S.P.C.

Cole
Monoclonal Antibodies
SUMMARY SOMMAIRE
The development of hybridoma monoclonal Le developpement de la technologie utilisant
l'hybridation des anticorps monoclonaux (MAb) a eu
antibody (MAb) technology has had a major un impact majeur en medecine clinique et de
impact on clinical and laboratory medicine. It laboratoire. Elle a aussi permis la production de
has allowed the production of essentially quantites virtuellement infinies d'anticorps purs et
unlimted quantities of pure, homogeneous homogenes contre une grande vari6t6 d'antigenes.
antibodies against a large variety of antigens. Malgr6 les nombreux avantages des MAb, les efforts
Despite the many advantages of MAbs, the et les cotits requis pour leur production ne sont pas
toujours justifies et, dans certains cas, l'antiserum
effort and expense required for their conventionnel, moins couteux et plus facilement
production is not always justified. Less disponible, demeure parfaitement approprie.
expensive and more easily obtained Neanmoins, l'utilisation des MAb murins est tres
repandue dans les laboratoires cliniques et en
conventional anti-serum is sfill perfectly recherche, particulierement dans le domaine de
adequate in some instances. Nevertheless, l'oncologie. Des efforts considerables ont e
murine MAbs have found widespread use in consacres au developpement de MAb humains, car
clinical and research laboratories, particularly on s'attend a ce que les anticorps d'origine humaine
in the field of oncology. Considerable effort soient superieurs dans les contextes therapeutiques
has been devoted to the development of et diagnostiques in vivo. Actuellement, des
human MAbs, since antibodies of human difficultes techniques font obstacle a leur utilisation
sur une plus grande echelle. Finalement, l'auteur
origin are expected to be superior in decrit de nouvelles approches bastes sur les
therapeutic and in vivo diagnostic settings. techniques de genie genetique qui laissent presager
However, technical difficulties currently de nouveaux developpements prometteurs dans un
prevent their widespread use. Finally, novel avenir rapproch6.
approaches using genetic engineering
techniques are described, which portend
exciting new developments in the near future.
(Can Fam Physician 1987; 33:369-372.)
Key Words: hybridoma monoclonal antibody technology, antibodies, genetic engineering
1- I M
S.P.C. Cole, PhD, is a career Institute and the Medical Research "Such cultures could be valuable for
scientist at the Ontario Cancer Council. Her program includes medical and industrial use."
Treatment and Research work on the development of human
Foundation, Kingston Regional and murine monoclonal antibodies
Cancer Centre. She is also assistant to lung tumour antigens. Reprint
professor of Oncology and requests to: Dr. S.P.C. Cole, The T HE SENTENCE QUOTED
Microbiology and Immunology at Ontario Cancer Treatment and above concludes the first report
Queen's University, Kingston. Dr. Research Foundation, Kingston on the production of hybridoma mo-
Cole's current research is being Regional Cancer Centre, King St. noclonal antibodies (MAbs).1 It truly
funded by the National Cancer W., Kingston, Ont. K7L 2V7 must stand as one of the more ex-
CAN. FAM. PHYSICIAN Vol. 33: FEBRUARY 1987 369
treme examples of British understate-
ment. In fact, MAbs have made an
enormous impact on virtually every
health science discipline. The magni-
tude of this impact was recognized in
1984, when the Nobel Prize was
awarded to the authors of this paper,
Georges Kohler and Cesar Milstein.
History
Like many other great discoveries
in science and medicine, the produc-
tion of monoclonal antibodies was
partly accidental. Before 1975, the re-
search in Dr. Milstein's laboratory
had focused on the regulation of im-
munoglobulin expression, using so-
matic cell hybridization techniques.
With the creation of the first hybri-
domas, the pertinent question was: If
a specific, antibody-producing, mortal
plasma cell is hybridized or fused
with an immortal, non-specific, anti-
body-producing, myeloma cell, will
the hybrid continue to produce spe-
cific antibody? The answer was yes,
and thus the era of hybridoma tech-
nology was launched.
