From the
Induction of an immune response is strongly dependent on the
phylogenetic distance between antigen and recipient. In general,
antibodies will not be raised against self-antigens nor against highly
conserved domains. In the present study we describe the production and
characterization of murine monoclonal ``auto-antibodies''
against murine tissue-type plasminogen activator (t-PA) raised in
``knock-out'' mice, homozygously deficient of the functional
gene. 203 stable hybridomas were obtained producing murine monoclonal
antibodies against murine t-PA. Analysis of the species reactivity
revealed that 182 cross-reacted with one or more (t-)PAs originating
from other species including rat t-PA, human t-PA, and vampire bat-PA.
121 reacted with epitopes conserved among murine, rat, and human t-PA.
In addition, 31 of the monoclonal antibodies were directed against
domains present in all four species. Epitope mapping indicated a high
frequency of specificity toward diverse epitopes that are highly
conserved across species. Comparative analysis of their influence on
the enzymatic activity of t-PA and their species cross-reactivity
clearly demonstrated that the domains required for the biological
activity of plasminogen activators are more conserved ( p <
0.02) than non-functional domains.
The availability of such unique
antibodies against a wide variety of conserved epitopes may facilitate
studies on the structural homologies between (t-)PAs isolated from
various species. The present approach should also apply to various
other classes of proteins, allowing the generation of monoclonal
antibodies, against conserved epitopes, which could not be raised in
wild-type animals because of their ``self-antigen'' nature.
Since the development of the hybridoma technology by Kohler and
Milstein
(1) , the production of murine monoclonal antibodies
against foreign antigens has become a routine technique. However, it is
generally accepted that the greater the phylogenetic distance between
antigen and recipient, the more pronounced the immune response,
e.g. highly conserved mammalian proteins usually evoke a weak
immune response
(2) . In this same view, generation of murine
monoclonal antibodies against murine proteins ( i.e. self-antigens) is not to be expected due to an absence, by active
elimination or functional inactivation, of lymphocytes bearing
receptors for self-antigens
(3, 4) . For similar
reasons, the immunological system will also be unable to recognize an
epitope present in a protein obtained from another species, but showing
a high degree of homology with an epitope in the corresponding protein
of the immunized species. As a consequence all available monoclonal
antibodies of murine origin, raised against a particular non-murine
protein, will not cross-react (or only to a low extent) with the
corresponding murine protein. From a practical viewpoint, this
eliminates the possibility of detection or quantitation of murine
proteins with highly specific murine monoclonal antibodies. More
importantly, none of the currently available murine monoclonal
antibodies should react with domains or epitopes that are conserved
between various species.
We therefore hypothesized that immunization
of transgenic mice, in which the expression of specific proteins is
abolished by homologous recombination in embryonic stem cells
(5) , could result in the generation of murine antibodies
against the ``knocked-out'' protein and against epitopes
conserved across species. In the present study we describe the
generation and characterization of a wide variety of murine monoclonal
antibodies raised against murine tissue-type plasminogen activator
(mt-PA)(
Administration of mt-PA evoked a strong immune response in
t-PA
Two fusions of myeloma
cells with spleen cells isolated from immunized
t-PA
The cross-reactivity of the anti-mt-PA
monoclonal antibodies was further studied using rat t-PA (rt-PA) and
vampire bat-PA (bat-PA). Table I illustrates that 10% of the monoclonal
antibodies reacted exclusively with mt-PA, 10% cross-reacted with
rt-PA, 3% with ht-PA, and 0.5% with bat-PA. However, 61% cross-reacted
with three of the four PAs tested, while 15% reacted with all four PAs.
None of the monoclonal antibodies raised with mt-PA cross-reacted with
mu-PA.
All antibodies cross-reacting with ht-PA ( i.e. n = 158) could be further evaluated with respect to their
domain reactivity using a set of deletion/insertion mutants of ht-PA.
