NOTE: Click here to jump ahead on the page for information on aborted fetal cell line PER C6
HEK (human embryonic kidney) 293 - the number of aborted fetal experiments prior to establishing the cell line.
HEK 293 is used to deliver the lentivirus gene in Dr Yamanaka's adult skin cell reprogramming, as a cell line and reagent for testing many research products and in the drug Xigris. But one would never know by looking at the package insert. In order to obtain the truth, Children of God for Life filed a Freedom of Information Act Request with the FDA.
“Xigris is a recombinant form of human activated protein C. An established human cell line possessing the complementary DNA for the inactive human protein C zymogen secretes the protein into the fermentation medium.”
About the 293 Cell Line
The protein C is produced in the HEK 293 aborted fetal cell line. 293 cells are available from the American Type Culture Collection. There are variants of the cell line that derive from the parent.
ATCC Number: CRL-1573 Price:
The 293 cell line is a permanent line of primary human embryonal kidney transformed by sheared human adenovirus type 5 (Ad 5)DNA. [RF32725]
The cells express the transforming gene of adenovirus 5. Although an earlier report suggested that the cells contained Adenovirus 5 DNA from both the right and left ends of the viral genome [RF32764], it is now clear that only left end sequences are present. [RF50113]
The cells express an unusual cell surface receptor for vitronectin composed of the integrin beta-1 subunit and the vitronectin receptor alpha-v subunit. [RF33793] Purified DNA from this line is available as ATCC 45504 (25 micrograms) and ATCC 45505 (100 micrograms).
The Ad5 insert was cloned and sequenced, and it was determined that a colinear seqment from nts 1 to 4344 is integrated into chromosome 19 (19q13.2). [RF50113]
United States Patent 5,681,932
1. The recombinant human protein C molecule produced by inserting a vector comprising the DNA encoding human protein C into an adenovirus-transformed host cell then culturing said host cell under growth conditions suitable for production of said recombinant human protein C.
2. The recombinant human protein C molecule of claim 1 wherein the adenovirus-transformed host cell is selected from the group consisting of AV12 cells and human embryonic kidney 293 cells.
3. The recombinant human protein C molecule of claim 2 wherein the adenovirus-transformed host cell is an AV12 cell.
4. The recombinant human protein C molecule of claim 2 wherein the adenovirus transformed host cell is a human embryonic kidney 293 cell.
In the FDA report found at http://www.fda.gov/ohrms/dockets/ac/01/transcripts/3750t1_01.pdf
81 lines 14-22
"So the Kidney
material, the fetal kidney material was as follows.
The kidney of the fetus was, with an unknown family history, was obtained
in 1972 probably. The precise date
is not known anymore.
The fetus, as far as I can remember was completely normal. Nothing was wrong. The reasons for the abortion were unknown to me. I probably knew it at the time, but it got lost, all this information."
Key excerpts from
the above document: Dr. Van Der Eb, Crucel NV is speaking...
"So I isolated retina from a fetus, from a healthy fetus as far as could be seen, of 18 weeks old. There was nothing special with a family history or the pregnancy was completely normal up to the 18 weeks, and it turned out to be a socially indicated abortus - abortus provocatus, and that was simply because the woman wanted to get rid of the fetus."
father was not known not to the hospital anymore, what was written down was
unknown father, and that was, in fact, the reason why the abortion was
was permission, et cetera, and that was, however, was in 1985, ten years before
this. This shows that the cells
were isolated in October 1985, Laeiden University in my lab.
At that time already '85, I should say the cells were frozen, stored in
liquid nitrogen, and in 1995 one of these was thawed for the generation of the
"And this is
the final slide just showing you some comparisons between 293 and PER.C6.
Again, I remind you that both cell lines were made in my lab for
different reasons. The objective, as I indicated, is for 293--was basic
research, and we have done many different transformation studies after that, not
transformation studies, but gene expressions studies with human embryonic kidney
cells in the years following that up to now, I would say.
"PER C6 was
made JUST FOR PHARMACEUTICAL MANUFACTURING OF ADENOVIRUS VECTORS
As to RCA free, PER.C6 are RCA Free.
The history documentation of the cell line has been carried out
completely for PER. C6 and was not done at that time for 293.
About the Cell
Lines and Adenovirus (AD5) Safety issues...