Two problems were quickly identi-
fied. The first was that the original
fusing agent proved inefficient; poly-
ethylene glycol was found to be a bet-
ter agent. The second problem was
that the continued production of non-
specific immunoglobulin by the mye-
loma cell line caused the hybridoma
to produce a mixture of mixed chain
immunoglobulin molecules; thus, the
specific antibody was only a fraction
of the immunoglobulin produced.
This problem was circumvented by
the generation of mutant myeloma
cell lines which have lost the ability
to synthesize or secrete their own im-
munoglobulin. These modifications of
the original procedure described by
Kohler and Milstein have resulted in a
widely successful methodology that is
easily employed in most tissue-culture
laboratories.
Methods
Mouse hybridomas are constructed
conventionally by the fusion of
murine myeloma cells with specific
antibody-forming B lymphocytes ob-
tained from the spleens of immunized
mice. The myeloma cell confers im-
mortality on the antibody-pro«ducing B
lymphocyte, and allows it to grow in
culture and as ascites tumours in
mice, whereas the B lymphocyte de-
370
termines the antigen specificity of the
antibodies produced by the hybrid.
The hybrid cell lines can be grown
continuously in culture, or frozen and
recovered when desired. This technol-
ogy has permitted the production of
essentially unlimited quantities of
pure homogeneous antibodies against
a large variety of antigens, including
haptens, hormones, enzymes and pro-
teins, as well as viruses, bacteria and
protozoa.
Despite the apparent simplicity of
hybridoma technology, it remains an
expensive and labour-intensive
process. Whether a MAb is essential
for the intended application, or
whether a cheaper, more easily pro-
duced, conventional anti-serum would
suffice are questions that should
therefore be carefully considered.
To appreciate the complexity of the
problems facing laboratory scientists
engaged in work with hybridomas it is
helpful to understand how antibody-
producing hybridomas are generated.
First, mice are immunized with the
antigen of interest. If the antigen is
known and well characterized (e.g.,
tetanus toxoid), conventional immuni-
zation schedules, known to evoke a
high-titred, secondary immune re-
sponse, are followed. In many cases,
however, the biochemical nature of
the antigen is unknown.
Tumour-associated antigens belong
to this second group. In this instance,
and in many others, the researcher
must consider the ultimate intended
use of the MAbs when deciding how
to immunize. If, for example, the re-
searcher is interested in developing
MAbs for therapeutic purposes, the an-
tigens of most interest are those on
the surface of the cell; therefore, the
researcher should consider immuniz-
ing with intact tumour cells obtained,
ideally, from a patient. However,
such samples are often difficult to ob-
tain, and so established tumour cell
lines are frequently used. The prob-
lem with this approach is that such
cell lines seldom represent the hetero-
geneous nature of most human tu-
mours; therefore, the MAbs obtained
using tumour cell lines as immuno-
gens may be of limited interest. On
the other hand, if the researcher in-
tends to use the MAbs as diagnostic
probes on formalin-fixed biopsy sam-
ples, she or he should consider using
formalin-fixed tissue to immunize the
mice. It is difficult to generalize on an
optimal immunization schedule when
the antigen is unknown, and so, in
many cases, hitting on an effective
schedule is largely a matter of trial
and error or of luck.
Once the mouse is optimally im-
munized, the spleen cells are isolated
and mixed with murine myeloma
cells; polyethylene glycol is then
added to promote fusion of the cell
membranes. The efficiency of this fu-
sion process is about 1 in 105 cells.
Therefore, one mouse spleen, which
consists of approximately 108 lym-
phocytes, will yield about 1,000
hybrids. Recently, electrofusion
methods have been developed which
increase the fusion frequency,2 but
because they require costly equip-
ment, they are not yet widely used.
