Bioconjugate Chem. 1995, 6, 166-1 73
166
ARTICLES Biochemical and Cytotoxic Properties of Conjugates of Transferrin with Equinatoxin 11, a Cytolysin from a Sea Anemone Cecilia Pederzolli,+J Giovanna Belmonte,'J Mauro Dalla Serra,tJ Peter MaEek,§and Gianfranco Menestrina*,$ Dipartimento di Fisica, Universita' di Trento, 38050 Povo, Trento, Italy, CNR Centro di Fisica degli Stati Aggregati, 38050-Pov0, Trento, Italy, and Department of Biology, Biotechnical Faculty, University of Ljubljana, 61000-Ljubljana, Slovenia. Received June 6, 1994@
Transferrin, a serum glycoprotein, is a major regulator of cellular growth via its cellular receptor. Because transferrin receptors are absent from the plasma membranes of most normal adult resting cells, but are present on transformed, activated, and malignant cells, it can be used to address a toxin toward these cells. The cytolysin equinatoxin 11, isolated from the sea anemone Actinia equina L., was coupled to human apo or diferric transferrin by using a heterobifunctional cross-linking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate(SPDP). The conjugates were separated by column chromatography, and their composition was demonstrated by electrophoresis, antibody staining, and determination of the hemolytic activity in the absence or presence of a reducing agent. The average molar ratio of equinatoxin I1 to transferrin for the studied conjugates was found to be ~3.4.The activity of the conjugates against human erythrocytes and human tumor cells (Raji and Jurkat) was assessed. The conjugate is very active on tumor cells in vitro; however, the hybrid molecule maintains an unspecific hemolytic activity. This unspecific toxicity is due to the fact t h a t transferrin-bound toxin partially retains its original ability to bind to the cell membrane directly. It could be strongly reduced (and even eliminated) by pretreating the conjugates with sphingomyelin, the natural ligand of sea anemone cytolysins. These conjugates were stable versus temperature (up to a t least 40 "C), versus time (up to several weeks a t 4 "C and a t least 1year a t -80 "C), and versus repeated freezethaw cycles with liquid nitrogen (but not with -80 "C).
INTRODUCTION
Anticancer immunotoxins, built by chemically or genetically linking a toxin to an antibody directed against a transformed cell line, have been extensively studied in the last few years (I, 2 ) . In most cases, toxins with a n intracellular cytosolic target (so-called A-B type) have been used (I,2 ) . One reason for this is that it is possible to remove the receptor binding part from these toxins, thus strongly enhancing the specificity of the conjugate, although a t the expenses of its overall cytotoxicity (11. In many cases, human growth factors have been used in place of antibodies of animal origin as a means to target cytotoxic agents while avoiding human anti-mouse antibody (HAMA) response ( 3 ) . Toxins with an intracellular target require endocytosis to become effective. In principle, other cellular compartments could be addressed with some advantage (41, in particular the cell membrane. One way to achieve this is by using a hemolytic toxin in place of a n A-B toxin.
Immunotoxins, built by linking a hemolysin from a sea anemone to an antibody, have been described (5,6). We decided to readdress this question by conjugating a hemolysin to a mitogenic molecule like human transferrin (Tfn),2which is often used in place of antibodies (7). As toxin we chose equinatoxin 11, recently isolated from the sea anemone Actinia equina and purified to homogeneity (8). It is a single polypeptide chain with a molecular weight of about 19 kDa and a PI of 10.3. It shows hemolytic, cytotoxic, and cardiotoxic activity and causes platelet aggregation and lung damage a t concentrations ranging from to 10 -lo M. At least in part, these effects are due to its ability to form ion channels in the membrane of the attacked cells (9, 10). Tfn is a major regulator of cellular growth ( I I - I 3 ) , but also a potent mitogen for a variety of tumors (1417). It may induce proliferation even independently of its iron carrying properties (12);in fact, a regulatory role of apo-Tfn on the growth of pituitary tumor cells was
* To whom correspondence should be addressed. Tel: ++39 461 881588. Fax: ++39 461 810628. E.mai1: Menestrina@ itnvax.science.unitn.it.BITnet: MenestrinaCOitncisca. Universita' di Trento. CNR Centro di Fisica degli Stati Aggregati. University of Ljubljana. Abstract published in Advance ACS Abstracts, December 1, 1994. C.P. and G.B. were the recipients of a fellowship from the Fondazione Trentina per la Ricerca sui Tumori, and M.D.S. was the recipient of a fellowship from the Consiglio Nazionale delle Ricerche.
* Abbreviations: EqT 11,Actinia equina equinatoxin 11; Tfn, transferrin; TR, transferrin receptor; BSA, bovine serum albumin; AP, alkaline phosphatase; SPDP, N-succinimidyl 3-(2pyridy1dithio)propionate;DTT, dithiothreitol; PLP, pyridoxal 5'phosphate; BCIP, 5-bromo-4-chloro-3 indolyl phosphate; NBT, nitroblue tetrazolium; MTT, 3-(4,5-dimethylthiazol-2-y1)-2,5diphenyltetrazolium bromide; P-ME, P-mercaptoethanol; SM, sphingomyelin; PC, phosphatidylcholine; S W , small unilamellar vesicles; HRBC, human red blood cells; FCS, fetal calf serum; TLC, thin layer chromatography; SDS, sodium dodecyl sulfate; PVDF, hydrophobic polyvinylidene difluoride; PBS, phosphatebuffered saline; LNa, liquid nitrogen.
