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Monoclonal Antibodies Completo
Monoclonal Antibodies Completo
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ever, whose rapidly proliferating cells ma cells in tissue culture at the Salk In
W
hen a foreign substance enters
the body of a vertebrate ani produce large amounts of abnormal im stitute for Biological Studies, but the
mal or is injected into it, one munoglobulins called myeloma pro line was then lost. Eventually Horibata
aspect of the immune response is the teins. A tumor is itself an immortal and A. W. Harris were able to establish a
secretion by plasma cells of antibodies: clone of cells descended from a single number of lines, which they distributed
immunoglobulin molecules with com progenitor, and so myeloma cells can be to other laboratories. My group at the
bining sites that recognize the shape of cultured indefinitely, and all the immu Medical Research Council Laboratory
particular determinants on the surface noglobulins they secrete are identical in of Molecular Biology in Cambridge
of the foreign substance, or antigen, and chemical structure. They are in effect subjected a line derived from one of
bind to them. The combination of anti monoclonal antibodies, but there is no Potter's tumors to intensive study.
body with antigen sets in train processes way to know what antigen they are di At that time we were not thinking
that can neutralize and eliminate the rected against, nor can one induce mye about monoclonal antibodies. We were
foreign substance. Quite apart from the lomas that produce antibody to a specif studying how somatic (body) cells diver
natural function of antibodies in the im ic antigen. sify in culture and how mutations modi
mune response they have long been an In 1975 my colleagues and I learned fy the combining specificity of antibod
important tool for investigators, who how to fuse mouse myeloma cells with ies, and the mouse myeloma line was for
capitalize on their specificity to identify lymphocytes from the spleen of mice us simply another appropriate tissue
or label particular molecules or cells immunized with a particular antigen. culture line. By 1973 Richard G. Cot
and to separate them from a mixture. The resulting hybrid-myeloma, or "hy ton, David S. Secher and I were able for
The antibody response to a typical an bridoma," cells express both the lym the first time to produce structural mu
tigen is highly heterogeneous. There are phocyte's property of specific-antibody tants of a mouse myeloma protein se
perhaps a million different lines of B production and the immortal character creted by a cultured cell line. That work
lymphocytes, the precursors of plasma of the myeloma cells. Such hybrid cells and parallel investigations by Matthew
cells, in the spleen of a mouse or a man. can be manipulated by the techniques D. Scharff of the Albert Einstein Col
All are derived from a common stem applicable to animal cells in permanent lege of Medicine in New York demon
cell, but each line develops an indepen culture. Individual hybrid cells can be strated spontaneous mutations in cul
dent capacity to make an antibody that cloned, and each clone produces large tured cells that affected the structure
recognizes a different antigenic determi amounts of identical antibody to a sin of the proteins they manufactured, and
nant. When an animal is injected with an gle antigenic determinant. The individu also told something about the molecular
immunizing agent, it responds by mak al clones can be maintained indefinitely, nature of the mutations and their fre
ing diverse antibodies directed against and at any time samples can be grown in quency. The search for mutants was la
different antigen molecules on the in culture or injected into animals for the borious, however, because the proteins
jected substance and different determi large-scale production of monoclonal made by the parental cells lacked recog
nants on a single antigen, and even dif antibody. Highly specific monoclonal nizable antibody activity, changes in
ferent antibodies that fit, more or less antibodies produced by this general which would be the most effective in
well, a single determinant. It is next to method have proved to be a remarkably dication of slight differences caused
impossible to separate the various anti versatile tool in many areas of biologi by mutations. Clearly what was needed
bodies, and so conventional antiserums cal research and clinical medicine. was a cell line that secreted an immu
contain mixtures of antibodies, and the noglobulin exhibiting antibody activity
mixtures vary from animal to animal. uman myelomas have been known that could be easily assayed. No such
Each antibody is made, however, by a
different line of lymphocytes and their
H to physicians for a long time, but it
was not until the early 1960's that the
line existed.