These experiments revealed that the majority of these antibodies were
directed against an epitope in kringle 1 ( n = 48),
kringle 2 ( n = 33), or the protease domain ( n = 52) (Table II). 16 monoclonal antibodies required the
simultaneous presence of at least two domains
(K
Evaluation of the
interference of the monoclonal antibodies with the plasminogen
activation potential of mt-PA revealed that 111 out of 203 exhibited
inhibitory properties toward mt-PA activity (). Of the 21
monoclonal antibodies reacting exclusively with mt-PA, only 6 (29%)
were inhibitory, whereas out of the 182 monoclonal antibodies
cross-reacting with one or more other (t-)PAs, 105 (58%) were
inhibitory. shows a subanalysis in which the inhibitory
properties of the 158 monoclonal antibodies cross-reacting with ht-PA
were compared to their domain reactivity in ht-PA. From these data it
appeared that the majority (26 out of 33, 81%) of the
kringle-2-reacting antibodies had an inhibitory effect while only 35%
(17 out of 48) of the kringle-1-reacting antibodies interfered with the
activity. 32 out of 52 (62%) monoclonal antibodies reacting with the
protease domain exhibited inhibitory properties ().
Interestingly, the kringle-1-reacting antibodies that cross-reacted
with the four (t-)PAs were all inhibitory.
26 purified monoclonal
antibodies were further subjected to a detailed epitope mapping
analysis based on their mutual competition for binding to mt-PA. These
data revealed the presence of at least six distinct clusters. Fig. 1
shows a schematic diagram of the various epitopes and the association
with their domain and/or species reactivity. At least three distinct
epitopes were localized in the kringle 1 domain, two in the protease
domain and one in the kringle 2 domain. At least three non-overlapping
epitopes, two located in the kringle 1 domain and one located in the
protease domain, were conserved in all four species and are involved in
the t-PA activity.
In general, antibodies are prepared either from antisera
collected from immunized animals or by hybridoma technology using
spleen cells from immunized mice and formation of stable hybridomas
producing monoclonal antibodies. Recently strategies have been
developed for expression of antibody fragments in Escherichia coli and for the generation of combinatorial libraries in
bacteriophages, using phagemid vectors allowing the formation of
``phage-displayed'' antibodies
(15, 16) . The
efficiency ( i.e. number of antibodies as well as their
affinity) of the latter procedures is also dependent on the
immunization of the animal prior to construction of the library
(15) . In addition, different approaches have been described to
mimic, in vitro, the naturally occurring process of affinity
maturation
(17, 18) . However, because of the clonal
selection, resulting in an elimination of functional lymphocytes
bearing receptors for self-antigens
(3, 4) , the
currently available methods do not allow consistent generation of
antibodies against self-antigens. In addition, all induced and/or
selected antibodies will be directed against epitopes that are not
conserved between the two respective species. We hypothesized that the
use of t-PA-deficient mice for the production of monoclonal antibodies
against mt-PA should not only yield, with a high efficiency, an immune
response toward mt-PA, but should also result in the generation of a
panel of monoclonal antibodies cross-reacting with t-PA from other
species. Immunization with mt-PA and mu-PA indeed caused a
significantly higher immune response in gene-inactivated than in
wild-type mice.
Out of two fusions using spleen cells isolated from
the immunized t-PA
More
interestingly, 15% (31 out of 203) of the monoclonal antibodies
cross-reacted with mt-PA, rt-PA, ht-PA, and bat-PA. Localization of the
domain reactivity of these antibodies revealed that none were directed
against the kringle 2 domain. This observation is consistent with the
hypothesis, based on its primary structure, that the kringle of bat-PA
is more similar to kringle 1 (77% amino acid identity) than to kringle
2 (56% amino acid identity) of human t-PA
(21) .
From the
observation that none of the currently described anti-mt-PA antibodies
cross-reacted with mu-PA, one may not conclude that mt-PA and mu-PA
would not have any common epitopes since the t-PA
Monoclonal antibodies can be directed against
epitopes involved in the biological or enzymatic activity of proteins,
and it is not unlikely that homologous proteins with similar activities
have these particular epitopes in common or at least share a
significant degree of similarity. In the present study, out of the 203
monoclonal antibodies, 111 inhibited plasminogen activation by mt-PA.