@
1043-1802/95/2906-0166$09.00/0
0 1995 American Chemical Society
Cytotoxicity of Transferrin-Equinatoxin I1 Conjugates
observed (18). The presence of a larger number of Tfn receptors (TR) on growing tumor cells (12, 13), particularly on highly metastasizing (19)and drug resistant (20) cells, as compared to the normal resting counterparts, suggests t h a t Tfn conjugates, such as those described here, could have some use a s antitumoral drugs. I t is expected that upon interaction with tumor cells the toxin might be liberated a t the cell surface or inside the endocytic vacuole where intracellular membranes might then become targets. Furthermore, the presence of Tfn binding proteins on the outer membrane of pathogenic parasites (21,22)and pathogenic bacteria (23, 24) indicates that these conjugates may also have, a t least in some cases, a n antimicrobial effect. Since microbial Tfn-binding proteins are genetically unrelated to the TR (211,conjugates employing anti-TR antibodies would be useless in this application. EXPERIMENTAL PROCEDURES
Materials. Human iron-saturated and apo-Tfn were obtained from Miles and Sigma, respectively. Tfn has a molecular weight around 80 kDa (11)and a PI between 5.2 and 5.7 dependent on the microheterogeneity of its glycan chains (25). Bovine serum albumin (BSA), antihuman Tfn polyclonal IgG antibodies, alkaline phosphatase (AP)conjugated anti-IgG antibodies, N-succinimidyl 3-(2-pyridyldithio)propionate(SPDP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and dithiothreitol (DTT) were all obtained from Sigma. SM was from Fluka and PC from Calbiochem. All other chemicals were commercial products of sequential or analytical reagent grade. EqT 11. EqT I1 was isolated and assayed as described elsewhere (8). I t has a molecular weight of 19 kDa, a PI of 10.3, and a molar extinction coefficient of 3.61 x lo4 M-l cm-I a t 280 nm (26). Chemical modification of the lysine residues by PLP was performed according to (27) exactly as described earlier (28). The number of moles of lysine residues modified per mole of toxin was determined spectrophotometrically (29). EqT 11-Tfn Conjugation and Purification. The toxin was conjugated to Tfn by means of an artificial disulfide bridge introduced via the heterobifunctional cross-linking reagent N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) (30). A three-step scheme was followed. Step 1: Tfn and toxin were separately converted into 2-pyridyl disulfide derivatives using a molar excess of SPDP of 8:l and 2:1, respectively; after 60 min incubation a t room temperature the excess of reagent was removed by gel filtration on a Sephadex G-25 column. Step 2: SPDP-modified toxin was reduced with 10 mM DTT for 1h and the sample dialyzed to remove the excess of DTT; we found that a 2-fold molar excess of SPDP introduces an average of 1.75 of 2-pyridyldisulfide groups into the toxin, as determined by the release of pyridine2-thione followed a t 343 nm, and confirmed by ionexchange HPLC. Step 3: freshly reduced SPDP-modified toxin was mixed with SPDP-modified Tfn (with a n average of 4.1 2-pyridyldisulfide groups per molecule) in a molar ratio of 2:l and incubated overnight a t 4 "C. The extent of conjugation was again estimated by following the release of pyridine-2-thione a t 343 nm, as a result of the thiol disulfide exchange reaction. Either apo or diferric Tfn was used (as specified in the text); conjugates were also made with a PLP-modified toxin prepared as described (27). Conjugates were purified by liquid chromatography in two steps. First, the coupled reaction mixture was applied to a size exclusion column (10 mm i.d., 450 mm
Bioconjugate Chem., Vol. 6,No. 2, 1995 167
length), loaded with Sephacryl S-200 HR from Pharmacia to remove unconjugated toxin. Fractions of 1 mL were collected a t a rate of 15 m u . Thereafter, free Tfn was removed by passage through a n ionic exchange column (10 mm i.d., 70 mm length, loaded with CM-Sepharose Fast Flow, from Pharmacia). Fractions of 1 mL were collected a t a rate of 40 m L h The presence of the conjugate in the different fractions was estimated by the absorption a t 280 nm and by titrating their hemolytic activity before and after reduction with 2.5 mM DTT.
Electrophoretic Analysis of EqT 11-Tfn Conjugates. SDS-Page. Denaturing gel electrophoresis was performed according to Laemmli (31) using precast minigels (Pharmacia, Uppsala, Sweden). Density gradients ranging from 8 to 25% or 10 to 15% were both used (as it will specifically detailed in the text). A semiautomatic horizontal unit, PhastSystem by Pharmacia, was employed. Gels were stained with either Coomassie brilliant blue or silver stain, and the amount of protein was quantitated by bidimensional densitometry using a PhastImage densitometer (Pharmacia) with a band-pass filter a t 613 nm, for Coomassie-stained gels, or a t 546 nm, for silver-stained gels. Western Blot. After SDSpage the separated bands were transferred to PVDF (hydrophobic poly(viny1idene difluoride)) membrane (Immobilon-P from Millipore) with a semidry blotter (PhastTransfer by Pharmacia) using 20 V for 20 min a t 15 "C. The transfer was checked by reversible prestaining of the blotted bands with ponceau S (from Sigma) and crosschecked by Coomassie staining of the original gel. After ponceau red was removed the membranes were incubated with either antitoxin polyclonal antibodies (mice IgG raised as described (32))or anti-Tfn polyclonal antibodies (goat IgG from Sigma), and the bound proteins were detected with AP conjugated to a secondary antibody (anti-IgG) (33,34). The AP complex was developed using the substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT), which form a brown insoluble precipitate (35). Hemolytic Assays. Hemolytic activity of EqT I1 and conjugates was determined turbidimetrically a t 650 nm with a microplate reader (UVmax from Molecular Devices) supported by the computer program SOFTmax. Human RBC were prepared from fresh heparinized blood by washing it three times (700 g for 10 min) and resuspending it in the saline buffer: 160 mM NaC1, 10 mM TrisHC1, pH 7.5. Finally, the concentration of HRBC was adjusted with the buffer to a n apparent absorbance of 1.0 a t 650 nm in a 1 cm path length cuvette. Toxin and conjugates were 2-fold serially diluted in saline buffer (plus 0.2 mg/mL BSA, to saturate the unspecific protein binding sites of plastic which can reduce toxin activity) using flat-bottom 96-well microplates, and one volume of HRBC was added to each well. One hemolytic unit was arbitrarily defined as the reciprocal of the dilution of EqT I1 changing the optical density with a maximal rate of 0.01 OD/min, which in this assay corresponds to a lysis of around 50% HRBC after 30 min. Binding of EqT I1 and conjugates to S W and HRBC was determined indirectly by measuring the residual hemolytic activity after a preincubation with S W or HRBC as follows. SUV were prepared by sonication of SM and PC mixtures, exactly as described earlier (9).EqT I1 (5 x M) or conjugates B1 and B2 (2.6 x M or 3.5 x M, respectively, see Figure 2 for the sample definition) were incubated with (or without) S W of different composition for 20 min a t 30 "C or with (or without) HRBC in saline for 10 min a t 30 "C. In the case of SUV, unbound toxin (or conjugate) was recovered in a filtrate obtained by centrifuging the mixtures through a
Pederzolli et al.