When two cell lines are fused, it
is necessary to have the means of
eliminating the outgrowth of the
parent cells, while allowing the rare
hybrid cells to thrive. This result has
been achieved in the conventional
mouse-hybridoma system by using
myeloma cells that have been ren-
dered resistant to the purine antimeta-
bolites, 8-azaguanine or 6-thioguan-
ine; thus they are deficient in
hypoxanthine-guanosine phosphoribo-
syl transferase (HGPRT). This means
that the purine salvage pathway is
blocked, and the myeloma cells will
therefore die when cultured in me-
dium containing hypoxanthine, amin-
opterin, and thymidine (HAT), since
the aminopterin inhibits dihydrofolate
reductase and blocks de novo synthe-
sis of purines and pyrimidines. Ac-
cordingly, HGPRT-deficient parent
myeloma cells are HAT sensitive, and
the parent mouse-spleen cells die after
a few days in tissue culture, since
they are not immortal. Mouse x
mouse hybrid cells survive in HAT me-
dium because the spleen cell provides
the functional HGPRT enzyme neces-
sary to overcome the block by amin-
opterin, while the myeloma cell pro-
vides immortality.
Two to three weeks after a fusion,
hybrids can easily be seen on gross
examination. At this point, the la-
bour-intensive work of screening for
specific antibody begins. The impor-
tance of having screening assays de-
veloped and working well before fus-
ing cannot be overemphasized. Many
a disappointed and frustrated scientist
has been forced to abandon the hybri-
domas at this stage because of inade-
quate screening methods, since it is
impossible to propagate 600 or 700
CAN.-FAM. PHYSICIAN Vol. 33: FEBRUARY 1987
different clones growing at different
rates for any length of time. The ideal
initial screening method is sensitive,
simple, rapid and relatively inexpen-
sive. More complicated time-consum-
ing assays are usually reserved for
subsequent screens once the first
screen eliminates non-productive or
non-specific antibody producing hy-
bridomas. Enzyme-linked immuno-
sorbent assays (ELISAS) or radioim-
munoassays (RIAs) are the most
popular initial screening methods, and
the choice is often made on the basis
of personal preference. The supema-
tants of the hybridoma cells are thus
screened against the desired target,
which may be purified antigen, for
known antigens or antigen-bearing
cells, or membrane preparations if the
specific antigen is unknown. Follow-
ing this first screen, the number of
hybridomas is reduced to a more man-
ageable level of 40 or fewer. These
hybrids can then be tested, if desired,
in functional assays (e.g., agglutina-
tion, complement-mediated cytotoxi-
city assays).
It then becomes vital to clone the
individual hybridomas by initiating
cultures at one cell per well. This pro-
cedure allows the selection of vi-
gorously growing, stable clones of a
particular hybridoma and the elimina-
tion of unstable or non-producing hy-
bridomas from a whole population.
These cloning procedures take consid-
erable effort, since it is not unusual to
generate 100-200 clones from each
hybridoma. Of course, each clone
must be retested for specific-antibody
production. To ensure monoclonality
of the culture, the cloning procedure
is usually repeated twice. Once stable
specific antibody-producing clones
have been established, they can be in-
jected into syngeneic mice to produce
large amounts of ascites fluid contain-
ing high-titred antibody; stocks of
cells can also be frozen away in liquid
nitrogen. The entire process, from the
first immunization to the first freezing
down of hybridoma cells, can often
take as long as a year, depending on
the immunization schedule followed.
Applications in Oncology
It is rare today to hear anyone
speak of a tumour-specific antigen,
but the presence of tumour-associated
antigens has been established beyond
doubt; thus, the antigenic differences
between normal cells and malignant
CAN. FAM. PHYSICIAN Vol. 33: FEBRUARY 1987
cells are largely quantitative rather
than qualitative. In many cases, how-
ever, these quantitative differences
have been sufficient to allow the use
of MAbs for diagnostic and experi-
mental therapeutic procedures.3
Dozens of tumour-associated antigens
have been identified using MAbs, in-
cluding two of the more widely
known: carcinoembryonic antigen
(CEA) and oc1 25.