At that point a lucky circumstance led
derived plasma cells. What if one could precise nature of myeloma proteins was us to the hybrid-myeloma technique.
pluck out one such cell making a sin elucidated by immunologists. Michael While we were working on somatic mu
gle specific antibody and grow it in cul Potter of the National Cancer Institute tations Georges Kohler and I were also
ture? The single cell's progeny, or clone, then induced myelomas in mice, and following a quite different line of re
would be a source of large amounts of these too produced large amounts of search in an attempt to learn more about
identical antibody against a single anti monoclonal immunoglobulins. In spite the genetic control of the synthesis of
genic determinant: a monoclonal anti of much effort, however, it was not antibodies. The synthesis of antibodies
body. Unfortunately antibody-secreting possible to induce tumors that could is controlled by two sets of genes. One
cells cannot be maintained in a culture synthesize antibodies to an injected anti set encodes the "variable" region of the
medium. gen. Leo Sachs, Kenko Horibata, Edwin antibody molecule'S light and heavy
There are malignant tumors of the S. Lennox and Melvin Cohn did succeed chains, the region that controls antibody
immune system called myelomas, how- in establishing a line of mouse myelo- specificity; the other set encodes the
66
© 1980 SCIENTIFIC AMERICAN, INC
•
and C
ed by a single pair of V (for variable)
(for constant) genes out of a large
repertory of such genes in the cell, and
We fused two myeloma cells, one from
a mouse line and one from a rat line.
Analysis of the hybrid cells showed that
the
DNA DNA.
regions are separated by introns,
or intervening sequences of
entire stretch of
The
is transcribed
a b
..
•
,-
fill •.
I-
••
.
• ••
.�
' 't�
-
•,,•- •
.,.
1d
ANTIBODY-SECRETING CLONES of bybrid-myeloma cells loma) cells and plated tbe bybrids (top right). Hybrid-cell colonies de
were first detected by a test for antibodies to sbeep red blood cells. veloped (black spots). Wben a layer of sbeep red cells was added along
In tbe standard test (top left) tbe red cells and antibody-secreting cells witb complement, a few bybrid colonies gave rise to plaques (white
are incubated on agar, and complement (a protein complex from areas aroulld colollies), indicating tbat tbey were secreting specific
blood plasma) is added. Antibody diffusing from eacb secreting cell antibody. Individual cells were picked from a colony of antibody
binds to antigens on nearby blood cells, initiating a complement reac secreting cells and plated tbinly (bo om left); most of tbe clones de
tf
tion that kills blood cells, forming a plaque: a clear area (white spots) rived from tbem proved to be secretors of tbe anti-red-cell antibody.
around each secreting cell. Tbe autbor and Georges Kobler fused A pbotomicrograpb of a single secreting clone (bottom right) sbows
mouse cells secreting antibody to tbe blood cells witb tumor (mye- tbe individual cells of the clone and tbe area of dead cells around it.
67
© 1980 SCIENTIFIC AMERICAN, INC
> C
experiment, in other words, showed that
S"/ j \ '\
the splicing of the Vand the sequences
takes place within a single molecule of
RNA.
LYMPHOCYTES8
e e HYBRI
CELLSD-
both parents was "codominantly" ex
pressed by the fused cells. That finding
suggested to Kohler and me a possible
answer to our need for an antibody-pro
\
�
, !
..v
j / j \�
ducing cell in the mutation experiment.
