PERSPECTIVES

LANDMARK

Monoclonal antibodies: the story of a

discovery that revolutionized science
and medicine
Sefik S. Alkan
Monoclonal antibodies are unique molecules
that can be used equally well in research,
diagnosis and in the treatment of diverse
diseases, including rheumatoid arthritis and
cancer. In this article, I describe how three
scientists from different cultural backgrounds
made this remarkable discovery, which has
had such impact on medical research.
freedom is the oxygen without which
science cannot breath. David Sarnoff

The discovery of monoclonal antibodies has

changed the face of biomedicine and will
probably impact our lives greatly in the coming
centuries. One of the landmark studies in this
area was the development of lymphocyte
fusion for the production of monoclonal antibodies by Georges Khler (19461995) and
Csar Milstein (19272002) in 1975 (REF. 1).
However, few people know the background
of the ideas and questions that the scientists
pursued, as well as the theories and fortunate
coincidences that led to this remarkable discovery. As we enter a new phase in which
monoclonal antibodies are being used as
therapeutic agents for cancer and other diseases, it is time to reflect on these key events.
I hope that the history of this particular discovery will be illuminating for generations to
come. Here, as a fascinated witness and friend
of Khler and of Niels Jerne (19111994),
who also made an important contribution,
I highlight the key elements of their discovery
(FIGS 1 and 2).

Let me start with a pleasant scene from

Istanbul, Turkey. Three years after the discovery of monoclonal antibodies, Khler, Zelig
Eshhar, myself and our wives were sailing on a
boat along the Bosporus. On a luminous day
with blue skies, encompassed by thousands of
years of history to the east (Asia) and the west
(Europe) of us, Khler turned to me and said,
I would like to spend the rest of my life here
in Istanbul. Can you find me a University job?
I will need a helicopter though with this
traffic!. We laughed. And I responded,You
can only afford that after winning the Nobel
Prize, but for that you will have to wait for
Susumu Tonegawas. You want to bet?.
Yes, I replied. (I lost the bet in 1984;
Khler won the Nobel Prize first, followed
by Tonegawa in 1987.)

Figure 1 | Photograph of Csar Milstein.

NATURE REVIEWS | IMMUNOLOGY

To really appreciate the path that led to

the discovery of monoclonal antibody technology, we need to go back half a century
and understand the generation of antibody
diversity. How is it possible that the immune
system can make antibodies that can recognize almost any microorganism (viruses,
bacteria, fungi, yeast and parasites) or any
other foreign invaders that exist? Antibodies
were discovered in the 1890s and it took until
the 1960s for scientists to discover that only
lymphocytes could produce them2. Our
immune system has ~1 1012 lymphocytes,
which constitute ~1% of our body weight.
What mechanism allows these cells to generate
millions of specific proteins, each binding
perfectly to only one antigen?3
The three scientists

Lets start with Jerne. We met in Berkeley, a few

days after my arrival at the San Francisco
Medical Centre, California, USA, in 1969. A
colleague, Richard Pink, took me to the seminar in which Jerne talked about the immune
response, its diversity and regulation. In
particular, I remember the accompanying
discussion prompted by questions from
Berkeleys revolutionary students. It was
compelling. We met a second time in 1975 at
the Weizmann Institute, Rehovot, Israel. He
was interested in my work on TB-cell collaboration and invited me to join the Basel
Institute for Immunology in Switzerland. Jerne
a philosopher and proponent of systems
biology was the first scientist to put forward the natural selection theory of antibody formation in 1955 (REF. 4). In contrast to
the contemporary template theories, he
argued that antigens did not instruct antibody formation, but instead selected fitting
antibodies that pre-existed in the serum. This
revolutionary concept was Jernes first and
greatest contribution to immunology as it
caused a paradigm shift. Frank Macfarlane
Burnet refined Jernes hypothesis into the
clonal selection theory, proposing that
antibodies were cellular receptors5. He turned
out to be correct and the Nobel Prize was