168 Bioconjugate Chem., Vol. 6, No. 2, 1995
a
b
c
d
“-‘I 1.0
I
A- 0.8
-
A
e f -
*
*
kDa
4
330
a
gi
4
36
4
18.5
4
--
0
I
10
0
E
0.6 0 A- 0.5
5
(D 0
2 0.6 -
0
o
-a
Y
Q)
5:
18.5
cy
0.4
0.2
.n 4
0.0
-
Q)
10
0.3
0
2
e
0.2
0
;0.1 a
I
0
0.4
20
30
40
50
0.0 0
10
20
30
40
50
60
Fraction number
Fraction number
Figure 1. Separation of EqT 11-Tfn conjugate from free toxin.
Figure 2. Separation of EqT 11-Tfn conjugate from free Tfn.
Lower panel. Toxin and apo-Tfn were linked via a n artificial disulfide bond introduced with the heterobifunctional crosslinking reagent SPDP. To remove unconjugated toxin the coupled reaction mixture was applied to a size exclusion gel filtration column (Sephacryl S-200 HR). The presence of the conjugate in the different fractions was estimated by the absorption a t 280 nm (open circles) and by measuring their hemol-ytic activity before and after reduction with DTT (closed and open triangles respectively). Upper panel. SDS-page of the different samples: lanes a-c, fractions 33, 22, and 17 ( a s indicated in the lower panel); lane d, unfractionated reaction mixture; lane e, EqT 11; lane f, apo-Tfn. The position of standard proteins of known molecular weight is shown on the right with full arrowheads. An asterisk marks the end of the stacking gel. Experimental conditions: polyacrylamide gradient 8-25c/r, SDS 1.374, silver staining.
polysulfone filter with a MW cutoff of 300 kDa (100OOg for 10 min) which retained vesicles and bound toxin (or conjugate). Before usage the filters ( Ultrafree-MC purchased from Millipore) were washed with a 0.2 mg/mL solution of BSA in saline to saturate unspecific protein binding sites. In the case of HRBC it was recovered in the supernatant after centrifugation at 4500g for 5 min. The hemolytic activity of toxin and conjugates was then tested as above. Cytotoxicity Tetrazolium-Based Assay. Toxicity “in vitro” was measured on human Burkitt lymphoma cells (Raji cells) by the 3-(4,5-dimethylthiazol-2-y1)-2,5diphenyltetrazolium bromide (MTT) reduction assay. Cells were maintained in RPMI-1640 medium (by Sigma) supplemented with 25 mM Hepes, 4 mM L-glutamine, 200 pg/mL of gentamicin, and 10% heat-inactivated fetal calf serum (FCS) at 37 “C in a humidified air atmosphere with 5% Con. M’M’ was dissolved in modified Dulbecco’s phosphate buffered saline (PBS, purchased by Sigma) at 5 mg/mL and filtered for sterility. This solution was stored at 4 “C in a dark bottle for 1week at most. Toxin and conjugates were serially diluted (10-fold at each step) with the RPMI-1640 medium on 96-well microtiter plates. Raji cells (4 x lo4)were added to each well in a final
Lower panel. High molecular weight fractions from gel filtration were pooled and applied to a weak cation exchange column (CMsepharose Fast Flow) to remove free apo-Tfn. The pH profile applied is reported. Fractions 20-40 and 41-48 were pooled and called conjugate B1 and B2, respectively. As in Figure 1, the presence of the conjugate was estimated by measuring the absorption a t 280 nm (open circles) and the hemolytic activity before and after reduction with D l l ’ (closed and open triangles, respectively). Upper panel. SDS-page of the different samples: lane a, EqT 11; lanes b-d, fractions 41-48, 20-40, and 7 (as indicated in the lower panel); lane e, apo-Tfn. The position of standard proteins of known molecular weight is shown on the right with full arrow-heads. An asterisk marks the end of the stacking gel. Other experimental conditions as in Figure 1.