It occurred to us that it might be possi
ble to fuse a normal lymphocyte or plas
/u-r.:!",
ANTIBODY,.. � ANTIGEN
\.. /
C"" � 1 �2 �3 �4
CLONE
ma cell with a myeloma cell and thus to
immortalize the expression of the plas
ma cell's specific-antibody secretion. We
would be applying the well-established
�
namely to fix in a permanent cell line a
function that is normally expressed only
� MONOCLONAL
���
in a "terminal" cell� the plasma cell de
is
family of proteins called immunoglobulins. The basic shape of an immunoglobulin molecule
that of a molecule of the class immunoglobulin G (lgG), a heterogeneous population of mole
cules sharing a Y-shaped structure composed of two kinds of molecular chain, heavy and light,
Eventually the basic problem was dis
covered: a toxic batch of one of the re
agents. Once that fault was remedied,
linked by disulfide bonds. The number and precise position of the disulfide bonds differ and are
with Jonathan C. Howard and Geof
characteristic of the IgG subclass. Each chain has two regions. In the variable region amino
acid sequences that differ from antibody to antibody provide differently shaped combining frey W. Butcher of the Agricultural
sites that bind specifically to different antigens. Constant region of the chains, with the same Research Council Institute of Animal
amino acid sequence in all antibodies of a given subclass, is responsible for other functions. Physiology in Cambridge we achieved a
68
© 1980 SCIENTIFIC AMERICAN, INC
body-secreting
about one in 10
plasma cell, and yet
of our hybrid clones FUSE
we had 10
turned out to secrete antibody. That is,
times as many positive, im
mortal hybrids as one. would expect if 1
immortality were randomly transferred
to the heterogeneous spleen-cell popula
tion; apparently we were achieving se
lectivity along with immortality. The
explanation for this selectivity is not
HAT MEDIUM
completely established, but according
to recent evidence it probably has two
components. On the one hand, secretion
seems to be amplified, with lymphocytes
that synthesize antibody but do not nor
1
�:):::,:.\. :;)-.-1- 50"''',,,,,
mally secrete it giving rise to hybrids
- - - -W) -----.
that both synthesize and secrete anti
body. Probably the myeloma parent
provides the secreting machinery some
antibody producers lack. On the other
hand, the conditions under which the
HYBRID MOLECULES SCRAMBLED MOLECULES
fusion takes place apparently make it
ARE MADE ARE NOT MADE
unlikely that spleen cells other than B
yy
lymphocytes will give rise to long-lived
hybrids.
GENE
A' B
GENE
B'
� �
TRANSCRIPTION
spleen-cell parent and is expressing both
A A' B B'
sets of chromosomes. Potentially a hy RNA
A' B
� B'
one kind of light chain), can produce
MESSENGER RNA
two heavy chains and two light ones. We
refer to such a cell as HLGK because it
secretes the heavy and the light chains of
the spleen-cell parent and the corre
ANTIBODY
A �I U\I\I\,,-
�I
TRANSLATION
A' B
-- B'
from the other animal (right). These results indicated that the variable and the constant regions
secretion pattern is HLK or GLK, and are transcribed from DNA into RNA, and the RNA is processed and translated into protein, as
these in turn to such variants as HL,
HK, LK, L and K.
It is the clone of HL cells, expressing
is indicated (b), and also that the genetic information of both parental cells is "codominantly"
expressed by the hybrids. More recent studies on DNA show that the
selves are interrupted by intervening sequences that are excised in the course of processing.
V and C genes them
69
© 1980 SCIENTIFIC AMERICAN, INC
�
eliminating the distinction between the
A and the B groups.)
We have been able to establish that a
monoclonal antibody to the individual
l ---
test we could think of. The substance of
choice was interferon, which is notori
r--- ISOLATED CLONES ously difficult to purify and to obtain in
significant quantity. When Secher and
Derek C. Burke of the University of
Warwick set out to purify interferon by
immunoadsorption, the best interferon
preparations to which they had access
were about 1 percent pure. They immu
nized mice with a preparation of this
crude interferon, fused spleen cells from
the immunized mice with mouse mye
IgG1 IgA2b IgM
ANTI-SRBC ANTI·SRBC ANTI·SRBC loma cells and then tested the hybrid
clones for their production of anti-inter
FIRST HYBRID-MYELOMA CELLS were prepared by immunizing mice with sheep red feron antibody, supplementing the unre
blood cells (SRBC). The cells involved in the experiment and their immunoglobulins are de
liable and laborious biological assay for
picted here schematically. Cells from the spleen of immunized mice (which die in any tissue
anti-interferon activity with tests for im
culture) were fused with mouse myeloma cells deficient in HPGRT (which die in the selective
70
© 1980 SCIENTIFIC AMERICAN, INC
�
..........
