Since he discovered the work of the American virologist W.P. Rowe's on the identification of “a possibly new, tissue culture cytopathogenic agent” in tonsil tissues (1), Alex has only one idea in mind. And this thought obsesses him all day long in his laboratory: to leave its mark on adenovirus-type viruses’ research.
In 1966, after many years, Alex van der Eb finally characterized the adenovirus DNA - composed of 35,000 base pairs in a double strand - simultaneously with Dr. Green in his thesis work and so obtained the title of doctor in 1968 from Leiden University, Netherlands.
But this is just the beginning, and Alex wants to take the research a step further. There is no shortage of work to explore the adenovirus field, and he needs help. This is how Frank applied for a post-doc internship position in his laboratory. Originated from Canada, Frank had just successfully completed his Ph.D. in Toronto. The young man knows very little about adenovirus but is curious, and has already demonstrated an insatiable thirst for learning.
Oncogene theory and first experiments
Enthusiastic about Huebner and Todaro recent theories on oncogenes, Frank Graham submitted a subject to Alex: he decided to investigate the correlation between low GC content in adenoviral DNA and oncogenicity. As Frank will recall us a few years later during an interview at McMaster University “At that time, nobody knew much about how or why cancers started, so using oncogenic viruses to transform normal cells into cancerous cells in culture seemed a good in vitro model for tumour induction”.
Graham achieved to transfect HK cells (subline of the ubiquitous keratin-forming tumor cell line HeLa) with adenovirus type 5 DNA, thanks to the forming of a calcium phosphate-DNA precipitate, a technique still in use today in many viral vector companies. Calcium phosphate facilitates the binding of the DNA to the cell surface and DNA then enters the cell by endocytosis (2).
But overseas, Rowe and his team are used to inoculate SV40 derived-viruses in primary cultures of human embryonic kidney (HEK) cells, purchased from Microbiological Associates, Inc. (today BioReliance, part of Sigma group). Van der Eb found a way to gain access to a healthy, legally aborted foetus (3) and isolated embryonic kidney cells from it. He proposed to Frank to test its calcium phosphate transfection technique on HEK cells.
A man with dreams needs a woman with vision
Galvanized by his success with HK cells, Frank is confident that he will succeed in transfecting HEK cells with type 5 adenoviruses. Starting his work in 1972, he quickly falls apart after the first attempts. Only one clone produced a single transformed colony, and even that subsequently failed to grow. In 1973, Frank identified a pair of transformed colonies but the cells double only every 10 days, and he collected sufficient cells for one vial after one year of work.
After a few months, cells totally stop to grow. And Frank went through moments of doubt, not knowing why the cells suddenly stopped growing.
But as they say, "behind every great man, there is a woman".
Silvia Bacchetti, Frank's wife, was also brilliant researcher on genetics. After a long evening of discussion about the problem of HEK growth, Silvia presented Frank with a hypothesis.
“What if HEK cells are not expressing telomerase at the current moment?”
Telomerase is the enzyme that protects telomeres (chromosomes tips) from shortening with each cell division. Having found an explanation for his problems, Frank perseveres, hoping to go beyond this cell crisis period. In fact, if you look up the word "perseverance" in the dictionary, I’m pretty sure you’ll come across Frank L. Graham.
After 292 failures, Frank finally succeeded in 1977 in creating an immortal HEK lineage incorporating the adenovirus genome. As a tribute to his iterative learning from his failures, Frank named this line HEK293 (4). The cell line was later confirmed to have incorporated the Ad5 E1A and E1B genes into chromosome 19, E1 region being necessary for activation of viral promoters and expression of both early and late genes.
Beyond Pros and Cons of HEK293
HEK293 cell lineage rapidly showed numerous advantages for research:
- it grows pretty fast, with a doubling time of 36 hours
- it’s robust, semi-adherent and can be cultivated in suspension or monolayer mode,
- it can be used for transient or stable transfection with simple protocols,
- it is able to produce large amount of recombinant proteins from plasmid vectors carrying the CMV promoter region,
- it is able to perform human post-translational modifications on secreted recombinant proteins.
But in life, not everything falls under a pink world, and HEK lineage may have some drawbacks:
- due to its human origin, HEK293 is sensitive to human viral contaminations. Mycoplasma infections are also not to be neglected.
- if cultures are too extensive, with too many passages, HEK293 tends to become less and less reliable in terms of cell growth and transfection efficiency.
More importantly, for viral vector production purposes, recombination between the E1 region sequences in the complementing cell line and the recombinant virus can give rise to viral progeny with functional E1 genes that are replication competent (5).
In order to minimize this last occurrence, Alex van der Eb and his team wanted to develop a new tool: a cell line dedicated to pharmaceutical manufacturing whose the raw materials and elements offer a full traceability (not really the case for HEK293), and free of replication competent adenoviral (RCA).
In 1985, Alex Van der Eb gained access to a 18 week gestation aborted foetus but this time isolated retinal cells from it. Human embryonic retina was chosen because Byrd (6) showed not long before that that human embryonic retina was permissive to transformation, and didn’t show any crisis period.
Brahm Bout from Introgene company, the spin-off of Leiden University, and Frits Fallaux from the Gene Therapy Group of Leiden University collaborated to create this new cell line under the supervision of Alex. Of the potential RCA-free clones of interest, clone 6 gave the highest yield of viruses and a high expression of E1A and E1B. Created from Primary human Embryonic Retinal cells, this clone led to the Master Cell Bank and Working Cell Bank of PER.C6.
Introgene became Crucell after the merger with U-Bysis.
The rest is licensing history.
(1) Rowe, W. P.; Huebner, R. J.; Gilmore, L. K.; Parrott, R. H.; Ward, T. G. (1953). "Isolation of a Cytopathogenic Agent from Human Adenoids Undergoing Spontaneous Degeneration in Tissue Culture". Experimental Biology and Medicine. 84 (3): 570–573. doi:10.3181/00379727-84-20714
(2) F.L.Graham, A.J.van der Eb. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology volume 52, Issue 2, April 73, Pages 456-467. https://doi.org/10.1016/0042-6822(73)90341-3
(3) Alvin Wong, M.D. The Ethics of HEK 293. National Catholic Bioethics Quarterly 6.3 (Autumn 2006): 473–495 by The National Catholic Bioethics Center.
(4) Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36, 59–74
(5) H. Lochmüller, A. Jani, J. Huard, S. Prescott, M. Simoneau, B. Massie, G. Karpati. Emergence of Early Region 1-Containing Replication-Competent Adenovirus in Stocks of Replication-Defective Adenovirus Recombinants (ΔE1 + ΔE3) During Multiple Passages in 293 Cells. Human Gene Therapy VOL. 5, NO. 12. https://doi.org/10.1089/hum.1994.5.12-1485
(6) Byrd, P., Brown, K. & Gallimore, P. Malignant transformation of human embryo retinoblasts by cloned adenovirus 12 DNA. Nature 298, 69–71 (1982). https://doi.org/10.1038/298069a0