In 1954, during
discussions surrounding the development of adenovirus vaccines for use in the
military, the U.S. Armed Forces Epidemiology Board (AFEB) recommended the use of
"normal cells" as the substrate for vaccine production rather than
cell lines established from human tumors. This decision was based on concerns
about the possibility that human tumor cells might be contaminated with occult
oncogenic agents that might be transferred to vaccine recipients, an event which
might in turn increase the risk of cancer and other neoplastic diseases in
vaccinees. As evidenced by current regulatory guidelines and activities of
control authorities worldwide, the precedent set in 1954 by the AFEB remains an
important factor in the acceptance of all substrates for vaccine manufacture.
Currently, the only cultured animal cells that have been used as substrates in
U.S. licensed viral vaccines have been primary cells (e.g., derived from monkey,
chick, mouse), diploid cell lines (e.g., WI38, MRC-5, FRhL-2), or immortalized
(continuous), non-tumorigenic cell lines (e.g., VERO).
Over the past 47
years, two important factors have emerged that warrant serious consideration of
the use of immortalized tumorigenic cell lines for viral vaccine production. The
first of these factors is that certain novel virus vectors that are presently
under development for high-priority target diseases, most notably AIDS, cannot
feasibly be propagated in traditionally acceptable cell substrates. The second
factor is that scientific understanding of neoplastic processes and
viral-induced carcinogenesis has rapidly advanced, as has the ability to detect
and identify infectious, oncogenic agents and other types of adventitious agents
that may potentially contaminate cell substrates. These factors underscore the
need for developing a regulatory framework in which the relative benefits and
risks in using tumorigenic cell lines for vaccine production can be carefully
and cautiously revisited.
FDA would like the
VRBPAC to consider the potential risks in using two novel cell substrates, 293
cells and PER.C6 cells. These cell lines were developed by transforming human
embryonic kidney cells (293) and human embryonic retinal cells (PER.C6) with the
transforming early region 1 (E1) of adenovirus type 5 (Ad5). Since cell lines
such as 293 and PER.C6 express the Ad5 E1 region, they are able to complement
the growth of defective Ad5 vectors which have been "crippled" by
deletion of E1. Defective Ad5 vectors have been engineered to express foreign
genes such as those from human immunodeficiency virus (HIV), the causative agent
of AIDS, and vectors of this type are thought to have significant potential for
vaccine development because of their demonstrated ability to generate
cell-mediated immune responses to HIV. However, a feature of regulatory
importance associated with Ad5-transformed cells is their capacity to form
tumors in immunodeficient animals such as nude mice.
potential risks associated with the use of these so-called Designer Cell
Substrates – i.e., neoplastic cells derived from normal human cells
transformed by defined viral or cellular oncogenes or by immortalizing cellular
genes (e.g., telomerase) – OVRR/CBER is considering the approach
outlined below within the framework of a "defined-risks" assessment
(see enclosed reference Lewis et al., "A defined-risks approach to
the regulatory assessment of the use of neoplastic cells as substrates for viral
vaccine manufacture", In Evolving Scientific and Regulatory Perspectives on
Cell Substrates for Vaccine Development. Brown, Lewis, Peden, Krause (eds.)
Develop. Biol. Stand. [in press]). This framework is intended to examine, and
wherever possible, to quantify the potential risk of "transmitting"
the tumorigenic components of the cell substrate used for vaccine production,
and determine whether that "transmission" might pose a risk,
particularly an oncogenic risk, to vaccinees. Factors that could influence the
risk associated with the use of Designer Cell Substrates include (1) the known
mechanism of cell transformation leading to the development of tumorigenic
cells; (2) residual cell substrate DNA; and (3) the presence of adventitious
agents, especially oncogenic viruses. These three factors are discussed in more
Adenovirus 5-Transformed Designer Cell Substrates
The purpose of
tumorigenicity testing as applied to cell substrates used for viral vaccine
manufacture is to discriminate between cells that have the capacity to form
tumors and cells that do not form tumors. The potential risk of oncogenic
activity is thought to be higher for cell substrates that have the capacity to
form tumors, whereas the potential risk is thought to be low for cell substrates
that are unable to form tumors. In considering the risk of tumorigenicity of
Ad5-transformed Designer Cell Substrates, it is important to consider the
molecular processes that determine the ability of the cells to form tumors.