antibody was attached to carbohydrate
beads to prepare an immunoadsorbent
column. Passing a totally crude interfer
on preparation through the column pu
-----
rified it 5,OOO-fold in a single step. Pu
rification on an industrial scale is now
FUSE IN
being explored. POLYE THYL EN E GLYCOL
Monoclonal antibodies can be pre
pared that are specific for individual
components of any complex mixture,
and unlimited amounts of each anti
•••. . .•• ..
MYELOMA CELLS
SPLEEN CELLS
body can be produced for immunoad (HPGRT-)
/Ij \\�
to dissect a mixture of completely un
known substances into its components.
Animals are immunized with the mix
ture to be analyzed; hybrid-myeloma
clones are derived and the antibodies
HAT MEDIUM � SELECT HYBRID CELLS
@ @@
gy. Membrane proteins are hard to puri
CLONE
fy. They are present in cells in small
amounts; often they have no easily mea
sured biological activity, or else their ac
tivity is destroyed when the membranes
are solubilized for analysis. One way to
overcome these problems is to charac
I \ J � I1
terize cell-surface molecules by immu
nological methods, an approach that has �
been fruitful in the recognition of sur
face antigens that characterize particu 1 ASSAY FOR ANTIBODY
1
showed how the hybrid-myeloma tech
nique could identify individual differ
entiation antigens. We immunized a ANALYZE TO SELECT VARIANTS
THAW
•
clones producing different specific anti
bodies. In that one attempt we defined
three new antigenic specificities, a task
that might have required years of so
phisticated immunology by convention
GROW IN
MASS CULTURE /� �� rg INDUCE
T UMORS
71
© 1980 SCIENTIFIC AMERICAN, INC
b
0
SPLEEN CELL MYELOMA CELL
VT.K
f::\
c
FUSE
0
SPLEEN CELL MUTANT
MYELOMA CELL
8� f'::\
VT-
FUSE
\
SPLEEN CELL MUTANT
MYELOMA CELL
88
(a) synthesizes not only the heavy
cell parent but also the gamma
((HG)) (L)
HYBRID-MYELOMA CELL produced by the usual fusion process
and light
heavy and the kappa (K)
chains of its spleen
ever, are not specific for a single subset ration, but not always. One monoclonal so that no such lattice can be formed by
of cells. Even the B cell's characteristic antibody seems to recognize an antigen a monoclonal antibody and most anti
immunoglobulin may be present in sev characteristic of certain bone-marrow gens. It can be formed only if the anti
eral functionally different members of cells in the rat, whereas in the peripheral gen is a polymer composed of repeated
the B-cell lineage, from the so-called lymphoid organs it recognizes lympho identical structural elements.
memory cells (which respond to reim cytes and in nervous tissue it recognizes Monospecificity has also revealed
munization with an antigen the organ some component that is as yet unidenti some hitherto unsuspected phenomena
ism has previously been exposed to) to
plasma cells (which secrete antibody).
