HYBRIDOMA Volume 11, Number 4, 1992 Mary Ann Liebert, Inc., Publishers
Production and Characterization of a Monoclonal Antibody Against the Ribosome Inactivating Protein Alpha Sarcin ANDRÉS J.M. FERRERI,1 ELENA FERRARIS
di CELLE,1 CALDERA,1 SATURNINO MUÑOZ,2 STÉPHANIE SALVARELLI,2 FRANCISCO CONDE,2 SILVANA CANEVARI,1 EMANUELA TOSÍ,1 MARIA INES COLNAGHI1
MARISTELLA
'Division of Experimental Oncology E, 2Section of Immunochemistry,
Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian I, 20133 Milan, Italy Department of Investigation, Hospital Ramón y Cajal, Madrid 34, Spain
ABSTRACT
improve the purification of immunoconjugates containing alpha sarcin, a ribosomeinactivating protein, and in the attempt to define the enzymic region of the toxin, MAbs against alpha sarcin were produced. From 5 fusions, by adopting a short period of immunization and very low doses of the immunogen, 10 anti-toxin-producing clones were obtained. One of them, named MAsg2 (IgG2b), due to its specific reactivity and secreting properties, was selected for further characterization. MAsg2 was found to recognize an epitope which is common to two, i.e. alpha sarcin and clavatin, of the three different aspergillins tested, but is not involved in the In order to
active site of the toxins.
INTRODUCTION
Alpha sarcin is a single polypeptide chain with a molecular weight of 17.000 Daltons produced by Aspergillus giganteus (1). It shows a high level of sequence homology and immunological cross-reactivity (2-6) with restrictocin (7) and mitogillin (8), both of which areisolated from the filtrate of aspergillus strains. These molecules, hereafter named Aspergillins, like other plant and bacterial toxins, are ribosome-inactivating proteins (RIPs) (9), and their mechanism of action has been widely studied. Alpha sarcin makes a specific cut in 28S rRNA by hydrolysis of a single phosphodiester bond (10). Despite this knowledge, the region of the molecule devoted to the enzymic activity has not yet been fully defined. Several plant and bacterial RIPs, including members of the aspergillin family, have been coupled to monoclonal antibodies (MAbs) to generate new immunotherapeutic agents (11-15). One of the problems in conjugate production is represented by the removal of the unconjugated antibodies from the final preparation. Cibacron Blue chromatography (16) has been applied for this purpose, but controversial data have been reported in the case of aspergillin conjugates (14,15). Alternative methods such as ion exchange or affinity chromatography (17,18) are now being studied. In order to improve the purification of immunoconjugates containing aspergillins and in the attempt to define the aspergillin enzymic region, MAbs against alpha sarcin have been produced. The present report deals with the manipulation of alpha sarcin as immunogen and with the characterization of a monoclonal antibody, MAsg2, directed against an epitope of alpha sarcin common to different aspergillins.
437
MATERIAL AND METHODS
Toxins
Alpha
sarcin and clavatin
were
respectively
isolated from the culture filtrate of the mould
Aspergillus giganteus strain MDH 18894, and of the Aspergillus clavatus strain IFO 8605 (Institute for Fermentation, Osaka, Japan) and were purified according to the Olson and Goerner's procedure (1). Aspergillus fumigatus allergen I (Asp fl) (19) was kindly provided by K. Amida M.D. (University of Virginia, USA). Ricin A-chain (RTA) and pokeweed antiviral protein (PAP1) were obtained from Calbiochem (La Jolla, CA, USA). Saporin and gelonin were obtained from Inland Laboratories (Austin, Texas, USA). Mice immunization Different manipulations of the immunogen and protocols of immunizations were adopted as
summarized in Table 1.
TABLE 1. SUMMARY OF IMMUNIZATION PROTOCOLS
Protocol
Injections
Immunogen
preparation
/xg/dose
Response before fusion' n°
positive
B.I.
sera
FA
80
3/3
4.2
B
Glut+FA
80
3/3
6.5
C
Glut+KLH+FA
7
80
3/3
9.0
D
MRBC +FA
5
N.E.