volume of 100 pL. Each well also contained 0.1 mg/mL of BSA and, if indicated, Tfn (0.4 mg/mL) or SW of pure SM (10pglmL). Plates were then incubated (37 “C, 5% COS)for either 1h (acute cytotoxicity test) or 24 h (long term cytotoxicity test). In the last test 5% FCS was added after the first 6 h. MTZ‘ was finally added to each well (0.2 mg/mL), and the cells were cultured for another 2 h before 100 pL of 10% SDS in 0.01 N HCl was added to dissolve the dark blue crystals. After a n overnight incubation at room temperature, the optical densities at 575 nm of each well were measured with a microplate reader ( W m a x from Molecular Devices). RESULTS AND DISCUSSION
Construction and Purification of Covalent EqT 11-Tfn Conjugates. EqT I1 and PLP-modified EqT I1 were conjugated to either apo or diferric Tfn by crosslinking with SPDP, as explained in the Experimental Procedures. Conjugates were purified by liquid chromatography in two steps. In the first step unreacted toxin, MW 19 kDa, was separated from Tfn, MW around 80 kDa, and higher molecular weight conjugates by highresolution gel filtration (Figure 1). The presence of the free and conjugated toxin in the different fractions was estimated by measuring their hemolytic activity before
Bioconjugafe Chem., Vol. 6, No. 2, 1995 169
Cytotoxicity of Transferrin-Equinatoxin II Conjugates
a
b
c
d
e
f
*
4 4
ertl-:fans:errin
+ - + - + -
B-ME
Figure 3. SDS-page of EqT 11-Tfn conjugate under native and reduced conditions: lanes a, b, EqT 11; lanes c, d, conjugate with diferric Tfn; lanes e, f, Tfn. Lanes a, c, and e also had 3% /j-mercaptoethanol. I t should be noted that in the presence of /j-mercaptoethanol bands are stained heavier. The stacking gel is marked by a n asterisk. Other experimental conditions: polyacrylamide gradient 10-15'2, SDS 1.3%, silver staining.
and after reduction with DTT. Fractions eluting later (molecular weight around 18 kDa) had a high hemolytic activity independent of the reducing conditions, as is expected for the free toxin which lacks any disulfide bridge. On the contrary, fractions eluting a t high molecular weight (between 70 and 200 kDa) displayed a relevant hemolytic activity only under the reducing conditions, which are expected to release free toxin from the conjugates. Residual unconjugated Tfn was removed by a second step through an ionic exchange column, taking advantage of the widely different isoelectric point of Tfn and toxin, PI 5.9 and 10.3,respectively. By using a cation exchange column only the conjugates were retained at a pH lower than 7.0 (Figure 2). As expected, fractions eluting in the void volume, supposed to contain free Tfn, were devoid of any hemolytic activity, whereas fractions eluting at higher pH had a high hemolytic activity only in the presence of DTT.
Electrophoretic Characterization of EqT 11-Tfn Conjugates. SDS-page analysis of EqT 11-Tfn conjugates, under nonreducing conditions (Figure 31,confirmed the presence of high molecular weight compounds and the absence of polypeptides with MW corresponding to either free toxin or free Tfn. Contamination by the free components was below the resolution of the silver staining. By using reducing conditions, we confirmed that the high molecular weight compounds were actually disulfide-linked conjugates containing only one low MW component, around 17 kDa, and one high MW component, around 78 kDa. Apparently, conjugates containing a variable number of toxin molecules were obtained (Figures 1-3). From a densitometric analysis of lane d of Figure 3, it appears that the principal conjugates had molecular weight of 110 and 125 kDa, corresponding to two and three toxin molecules bound per conjugate. The average value in different preparations was 2.4. Interestingly, the minimum number appeared in any case to be two. A possible explanation for this is that Tfn is a bilobate molecule (36, 37), and thus, if a highly reactive site is created with SPDP it is conceivably present in two copies per molecule. From the densities of the toxin and Tfn bands, obtained after reduction of the conjugates (lane c), and comparing to the relevant control lanes (a,e), we evaluated an average number of 3.1 EqT I1 molecules bound per Tfn in Figure 3. With different preparations the average
2"'',- :ox-
36 18.5
s lver staired
Figure 4. Western blotting of EqT 11-Tfn conjugate. Toxin, Tfn, and conjugates (as indicated) were applied in parallel to three polyacrylamide gels (gradient 10-15%) and run a s in Figures 1-3. Proteins from two of the gels were then transferred to blotting membranes and stained with either anti-Tfn (left panel) or anti-toxin (middle panel) antibodies, under nonreducing condition. The third sample was silver-stained (right panel). Arrows on the right indicate the position of standard proteins of different molecular weight. "conjugate-lys" indicates a conjugate made with a lysine modified toxin (PLP modification).
number was 3.4 f 0.4. This number is slightly higher than that obtained under nonreducing conditions. The reason for this is that some conjugates of very high molecular weight exist, which enter the gel only under reducing conditions, otherwise remaining trapped at the end of the stacking gel (lane d). These high MW compounds concur to raise the average toxidconjugate ratio under reducing conditions.