What characterizes a particular differ
the antigen is present on T
fied. Among the peripheral lymphocytes
cells but not
on B cells; yet it reappears on plasma
that call for new interpretations of anti
gen-antibody reactions. To take just one
example, it appears that the binding of
entiated state is the presence of a partic cells, which are derived from B cells. We different antibodies to neighboring sites
ular ensemble of surface antigens and say this monoclonal antibody recogniz on the same antigen is an important fac
their quantitative expression. es a kind of "jumping" antigenic deter tor in the rupture of a cell membrane by
To establish a unique antigenic profile minant. complement. This synergistic effect was
for each of many cell types will require a The monoclonal approach to char discovered as we were isolating the rat
vast collection of monoclonal antibod acterizing differentiation antigens thus antibodies to histocompatibility anti
ies and will take a long time. A good makes it possible to probe for the partic gens. We assayed for the presence of an
start has been made with the few re ular stage at which an antigen is ex tibody-secreting hybrid myelomas by
agents already available and with the pressed as well as for the line of cells measuring the cytotoxic, or cell-killing,
help of cytofluorometers and fluores that expresses it. The cascade purifica activity of their culture mediums. The
cence-activated cell sorters: instruments tion method I described above can be supernatants of the uncloned cultures
that can quickly measure both the size applied' not only to characterize the anti were consistently cytotoxic, but once we
and the fluorescent intensity of large genic complexity of the cell surface but had cloned individual cells their super
numbers of cells to which monoclonal also to dissect functional as well as natants showed no such activity. It oc
antibodies, tagged with a fluorescent structural components of other biologi curred to Howard to measure the activi
dye, have been attached. A large cell cal materials such as cell organelles and ty of a mixture of the supernatants of
population can thus be fractionated into pharmacologically active cell extracts. these apparently negative clones. To our
subpopulations on the basis of their size delight the mixture was active, and then
and surface-antigen pattern, and then
the function of each subpopulation can
be studied. Monoclonal antibodies, in
T he "monospecificity" of antibodies
from hybrid-myeloma clones has
thrown new light on some well-known
it was easy to purify two complementa
ry components.
Once the synergistic effect was under
other words, are standard reagents that phenomena of antigen-antibody reac stood the "silent" activity of the isolated
can identify new surface molecules and tions. One indication of the binding of components could be exploited in a spe
at the same time distinguish among cell antigen to conventional antibodies in a cial way. Test cells could be "sensitized"
populations. So far the best results have test tube, for example, is the formation by exposure to one monoclonal anti
been reported with various blood-form of a precipitate. The effect is not gen body and then exposed to the antibodies
ing and lymphoid cells; one directly erally observed when the antibody is from other clones, thus revealing entire
practical application has been the dif· monoclonal. This is perhaps the first repertories of antibodies that act syner
ferential diagnosis of various leukemias
and related disorders.
The pattern of reactivity of mono
more than 40
formal proof of the theory, advanced
years ago, that the precipi
tate is a three-dimensional lattice of an
gistically. Clearly there are cases where
mixtures of monoclonal antibodies will
be essential to produce a desired effect.
clonal antibodies against subpopula tigens and antibodies. A monoclonal In each case a decision will have to be
tions of cells is sometimes consistent antibody binds only to a single antigen made whether the advantages of blend
with a given cell line's pattern of matu- ic determinant on an antibody molecule, ing monoclonals in specific proportions
72
© 1980 SCIENTIFIC AMERICAN, INC
o
'c"�,'� 'IC �
many commercial companies are mar
keting them. Because they can be pro
duced in large quantities they will make
possible widespread use of kits of di ,
N
'I' qp
MYELOMA
CELLS
stance P,
body to the neurotransmitter called Sub
derived recently by A. Claudio
Cuello and me. The impact of mono
�t
SELECTED CLONE
1
CRUDE INTERFERON
to active immunization with an antigen
that stimulates the patient's own anti
body response). Given the impurity of
conventional antibodies, passive immu IMMUNOADSORBENT
nization is not a common method of COLUMN
1h\\
kinds of role are foreseen for monoclon- WASH
'
terferon activity and immunoglobulin secre
tion was injected to induce myelomas: puri
fied antibody from the serum of tumor-bear
.-Jt.
ing mice was attached to carbohydrate beads '_' WASH ELUTE
to prepare immunoadsorbent columns. When "
crude interferon was passed through such a
column, it bound to the antibody and was re
"01o\'
tained when other components of the crude t('JI
mixture were washed out; then the interferon
was eluted. One passage through a column in
creased the preparation's interferon activity
W0:�: -
about S,OOO-fold. Electrophoresis of the pro
::i
teins (radioactively labeled to increase their
visibility) made the results visible (bottom). ()::UJ-'o::J
(A),
The partially enriched preparation showed a
73
© 1980 SCIENTIFIC AMERICAN, INC
���
ANTIGEN
©
®@ �
�
®
@
� �
� �
SPLEEN � °8 leor"
&
f FUSE
, ..•
-- -
CELLS _e
.