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Figure 2 | Photograph of Georges Khler

and Niels Jerne. Photographed celebrating the
announcement of the Nobel Prize in Physiology
or Medicine awarded to Jerne, Khler and
Milstein on 15 October 1984. Reprinted with
permission from REF. 18 F. Hoffman-La Roche,
Basel, Switzerland (1996).

awarded to Burnet in 1960 (REF. 5). Jerne

believed that B cells must undergo somatic
changes to produce antibodies. So, after
founding the Basel Institute for Immunology,
he strongly supported Tonegawa, who
thought, if you want to solve the antibody
gene problem, you have to study antibody
genes. He successfully followed this approach
and won the Nobel Prize in 1987 (REF. 6).
Jernes second important contribution
was unexpectedly practical. He developed an
elegant haemolytic plaque assay that allowed
immunologists to visualize and determine the
number of antibody-producing B cells by the
naked eye7. Without the Jerne plaque assay,
Khler could not have detected hybridoma
cells producing monoclonal antibodies so
conveniently. I also admired Jerne for his
role in founding the Basel Institute for
Immunology, which trained hundreds of
immunologists from around the world. He
was a well-read man and strived to see the big
picture8. In his office, there was a long table
adorned by dozens of scientific journals; all
were being read regardless of language
(English, Dutch, Danish, French and
German). For example, he translated an article
for me from Danish on the development of a
new sensitive assay for antibody detection
using an antigen that I was working with. Was
Jerne always right? Almost always. In his
somewhat controversial network theory, he
proposed that, owing to the large diversity of
the antibody repertoire, some antibodies
eventually might recognize other antibodies as
antigen (idiotypes); he predicted that in the
following sequence of events, antigen antibody 1 antibody 2, the second antibody

154

should look like the antigen. Although this

concept has been shown experimentally, the
role of idiotypes in regulating the immune
response remains elusive and impossible
to test, as he once told me. One day he asked
me, I hope you are not wasting your time
with the stupid cells (macrophages), are
you?. He somehow heard that I was using
macrophages as antigen-presenting cells and
indicator cells for T-cell activation. He strongly
disliked any cell that did not have specific
receptors. Also, Jerne remained ignorant of the
importance of antigen-presenting cells and
innate immunity.
We can now consider Milstein, the second
discoverer. He was also interested in the mechanism of antibody diversity; unlike Jerne, he
believed that by determining the chemical
structure of different antibodies, the antibody
diversity problem could be solved9. He started
using transformed B cells (myelomas) to
obtain sufficient antibodies for chemical characterization. His group carried out myeloma
myeloma fusions to generate hybrid cells that
secreted different (or even hybrid) antibodies9.
Myelomas, however, were making antibodylike molecules without specificity. Numerous
attempts to find myelomas with somatic
mutations that had antigen-binding activity
had failed, but in this process Milsteins group
had developed a technology and wealth of
knowledge that later made the discovery possible. Armed with these results, Milstein came
to the Basel Institute in the 1970s to give a
seminar. After hearing Milsteins talk, the life of
Khler, the third scientist in this story, was
changed forever.
The discovery

The young, independent mind of Khler was

struggling to find a way to obtain enough antibodies from mortal B cells in culture. After
hearing Milsteins seminar, he worked out a
solution to this problem. According to Khler,
I was trying to find out how the variable and
constant regions of heavy and light chains of
antibody (genes) get together to generate antigen specificity. He considered fusing B cells,
which make antibodies against a known antigen, with Milsteins myelomas to make them
immortal, so that they could grow in culture
indefinitely and secrete antibodies of desired
specificity. A mutual friend, the late Charlie
Steinberg, confirmed that Khler nurtured
this idea before going to the Medical Research
Council (MRC). Khler joined Milsteins lab
as a postdoctoral fellow in 1973. There, he
used all of the important groundwork that
had been put in place by Milstein and his
group. He began by immunizing a mouse
with antigen (sheep red-blood cells). Then he