3/3
3.2
complex3
1. See Material and Methods and reference n° 20. 2. Evaluated by solid-phase RIA after 7 immunizations. Positive cut -off B.I. _> 3. The Binding Index (BI) was the ratio between cpm bound with the sample and cpm bound with the medium alone. The reported data refer to the 1:125 serum dilution. 3. See Material and Methods and reference n° 21. 4. N.E. not évaluable. FA: Freund's adjuvant. Glut: Glutaraldehyde polymerization. KLH: Keyhole limpet haemocyanin. MRBC: Mouse red blood cells. =
The immunogen (alpha sarcin in NaCl 0.9%) was prepared essentially as in Gullick et al. (20), A) emulsified with Freund's adjuvant (GIBCO LAB., Grand Island, New York, U.S.A.); B) polymerized with glutaraldehyde and then treated as in A; C) as in B but in the presence of Keyhole limpet haemocyanin (KLH). As an alternative, protocol D, previously adopted to obtain second generation MAbs (21), was applied. Briefly, the sera of immunized mice from group B after the 7th injection were pooled and passed on an Affigel blue affinity column (Biorad Laboratories, Richmond, CA) to remove the majority of albumin. Serum proteins (6 150 /A of autologous mouse red blood cells mg/ml) in 0.15M of NaCl were incubated with (MRBC) vol. to vol. in the presence of 1.5 ml of 0.0002 M CrClß.o^O. After 1 hour of incubation at 37°C with shaking and three washings with PBS, the serum coated MRBC were incubated with alpha sarcin (160 /ig) for 45 min. at 37°C. After further washings 25 /i\ of pellet of MRBC-serum-alpha sarcin complex were injected into the mice. Four groups of 3 female BALB/c mice, 8-10 weeks old, were injected s.c. and i.p. 5 (protocol D) or 7 times (protocols A, B and C), once a week, with the immunogen. After the fourth injection the sera from the mice were tested by solid-phase RIA (see below). On the basis of the binding results, five days after the last injection for each immunization protocol, the mouse with the highest response was selected. The selected mice were injected i.p. and i.v. once again with the proper dose of alpha sarcin in saline and three days later killed for spleen removal. In the last immunization protocol
438
(E), not reported in Table 1, the scheme reported in Fig 1.
alpha sarcin was prepared and administered according to
the
Route:
sc/ip
sc/ip
sc/ip
0,5 ug
0,5 ug
¡p/iv
ip/iv
Dose/route:
0,5 ug
Days: O
F
0,5 ug 0,5 ug
FUSION 28
14
T
35
37
40
T TT
Fig. 1: Scheme of immunization protocol E. The alpha sarcin was administered in complete Freund's adjuvant on day 0, incomplete Freund's adjuvant on days 14 and 28 and in phosphate buffered solution on days 35 and 37. Monoclonal antibodies The spleen cells were fused with NSO myeloma, as described (22). The selection of growing hybrids was carried out in HAT medium (Hypoxantin, Aminopterin, Thymidine). Hybrids secreting antibodies were cultured "in vitro" and the isotype of the produced MAb was determined by indirect immunofluorescence, as described (22). For the production of the selected MAb, hybridoma cells were grown as ascites in the peritoneal cavity of pristane primed nu/nu BALB/c mice. The MAb 4A6 (IgGl), raised against Asp fl (19), was kindly provided by Dr. Arruda.
Indirect Solid-Phase Radioimmunoassay (SP-RIA) RIPs were seeded in polystyrene plates (Greiner, Labor Thecnik), dried overnight at 37° C and then fixed with glutaraldehyde 0.2%. Then the wells were filled with 50 /u\ of a titrated dose of the hybridoma supernatant, ascite or serum (in duplicates). After a 45 min. incubation at 37°C, a tracer amount of 125I-labelled Anti-mouse immunoglobulin (Amersham International Buckinghamshire, England) was added and the plate was incubated for another 45 min. at 37°C. In each test untreated
BALB/c serum (diluted 1:5) and rabbit anti-alpha sarcin serum (diluted 1:125) were respectively added as a negative and positive control. The latter was obtained by i.m. injection of alpha sarcin (1 mg) emulsified with complete Freund's adjuvant followed one month later by a boost with incomplete adjuvant. Its binding was detected by 125I-anti-rabbit immunoglobulin (Amersham). The results were expressed as the binding index, calculated as the ratio between cpm bound with the tested sample and cpm bound with the medium alone, or as specific cpm bound, i.e. cpm bound with the tested sample minus the cpm bound with the
medium alone. SDS-PAGE and Western Blotting Precast slab gels (Phastgel; 20% of acrylamide, Pharmacia, Piscataway, N.J., USA) were used to analyze the toxins, employing the automated microprocessor driven Phastsystem following the manufacturer's suggested procedure (Pharmacia). The blotting was carried out as described by Towbin et al. (23): the nitrocellulose was reacted with undiluted hybridoma supernatant or ascitic fluid (1:100) and then with 250,000 cpm/ml of 125I-labelled anti-mouse immunoglobulin. The immunoreacted paper was autoradiographed at 80°C using an intensifying screen (Amersham). Labelled-antigen IEF-Affinity Immunoblot Analysis This method was performed essentially as described by Hamilton et al. (24) and used to screen hybridoma supernatants from fusion E. For immunoreaction of blotted papers, alpha sarcin was 125I-radiolabefled by the Iodogen method (25) to a final specific activity from 0.75 to 1.5 /iCi//ig. -
439
Seven ml of 125I-alpha sarcin (l^Ci/ml) were incubated with the blotted papers for 3 hours at 23°C. The paper was washed in PBS and allowed to air dry. After an autoradiography using high-performance autoradiography film (Hyperfilm-MP, Amersham, Sweden) for 16-18 hours, the blot was reacted with 125I-anti-mouse immunoglobulin (0,25 /iCi/m\) and exposed for 12 hours again. This procedure allowed to evidence the presence of immunoglobulin independently of the antigen reactivity. Inhibition of Protein Synthesis in a Cell-Free Translation System Inhibition of the cell-free translation of the Tobacco Mosaic Virus mRNA (Amersham) was measured by the Translation Kit Reticulocytes Type II (Boehringer Mannheim, GmbH, W. Germany). In all cases the concentration required to inhibit 50% of protein synthesis (IC50) was derived from dose-response curves.
