©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Generation of Monoclonal Antibodies against Autologous Proteins in Gene-inactivated Mice (*)

Paul J. Declerck (§) , Peter Carmeliet , Maria Verstreken , Frans De Cock , Désiré Collen (¶)

From the (1) Center for Molecular and Vascular Biology, University of Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)()in transgenic mice in which the gene encoding tissue-type plasminogen activator had been inactivated (6) .


MATERIALS AND METHODS

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 KKP (lacking the finger-like and growth factor domain) (11) , EKKP (lacking the finger-like domain) (11) , KKP (lacking the finger-like, growth factor, and kringle 1 domains and containing an extra kringle 2 domain) (12) , FEKKP (lacking the kringle 1 domain and containing an extra kringle 2 domain) (12) , and KP (consisting of kringle 2 and the protease domain of human t-PA) (13) were produced and characterized as described. A hybrid molecule (KKP) 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.


RESULTS

Administration of mt-PA evoked a strong immune response in t-PAmice, 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).

Two fusions of myeloma cells with spleen cells isolated from immunized t-PAmice yielded 203 hybridomas producing monoclonal antibodies against mt-PA, whereas one fusion with spleen cells of an immunized u-PAmouse 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.

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-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 ().

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.


DISCUSSION

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-PAmice, 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.

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-PAmice 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.

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).


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.

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.

  
Table: Cross-reactivity of anti-murine t-PA monoclonal antibodies with t-PA obtained from various species

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.


  
Table: Domain localization of the epitopes recognized by monoclonal antibodies that cross-react with human t-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 (Kand 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



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Senior Research Associate of the National Fund for Scientific Research (Belgium). Current address: Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, University of Leuven, B-3000 Leuven, Belgium.

To whom correspondence should be addressed: Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven. Tel.: 32-16-345772; Fax: 32-16-345990.

The abbreviations used are: mt-PA, murine t-PA; t-PA, tissue-type plasminogen activator; u-PA, urokinase-type plasminogen activator; ht-PA, human t-PA; rt-PA, rat t-PA; bat-PA, vampire bat plasminogen activator; mu-PA, murine u-PA; hu-PA, human u-PA; t-PA, t-PA-deficient; u-PA, u-PA-deficient; ELISA, enzyme-linked immunosorbent assay.

P. J. Declerck, M. Verstreken, P. Carmeliet, D. Collen, unpublished observations.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.