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collected spleen cells (full of activated

B cells) and fused them with an available
myeloma cell line. (I was told that by pure
luck he had picked the best myeloma fusion
partner.) He then used Jernes plaque-forming B-cell assay to detect hybridomas
(B cellmyeloma hybrids) that secreted sheep
red-blood-cell-specific antibodies (FIG. 3). The
experiment worked well because the basics of
the hybridoma technology and selective culture conditions that allowed growth of only
the hybrid cells, for example, were available in
the laboratory. He was also lucky with his
experimental protocol; his timing of cell
fusion after immunization was perfect. The
use of the mouse model and not the human
cells for fusion was also fortuitous, as numerous previous attempts to use the latter had
been unsuccessful. The title of his Nature
publication was Continuous cultures of
fused cells secreting antibodies of predefined
specificity1,10. These immortalized B cells
secreted a single type of antibody; for this
reason, we now routinely use the term monoclonal antibody. It is interesting to note that
Milstein and Khler did not attempt to
patent their findings.
After the discovery

Khler returned to the Basel Institute for

Immunology when I joined (1976). I was
fascinated by Khlers personality as well as
his cell-fusion method. He subsequently
taught me how to generate hydridomas.
However, I still thought Tonegawas approach
was a better tool for solving the issue of antibody diversity. As Tonegawa showed that
antibody diversity is generated by somatic
recombination6, I thought that the Nobel
Prize would go to him first. I thought monoclonal antibodies were better tools for
other things such as epitope mapping of
antigens11, affinity purification of mixed
molecules, development of diagnostic tests
and monoclonal antibody-mediated therapy.
But neither Khler nor the rest of the world
were interested in these experimental tools in
those days.
By the 1980s, we were both looking for
new jobs. Khler even approached Jerne for
a small salary increase, only to hear, What
is the matter with you; are you not interested in science anymore?. As both Khler
and I had exactly the same salary, I kept
quiet. Khler received several job offers
from the industries in Basel; however, he
told me that he could not lose freedom
even if it was only for a little while. As for
myself, I accepted a temporary position at
Ciba-Geigy, Basel, where I produced monoclonal antibodies specific for interferon-

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2004 Nature Publishing Group

PERSPECTIVES
Spleen cells (HGPRT+)
(Antibody producing)

Myeloma cells (HGPRT)

Freiburg. Until his sudden death in 1995, we

remained good friends and neighbours, he in
Germany and I in Switzerland, only a few
miles apart.
The 1984 Nobel Prize

Fusion in polyethylene glycol

Unfused
spleen
cells

Fused
spleen
cells

Hybridoma

Unfused
myeloma
cells

Fused
myeloma
cells

Cell death

Cell death
Proliferation

It took nearly ten years for the importance of

this discovery to be recognized by the Nobel
Committee. In 1984, Jerne, Milstein and
Khler shared the Nobel Prize for Physiology
or Medicine for theories concerning the
specificity in development and control of
the immune system and discovery of the
principle for production of monoclonal
antibodies. Clearly, none of these scientists
had any intention of generating monoclonal
antibodies. Instead, they were trying to
satisfy a century-old scientific curiosity
about the ability of our immune system to
cope with unpredictable invaders. The
crucial role of Milstein in this discovery can
not be overstated. However, I believe the
scientific atmosphere at the Basel Institute
for Immunology also had a unique role in
this and other discoveries. Bill Paul (National
Institutes of Health) recently asked me in
New York,Why do you think the Stockholm
Committee included Jerne in the 1984 Nobel
Prize?. My answer was simple: without Jerne,
there would have been no theories about
antibody diversity, no Basel Institute for
Immunology, no education of Khler, no
antibody-forming B-cell assay, no Milstein
seminar. However, without Milstein there
would have been no hybridoma technology,
and no unconditional support of a talented,
young scientist. Without Khler, we might
have had to wait decades to put all this
together.
The monoclonal antibody industry