RESULTS The alpha sarcin immunogenic activity was studied in BALB/c mice using different immunization protocols. As a first approach the native protein was injected alone for several times, but even after seven injections it was not possible to observe an immune response in the injected animals (data not shown). The results of 4 other immunizzation protocols are reported in Table 1. When Freund's adjuvant was used (protocol A) the mouse sera exhibited a detectable but low level of reactivity (B.I.= 4.2). In the same experimental conditions the rabbit antiserum gave a higher B.I. (13.5). Only when the protein was polymerized with glutaraldehyde, in the presence (protocol C) or absence (protocol B) of KLH, a positive response was obtained since the B.I. increased from to 6.5 (B) or to 9.0 (C) respectively. TABLE 2. SUMMARY OF THE FUSIONS AGAINST ALPHA SARCIN
Number of
Protocol
Seeded wells
grown clones
reactive1 clones
_(%r_(%T_ Ä Ö 9 (2.5) 354 9
(0.6)
11
(2.1)
B
1374
60
C
508
202
D
362
69
(19)
0
E
887
7034
(80)
10
(4.3)
(39)3
(1.1)
1. On alpha sarcin by SP-RIA (A through D) or IEF-IB (E). 2. % relative to seeded wells. 3. Among the grown clones 120 (23%) were reactive on KLH and 22 (4.3 %) on both alpha sarcin and KLH. 4. Only 411/703 grown clones could be tested by IEF-IB.
reported in Table 2, no reactive clones could be obtained from fusion A. As regards fusion B, wells, only 60 clones grew and 15% of them (9/60) were reactive on alpha sarcin. On the contrary, from fusion C about 40% of the hybrid cells grew, but when their supernatants were tested by solid-phase RIA, only 5% of the grown clones (11/202) were reactive on alpha sarcin, whereas the large majority of them showed a good response against KLH. However, none of the selected clones, all of IgM isotype, after subcloning consistently produced antibodies which were reactive to alpha sarcin. The immunization protocol D was also As
out of 1374 seeded
unsuccessful in spite of the detectable selected for fusion.
antibody titer (see Table 1)
in the
serum
of the animal
In the 5th fusion
(E) alpha sarcin was used in its native form at a very low dose, 1 /ig for each single immunization, and the screening was performed as the reactivity on the native protein. Out of the 411 tested clones, 61 (15%) were Ab-producing by immunoreaction with 125I-antimouse Ig, whereas only 10 (2.4%) were reactive with the 125I-labelled immunogen (see Table 2). As shown in Fig. 2, in 5 of the 7 examined supernatants, immunoglobulins could be detected by antimouse Ig, but only one of them (lane 6: the 7B9 supernatant) was specifically reactive with alpha sarcin.
A» •*
• 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Fig. 2: Example of the reactivity of supernatants from growing clones of fusion E by IEF-IB (lanes 1 to 7). Immunoreaction with 125I-alpha sarcin (A) followed by a further reaction with 12ïanti-mouse Ig (B). In the lane 8: purified IgGl irrelevant MAb (pi: 6) was used as a control. Seven of the ten clones which were positive by IEF-IB were selected to test their reactivity by solid-phase RIA (Table 3). Six of them were of IgM(k) isotype and one was of IgG2b(k). The latter hereafter named MAsg2, due to its specific reactivity and consistent immunoglobulin production, was selected for further characterization.
TABLE 3. REACTIVITY OF SUPERNATANTS FROM SELECTED CLONES FROM FUSION E BY SOLID-PHASE RIA ON ALPHA SARCIN (RELEVANT ANTIGEN) OR RTA
(IRRELEVANT RIP)
Clone
specific
Isotype
alpha IgM (K)
cpm bound
sarcin
RTA
5000
1784
7C12
3950
1441
2D7
2702
ND
2F1
2784
ND
9B7
2922
ND
9D8
2781
ND
6H12
7B9
IgG2b (K)
11200
1. At 1:100 dilution.
441
905
study the MAsg2 specificity, besides alpha sarcin and RTA, several other RIPs were tested by SP-RIA and the immunoblotting assay. The rabbit serum, which specifically recognized all the aspergillins, and MAb 4A6, which according to previously reported data (19) bound to Asp fl and mitogillin, were used as controls (data not shown). MAsg2, either as hybridoma supernatant (Fig. 3 A)or ascitic fluid (Fig. 3 B), recognized alpha sarcin and clavatin, whereas Asp fl, was not recognized. Other plant RIPs or irrelevant proteins were completely unreactive. The same results can be seen in the immunoblotting assay (Fig. 4), with the only exception of Asp fl which could be labelled by MAsg2 with this assay. Furthermore, MAsg2 also recognized the dimers and polymers of the aspergillins and an isomeric form of alpha sarcin with an apparent lower molecular weight. To
Fig. 3: Binding reactivity by SP-RIA of MAsg2 hybridoma supernatant (A) or ascitic fluid (B) on different glutaraldehyde-fixed toxins. Aspergillins: alpha sarcin (*), clavatin (•), Asp fl (x); other RIPs: RTA ( + ), PAP ( ), gelonin (-), saporin (o); other irrelevant proteins: cytocrom C (a), plant irrelevant IgGl MAb (a). MAsg 2 maintained, after various single-cell cloning, a range of pi between 5.8 and 6.4 as determined by IEF-blotting assay (Fig. 2). This finding is compatible with monoclonal antibody micro heterogeneity, as reported (24). Finally, we investigated the functional relationship between alpha sarcin and MAsg2 by
Fig 4: Different RIPs were separated by SDS-PAGE in a homogeneous 20% gel and then: A) transferred to nitrocellulose paper and immunoreacted with MAsg2 containing cell culture supernatant or B) stained with brilliant blue Coomasie. The position of the molecular weight standards is indicated. 1) gelonin, 2) PAP, 3) RTA, 4) alpha sarcin, 5) Asp fl, 6) clavatin.