CEA is a glycoprotein found in em-
bryonic colonic mucosa and carci-
nomas of the gastrointestinal tract.4
Numerous MAbs have been described
by a number of laboratories which de-
fine a repertoire of epitopes on CEA.
These MAbs are now being used in
blood assays by a number of centres,
to monitor tumour burden, and in im-
munohistopathological analysis of tis-
sue samples. The most extensively
used immunoassay to monitor carci-
noma patients has been that to detect
serum levels of CEA.
A problem which limits the wider
use of most of these MAbs is their un-
acceptably high degree of cross-reac-
tivity with normal adult tissues. Re-
cently, however, a small panel of
MAbs of greater tumour specificity
have been described.5 These MAbs
may eventually be used for in vivo
diagnosis or possibly for a therapeutic
purpose.
Oci25 is a MAb that detects a de-
terminant, designated CA 125 that is
found on more than 80% of non-mu-
cinous epithelial ovarian carcinomas.'
Using MAb OC1 25, a radioimmunoas-
say has been developed that detects
active CA 125 antigen in serum. Sev-
eral large studies have demonstrated
that elevated levels of CA 125 correlate
*with the presence of malignant dis-
ease. In other studies, rising or falling
antigen levels correlated with progres-
sion of disease in more than 85% of
patients tested. Thus MAb ocl25 may
be useful in monitoring the response
to treatment of patients with ovarian
cancer.
Human Monoclonal
Antibodies
Human monoclonal antibodies
(HuMAbs) are expected to have many
advantages over their murine counter-
parts, in terms of therapeutic potential.
A host-immune response in patients
treated with murine MAbs may well
limit the effectiveness of these
agents.7-9 This anti-immunoglobulin
response is expected to decrease with
HuMAbs. It is also reasonable to as-
sume that HuMAbs will interact more
effectively than murine MAbs with
human effector cells and/or comple-
ment. For these reasons, considerable
effort has been devoted to optimizing
the production of HuMAbs. I
HuMAbs will likely find their first
application in clinical situations where
passively administered human anti-
sera are already in use. "'l 12 A good
example might be the postpartum ad-
ministration of anti-Rh(D) human im-
munoglobulin to Rh-negative unsensi-
tized women who bear an Rh-positive
infant. Using purified HuMAbs will re-
duce, if not eliminate, the risk of
transfer of infectious agents. Further-
more, because of the essentially un-
limited supply of these laboratory-
produced antibodies, the continuous
availability of suitable serum donors is
no longer a concern.
Since murine MAbs were first de-
scribed in 1975, attempts have been
made to use similar somatic cell-hy-
bridization techniques to produce
HuMAb. Two major problems have
been encountered: the unexpected dif-
ficulty of obtaining a suitable human
myeloma fusion partner, and lack of
methods to enrich or select for antigen-
specific human lymphocytes. Human
myeloma cell lines are notoriously dif-
ficult to establish. Those that have
been described tend to be slow grow-
ing and are fastidious in their nutrient
requirements. Another problem is that
all of these cell lines secrete their own
immunoglobulin; in contrast to mouse
myelomas, and in spite of considerable
effort by a number of laboratories, a
non-secretory human myeloma cell
line has not yet been isolated. Thus all
hybridomas presently generated by
human x human fusion methods se-
crete mixed immunoglobulin mole-
cules. Another approach has been to
fuse human lymphocytes with a
murine myeloma cell line. This
method is less popular because inter-
species hybridomas tend to be less
stable than intraspecies hybrids. The
second problem of insufficient anti-
gen-specific B cells is particularly dif-
ficult to solve, since it is not always
possible or ethically feasible deliber-
ately to immunize humans with anti-
gen. Notable exceptions are tetanus
toxoid-vaccinated donors. On the
other hand, there are unlikely to be
many healthy immunocompetent indi-
viduals who would volunteer for im-
371
munization with human tumour anti-
gens.