However, of the 21 antibodies reacting exclusively with mt-PA, only 6
(29%) exhibited neutralizing properties, whereas out of the 182
monoclonal antibodies cross-reacting with at least one other PA, 105
(58%) were inhibitory. The difference between these ratios is
significantly different ( p < 0.02), suggesting that
epitopes required for the biological activity of plasminogen activators
are more conserved across species than other regions of the molecule.
Three different epitopes (Fig. 1), conserved among all four
species, were found to be involved in the plasminogen activation
potential of t-PA. Evaluation of their presence in plasminogen
activators other than those used in the present study may reveal how
far this conservation extends over various other species. In the
current study, the inhibiting properties were evaluated using a
plasminogen-coupled chromogenic substrate assay, in the presence of
fibrin. Using this method, antibodies interfering with the catalytic
activity as well as antibodies interfering with plasminogen or fibrin
binding are detected. In addition, evaluation of the inhibitory
properties of 23 purified antibodies, using a direct assay for t-PA
activity, revealed that one cluster of monoclonal antibodies directed
against the protease domain (Fig. 1, MA-H2B1, MA-H5F10, MA-H27B6,
MA-H31A1, and MA-H32B8), and recognizing an epitope that is conserved
among murine, rat, and human t-PA, also interfered with the catalytic
activity of murine as well as human t-PA (data not shown).
In conclusion, a large panel of murine
monoclonal antibodies directed against mt-PA was obtained using
t-PA-deficient mice as immunizing target. These antibodies cover a wide
range of epitopes localized in different domains of the molecule and
conserved among the various species studied. By extrapolation, it is
assumed that immunization of any type of gene-inactivated mice with the
respective knocked-out proteins could provide a general means to obtain
unique monoclonal antibodies against structurally and functionally
conserved domains within other protein families.
The data represent the number of monoclonal antibodies that
cross-react with t-PA from the respective species. Numbers in
parentheses represent the number of monoclonal antibodies that inhibit
plasminogen activation by mt-PA.
The data represent the number of monoclonal
antibodies reactive with the indicated species and with the indicated
domains of human t-PA. Numbers in parentheses represent the number of
antibodies that inhibit plasminogen activation. Based on the homology
between distinct portions of the amino acid sequence of t-PA with
corresponding domains of other proteins, five subdomains
(``modules'') are distinguished: a fingerlike domain (F), an
epidermal growth-factor domain (E), two kringle domains (K
)
in transgenic mice in which the gene
encoding tissue-type plasminogen activator had been inactivated
(6) .
Monoclonal Antibodies
Monoclonal antibodies were
produced essentially as described by Galfré and Milstein
(7) . Homozygous t-PA-deficient (t-PA)
mice
(6) were immunized by subcutaneous injection of 10 µg
murine t-PA in complete Freund's adjuvant, followed 2 weeks later
by intraperitoneal injection of 10 µg of mt-PA in incomplete
Freund's adjuvant. Antisera were collected 1 week later and were
analyzed in a micro-ELISA using microtiter plates coated with mt-PA (1
µg/ml) and detection of bound immunoglobulins with horseradish
peroxidase-conjugated rabbit anti-mouse IgG. The specific antibody
concentration in these antisera (see ``Results'') was
retrospectively calculated by ELISA on microtiter plates coated with
the respective antigen using purified monoclonal antibodies for
calibration. An identical procedure was used for the evaluation of the
immune response in wild-type mice. After an interval of at least 4
weeks, the mice were boosted intraperitoneally with 10 µg of mt-PA
in saline on days 4 and 2 before the cell fusion. Spleen cells were
isolated and fused with either P3x63.Ag.8-6.5.3 or Sp2/0-Ag14
myeloma cells. After selection in hypoxanthine-aminopterine-thymidine
medium, the supernatants were screened for specific antibody production
with a one-site non-competitive micro-ELISA using microtiter plates
coated with mt-PA and detection of bound immunoglobulins as described
above. Positive clones were used for the production of ascites in
pristane-primed mice. The IgG fraction of the monoclonal antibodies was
purified from ascites by affinity-chromatography on protein
A-Sepharose. A similar procedure was followed for the generation of
monoclonal antibodies against murine urokinase-type plasminogen
activator (mu-PA) in homozygous u-PA-deficient
(u-PA
) mice.