442
inhibition of the protein synthesis in a cell-free translation system (Fig. 5). Alpha sarcin at a concentration of 101(M exerted an 85% inhibition of the protein synthesis. This inhibition was not blocked by MAsg2 neither as cell culture supernatant (data not shown) nor as ascitic fluid diluted in 1:1000 (Fig. 5). 180
Fig. 5: Inhibition of the protein synthesis in a cell-free system expressed as in corporation of 3H-leucin in lysated reticulocytes from rabbit. MAsg2 does not block the inhibition produced by alpha-sarcin at 101(M. Control ( •), alpha sarcin (o), alpha sarcin and MAsg2 (*).
Cpm (x
-r
O
10"3)
60
30
90
Minutes
DISCUSSION
Long-term immunization of mice with high doses of alpha sarcin, manipulated in different ways, elicited a detectable titer of circulating antibodies. However, no stable clones against the immunogen could be obtained from these animals. On the contrary, by using shorter periods and very low doses, a good yield of MAbs which reacted with alpha sarcin was obtained. These findings are in keeping with previously reported data (26,27) and could probably be attributed to a dose-related immunosuppressive effect of the used RIP. In fact, some plant RIPs, like other cytotoxic substances, in spite of their immunogenicity, exert a selected toxicity against the lymphoid system (9,28). To confirm this hypothesis, studies regarding the immunointerfering effects of aspergillins are now underway. One of the obtained MAb (MAsg2) has been characterized. It specifically reacts by IEFblotting with alpha sarcin and clavatin in their native forms by using the toxins as radiolabelled antigens in solution. Moreover, MAsg2 reacted by SP-RIA on dried glutaraldehyde-fixed alpha sarcin and clavatin. On the contrary, no cross-reactivity was detected with the other plant RIPs. When the toxins were denaturated (see immunoblotting results), the MAb enlarged its spectrum of reactivity to another aspergillin toxin, i.e. the Asp f 1, but still maintained its specificity. These findings suggest that the recognized sequence is not conformational but can be masked in the native structure of some members of the aspergillin family. Moreover, MAsg2 does not block the RIP-induced inhibition of protein synthesis as assayed in a cell-free translation system. Altogether these data suggest that MAsg2 does not recognize the active site of the aspergillins. Interestingly none of the anti-RIP MAbs obtained so far against native toxins were able to inhibit their enzymic activity (19,29,30), thus indicating that this region is either nonimmunogenic or unaccessible in the native molecule to antibody recognition. At least for the aspergillin toxins, we consider the second hypothesis to be more likely since the active site seemed to be related to the more hydrophobic region of the molecule (31). The MAsg2 MAb, due to its fine specificity, could be useful together with other MAbs such as 4A6 (19) for epitope mapping of different members of the same family of toxins. The availability of the aminoacid sequence of some aspergillins (3,5,6) and of specific peptide fragments by recombinant DNA technology (32) could help in this task. Moreover, due its specific reactivity on native alpha sarcin, MAsg2 can be considered a good reagent for affinity chromatography purification. Finally, the MAsg2 MAb or its relevant hybridoma should be used to obtain anti-tumor antitoxin bispecific MAbs. These kind of reagents, obtained either by somatic fusion or by chemical linkage (33-35) have been reported to increase the toxicity of saporin (34) and could -
represent an attractive alternative to the chemical linkage between toxin and MAb.
