[Cancer Research 59, 4018-4022, August 1, 1999]
© 1999 American Association for Cancer Research
[Cancer Research 59, 4018-4022, August 15, 1999]
© 1999 American Association for Cancer Research
Experimental Therapeutics |
Pharmacokinetics and Antitumor Activity of a Bivalent Disulfide-stabilized Fv Immunotoxin with Improved Antigen Binding to erbB2
Tapan K. Bera,
Jaye Viner,
Elisabeth Brinkmann and
Ira Pastan1
Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255
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ABSTRACT
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We have generated a stable bivalent Fv molecule [(dsFv)2] of the anti-erbB2 monoclonal antibody e23 in which the VH and VL domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a 15-amino acid linker (T. K. Bera et al., J. Mol. Biol., 281: 475–483, 1998). The e23 (dsFv)2 molecule is linked to a truncated form of Pseudomonas exotoxin (PE38) to generate a bivalent disulfide-stabilized immunotoxin e23 (dsFv)2-PE38. Compared to the monovalent immunotoxin, the (dsFv)2 immunotoxin showed greatly increased cytotoxicity to four cancer cell lines expressing low levels of erbB2 but not to four other cell lines with high erbB2 expression. e23 (dsFv)2-PE38 was administered i.v. to mice, and its half-life was determined. The t1/2
and t1/2ß were 20 and 325 min, respectively, whereas the corresponding values for the monovalent dsFv immunotoxin were shorter, 6 and 52 min. The antitumor activities of the monovalent and bivalent immunotoxin were compared using mice bearing A431 tumors. Despite the fact that e23 (dsFv)2-PE38 was 13-fold more active than e23 dsFv-PE38 on A431 cells in cell culture, its antitumor activity in mice was <2-fold that of the monovalent immunotoxin. These data show that a large increase in avidity does not always lead to an increase in cytotoxic activity. Furthermore, in one of the cases in which cytotoxic activity in vitro was greatly enhanced, there was only a small increase in antitumor activity.
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INTRODUCTION
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MAbs2
that recognize antigens on cancer cells are now being used for the targeted therapy of cancer. The antibodies can be used alone or as conjugates for the delivery of toxins, radioisotopes, or drugs (1, 2, 3, 4)
. Immunotoxins are produced by the linkage of an antibody to a toxin made by a bacteria or a plant. In our laboratory, we have used mutant forms of PE to produce immunotoxins (5)
. PE has three domains, each with a different function. Domain I is responsible for cell recognition, domain II is responsible for translocation across the cell membrane, and domain III is responsible for the ADP ribosylation activity of the toxin (6)
. Using recombinant DNA technology, we have deleted portions of domain I of PE to make a Mr 38,000 fragment (PE38) that retains the full translocation and ADP ribosylation activity of the toxin (7
, 8)
.
Current recombinant DNA technology enables the isolation and cloning of the Fv fragments of most MAbs (4
, 9
, 10)
. Fv fragments are the smallest functional portions of antibodies required for high-affinity antigen binding. Fv domains have been successfully used for tumor imaging and have been fused to toxins to make immunotoxins in which the Fv domain serves as a cell-targeting moiety for a potent toxin. Although single-chain Fvs are the smallest functional modules that confer specific antigen binding, they lack one vital property of whole antibodies. The Fvs are monovalent, as compared to bivalent IgG or decavalent IgM. Recently, we described a new approach to the generation of a bivalent disulfide-stabilized Fv fragment of MAb e23, which binds to erbB2 (11)
. The erbB2 proto-oncogene belongs to the epidermal growth factor receptor family and has been implicated in malignant transformation. Amplification and overexpression of the erbB2 gene has been shown to occur in many human cancers including lung, breast, ovary, and stomach (12, 13, 14)
. Overexpression of erbB2 has been directly linked to the malignant conversion of cancer cells, and thus, it makes an excellent target for immunotoxin therapy because cancer cells are unlikely to escape treatment by loss of the antigen (15
, 16)
. We have used Fv fragments of the anti erbB2 MAb e23 to generate a bivalent dsFv immunotoxin (11)
. Binding studies demonstrated that the (dsFv)2 molecule has a much higher affinity for the erbB2 than the monovalent dsFv molecule and a binding affinity that is similar to that of the parental antibody e23. The bivalent e23 immunotoxin was 10–20-fold more cytotoxic to two e23 antigen-positive cell lines tested than the corresponding monovalent dsFv immunotoxin. In this study, we have tested the activity of this immunotoxin toward a wide variety of e23 antigen-positive and -negative cell lines. We also analyzed the antitumor activity and pharmacokinetic behavior of this molecule in mice. Surprisingly, despite its higher affinity and prolonged half life in the circulation of mice, the bivalent immunotoxin showed very little increase in antitumor activity, although it was 13-fold more cytotoxic against cultured tumor cells.