Immunological Characterization of the Conjugate. The nature of these compounds was made unequivocal by Western blotting experiments (Figure 4). Bands, separated by SDS-page under non reducing conditions, were transferred to a PVDF membrane and stained with either anti-toxin or anti-Tfn polyclonal antibodies. The membrane was then developed using the corresponding AP-conjugated anti-IgG antibodies. Antitoxin antibodies stained either the free toxin or the high molecular weight bands but not free Tfn. Correspondingly, anti-Tfn antibodies stained either the free Tfn or the high molecular weight bands but not free toxin. This demonstrates that the high molecular weight bands are disulfide-linked conjugates of Tfn and toxin. Finally, PLP-modified toxin, in which the amino groups of most exposed lysine residues were modified, cross-linked only to a minor extent with Tfn (see Figure 4). This confirms that accessible amino groups are used for conjugation by SPDP. Residual Hemolyticity of the Conjugate. A titration of the hemolytic activity of free EqT I1 and a purified Tfh conjugate under reducing and nonreducing conditions is shown in Figure 5. HRBC lack the TR and should be resistant to the conjugate. However, despite the removal of all free toxin, we found that, under native conditions, the conjugate was still hemolytic although 70-fold less than the toxin. Its hemolytic activity increased 9-fold when reduced, whereas that of the toxin decreased by a factor of 1.5. Although this residual, unspecific, hemolytic activity is small (less than 1.5% that of free toxin) i t might be harmful and should be further investigated. I t is probably due to the fact that the toxin in the conjugate, while covalently bound to Tfn, still retains, at least in part, its ability to interact with, and bind to, cell membranes. Conjugates using apo-Tfn gave results which were qualitatively similar. To demonstrate the binding of conjugates to HRBC we titrated the amount of D?T-dependent hemolytic activity
-
170 Biuconjugafe Chem., Vol. 6,No. 2, 1995 120 4 100 -
80-
.-ea
-Z 5 c
60-
0
40-
20
.1
10’”
10”O
10-9
1 0-e
molar concentration Figure 5. Hemolytic activity of the EqT 11-Tfn conjugate. The hemolytic activity of free EqT I1 (circles)and a purified diferric Tfn conjugate (squares)were estimated under reducing (2.5 mM DTT, filled s?rmbols)and nonreducing conditions (open slymbols). The percentage of hemolysis of HRHC was determined with a microplate reader as explained in the Experimental Procedures. Molar concentrations indicated are those equivalent to the toxin content, and in the case of the conjugate, they are lower limits which were calculated assuming an average of two toxin molecules per conjugate. n
control PC PC/SM SM HRBC Figure 6. Binding of the EqT 11-Tfn conjugate to lipid vesicles and HRRC. Binding of toxin and conjugates to SW of different composition and to HRRC was estimated by titrating the amount of hemolytic activity remaining in solution after a preincubation with SUV or HRRC. The hemolytic activity was calculated as the reciprocal of the concentration required for 5OC4 hemolysis in the presence of DTT and reported in percentage. One hundred percent was the activity of a pertinent sample incubated only with buffer. Incubation was with S W for 20 min at 30 “C or with HRRC for 10 min at 30 “C. In the case of SLV, unbound toxin and conjugates were recovered in the filtrate after ultrafiltration through filters with cut off at MW 300 kDa pretreated with RSA. With HRBC they were recovered in the supernatant after centrifugation.
remaining in solution after a preincubation of the conjugate with HRBC and compared to that of the toxin (Figure 6). Assuming a linear dependence between hemolytic activity and toxin concentration, i t appears that 100%of free toxin, about 90% of conjugate B1, and 98% of conjugate B2 are bound to the cells and removed from the supernatant during this step. To demonstrate that binding is mediated by the toxin we investigated whether SM, the best known substrate for toxin action, was also able to bind the conjugate. Binding of toxin and conjugates to small unilamellar vesicles (SW)of different composition was again determined indirectly by titrating the residual D’IT-dependent hemolytic activity remaining in solution after the incubation with SW (Figure 6 ) . We found that indeed the free
Pederzolli et al.
conjugate concentration was decreased by preincubation with sphingolipids in a way qualitatively similar to free EqT 11. The nonspecific (D’IT-independent) activity of the recovered conjugate is best estimated through the ratio C X - (where C - and C-are the concentration of conjugate necessary to obtain 50% hemolysis in the presence or in the absence of DTT,respectively). The larger this value the lower is the nonspecific activity. Free toxin has a value of about 1 in all cases. The B1 and B2 conjugates instead have values of 8 and 10, respectively, which then slightly increase after incubation with SMcontaining SW (to 9 and 11). This indicates that more specific complexes were recovered after the incubation with lipid, suggesting the enrichment in a population of conjugate molecules with a lower affinity for SM. Such conjugates might have improved specificity in vivo due to reduced interaction with SM. Cytotoxicity on Tumor Cells. To test the cytotoxicity of these conjugates against cancer cells expressing the TR, a human lymphoblastoid cell line was used (Raji cells from Burkitt lymphoma). Acute toxicity (appearing within 1 h) and long-term toxicity (developed after 24 h) were determined (Figure 7). In the acute test, 50% reduction of viability by EqT I1 occurred a t =3.5 x M, whereas with the diferric and apo conjugates at about 2 x loy9and 3 x lo-” M, respectively (data for the apoferric conjugate are not shown). While the cytotoxic activity of EqT I1 was slightly enhanced by the presence of a n excess of free ferric Tfn, that of the ferric conjugate was inhibited by a factor of about 3 (i.e., 66%),suggesting i t was, a t least in part, dependent on the expression of the