443
ACKNOWLEDGMENTS The research activities leading to this work were partially developed in relation to a contract of the National Program of Pharmacological Research (Rif. 1160/218 088707), by Sudbiotec for the Italian Ministry of University and Scientific and Technological Research. We are grateful to Dr. D. Flavell for helpful suggestions regarding the immunization protocol E. We thank Ms P. Alberti and Ms E. Luisón for excellent technical assistance; Ms L. Mameli and Ms M. Hatton for manuscript preparation and Mr M. Azzini for photographic reproduction. Dr. S. Muñoz's work was supported by a grant from the European Association for Cancer Research and from the Associazione Italiana per la Ricerca sul Cancro. -
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Address reprint requests to: Dr. Maria I. Colnaghi Division of Experimental Oncology E Istituto Nazionale dei Tumori Via G. Venezian 1 20133 Milan, Italy. Fax n° +39-2-2362692 -
Received for publication: Accepted after revision:
12/9/91 3/31/92
446
Production and Characterization of a Monoclonal Antibody Against the Ribosome Inactivating Protein Alpha Sarcin ANDRÉS J.M. FERRERI,1 ELENA FERRARIS
di CELLE,1 CALDERA,1 SATURNINO MUÑOZ,2 STÉPHANIE SALVARELLI,2 FRANCISCO CONDE,2 SILVANA CANEVARI,1 EMANUELA TOSÍ,1 MARIA INES COLNAGHI1
MARISTELLA
'Division of Experimental Oncology E, 2Section of Immunochemistry,
Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian I, 20133 Milan, Italy Department of Investigation, Hospital Ramón y Cajal, Madrid 34, Spain
ABSTRACT
improve the purification of immunoconjugates containing alpha sarcin, a ribosomeinactivating protein, and in the attempt to define the enzymic region of the toxin, MAbs against alpha sarcin were produced. From 5 fusions, by adopting a short period of immunization and very low doses of the immunogen, 10 anti-toxin-producing clones were obtained. One of them, named MAsg2 (IgG2b), due to its specific reactivity and secreting properties, was selected for further characterization. MAsg2 was found to recognize an epitope which is common to two, i.e. alpha sarcin and clavatin, of the three different aspergillins tested, but is not involved in the In order to
active site of the toxins.
INTRODUCTION
Alpha sarcin is a single polypeptide chain with a molecular weight of 17.000 Daltons produced by Aspergillus giganteus (1). It shows a high level of sequence homology and immunological cross-reactivity (2-6) with restrictocin (7) and mitogillin (8), both of which areisolated from the filtrate of aspergillus strains. These molecules, hereafter named Aspergillins, like other plant and bacterial toxins, are ribosome-inactivating proteins (RIPs) (9), and their mechanism of action has been widely studied. Alpha sarcin makes a specific cut in 28S rRNA by hydrolysis of a single phosphodiester bond (10). Despite this knowledge, the region of the molecule devoted to the enzymic activity has not yet been fully defined. Several plant and bacterial RIPs, including members of the aspergillin family, have been coupled to monoclonal antibodies (MAbs) to generate new immunotherapeutic agents (11-15). One of the problems in conjugate production is represented by the removal of the unconjugated antibodies from the final preparation. Cibacron Blue chromatography (16) has been applied for this purpose, but controversial data have been reported in the case of aspergillin conjugates (14,15). Alternative methods such as ion exchange or affinity chromatography (17,18) are now being studied. In order to improve the purification of immunoconjugates containing aspergillins and in the attempt to define the aspergillin enzymic region, MAbs against alpha sarcin have been produced. The present report deals with the manipulation of alpha sarcin as immunogen and with the characterization of a monoclonal antibody, MAsg2, directed against an epitope of alpha sarcin common to different aspergillins.
437
MATERIAL AND METHODS
Toxins
Alpha
sarcin and clavatin
were
respectively
isolated from the culture filtrate of the mould
Aspergillus giganteus strain MDH 18894, and of the Aspergillus clavatus strain IFO 8605 (Institute for Fermentation, Osaka, Japan) and were purified according to the Olson and Goerner's procedure (1). Aspergillus fumigatus allergen I (Asp fl) (19) was kindly provided by K. Amida M.D. (University of Virginia, USA). Ricin A-chain (RTA) and pokeweed antiviral protein (PAP1) were obtained from Calbiochem (La Jolla, CA, USA). Saporin and gelonin were obtained from Inland Laboratories (Austin, Texas, USA). Mice immunization Different manipulations of the immunogen and protocols of immunizations were adopted as
summarized in Table 1.
TABLE 1. SUMMARY OF IMMUNIZATION PROTOCOLS
Protocol
Injections
Immunogen
preparation
/xg/dose
Response before fusion' n°
positive
B.I.
sera
FA
80
3/3
4.2
B
Glut+FA
80
3/3
6.5
C
Glut+KLH+FA
7
80
3/3
9.0
D
MRBC +FA
5
N.E.
3/3
3.2
complex3
1. See Material and Methods and reference n° 20. 2. Evaluated by solid-phase RIA after 7 immunizations. Positive cut -off B.I. _> 3. The Binding Index (BI) was the ratio between cpm bound with the sample and cpm bound with the medium alone. The reported data refer to the 1:125 serum dilution. 3. See Material and Methods and reference n° 21. 4. N.E. not évaluable. FA: Freund's adjuvant. Glut: Glutaraldehyde polymerization. KLH: Keyhole limpet haemocyanin. MRBC: Mouse red blood cells. =
The immunogen (alpha sarcin in NaCl 0.9%) was prepared essentially as in Gullick et al. (20), A) emulsified with Freund's adjuvant (GIBCO LAB., Grand Island, New York, U.S.A.); B) polymerized with glutaraldehyde and then treated as in A; C) as in B but in the presence of Keyhole limpet haemocyanin (KLH). As an alternative, protocol D, previously adopted to obtain second generation MAbs (21), was applied. Briefly, the sera of immunized mice from group B after the 7th injection were pooled and passed on an Affigel blue affinity column (Biorad Laboratories, Richmond, CA) to remove the majority of albumin. Serum proteins (6 150 /A of autologous mouse red blood cells mg/ml) in 0.15M of NaCl were incubated with (MRBC) vol. to vol. in the presence of 1.5 ml of 0.0002 M CrClß.o^O. After 1 hour of incubation at 37°C with shaking and three washings with PBS, the serum coated MRBC were incubated with alpha sarcin (160 /ig) for 45 min. at 37°C. After further washings 25 /i\ of pellet of MRBC-serum-alpha sarcin complex were injected into the mice. Four groups of 3 female BALB/c mice, 8-10 weeks old, were injected s.c. and i.p. 5 (protocol D) or 7 times (protocols A, B and C), once a week, with the immunogen. After the fourth injection the sera from the mice were tested by solid-phase RIA (see below). On the basis of the binding results, five days after the last injection for each immunization protocol, the mouse with the highest response was selected. The selected mice were injected i.p. and i.v. once again with the proper dose of alpha sarcin in saline and three days later killed for spleen removal. In the last immunization protocol
438
(E), not reported in Table 1, the scheme reported in Fig 1.