Monoclonal antibodies with single antigen specificity

Figure 3 | Cell fusion and monoclonal antibody production. A schematic representation of hydridoma
technology. Spleen cells from an immunized mice are fused, using polyethylene glycol, with myeloma cells
that were rendered drug sensitive by a mutation in a growth essential gene HGPRT. The cell mixture is
then cultured in a medium containing the selective drug. As immune cells, although not sensitive to
HGPRT, survive for only about one week in culture and the myeloma cells are drug sensitive they will all die
within a week or so. The only cells that can survive are those hybrid myeloma cells that obtained a normal
HGPRT gene from the immune cells. These hybridomas can grow continuously in vitro and some secrete
antibody. By using appropriate screening technology, clones of cells that secrete antibody of interest can
be identified and expanded in vitro or in vivo to obtain large quantities of monoclonal antibody that can
subsequently be purified to homogeneity. HGPRT, hypoxanthine-guanine-phosphoribosyltransferase.
Modified, with permission, from REF. 19 Johns Hopkins University (2003). www.hopkins-arthritis.org.

molecules and many other molecules of

interest 11,12. However, I was mainly interested in finding a myeloma partner to fuse
with human B cells to produce human
monoclonal antibodies. Unfortunately,
similar to many other scientists across the
world, all my years of efforts failed13.

Khler almost accepted a job at the Red

Cross Blood Centre in The Netherlands. At
the same time, the press in his native country
Germany started a publicity campaign about
his contribution to the discovery of monoclonal antibodies. Only then was he offered a
position at the Max Planck Institute in nearby

NATURE REVIEWS | IMMUNOLOGY

Today, mass production of thousands of

different monoclonal antibodies by hundreds
of biotech companies has enabled us to carry
out unparalleled biomedical research. In
addition, monoclonal antibodies have
increased the quality and speed of antibodybased diagnostic tests for numerous
diseases3,14. But, there is still more excitement
to come: monoclonal antibody-mediated therapy (my dream in the early 1980s) started
with mouse monoclonal antibodies, moved
to mousehuman chimaeras and later to
humanized monoclonal antibodies. Antibody
engineering has evolved into its own industry.
Today, monoclonal antibodies can be produced in bacteria, yeast, mammalian cells and
transgenic animals15. A herd of 75 transgenic
goats can produce in its milk 300 kilograms of
purified monoclonal antibody per year16.
Recently, a completely human monoclonal

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antibody (Abbott Humira, adalimumab)
entered the market, which should be good
news for individuals with rheumatoid arthritis.
When Khler died in 1995, the monoclonal
antibody market was worth a few million US
dollars; in 2002 the monoclonal antibody
market was valued at four billon US dollars17.
Wood Mackenzies forecast for 2005 is nearly
nine billon US dollars. Monoclonal antibodymediated therapy now covers the fields of
cancer, infectious diseases, transplantation,
allergy, asthma and some autoimmune
diseases. The major therapeutic advantages of
monoclonal antibodies are their high specificity, the high affinity with which they bind to
targets and the limited side effects associated
with their use.
The beauty of science is in its unpredictability. The wondering scientist needs to
be protected, to be supported by institutions
such as the Basel Institute for Immunology
and the MRC; there, he/she can learn how to
be open minded, to be critical of data and to
question paradigms. Also, as Jerne told me, it
is important to avoid doing experiments
before understanding the meaning of the last
experiment. The late Steinberg used to tell
me, You are better off by publishing few
articles; at least you are saving some trees
(paper) and other peoples time. I believe it
was the freedom and multicultural atmosphere at the Basel Institute for Immunology
and the MRC that fostered collaboration
between scientists that shaped this major

156

breakthrough, earned three Nobel prizes,

revolutionized medical science and enhanced
the quality of our lives.
Note added in proof

A recent publication describes a new technique

for the production of monoclonal antbodies:
Pasqualini, R. & Arap, W. Hybridoma-free
generation of monoclonal antibodies. Proc.
Natl Acad. Sci. USA 101, 257259 (2004).
Sefik S. Alkan is presently Head of Immunology at
Pharmaceuticals division, 3M Centre, St Paul,
Minnesota 55144, USA.
e-mail: ssalkan@mmm.com
doi:10.1038/nri1265
1.