Cross-reactivity
Cross-reactivity of the obtained
anti-mt-PA monoclonal antibodies with (t-)PA from other species (rat,
human, or vampire bat) or with murine urokinase-type plasminogen
activator, was evaluated by comparative analysis of the reactivity of
hybridoma supernatant or purified antibody in a micro-ELISA system
using microtiter plates coated with 1 µg/ml of the respective
antigen and detection of bound immunoglobulins as described above.
Parallel control reactions were carried out using microtiter plates
coated with bovine serum albumin to exclude false positive reactions.
Domain Localization
Domain localization of the
epitope recognized by antibodies cross-reacting with human t-PA (ht-PA)
was evaluated by comparative analysis of their reactivity in various
micro-ELISAs using microtiter plates coated with either one of the
domain deletion and/or insertion variants of human t-PA (see below).
Determination of Overlapping
Epitopes
Determination of overlapping epitopes was carried out
through competition of purified antibodies for binding to mt-PA using
the Biacore(Pharmacia, Uppsala, Sweden). Briefly, mt-PA (10
µg/ml, in 10 m
M acetate, pH 5.0, 30 µl) was coupled to
the sensor chip, followed by incubation with one antibody and
evaluation of its binding. Subsequently, the binding of a second
antibody was evaluated. Lack of binding of the second antibody
indicates that both antibodies are directed against the same epitope,
or that a significant portion of the epitopes overlap. All pairs of
antibodies evaluated were analyzed in both sequences. After each cycle
the sensor chip was regenerated using 10 m
M HCl. Positive and
negative control samples were included at the beginning and at the end
of each set of experiments.
Influence on Enzymatic Activity of t-PA
mt-PA (4
ng/ml) was preincubated (1 h at room temperature) with conditioned
medium diluted 1:5 or with a 20-fold molar excess of purified
monoclonal antibody. Subsequently residual t-PA activity was measured
by a plasminogen-coupled chromogenic substrate assay as described
(8) .
Proteins
Rat t-PA
(9) , vampire bat-PA
(DSPA1)
(10) , murine t-PA
(6) , and the human t-PA
variants K
K
P (lacking the finger-like and
growth factor domain)
(11) , EK
K
P
(lacking the finger-like domain)
(11) ,
K
K
P (lacking the finger-like, growth factor,
and kringle 1 domains and containing an extra kringle 2 domain)
(12) , FEK
K
P (lacking the kringle 1
domain and containing an extra kringle 2 domain)
(12) , and
K
P (consisting of kringle 2 and the protease domain of
human t-PA)
(13) were produced and characterized as described.
A hybrid molecule (K
K
P
)
consisting of kringle 1 and kringle 2 of human t-PA and the protease
part of human u-PA was produced and characterized as described
(14) . Human t-PA was a kind gift from Genentech Inc. (San
Francisco, CA).
Statistical Analysis
The statistical significance
of differences was assessed using the Mann-Whitney nonparametric test
for unpaired values and Fisher's exact test for proportions.
p values > 0.05 were considered to be not significant.
mice, resulting in a specific antibody
concentration in serum of 675 µg/ml (median; range 100-3,000,
n = 4) against mt-PA and of 100 µg/ml (median;
range 25-500, n = 4) against human t-PA (ht-PA).