alpha sarcin was prepared and administered according to
the
Route:
sc/ip
sc/ip
sc/ip
0,5 ug
0,5 ug
¡p/iv
ip/iv
Dose/route:
0,5 ug
Days: O
F
0,5 ug 0,5 ug
FUSION 28
14
T
35
37
40
T TT
Fig. 1: Scheme of immunization protocol E. The alpha sarcin was administered in complete Freund's adjuvant on day 0, incomplete Freund's adjuvant on days 14 and 28 and in phosphate buffered solution on days 35 and 37. Monoclonal antibodies The spleen cells were fused with NSO myeloma, as described (22). The selection of growing hybrids was carried out in HAT medium (Hypoxantin, Aminopterin, Thymidine). Hybrids secreting antibodies were cultured "in vitro" and the isotype of the produced MAb was determined by indirect immunofluorescence, as described (22). For the production of the selected MAb, hybridoma cells were grown as ascites in the peritoneal cavity of pristane primed nu/nu BALB/c mice. The MAb 4A6 (IgGl), raised against Asp fl (19), was kindly provided by Dr. Arruda.
Indirect Solid-Phase Radioimmunoassay (SP-RIA) RIPs were seeded in polystyrene plates (Greiner, Labor Thecnik), dried overnight at 37° C and then fixed with glutaraldehyde 0.2%. Then the wells were filled with 50 /u\ of a titrated dose of the hybridoma supernatant, ascite or serum (in duplicates). After a 45 min. incubation at 37°C, a tracer amount of 125I-labelled Anti-mouse immunoglobulin (Amersham International Buckinghamshire, England) was added and the plate was incubated for another 45 min. at 37°C. In each test untreated
BALB/c serum (diluted 1:5) and rabbit anti-alpha sarcin serum (diluted 1:125) were respectively added as a negative and positive control. The latter was obtained by i.m. injection of alpha sarcin (1 mg) emulsified with complete Freund's adjuvant followed one month later by a boost with incomplete adjuvant. Its binding was detected by 125I-anti-rabbit immunoglobulin (Amersham). The results were expressed as the binding index, calculated as the ratio between cpm bound with the tested sample and cpm bound with the medium alone, or as specific cpm bound, i.e. cpm bound with the tested sample minus the cpm bound with the
medium alone. SDS-PAGE and Western Blotting Precast slab gels (Phastgel; 20% of acrylamide, Pharmacia, Piscataway, N.J., USA) were used to analyze the toxins, employing the automated microprocessor driven Phastsystem following the manufacturer's suggested procedure (Pharmacia). The blotting was carried out as described by Towbin et al. (23): the nitrocellulose was reacted with undiluted hybridoma supernatant or ascitic fluid (1:100) and then with 250,000 cpm/ml of 125I-labelled anti-mouse immunoglobulin. The immunoreacted paper was autoradiographed at 80°C using an intensifying screen (Amersham). Labelled-antigen IEF-Affinity Immunoblot Analysis This method was performed essentially as described by Hamilton et al. (24) and used to screen hybridoma supernatants from fusion E. For immunoreaction of blotted papers, alpha sarcin was 125I-radiolabefled by the Iodogen method (25) to a final specific activity from 0.75 to 1.5 /iCi//ig. -
439
Seven ml of 125I-alpha sarcin (l^Ci/ml) were incubated with the blotted papers for 3 hours at 23°C. The paper was washed in PBS and allowed to air dry. After an autoradiography using high-performance autoradiography film (Hyperfilm-MP, Amersham, Sweden) for 16-18 hours, the blot was reacted with 125I-anti-mouse immunoglobulin (0,25 /iCi/m\) and exposed for 12 hours again. This procedure allowed to evidence the presence of immunoglobulin independently of the antigen reactivity. Inhibition of Protein Synthesis in a Cell-Free Translation System Inhibition of the cell-free translation of the Tobacco Mosaic Virus mRNA (Amersham) was measured by the Translation Kit Reticulocytes Type II (Boehringer Mannheim, GmbH, W. Germany). In all cases the concentration required to inhibit 50% of protein synthesis (IC50) was derived from dose-response curves.