Many laboratories have directed
strong efforts toward developing
human MAbs specific for tumour-cell
antigens.13 Our studies with lung tu-
mour cells indicate that B cells specific
for tumour cells are extremely rare.
The chances of finding human MAbs
with tumour-antigen specificity would
be greatly enhanced if lymphocytes
could be specifically stimulated in
vitro. Various methods have been de-
scribed for in vitro immunization of
mouse-spleen cells.14 These methods
have been applied with rather limited
success in human systems with defined
antigens. Further advances in this area
will likely be necessary before HuMAb
technology becomes widely used.
Human MAbs can also be produced
by means of a technique called EBV
immortalization. Epstein-Barr virus
(EBV) is able to infect only those cells
bearing EBV receptors, which are iden-
tical to the receptors for the comple-
ment protein c3d. Most mature B lym-
phocytes have receptors for EBV and
thus may be transformed by the virus.
The infection in vitro of B lymphocytes
leads to permanent stimulation of host-
cell growth, a phenomenon termed
'transformation', or 'immortalization'.
This 'immortalization' preserves the
characteristics of the original B cells,
including EBV receptors, complement
receptors, surface immunoglobulin
and secretory immunoglobulin. Those
cells which grow out in vitro and carry
EBV DNA are termed 'lymphoblastoid
cell lines' (LCL). Unfortunately, anti-
body-secreting LCL made this way tend
to produce low amounts of antibody,
predominantly of the igM isotype, for
only limited periods of time. One not-
able exception has been an IgG MAb
against Rh antigen, produced by an
EBV-immortalized B cell line which
may become commercially available
fairly soon. I5
Ongoing Developments
While awaiting the further develop-
ment of techniques to enrich antigen-
specific human B cell populations,
some laboratories have used recombi-
nant DNA technology to construct chi-
meric antibodies. 16-18 Trhis involves
cloning the immunoglobulin genes of a
murine MAb of desired antigen specif-
icity and then substituting the murine
constant-region genes with human
372
constant-region genes. This chimeric
gene is then introduced or transfected
into an appropriate cell line where it is
then expressed. The antibody mole-
cules produced in this way are largely
of human origin, although they retain
the antigen-binding specificity of the
murine MAb. Such chimeric antibodies
are expected to be less immunogenic
than wholly murine MAbs and thus less
likely to cause hypersensitivity reac-
tions. A major limitation that prevents
the widespread application of this
technique is the fact that the cell lines
currently available permit less-than-
optimal expression of the chimeric im-
munoglobulin genes.
Immunoglobulin gene-manipulation
technology has recently been taken
one step further. One group, again,
using recombinant DNA technology,
has been able to replace a portion of
the antibody heavy chain gene with the
gene for a completely unrelated pro-
tein, thus producing a bifunctional chi-
meric molecule exhibiting both spe-
cific antigen-binding and nuclease
activity. 19
Finally, another application of hy-
bridoma technology is the immortal-
ization of cells secreting cellular prod-
ucts other than immunoglobulins, such
as lymphokines, by fusion with ma-
lignant cells. For example, human T-
cell hybridomas that secrete interleu-
kin-2 and chemotactic factor have
already been described.20
References
1. Kohler G, Milstein C. Continuous cul-
tures of fused cells secreting antibody of
predefined specificity. Nature 1975; 256:
495-7.
2. Vienken J, Zimmermann U. An im-
proved electrofusion technique for produc-
tion of mouse hybridoma cells. FEBS Lett
1985; 182: 278-80.
3. Schlom J. Basic principles and applica-
tions of monoclonal antibodies in the man-
agement of carcinomas. The Richard and
Hinda Rosenthal Foundation Award Lec-
ture. Cancer Res 1986; 46:3225-38.