Adenovirus 5 does
not produce tumors when injected into rodents, but it does transform rodent
cells in tissue culture. Like adenovirus 5 virions, adenovirus 5-transformed
cells do not produce tumors when injected into immunocompetent adult rodents,
but these cells can form tumors when injected into immunodeficient rodents such
as nude mice. The tumor-forming capacity of Designer Cell Substrates that are
produced by transforming normal human cells with adenovirus 5 can be evaluated
by comparing them with adenovirus 5-transformed rodent cells. The adenovirus 5
early region 1 (E1) is composed of the transcription units E1A and E1B, which
transform normal cells to neoplastic cells through a multi-step process. The E1A
transcription unit immortalizes the cells and establishes those characteristics
of the transformed cells that permit them to be eliminated by the antitumor
defenses of immunocompetent rodents. During the transformation process, E1A
sensitizes cells to apoptosis (programmed cell death) and increases their
susceptibility to killing by natural killer cells, macrophages, and cytotoxic
lymphocytes, as well as cytokines such as tumor necrosis factor (see Routes et
al., 2000a, 2000b). The adenovirus E1B region alone is unable to immortalize
cells, but its function during neoplastic transformation ensures cell survival
by inhibiting virus-induced cell killing. Thus, the E1A region immortalizes
cells and determines their limited capacity to form tumors in immunodeficient
rodents, whose antitumor immune defenses are compromised. The complexity of
these tumor-host processes and their action through nontransferable, immune
mechanisms of the host implies that the capacity of adenovirus 5 E1-transformed
mammalian cells to form tumors in immunodeficient rodents does not represent a
risk factor for the manufacture of viral vaccines provided the cells can be
shown to be devoid of adventitious agents (see additional discussion on
adventitious agents below).
can be considered in evaluating tumorigenicity of adenovirus 5-transformed human
cell substrates. These approaches include demonstration that the tumor-forming
capacity of the cells in rodents is adenovirus 5-like, and that the cells in the
master cell bank are devoid of known and occult adventitious agents.
of DNA in Vaccines
Residual DNA in
vaccines derived from tumorigenic cells, including those transformed by Ad5, can
pose potential risks to the vaccine recipient in two respects: oncogenicity and
infectivity. Each of these biological properties must be considered and
evaluated for each cell substrate.
The oncogenic risk
of cell substrate DNA has been considered to be due to several mechanisms.
First, the residual DNA could have dominant activated oncogenes that could exert
their effect following expression in recipient cells. In the case of
Ad5-transformed cells, the dominant oncogenes would include the E1A and E1B
genes. Second, the incoming DNA could integrate into the host genome in certain
genes, such as the p53 gene or the retinoblastoma susceptibility (RB) gene,
termed tumor suppressor genes, which are involved in cell cycle control among
other cellular processes. Loss of function of tumor suppressor genes has been
associated with certain human tumors. Third, integration of residual
cell-substrate DNA could result in the activation of cellular regulatory genes
by promoter/enhancer insertion, and this could result in the development of a
neoplastic phenotype; this mechanism for tumor development was initially
described in chickens for leukemia formation by avian leukosis viruses. Another
result of integration that has been described is an increased methylation of
adjacent DNA sequences as well as sequences on other chromosomes, although the
consequences of such changes in methylation patterns to a cell are unknown.
biological activity of DNA that should be considered is its potential
infectivity. If a genome of a DNA virus or the provirus of a retrovirus is
present in the cell substrate used for vaccine manufacture, then the residual
DNA has the potential, upon inoculation into the vaccine recipient, to produce
infectious virus from this DNA and thus establish a productive infection.
The assessment of
the risk of DNA — both the oncogenic risk and the infectious risk — needs to
be considered both in terms of (1) the amount of residual DNA inoculated; and
(2) the concentration of oncogene or infectious genome present in this DNA. One
assumption is that the biological activity of any DNA administered is directly
proportional to the amount of that DNA, and if the active component (oncogene or
infectious genome) is carried as part of the cell-substrate DNA, the amounts of
the oncogene or infectious genome will be present at a level of 10-5
to 10-6. This is because the haploid mammalian genome is 3 x 109
base pairs, whereas an average gene is between 3 x 103 and 104
base pairs. Thus, if the residual DNA is present at 10 ng, an oncogene in that
DNA would be present at between 0.00001 and 0.0001 ng. Currently there are no
data indicating that purified isolated oncogenes or any other DNA are
biologically active at these levels.