TR. The slightly lower activity of the apo-Tfn conjugate, which was also inhibited by free Tfn (=70%c), is in line with the fact that apo-Tfn has a lower affinity for the TR than diferric Tfn (11). However, the residual activity of the diferric conjugate, in the presence of excess Tfn, was still relatively high (50%viability at %0.6 x 10 -’ M in the acute test), confirming that the hybrid molecule could retain, at least in part, the ability to interact with cells via the toxin receptor. These considerations prompted us to examine whether pretreating the conjugate with SM, which potently inhibits free toxin, could remove its unspecific toxicity. We found that, while preincubation of free toxin with SM removes about 99% of its toxicity (and the residual activity is independent from the presence of free Tfn), the same treatment removes only about 90% of the toxicity of the diferric conjugate, and, more importantly, the residual toxicity is this time strongly reduced by free Tfn, indicating i t is mediated by the TR. This can be understood on the basis of the heterogeneity of the chemical conjugates. Those conjugates in which the toxin molecules are more exposed (and hence have a higher nonspecific toxicity) would be the first to become inactivated by SM, whereas conjugates in which the SM-binding region of the toxin is less accessible (and hence have a lower nonspecific toxicity) would remain active via the Tfn part. The long term test confirmed and extended these conclusions. As expected, the toxicities of all samples appeared at lower concentrations. Fifty percent inhibition of viability occurred at =3 x 10 M with EqT I1 and at about 10-1° and 3 x M with the diferric and the apo conjugate, respectively (data for the apo-ferric conjugate are not shown). Ferric Tfn in excess reduced the activity of the ferric and the apo conjugate (by a factor of *3), but not that of free toxin. Furthermore, in this case, preincubation of the ferric conjugate with SM produced a molecule with 50% activity around 2 x 10 !’ M, which was completely inactivated by an excess of free
Cytotoxicity of Transferrin-Equinatoxin II Conjugates
m
ACUTE TOXICITY
120 100
n
80
r
60 - o EqTll
am ,-
*
LONG TERM TOXICITY
120
1
I
100
80
60
tTfn
40
: I
I
Bioconjugate Chem., Vol. 6,No. 2, 1995 171
40
o +Tfn +SM
20
20
toxin
lo-'
0
1 0-12
10-11
10-10 10-0 10-8
10-7
0
10-12 10-11 10-10 10-0
10-8
10-7
10-6
io-'
io-'
1'
120
100 80 60
40 20 0 1042
10-11 10-10 IO-o
molar concentration
-8
molar concentration
Figure 7. Cytotoxicity of EqT I1 and an EqT 11-Tfn conjugate toward cancer cells. Left: acute cytotoxicity test. Raji cells (4 x lo4) were exposed to various concentrations of toxin (A) or of conjugate (B)for 1 h a t 37 "C, and thereafter their viability was determined by the MTT method. In experiments of protection of cells with SM and free ferric Tfn, the final concentrations used were 10 ,ug/mL and 0.4 mg/mL, respectively (but similar results were obtained with 0.1 mg/mL of Tfn). Symbols: circles, toxin or conjugate; squares, the same plus free Tfn; triangles, plus SM; diamonds, plus both Tfn and SM. Right: long term cytotoxicity test. All experimental conditions and symbols are the same as in the left panel, except that the incubation was prolonged for 24 h. Conjugate concentration was calculated as in Figure 5 . Solid lines were drawn by eye. Results are from a single experiment. The tests were repeated two or three times with qualitatively similar results.
Tfn, indicating its toxicity was now absolutely dependent on the presence of the TR. In some control experiments a different cell line expressing the TR, i.e., human lymphoblastoid (Jurkat), was used, and in this case, a protein synthesis assay, using I4C-leucine, was performed as in (38, 39). The results were consistent and are not shown here. Stability of the Conjugate. To measure the stability of the complex with different treatments we assayed its hemolytic activity with or without the reducing agent DTT and determined C+, C- and the ratio C-IC+ as explained above (Table 1). A decrease of C-, and thus of the ratio C-IC-, is expected if the toxin dissociates from the complex, becoming free. Stability with Temperature. To test thermal stability the conjugate was incubated for 1h a t different temperatures. It appeared to be stable, a t least until 40 "C. Stability with Storage at 4 "C. The conjugate is normally stored frozen a t -80 "C and retains its properties for a period of a t least 12 months (not shown). It appears that after thawing it is stable in the refrigerator a t 4 "C for a t least 2 weeks. Stability with Freezing and Thawing. Cycles of freezing and thawing either with liquid nitrogen (LN2) or a t -80 "C were tried. We found a different response with the two protocols. Freezing and thawing the conjugate in LN2 preserved all its activity, whereas freezing and thawing a t -80 "C decreased its activity progressively until a maximum decrease of a factor ~ 6 However, . the relative ratio of activity under reducing and nonreducing conditions (C-IC-) was constant in both cases, indicating that the covalent bonding was stable. It appears that repeatedly freezing and thawing a t -80 "C should be
Table 1. Stability of the EqT 11-Tfn Conjugate with Temperature, Time, and FreezingThawing
T,b"C
time; days no. of cyclesd LNz
no. of cyclese -80 "C
10 20 30 40 0 5 14 0 1 2 3 4 5 0 1 2 3 4 5
C-,"nM 8.0 7.0 9.0 8.0 3.3 5.2 4.0 3.9 4.5 6.5 4.9 4.0 4.4 3.8 4.9 16.0
21.0 28.0 25.0
C+;nM 0.72 0.98 0.72 0.89 0.30 0.40 0.32 0.34 0.45 0.59 0.50 0.45 0.45 0.50 0.71 1.90 2.70 3.50 3.20
C-IC+ 11.1
7.1 12.5 9.0 11.0 13.0 12.5 11.5 10.0 11.0 9.8 8.9 9.8 7.6 6.9 8.4 7.8 8.0 7.8
a C- and C+ are the concentration of conjugate necessary to obtain 50% hemolysis in the absence and in the presence of DTT, respectively. One hour incubation of the apo-Tfn conjugate (Bl) a t the given temperature. Storage of a thawed apo-Tfn conjugate (B1)at 4 "C for the given time. Freezing and thawing in LNz of the apo-Tfn conjugate (Bl) for the given number of cycles. e As in d but at -80 "C with the ferric conjugate.