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MATERIALS AND METHODS
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Production of Recombinant Protein.
The components of e23 dsFv and dsFv immunotoxins were expressed in Escherichia coli BL21(
DE3) and accumulated in IBs, as described previously for other recombinant immunotoxins (4)
. IBs were solubilized in guanidine-HCl reduced with dithioerythritol and refolded by dilution in a refolding buffer containing arginine to prevent aggregation and oxidized and reduced glutathione to facilitate redox shuffling (17
, 18)
. Active monomeric protein was purified from the refolding solution by ion exchange and size exclusion chromatography to near homogeneity, as described previously (17)
. Protein concentrations were determined by Bradford assay (Coomassie Plus; Bio-Rad, Richmond, CA).
Binding and Cytotoxicity Assays.
The affinities of the monovalent and bivalent e23 dsFv toxin molecules were determined by a binding competition assay on erbB2 expressing A431 and N87 cells, as described previously (11)
. The antibody e23 IgG, radioiodinated by Bolton-Hunter reagent (New England Nuclear, Boston, MA), was used as tracer in this assay.
The specific cytotoxicities of the e23 (dsFv)2 and dsFv immunotoxins were assessed by protein synthesis inhibition assays (inhibition of incorporation of tritium-labeled leucine into cellular protein) in 96-well plates, as described previously (7)
. The activity of the molecule was defined by the IC50, the toxin concentration that reduces incorporation of radioactivity by 50% compared to cells that are not treated with toxin. The specificity was obtained by comparing the activity toward antigen-positive cells versus the toxicity against antigen-negative cells.
To determine the cell viability, we performed cell proliferation reagent WST-1 (Boehringer Mannheim) assay on A431, MCF7, and SK-BR-3 cells. Experiments were carried out in 24-well plates. Wells were seeded with 50,000 cells/well in 0.5 ml of medium. Different amounts of immunotoxin in 0.5 ml of RPMI-10% FBS were added to each well and incubated for 3 days. The assays were performed by following the instructions provided with the kit.
Assay of Blood Levels of Bivalent e23 dsFv Immunotoxin in Mice.
Female BALB/c mice were injected with 10 µg of immunotoxin in the tail vein. Blood samples were drawn at various time intervals, and the level of the active immunotoxin was measured by the cytotoxicity assay using A431 cells. The A431 cells were incubated with the diluted serum samples from different time points, and their ability to inhibit protein synthesis was measured. A standard curve was made with each pure immunotoxin. The data were analyzed by an exponential curve-fitting program RSTRIP (Version 5; MicroMath Scientific Software, Salt Lake City, UT)
Antitumor Activities of Mono- and Bivalent Immunotoxins in Nude Mice.
The antitumor activities of the e23 dsFv-PE38 and e23 (dsFv)2-PE38 immunotoxins were determined in athymic nude mice bearing a human epidermoid carcinoma (A431 cell line). A431 cells (1.5 x 106) were injected s.c. on day 0 into athymic nude mice, and treatment was started at day 5 when the tumors measured 
50 mm3. Each mouse receive three doses of immunotoxin on days 5, 7, and 9 by i.v. injection. Each group consisted of five animals. Control group received only the diluent, which was 0.2% HSA in DPBS. Tumors were measured with a caliper every other day; the volume of the tumor was calculated using the formula 
.
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RESULTS
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Production of Bivalent e23 dsFv Immunotoxin.
E. coli BL21 (DE3) cells containing the plasmids pTKB22.19 and pYR40 for expression of the components of e23 (dsFv)2-PE38 were grown and induced with isopropyl-1-thio-ß-D-galactopyranoside separately (11)
. The fusion proteins accumulated in insoluble intracellular IBs. These IBs contained almost pure recombinant protein, but in an insoluble and aggregated form. Refolded, soluble monomeric protein was purified from improperly folded proteins and minor bacterial contaminants by ion exchange (Q-Sepharose, Mono Q) and size exclusion chromatography to near homogeneity (Fig. 1)
.
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Fig. 1. Gel analysis of recombinant e23 (dsFv)2 immunotoxin. SDS-PAGE of purified e23 (dsFv)2-PE38 immunotoxins. Lane 1, sample in nonreducing loading buffer; Lane 2, blank; Lane 3, sample in reducing loading buffer; Lane 4, prestain protein molecular weight standard.