Khler, G. & Milstein, C. Continuous cultures of fused cells

secreting antibodies of predefined specificity. Nature 256,
495497 (1975).
2. Silverstein, A. M. in Fundamental Immunology. 3rd edn (ed.
Paul, W. E.) 2141 (Raven Press, New York, 1993).
3. Janeway, C et al. Immunobiology. (Garland Publishers,
2001).
4. Jerne, N. K. The natural-selection theory of antibody
formation. Proc. Natl Acad. Sci. USA 41, 849857
(1955).
5. Burnet, F. M. Immunological recognition of self. Nobel
lecture, 589701 (12 December 1960).
6. Tonegawa, S. Somatic generation of immune diversity.
Nobel lecture, 381405 (8 December 1987).
7. Jerne, N. K. & Nordin, A. A. Plaque formation in agar
by single antibody producing cells. Science 140, 405
(1963).
8. Jerne, N. K. The generative grammar of the immune
system. Nobel lecture, 211225 (8 December 1984).
9. Milstein, C. From the structure of antibodies to the
diversification of the immune response. Nobel lecture,
248270 (8 December 1984).
10. Khler, G. Derivation and diversification of monoclonal
antibodies. Nobel lecture, 228243 (8 December 1984).
11. Alkan, S. S. & Braun, D. G. In Synthetic Peptides as
Antigens. 264278 (Ciba Foundation, Wiley, Chichester,
1986).

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12. Asselbergs, F. et al. Localization of peptides recognized by

monoclonal antibodies on tissue-type and urokinase-type
plasminogen activators using recombinant hybrid
enzymes. Fibrinolysis 7, 114 (1993).
13. Alkan, S. S. et al. Estimation of heterokaryon formation and
hybridoma growth in murine and human cell fusions.
Hybridoma 6, 371379 (1987).
14. Monoclonal Antibody-Based Diagnostics. Global Industry
Analysts, 1998.
15. Antibody Engineering, special edition, J. Immunol. Methods
231 (1999).
16. Pollock, D. P. et al. Transgenic milk as a method for the
production of recombinant antibodies. J. Immunol.
Methods 231, 147157 (1999).
17. W. Mckenzies PharmaQuantTM. Monoclonal antibodies: on
the crest of a wave. Horizons, Pharmaceuticals Issue 6
(January 2003).
18. Jerne, N. K. & Melchers, F. 25 Years Basel Institute for
Immunology Annual Report Introductions. (Roche, Basel,
Switzerland, 1996).
19. Soloski, M. J. What on Earth is a Monoclonal Antibody?
Johns Hopkins Arthritis Information on monoclonal
antibody development (2003).

Acknowledgements
I am grateful to F. Cochran and C. Akdis for critical reading of the
manuscript.

Competing interests statement

The author declares that he has no competing financial interests.

Online links
FURTHER INFORMATION
The Nobel Prize in Physiology or Medicine 1984:
http://www.nobel.se/medicine/laureates/1984/
Csar Milstein autobiography:
http://www.nobel.se/medicine/laureates/1984/milsteinautobio.html
Niels K. Jerne autobiography:
http://www.nobel.se/medicine/laureates/1984/jerneautobio.html
Georges J.F. Khler CV:
http://www.nobel.se/medicine/laureates/1984/kohler-cv.html
FURTHER READING
Brekke, O. H. & Sandlie, I. Therapeutic antibodies for human
diseases at the dawn of the twenty-first century. Nature Rev.
Drug Discovery 2, 5262 (2003)
Access to this interactive links box is free online.

www.nature.com/reviews/immunol
2004 Nature Publishing Group