It is also important
to note that an additional safety margin for oncogenic activity is provided by
the multi-step nature of cancer. This is because if more than one gene or event
is required, the risk is diminished and is given by the product of the risk for
each event. Thus, if the risk of a neoplastic event being induced by one
oncogene is 1 in 106, then if two oncogenes are required, the risk is
reduced to 1 in 1012.
Strategies that can
be considered in evaluating residual DNA for vaccine products manufactured in
adenovirus 5-transformed cell substrates include restricting the level of
residual DNA to 10 ng or less. If these levels of residual DNA are not feasible,
other methods can be considered to demonstrate the safety of higher levels of
DNA, such as the inoculation of cell-substrate DNA into neonatal rodents. In the
future, more sensitive animal models and in vitro assays may be developed to
assess the oncogenic activity of DNA.
The experience in
the early 1960s with SV40 contamination of poliovirus and adenovirus vaccines
and the continuing questions regarding whether SV40 could be responsible for
some human neoplasms underscore the importance of keeping viral vaccines free of
adventitious agents. This is particularly important when there is a theoretical
potential for contamination of a vaccine with viruses that might be associated
It is unclear
whether neoplastic cells have a greater or lower adventitious agent risk than
other types of cells. Because they can be grown for long periods in tissue
culture, there may be greater opportunities for any adventitious agents to be
detected. Because neoplastic cells survive indefinitely, it is easier to qualify
and bank cells that have passed all tests, especially as compared with primary
cells (which are derived repeatedly from live tissue and must be re-qualified
with each use). Moreover, many neoplastic cells can be grown in serum-free
medium, potentially reducing the likelihood of contamination with bovine
adventitious agents. However, if their growth in tissue culture is not well
controlled, there may exist additional opportunities for contamination of cells
with a longer lifespan. In cases of neoplastic cells for which the transforming
event is unknown, there is also a theoretical possibility that transformation
occurred as a result of a previous viral infection. Because some mammalian
tumors and some cells transformed by viruses contain infectious virus, cells
transformed by an unknown mechanism have a theoretical risk of containing a
transforming virus. Cells for which specific knowledge of the transforming event
exists (and can be shown not to be a virus that persists in the cells) may be
more easily characterized than cells for which there is no specific knowledge of
the transforming event (which could theoretically have been due to an infection
with a known or an unknown virus).
adventitious agent testing is required for all cells that are proposed for use
in vaccine production. This includes testing in various tissue culture systems,
inoculation of animals followed by observation or detection of pathogen-specific
antibodies, observation by electron microscopy, and molecular tests as is
appropriate based on the history and type of cell to be tested. Specific
polymerase chain reaction assays are used to rule out the presence of many
different viruses. PCR-based reverse transcriptase assays are used to rule out
the presence of retroviruses. The most sensitive of these assays include
amplification steps, either as a result of viral growth in culture or in an
organism, or molecular amplification such as in PCR.
Ad5-transformed cells are thought to be transformed by a known mechanism, the
consequences of overlooking an occult oncogenic agent are significant. As these
are the first cells in their class to be considered for vaccine production,
evaluating them for the presence of occult oncogenic agents could enhance
confidence in their use. One relevant animal assay that could be used for such
an evaluation is the inoculation of cell-free lysates into susceptible newborn
rodents, followed by observation for 5-6 months. This assay would detect most
known tumor viruses, as well as potentially detect unknown tumor viruses.
areas of research suggest that experimental assays to detect unknown
adventitious agents could soon become more generally available. As such assays
become available, they could be considered for use in qualifying novel cell
substrates, including neoplastic cell substrates.