avoided, possibly because it might cause irreversible aggregation of the molecules. CONCLUSIONS
The use of a cytolytic toxin from a sea anemone to build immunoconjugates was described recently (5, 6). We
172 Bioconjugate Chem., Vol. 6,No. 2, 1995
have now used a similar approach to create a mitotoxin by linking EqT I1 to the mitogenic factor, Tfn. Coupling a toxin which acts upon cell membranes with a n internalizing targeting ligand might seem to be a paradox. However, our experiments on human lymphoblastoid cells (Raji or Jurkat) showed that such conjugates are very active. We believe that after interaction with these cells the conjugate undergoes reduction, either on the surface of the cell or, more probably, inside the endocytic compartment, and the toxin is liberated. In this way all the internal membranes might become targets. The fact that long-term toxicity is a t least 10 times higher than acute toxicity would confirm this. Furthermore, because the mode of action of EqT I1 is not yet completely understood, we cannot exclude the existence of other intracellular targets. The fate of the conjugate on the cell is one of our goals in the continuing of this research. The produced mitotoxins exhibit stability versus temperature (up to a t least 40 "C), time (up to several weeks a t 4 "C and 1year at -80 "C), and freeze-thawing in LNZ. One particular problem that is foreseen in the use of cytolytic toxins to create immuno- o r mitotoxins is their unspecific toxicity toward innocent bystander cells. We found that indeed the hybrid Tfn-EqT I1 molecule maintains an unspecific hemolytic activity, and the presence of an excess of free Tfn could not completely prevent toxic effects of the conjugate on tumor cells. We demonstrated that this behavior is probably due to the ability of the toxin part of the conjugate to retain partly its ability to interact directly with cells. However, in the case of sea anemone cytolysins such unspecific toxicity can be strongly reduced (and even completely eliminated) by treating the conjugate with SM, the natural target lipid for these toxins. This suggests that, as in the case of A B toxins, it should be possible, by chemical or genetical modification of the cytolysin, to reduce its lipidbinding properties without impairing its cytolytic activity. Such binding-incompetent toxins would be more suited for the preparation of conjugated mitotoxins with a reduced nonspecific activity. ACKNOWLEDGMENT
The Italian authors were the recipients of grants from the Minister0 per 1'Universita e la Ricerca Scientifica and Consiglio Nazionale delle Ricerche, while P.M. was the recipient of a grant from the Ministry of Science and Technology, Republic of Slovenia. C.P. and G.B. were the recipients of fellowships from the Fondazione Trentina per la Ricerca sui Tumori, and M.D.S. was the recipient of a fellowship from the Consiglio Nazionale delle Ricerche (no. 201.02.45-21.02.05). We thank Dr. Marco Colombatti (Istituto di Scienze Immunologiche of the University of Verona) for advice and help with the Jurkat cells and the Blood Bank of S. Chiara Hospital in Trento for providing blood samples. LITERATURE CITED (1) Vitetta, E. S., Fulton, R. J., May, R. D., Till, M., and Uhr, J. W. (1987) Redesigning nature's poisons to create anti-tumor reagents. Science 238, 1098-1104. (2) Pastan, I., Chaudhary, V. K., and Fitzgerald, D. J. (1992) Recombinant toxins as novel therapeutic agents. Annu. Rev. Biochem. 61, 331-354. (3) Pietersz, G. A., and McKenzie, I. F. C. (1992) Antibody conjugates for the treatment of cancer. Zmmunol. Rev. 129, 57-80. (4)Tritton, T. R., and Hickman, J. A. (1990) How to kill cancer cells: membranes and calcium signaling as targets in cancer chemotherapy. Cancer Cells 2 , 95-105.
Pederzolli et al. (5) Avila, A. D., de Acosta, M. C., and Lage, A. (1988) A new immunotoxin built by linking a hemolytic toxin to a monoclonal antibody specific for immature T lymphocytes. Znt. J . Cancer 42, 568-571. (6) Avila, A. D., de Acosta, M. C., and Lage, A. (1989) A carcinoembryonic antigen-directed immunotoxin built by linking a monoclonal antibody to a hemolytic toxin. Znt. J . Cancer 43, 926-929. (7) Faulk, W. P., Harats, H., McIntyre, J . A., Berczi, A., Sun, I. L., and Crane, F. L. (1989) Recent advances in cancer research: drug targeting without the use of monoclonal antibodies. Am. J . Reprod. Zmmunol. 21, 151-154. (8) MaEek, P., and Lebez, D. (1988) Isolation and characterization of three lethal and hemolytic toxins from the sea anemone Actinia equina L . Toxicon 26, 441-451. (9) Belmonte, G., Pederzolli, C., MaEek, P., and Menestrina, G. (1993) Pore formation by the sea anemone cytolysin equinatoxin I1 in red blood cells and model lipid membranes. J. Membrane Biol. 131, 11-22. (10) MaEek, P., Belmonte, G., Pederzolli, C., and Menestrina, G. (1994) Mechanism of action of equinatoxin 11, a cytolysin from the sea anemone Actinia equina L. belonging to the family of actinoporins. Toxicology 87, 205-227. (11) Thortensen, K., and Romslo, I. (1990) The role of transferrin in the mechanism of cellular iron uptake. Biochem. J . 