4. Gold P, Freedman SO. Demonstration
of tumor-specific antigens in human co-
lonic carcinomata by immunological toler-
ance and absorption techniques. J Exp Med
1965; 121:439 -62.
5. Munaro R, Wunderlich D, Thor A,
Lundy J, Noguchi P, Cunningham R, et al.
Definition by monoclonal antibodies of a
repertoire of epitopes on carcinoembryonic
antigen differentially expressed in human
colon carcinomas versus normal adult tis-
sues. Cancer Res 1985; 45:5769-80.
6. Bast RC, Knapp RC. Monoclonal re-
agents in immunodiagnosis and immuno-
therapy of human epithelial ovarian carci-
noma. In: Roth JA, ed. Monoclonal anti-
bodies in cancer advances in diagnosis and
treatment. Futura Publishing Co,
1986:61-86.
7. Shawler DL, Bartholomew RM, Smith
LM, Dillman RO. Human immune re-
sponse to multiple injections of murine
monoclonal IgG. J Immunol 1985;
135:1530-5.
8. Goodman GE, Beaumier P, Hellstrom I,
Fernyhaugh B, Hellstrom K-E. Pilot trial
of murine monoclonal antibodies in pa-
tients with advanced melanoma. J Clin
Oncol 1985; 3:340-52.
9. Sears HF, Mattis J, Herlyn D, Hayry P,
Atkinson B, Ernst C, et al. Phase-I clinical
trial of monoclonal antibody in treatment
of gastrointestinal tumours. Lancet 1982;
1:762 -5.
10. Engleman EG, Foung S, Larrick J,
Raubitschek A, eds. Human Hybridomas
and Monoclonal Antibodies. New York:
Plenum Press, 1985.
11. Larrick JW, Bourla JM. Prospects for
the therapeutic use of human monoclonal
antibodies. J Biol Resp Mod 1986;
5:379-93.
12. Edwards PAW. Some properties and
applications of monoclonal antibodies.
Biochem J 1981; 200:1-10.
13. Cole SPC, Kozbor D, Roder JC. The
EBV-hybridoma technique and its applica-
tion to human lung cancer. In: Reisfeld
RA, Sell S, eds. Monoclonal Antibodies
and Cancer Therapy. New York: Alan R.
Liss Inc., 1985:77-96. (UCLA Symposia
on Molecular and Cellular Biology; Vol
27).
14. Borrebaeck CAK. In vitro immuniza-
tion for production of murine and human
monoclonal antibodies: present status.
Trends in Biotechnology 1986; 4:147-53.
15. Crawford DH, Barlow MJ, Harrison
JF, Winger L, Huehns ER. Production of
human monoclonal antibody to Rhesus D
antigen. Lancet 1983; 1:386-8.
16. Boulianne GL, Hozumi N, Shulman
MJ. Production of functional chimaeric
mouse/human antibody. Nature 1984;
312:643-6.
17. Sahagan BG, Dorai H, Saltzgaber-
Muller J, Toneguzzo F, Guindon CA, Lilly
SP, et al. A genetically engineered
murine/human chimeric antibody retains
specificity for human tumor-associated an-
tigen. JImmunol 1986; 137: 1066-73.
18. Neuberger MS, Williams GT, Mitchell
EB, Jouhal SS, Flanagan JG, Rabbitts TH.
A hapten-specific chimaeric IgE antibody
with human physiological effector func-
tion. Nature 1985; 314:268 -70.
19. Neuberger MS, Williams GT, Fox RO.
Recombinant antibodies possessing novel
effector functions. Nature 1984;
312:604-8.
20. Foon KA, Rossio IL, Schroff RW, et
al. The generation of stable human T-cell
hybridomas which constitutively produce
interleukin-2 and chemotactic factor. Hy-
bridoma 1985; 4:211-22.
CAN. FAM. PHYSICIAN Vol. 33: FEBRUARY 1987