RESULTS The alpha sarcin immunogenic activity was studied in BALB/c mice using different immunization protocols. As a first approach the native protein was injected alone for several times, but even after seven injections it was not possible to observe an immune response in the injected animals (data not shown). The results of 4 other immunizzation protocols are reported in Table 1. When Freund's adjuvant was used (protocol A) the mouse sera exhibited a detectable but low level of reactivity (B.I.= 4.2). In the same experimental conditions the rabbit antiserum gave a higher B.I. (13.5). Only when the protein was polymerized with glutaraldehyde, in the presence (protocol C) or absence (protocol B) of KLH, a positive response was obtained since the B.I. increased from to 6.5 (B) or to 9.0 (C) respectively. TABLE 2. SUMMARY OF THE FUSIONS AGAINST ALPHA SARCIN
Number of
Protocol
Seeded wells
grown clones
reactive1 clones
_(%r_(%T_ Ä Ö 9 (2.5) 354 9
(0.6)
11
(2.1)
B
1374
60
C
508
202
D
362
69
(19)
0
E
887
7034
(80)
10
(4.3)
(39)3
(1.1)
1. On alpha sarcin by SP-RIA (A through D) or IEF-IB (E). 2. % relative to seeded wells. 3. Among the grown clones 120 (23%) were reactive on KLH and 22 (4.3 %) on both alpha sarcin and KLH. 4. Only 411/703 grown clones could be tested by IEF-IB.
reported in Table 2, no reactive clones could be obtained from fusion A. As regards fusion B, wells, only 60 clones grew and 15% of them (9/60) were reactive on alpha sarcin. On the contrary, from fusion C about 40% of the hybrid cells grew, but when their supernatants were tested by solid-phase RIA, only 5% of the grown clones (11/202) were reactive on alpha sarcin, whereas the large majority of them showed a good response against KLH. However, none of the selected clones, all of IgM isotype, after subcloning consistently produced antibodies which were reactive to alpha sarcin. The immunization protocol D was also As
out of 1374 seeded
unsuccessful in spite of the detectable selected for fusion.
antibody titer (see Table 1)
in the
serum
of the animal
In the 5th fusion
(E) alpha sarcin was used in its native form at a very low dose, 1 /ig for each single immunization, and the screening was performed as the reactivity on the native protein. Out of the 411 tested clones, 61 (15%) were Ab-producing by immunoreaction with 125I-antimouse Ig, whereas only 10 (2.4%) were reactive with the 125I-labelled immunogen (see Table 2). As shown in Fig. 2, in 5 of the 7 examined supernatants, immunoglobulins could be detected by antimouse Ig, but only one of them (lane 6: the 7B9 supernatant) was specifically reactive with alpha sarcin.
A» •*
• 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Fig. 2: Example of the reactivity of supernatants from growing clones of fusion E by IEF-IB (lanes 1 to 7). Immunoreaction with 125I-alpha sarcin (A) followed by a further reaction with 12ïanti-mouse Ig (B). In the lane 8: purified IgGl irrelevant MAb (pi: 6) was used as a control. Seven of the ten clones which were positive by IEF-IB were selected to test their reactivity by solid-phase RIA (Table 3). Six of them were of IgM(k) isotype and one was of IgG2b(k). The latter hereafter named MAsg2, due to its specific reactivity and consistent immunoglobulin production, was selected for further characterization.
TABLE 3. REACTIVITY OF SUPERNATANTS FROM SELECTED CLONES FROM FUSION E BY SOLID-PHASE RIA ON ALPHA SARCIN (RELEVANT ANTIGEN) OR RTA
(IRRELEVANT RIP)
Clone
specific
Isotype
alpha IgM (K)
cpm bound
sarcin
RTA
5000
1784
7C12
3950
1441
2D7
2702
ND
2F1
2784
ND
9B7
2922
ND
9D8
2781
ND
6H12
7B9
IgG2b (K)
11200
1. At 1:100 dilution.
441
905
study the MAsg2 specificity, besides alpha sarcin and RTA, several other RIPs were tested by SP-RIA and the immunoblotting assay. The rabbit serum, which specifically recognized all the aspergillins, and MAb 4A6, which according to previously reported data (19) bound to Asp fl and mitogillin, were used as controls (data not shown). MAsg2, either as hybridoma supernatant (Fig. 3 A)or ascitic fluid (Fig. 3 B), recognized alpha sarcin and clavatin, whereas Asp fl, was not recognized. Other plant RIPs or irrelevant proteins were completely unreactive. The same results can be seen in the immunoblotting assay (Fig. 4), with the only exception of Asp fl which could be labelled by MAsg2 with this assay. Furthermore, MAsg2 also recognized the dimers and polymers of the aspergillins and an isomeric form of alpha sarcin with an apparent lower molecular weight. To
Fig. 3: Binding reactivity by SP-RIA of MAsg2 hybridoma supernatant (A) or ascitic fluid (B) on different glutaraldehyde-fixed toxins. Aspergillins: alpha sarcin (*), clavatin (•), Asp fl (x); other RIPs: RTA ( + ), PAP ( ), gelonin (-), saporin (o); other irrelevant proteins: cytocrom C (a), plant irrelevant IgGl MAb (a). MAsg 2 maintained, after various single-cell cloning, a range of pi between 5.8 and 6.4 as determined by IEF-blotting assay (Fig. 2). This finding is compatible with monoclonal antibody micro heterogeneity, as reported (24). Finally, we investigated the functional relationship between alpha sarcin and MAsg2 by
Fig 4: Different RIPs were separated by SDS-PAGE in a homogeneous 20% gel and then: A) transferred to nitrocellulose paper and immunoreacted with MAsg2 containing cell culture supernatant or B) stained with brilliant blue Coomasie. The position of the molecular weight standards is indicated. 1) gelonin, 2) PAP, 3) RTA, 4) alpha sarcin, 5) Asp fl, 6) clavatin.
