16                We also hope the discussion will include

    17    some consideration of the relative risks of using

    18    different types of cell substrates, including the role

    19    of species in cell type, whether it be primary,

    20    diploid or neoplastic, and for neoplastic cells, what

    21    the role of the transforming event and the known

    22    history of the cell plays in the safety assessment.

    23                I would like to close by putting this

    24    discussion into the context of the real world.  CBER

    25    currently has applications to study the use of various

                                                                         83

     1    types of neoplastic cells in vaccine development. 

     2    Some of these products could be amenable to production

     3    in other types of cells, but others might not.  By the

     4    time CBER gets an application, the manufacturer

     5    usually has already made a commitment to one cell

     6    substrate or another.  It is our hope that this

     7    meeting will both raise awareness of these issues

     8    among manufacturers in the academic community and will

     9    provide CBER with a sound scientific foundation to

    10    answer these critical regulatory questions, which

    11    include what types of data regarding these cells are

    12    necessary to ensure that vaccines, including

    13    unpurified ones that are produced in them, are safe. 

    14    And are there some vaccine cell substrate combinations

    15    for which current technology simply precludes an

    16    adequate safety assessment.  I thank you very much and

    17    good night.

    18                DR. RABINOVICH:  I think we should

    19    entertain some questions.  I think your approach will

    20    be provocative and will raise points for discussion

    21    throughout the meeting.  Are there any questions at

    22    this time?  Dr. Rubin?

    23                PARTICIPANT:  Well, I just thought this

    24    was an opportunity to raise a question about DNA that

    25    you, yourself, raised in a recent paper, of which you

                                                                         84

     1    were a co-author and which we were just talking about

     2    and which I haven't seen proposed for discussion at

     3    any point, which is that DNA are double-stranded RNA

     4    of as small as 25 base pairs when incorporated into

     5    the DNA of the cell and actually not incorporated even

     6    in the cytoplasm, I take it, of the cell can cause the

     7    production of protein molecules -- what are they MLH-1

     8    and 2 or something like that -- that are supposed to

     9    be very active in autoimmune reactions.  And I wonder

    10    how much you have thought about that and how much do

    11    you think that is relevant to the considerations that

    12    we have here.  It is a very surprising development to

    13    me that at least as little as 25 base pairs can be

    14    inserted and produce a fairly large biological effect.

    15                DR. KRAUSE:  Yes.  Of course, that was a

    16    study in which these small molecules were transfected

    17    into cells and the cells started to display some of

    18    the characteristics of antigen presenting cells.  And

    19    then the question then is, is that something which

    20    could subsequently be related to autoimmune responses

    21    or things like that.  I think those are legitimate

    22    kinds of questions.  I know that the world has a

    23    fairly long experience with unpurified viral vaccines

    24    which do contain eukaryotic DNA as residual DNA in

    25    quantities greater than 10 nanograms.  In particular

                                                                         85

     1    I am referring to WI38 or MRC5 DNA, which appear to be

     2    safe from that perspective.  I was on the committee

     3    that licensed the -- or actually I chaired the

     4    committee that licensed the chickenpox vaccine, and

     5    that was actually an issue that we considered at that

     6    time as well, and actually looked among recipients of

     7    the vaccine for evidence of an autoimmune response

     8    associated with the DNA that was included in that

     9    vaccine.  Actually, we didn't look, we asked the

    10    company to look and they did not find one.  Of course,

    11    in the context of DNA vaccines, we are talking about

    12    injecting even larger quantities of DNA into people

    13    which then -- and one might imagine that this response

    14    is at least related to some of the responses

    15    associated with the DNA vaccine.  

    16                I think in the context of the meeting, we

    17    had envisioned the discussion covering mostly issues

    18    that are specific to neoplastic cells, and at least in

    19    our initial thinking about the issue didn't think that

    20    this was an issue, given the small sizes of the

    21    molecules that were involved, that would be likely to

    22    be more of a problem with neoplastic than with other

    23    cells.  So we decided not to emphasize it at this

    24    time, in part because the plate is already very full. 

    25    But you raise a very interesting point.

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     1                DR. RABINOVICH:  Please identify yourself.

     2                PARTICIPANT:  Fried from ICRF London.  Do

     3    you think it is fair in your polyoma example to use

     4    linear DNA which can recircularize and then go into

     5    the mouse and replicate and amplify and compare it to

     6    integrated, where it can't come out and amplify?

     7                DR. KRAUSE:  You know, I guess the

     8    question isn't necessarily is it fair, but the

     9    question is what data does one need to know or to have

    10    in order to be certain that a given level of residual

    11    DNA from a certain cell type is safe. Now the

    12    experiment that I -- the trouble is that not a lot of

    13    information is available on this.  The only study that

    14    I am aware of where these data come from is that which

    15    was published by Marc Israel and Mel Martin's lab some

    16    20 or more years ago I think. And what they did is

    17    they looked at simply circularized DNA, and that was

    18    more infectious than linearized DNA.  And then

    19    depending on where you linearized the DNA, you then

    20    became more likely to either get infection or tumor. 

    21    But I think these kinds of experiments could certainly

    22    legitimately be done in other ways, whether

    23    linearizing with an enzyme which leaves overhangs to

    24    yield a different result than linearizing with an

    25    enzyme which leaves blunt ends and thereby potentially

                                                                         87

     1    precluding recircularization.  But I think --

     2                PARTICIPANT:  It doesn't preclude it.  I

     3    mean, it will recircularize even when you cut --

     4                DR. KRAUSE:  Okay, reduce the likelihood.

     5                PARTICIPANT:  I mean, you could put it in

     6    a plasmid, so you have a small piece of DNA and it is

     7    integrated in that, and then you can compare that to

     8    say cellular DNA, where the virus can't get out and

     9    replicate itself.

    10                DR. KRAUSE:  You are raising a very

    11    legitimate point.  To my way of thinking, these are

    12    the kinds of questions which need to be answered.  The

    13    question is do they need to be answered on an

    14    intuitive basis or do they need to be answered on an

    15    experimental basis.  

    16                DR. RABINOVICH:  Thank you. I think at

    17    this point, for those of us who have to coerce our

    18    children to go to school an hour earlier than usual,

    19    we will go ahead and close the session tonight and

    20    expect everyone to be here awake and coherent at 8:00

    21    in the morning to start.  Thank you very much.

    22                (Whereupon, at 9:53 p.m., the session was

    23    concluded.)

    24

    25