271, 1-10. (12) May, W. S. J., and Cuatrecasas, P. (1985) Transferrin receptor: its biological significance. J . Membrane Biol. 88, 205-215. (13) Gatter, K. C., Brown, G., Trowbridge, I. S., Woolston, R.E., and Mason, D. Y. (1983) Transferrin receptors in human tissues: their distribution and possible clinical relevance. J . Clin. Pathol. 36, 539-545. (14) Sutherland, R., Delia, D., Schneider, C., Newman, R., Kemshead, J., and Greaves, M. (1981) Ubiquitous cell-surface glycoprotein on tumor cells is proliferation-associated receptor for transferrin. Proc. Natl. Acad. Sei. U.S.A. 78, 4515-4519. (15) Cavanaugh, P. G., and Nicolson, G. L. (1991) Lung-derived growth factor that stimulates the growth of lung-metastasizing tumor cells: identification as transferrin. J . Cell. Biochem. 47, 261-271. (16) Denstman, S., Hromchak, R., Guan, X.-P., and Bloch, A. (1991) Identification of transferrin as a progression factor for ML-1 human myeloblastic leukemia cell differentiation. J . Biol. Chem. 266, 14873-14876. (17) Chakal Rossi, M., and Zetter, B. R. (1992) Selective stimulation of prostatic cell proliferation by transferrin. Proc. Natl. Acad. Sci. U.S.A. 89, 6197-6201. (18) Sirbasku, D. A., Pakala, R., Sato, H., and Eby, J. E. (1991) Thyroid hormone dependent pituitary tumor cell growth in serum-free chemically defined culture. A new regulatory role for apotransferrin. Biochemistry 30, 7466-7477. (19) Inoue, T., Cavanaugh, P. G., Steck, P. A., Brunner, N., and Nicolson, G. L. (1993) Differences in transferrin response and number of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potential. J . Cell. Physiol. 156, 212-217. (20) Barabas, K., and Faulk, W. P. (1993) Transferrin receptors associates with drug resistance in cancer cells. Biochem. Biophys. Res. Commun. 197, 702-708. (21) Schell, D., Evers, R., Preis, D., Ziegelbauer, K., Kiefer, H., Lottspeich, F., Cornelissen, A. W. C. A,, and Overath, P. (1991) A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site. EMBO J . 10, 1061-1066. (22) Voyiatzaki, C. S., and Soteriadou, K. P. (1990) Evidence of transferrin binding sites on the surface of Leishmania promastigotes. J . Biol. Chem. 265, 22380-22385. (23) Gerlach, G. F., Anderson, C., Potter, A. A,, Klashinsky, S., and Willson, P. J. (1992) Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae. Infect. Zmmun. 60, 892-898. (24) Williams, P., and Griffiths, E. (1992) Bacterial transferrin receptors- structure, function and contribution to virulence. Med. Microb. Zmmunol. 181, 301-322. (25) de Jong, G., van Noort, W. L., and van Eijk, H. G. (1992) Carbohydrate analysis of transferrin subfractions isolated by
Cytotoxicity of Transferrin-Equinatoxin II Conjugates preparative isoelectric focusing in immobilized pH gradients. Electrophoresis 13, 225-228. (26) Norton, R. S., MaEek, P., Reid, G. E., and Simpson, R. J. (1992) Relationship between the cytolysin tenebrosin-C from Actinia tenebrosa and equinatoxin-I1 from Actinia equina. Toxicon 30, 13-23. (27) Nishigori, H., and Toft, D. (1979) Modification of avian progesterone receptor by pyridoxal-5’-phosphate. J . Biol. Chem. 254, 9155-9161. (28) Turk, T., and MaEek, P. (1992) The role of lysine, histidine and carboxyl residues in biological activity of equinatoxin 11, a pore forming polypeptide from the sea anemone Actinia equina L. Biochim. Biophys. Acta 1119, 5-10. (29) Peach, C., and Tolbert, N. E. (1978) Active site studies of ribulose-1,5-bisphosphatecarboxylase/oxygenase with pyridoxal-5‘-phosphate. J. Biol.Chem. 253, 7864-7873. (30) Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. N succimidyl 3-(2-pyridyl-dithio)propionate,a new heterobifunctional reagent. Bi0chem.J. 173, 723-737. (31) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. (32) Narat, M., MaEek,, P., Kotnik, V., and Sedmak, B. (1993) The humoral and cellular immune response to a lipid attenuated pore-forming toxin from the sea anemone Actinia equina L. Toxicon 32, 65-71.
Bioconjugate Chem., Vol. 6,No. 2, 1995 173 (33) Bers, G., and Garfin, D. (1985) Protein and nucleic acid blotting and immunobiochemical detection. BioTechniques 3 , 276-288. (34) Pluskal, M. G., Przekop, M. B., Mark, R., Vecoli, C., and Hicks, D. A. (1986) Immobilon PVDF transfer membranes: a new membrane substrate for Western blotting of proteins. BioTechniques 4 , 272-283. (35) Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sei. U.S.A. 76, 4350-4354. (36) Bailey, S., Evans, R. W., Garratt, R. C., Gorinsky, B., Hasnain, S., Horsburgh, C., Jhoti, H., Lindley, P. F., Mydin, A,, Sarra, R., and Watson, J. L. (1988) Molecular structure of serum transferrin at 3.3-A resolution. Biochemistry 27, 5804-5812. (37) Anderson, B. F., Baker, H. M., Norris, G. E., David, W. R., and Baker, E. N. (1989) Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. J . Mol.Bio1. 209, 711-734. (38) Colombatti, M., Dell’Arciprete, L., Chignola, R., and Tridente, G. (1990) Carrier Protein-Monensin Conjugates: Enhancement of Immunotoxin Cytotoxicity and Potential in Tumor Treatment. Cancer Res. 50, 1385-1391. (39) Colombatti, M., Bisconti, M., Dell’Arciprete, L., Gerosa, M., and Tridente, G. (1988) Sensitivity of human glioma cells to cytotoxic heteroconjugates. Int. J. Cancer 42, 441 -448. BC9400884