Administration of murine urokinase-type plasminogen activator (mu-PA)
to urokinase-type plasminogen activator-deficient
(u-PA
) mice induced a specific antibody
concentration of 350 µg/ml serum (median; range 33-2,000,
n = 4) against mu-PA. However, no significant
cross-reactivity was observed with human u-PA (hu-PA). These findings
suggest that there are common (conserved) epitopes on mt-PA and ht-PA
but not on mu-PA and hu-PA; they are consistent with our previous
experience that polyclonal antisera raised in rabbits against ht-PA
cross-react with mt-PA, whereas antisera raised against hu-PA do not
cross-react with mu-PA.(
)
Administration of mt-PA
or mu-PA to wild-type mice resulted in a specific antibody
concentration in serum of 15 µg/ml (median; range 9-130,
n = 4) against mt-PA and 4 µg/ml (median; range
3-30, n = 4) against mu-PA, respectively ( p = 0.029 and p = 0.014 versus gene-inactivated mice, respectively).
mice yielded 203 hybridomas producing
monoclonal antibodies against mt-PA, whereas one fusion with spleen
cells of an immunized u-PA
mouse yielded 38
hybridomas producing monoclonal antibodies against mu-PA. Of the
antibodies against mt-PA, 78% cross-reacted with ht-PA, whereas none of
the anti-mu-PA antibodies cross-reacted with hu-PA, confirming the
differential cross-species reactivity of polyclonal antisera raised
with the respective proteins.
-K
, K
-P, or FEK
).
Only two antibodies were directed against an epitope in the finger-like
domain, while none recognized the growth factor-like domain. Further
analysis revealed that the epitopes recognized by the subgroup of 31
monoclonal antibodies reacting with all four PAs were exclusively
localized in the finger-like, kringle 1, or the protease domain but not
in the kringle 2 domain ().
mice, 203 stable
hybridomas were obtained producing murine monoclonal antibodies against
mt-PA, out of which 182 exhibited a cross-reactivity with at least one
of the three other PAs tested. Up to 60% cross-reacted with murine,
human, and rat t-PA. Of the currently available data regarding
monoclonal antibodies raised against ht-PA in wild-type mice, only a
few reports mentioned the occurrence of a cross-reactivity with either
rt-PA
(19) or mt-PA
(20) . The efficiency of the current
approach and its correlation with the extend of homology is further
illustrated in Table III. Indeed, the higher the percentage of
homology, the higher the number of cross-reacting antibodies.
mice used had normal u-PA levels
(6) . Consequently,
producton of antibodies against epitopes conserved between mt-PA and
mu-PA would require the use of ``double knocked-out'' mice.
Because such mice are however severely handicapped
(6) , this
was not attempted.
Figure 1:
Epitope mapping of purified antibodies.
Cross-reactivities: (), murine and rat t-PA; (
),
murine, rat and human t-PA; (
), murine, rat t-PA and vampire
bat-PA; (
), murine, rat, human t-PA and vampire bat-PA.
Codes in italic represent antibodies with inhibitory
properties.
The
observation that the 38 anti-mu-PA antibodies raised in
u-PAmice did not cross-react with hu-PA,
even though in line with our previous observations that polyclonal
antisera raised in rabbits against hu-PA do not cross-react with mu-PA,
should be interpreted with some caution. ( a) Only a limited
number of monoclonal antibodies could be studied that ( b) were
obtained out of only one fusion, and ( c) no u-PAs from other
species were included.
Table: Cross-reactivity of anti-murine t-PA
monoclonal antibodies with t-PA obtained from various species
Table: Domain localization
of the epitopes recognized by monoclonal antibodies that cross-react
with human t-PA
and K
), and a protease domain (P). Presentation of
one domain indicates that the epitope is localized entirely in the
respective domain. Presentation of two or three domains indicates that
the epitope is composed of parts of two or three domains. Undefined
indicates that the domain reactivity could not be deduced
unambiguously. mu, murine; hu, human; ra, rat; ba, vampire bat.
Table: Comparison of interspecies homology and
yield of cross-reacting antibodies obtained either from wild-type mice
or from t-PAmice
, t-PA-deficient;
u-PA
, u-PA-deficient; ELISA, enzyme-linked
immunosorbent assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.