CbO
. :
j'" : SPLEEN MYELOMA
�()
--7> .'I'1.
CLONE CELLS CELLS CELLS
�,.�
CELLS
ANTI·A
ANTIBODY
�j':" i(tl�.'©:.,
,:&V
� ::::.t;:;l
-:.:.�
. 2b .
.
HYBRID
CLONE
/ 0J .'
ANTI·e
ANTIBODY
ANTl-O
ANTIBODY
HYBRID
CLONE
ANTI·E
ANTIBODY
UNKNOWN MIXTURE can in effect be dissected by the mono The depleted mixture, now enriched in the remaining antigens, is in
clonal antibodies it engenders. A random set of antibodies, derived jected to produce more antibody, and so on in cascade fashion. Hy
by immunizing animals with the unknown mixture, are applied to im brid-myeloma cells therefore provide a tool for characterizing the
munoadsorbent columns, which remove the corresponding antigens. components and at the same time for separating and purifying them.
al antibodies. One role is the targeting when human cells are fused with animal been an impressive "spin-off " into many
of toxic drugs: antibodies to the tissues cells, there is a rapid preferential loss of other areas. It is always hard to define
of a particular organ or to specific tu human chromosomes from the resulting the boundary between basic and applied
mor antigens could be attached to drug interspecific hybrid cells. And so far the research, but to experience personally
molecules to concentrate 'the drug's ef search for a suitable human myeloma the transition from one to the other has
fect. Alternatively it may be possible to line that can be cultured and fused to made a deep impression on me. I can
produce antitumor antibodies that will make an intraspecific hybrid has not not think that if my research aim five or
themselves find and attack tumor cells. borne fruit. six years ago had been the production
For therapeutic applications antibod In this overview of the uses of hybrid of monoclonal antibodies, I would ever
ies derived from human lymphocytes myeloma antibodies I have referred have stumbled on the idea of attempting
rather than from the mouse or the rat only superficially to their obvious ap simultaneously to derive mutant anti
would be desirable. Contrary to early plications in basic immunological re body-secreting cells in one corner of the
hopes, this has proved to be difficult; search. I have preferred to emphasize laboratory and to fuse two myeloma
attempts to immortalize antibody-pro the fact that, although the technique cells in another corner. Yet that was
ducing human cells by fusing them with originated in our effort to understand the combination that led to the initial
mouse or rat myeloma cells have so far the genetic organization and expression production of monoclonal antibodies
been disappointing. The problem is that of immunoglobulins, there has already against sheep red blood cells.
l____ _________________ --
l CELLS
LYMPHOC YTES
DIFFERENTIATION ANTIGENS are cell-surface antigenic deter for four white'blood cells. The best definition of cell lineage and sub
minants that are either specific for individual cell types or common sets is the pattern of expression of such markers. Monoclonal anti
to sets or subsets of cell types. Here hypothetical antigens are shown bodies are an ideal tool for establishing such patterns of expression.
74
© 1980 SCIENTIFIC AMERICAN, INC
•
films.
users refrigerate their film and process
promptly anyway, out of business All this crafty scheming for fine 1880 1980
necessity. Their raw film inventory tuning of photographic properties to
justifies dedicating a refrigerator
to it. We take this into account in
balancing the relative sensitivities of
differences between use
fessionals and non-professionals is
just an example of the upgrading in
by pro
fi IOO-yEar start on tOlT19rrow