Transmissible Spongiform Encephalopathy (TSE)
In addition to
assessing the possibility of contamination of cell substrates with infectious
virus, it is important to consider other agents such as the agent of TSE. There
are several mechanisms by which vaccine cell substrates, including neoplastic
cells, could theoretically become infected with a TSE agent. First, viral
vaccines are developed and manufactured in cell substrates that may be derived
from humans, and all human cells represent a finite possibility of being derived
from individuals with a propensity to develop sporadic or familial
Creutzfeld-Jakob disease (CJD). Although the mechanism by which such individuals
develop CJD is not understood, CJD has in the past been transmitted to humans by
biological products derived from CJD patients, such as human growth hormone and
dura mater grafts. Second, vaccine cell substrates are usually exposed to
products derived from cattle during tissue culture. Bovine spongiform
encephalopathy (BSE) has been transmitted to humans in Europe in the form of
variant CJD, possibly due to ingestion of infected beef. Under certain
circumstances, cells in tissue culture can support the replication of certain
TSE agents, although it is not known whether human cells in tissue culture can
sustain the replication of the BSE agent. Assays to detect TSE/BSE agent
contamination exist, but they may not be sensitive enough to exclude low levels
of contamination. Until more is known about the replication of TSE/BSE agents in
tissue culture and until more sensitive assays to detect these agents become
available, the concern over the possible contamination of viral vaccines with
TSE/BSE agents will be at least a theoretical consideration in vaccine
The use of
immortalized, neoplastic human cells as substrates to develop recombinant viral
vectors as vaccines also raises theoretical concerns with regard to possible
contamination with TSE/BSE agents. These concerns include: (1) the implications
of the possible presence of a prion protein (PrP)-encoding gene (PRNP) that is
abnormal in the individual from whom the cells were derived; (2) the possibility
that the genomic instability attendant with neoplastic processes could produce
pathogenic alterations in the normal PRNP gene; (3) the possible exposure to
agents of BSE present in bovine serum used in the propagation of the cells; (4)
the possibility that an increased level of expression of either a normal or
abnormal PRNP gene or other unknown factors in neoplastic cells might, upon
exposure, sustain the replication of abnormal PrP proteins or otherwise
contribute to the development of TSE in humans; and (5) the possibility that
differences in the levels of expression of PRNP genes among clonal/subclonal
populations of neoplastic cells may make evaluation of these potential risks
more difficult. Since the lifetime risk of sporadic TSE in the population is
about one case per ten thousand people (see Brown, P. et al., , Potential
epidemic of Creutzfeldt-Jakob disease from human growth hormone therapy. N Engl
J Med. 1985, 313:728-731), there is a finite risk that random tissue samples
used for the development of neoplastic cell lines could contain abnormalities
that might be associated with TSE transmission.
can be considered for evaluation of neoplastic human cells for possible
contamination with TSE/BSE agents. These strategies include a determination of
the origin of the cells with respect to the possible family history and medical
history of the donor regarding TSE risk factors and the identification and
documentation of possible exposure of the cell line to bovine-derived materials,
such as fetal bovine serum from countries with BSE. Further, two validated
methods that could be used to evaluate the potential risk from the TSE agent
include sequencing of the PRNP gene from neoplastic cell substrates and
evaluating all such cell substrates by Western blot for the presence of protease
resistant PrP. Additional studies may become feasible in the near future and may
include evaluation of PRNP expression levels, determination of the ability of a
cell substrate to support replication of the BSE agent, and evaluation of for
the presence of infectious TSE agents by animal inoculation. As new assays for
the detection and evaluation of TSE agents become available, they should be
introduced as appropriate for cell substrate screening.
OVRR/CBER plans to
present these issues to the FDA TSE Advisory Committee for a comprehensive
discussion in the near future. In the interim, for cell substrates for which the
presence of TSE could be a risk, sponsors should evaluate this issue by a
combination of strategies, as may be technically feasible. Nevertheless, OVRR/CBER
would like this Committee to be aware of and consider those issues related to
the possible presence or exposure of cell substrates used for the development of
viral vaccines to agents associated with TSE/BSE.
experiments have demonstrated the utility of Ad5 vaccine vectors as means of
stimulating cell-mediated immunity against HIV-1. Based on the review of
available data, OVRR/CBER believes that there is an extremely low probability
that residual DNA from the adenovirus 5-transformed human cells could transfer
traits that could induce neoplastic development in vaccinees. OVRR/CBER also
believes that such cells may be considered for the development of HIV vaccines,
provided that the phenotype of the cells can be documented to be of an
adenovirus 5 E1 type, and that appropriate testing rules out the presence of
adventitious agents within the limits of state-of-the-art technology.
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