442
inhibition of the protein synthesis in a cell-free translation system (Fig. 5). Alpha sarcin at a concentration of 101(M exerted an 85% inhibition of the protein synthesis. This inhibition was not blocked by MAsg2 neither as cell culture supernatant (data not shown) nor as ascitic fluid diluted in 1:1000 (Fig. 5). 180
Fig. 5: Inhibition of the protein synthesis in a cell-free system expressed as in corporation of 3H-leucin in lysated reticulocytes from rabbit. MAsg2 does not block the inhibition produced by alpha-sarcin at 101(M. Control ( •), alpha sarcin (o), alpha sarcin and MAsg2 (*).
Cpm (x
-r
O
10"3)
60
30
90
Minutes
DISCUSSION
Long-term immunization of mice with high doses of alpha sarcin, manipulated in different ways, elicited a detectable titer of circulating antibodies. However, no stable clones against the immunogen could be obtained from these animals. On the contrary, by using shorter periods and very low doses, a good yield of MAbs which reacted with alpha sarcin was obtained. These findings are in keeping with previously reported data (26,27) and could probably be attributed to a dose-related immunosuppressive effect of the used RIP. In fact, some plant RIPs, like other cytotoxic substances, in spite of their immunogenicity, exert a selected toxicity against the lymphoid system (9,28). To confirm this hypothesis, studies regarding the immunointerfering effects of aspergillins are now underway. One of the obtained MAb (MAsg2) has been characterized. It specifically reacts by IEFblotting with alpha sarcin and clavatin in their native forms by using the toxins as radiolabelled antigens in solution. Moreover, MAsg2 reacted by SP-RIA on dried glutaraldehyde-fixed alpha sarcin and clavatin. On the contrary, no cross-reactivity was detected with the other plant RIPs. When the toxins were denaturated (see immunoblotting results), the MAb enlarged its spectrum of reactivity to another aspergillin toxin, i.e. the Asp f 1, but still maintained its specificity. These findings suggest that the recognized sequence is not conformational but can be masked in the native structure of some members of the aspergillin family. Moreover, MAsg2 does not block the RIP-induced inhibition of protein synthesis as assayed in a cell-free translation system. Altogether these data suggest that MAsg2 does not recognize the active site of the aspergillins. Interestingly none of the anti-RIP MAbs obtained so far against native toxins were able to inhibit their enzymic activity (19,29,30), thus indicating that this region is either nonimmunogenic or unaccessible in the native molecule to antibody recognition. At least for the aspergillin toxins, we consider the second hypothesis to be more likely since the active site seemed to be related to the more hydrophobic region of the molecule (31). The MAsg2 MAb, due to its fine specificity, could be useful together with other MAbs such as 4A6 (19) for epitope mapping of different members of the same family of toxins. The availability of the aminoacid sequence of some aspergillins (3,5,6) and of specific peptide fragments by recombinant DNA technology (32) could help in this task. Moreover, due its specific reactivity on native alpha sarcin, MAsg2 can be considered a good reagent for affinity chromatography purification. Finally, the MAsg2 MAb or its relevant hybridoma should be used to obtain anti-tumor antitoxin bispecific MAbs. These kind of reagents, obtained either by somatic fusion or by chemical linkage (33-35) have been reported to increase the toxicity of saporin (34) and could -
represent an attractive alternative to the chemical linkage between toxin and MAb.
443
ACKNOWLEDGMENTS The research activities leading to this work were partially developed in relation to a contract of the National Program of Pharmacological Research (Rif. 1160/218 088707), by Sudbiotec for the Italian Ministry of University and Scientific and Technological Research. We are grateful to Dr. D. Flavell for helpful suggestions regarding the immunization protocol E. We thank Ms P. Alberti and Ms E. Luisón for excellent technical assistance; Ms L. Mameli and Ms M. Hatton for manuscript preparation and Mr M. Azzini for photographic reproduction. Dr. S. Muñoz's work was supported by a grant from the European Association for Cancer Research and from the Associazione Italiana per la Ricerca sul Cancro. -
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Address reprint requests to: Dr. Maria I. Colnaghi Division of Experimental Oncology E Istituto Nazionale dei Tumori Via G. Venezian 1 20133 Milan, Italy. Fax n° +39-2-2362692 -
Received for publication: Accepted after revision:
12/9/91 3/31/92
446