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Cytotoxicities of Mono- and Bivalent e23 Immunotoxins toward Cell Lines Expressing Different Amounts of erbB2 Antigen.
PE38 is a truncated but enzymatically active form of PE (3
, 4) . Fusion proteins of antibody fragments with PE38 are cytotoxic to cells that bind and internalize the fusion protein but have very low cytotoxic activity against antigen-negative cells. Thus, cytotoxicity reflects specific antigen binding. In a previous study, we showed that the e23 (dsFv)2-PE38 is cytotoxic to antigen containing A431 and MCF7 cells but not to antigen-negative Hut102 (human) and SP2/Tac (mouse) cells. In addition, e23 (dsFv)2-PE38 was 10–15-fold more cytotoxic than its monovalent dsFv counterpart (11)
. To gain more information about the activity of the bivalent immunotoxin, we tested it on a large number of antigen-containing cancer cell lines (Table 1)
. As described previously and shown in Table 1
, e23 (dsFv)2-PE38 is 10–13-fold more active than its monovalent counterpart toward A431 and MCF7 cells. The dramatically increased cytotoxicity of the e23 bivalent immunotoxin is also evident on the LNCaP and HepG2 cell lines. The IC50s of e23 dsFv-PE38 on LNCaP and HepG2 cells were 14.5 and 10 ng/ml, respectively, whereas the corresponding IC50s for the (dsFv)2 immunotoxin were 0.6 and 1.1 ng/ml. Both the mono- and bivalent e23 dsFv immunotoxins are cytotoxic to BT-474, N87, and SK-BR-3 cells, but there is very little difference in IC50s between mono- and bivalent immunotoxins. The IC50s of the dsFv immunotoxin to the BT474, N87, and SK-BR-3 cells were 0.56, 0.4, and 2.9 ng/ml, respectively. The IC50s of the corresponding (dsFv)2 immunotoxin to BT474, N87, and SK-BR-3 cells were 0.72, 0.53, and 1.8 ng/ml, respectively. The ovarian cancer cell line SK-OV-3 has a high expression of the erbB2 antigen, but these cells are rather insensitive to both mono- and bivalent e23 immunotoxin, with the monovalent immunotoxin being somewhat more active. Neither immunotoxin affected antigen-negative cells (Hut102 and SP2/Tac), even at 1000 ng/ml (Table 1)
.
We also performed cytotoxicity assays by measuring cell viability using WST-1. In this type of assay, the cells were treated with immunotoxin for 3 days, and then the number of viable cells was measured. Table 2
shows that e23 (dsFv)2-PE38 is 12–18-fold more active than its monovalent counterpart on A431 and MCF7 cells, but there is much less difference in activity between the mono- and bivalent immunotoxin on SK-BR-3 cells. The IC50s of the e23 (dsFv)2-PE38 to A431, MCF7, and SK-BR-3 cells were 0.24, 0.18, and 1.1 ng/ml, respectively. The IC50s of the corresponding e23 dsFv immunotoxin to A431, MCF7, and SK-BR-3 cells were 2.9, 3.3, and 3.3 ng/ml, respectively.
Binding of e23 (dsFv)2-PE38 on erbB2-expressing Cells.
One very important factor in determining the cytotoxicity of an immunotoxin is the binding affinity or avidity of the immunotoxin toward its antigen (4)
. Because the bivalent e23 (dsFv) immunotoxin is 10–20-fold more active than its monovalent counterpart toward cell lines that expresses relatively low amount of e23 antigen, we determined the relative binding affinity of (dsFv)2 and dsFv molecule on cell lines that express higher or lower amount of e23 antigen. For competition binding assays, the binding affinities of e23 dsFv and of the newly constructed bivalent dsFv were measured on erbB2-expressing A431 (relatively low) and N87 (high) cells. Various amounts of recombinant e23 monovalent or bivalent immunotoxin were used to compete for binding of radiolabeled bivalent e23 IgG to erbB2-expressing A431 and N87 cells. Table 3
shows that bivalent immunotoxin competes 3.5-fold better for binding than its monovalent counterpart on A431 cells and 20-fold better on N87 cells. The concentrations of competitor that caused 50% inhibition of the binding of 125I-e23 IgG were 5, 8.1, and 160 nM for e23 IgG, e23 (dsFv)2-PE38, and e23 dsFv-PE38, respectively, on N87 cells. The concentrations of competitor needed for 50% competition on A431 cells were 35 and 100 nM for e23 (dsFv)2-PE38 and e23 dsFv-PE38, respectively. Thus, bivalency improves the binding of the recombinant e23 (dsFv)2 molecules on both N87 and A431 cells. It has previously been shown that the bivalent immunotoxin has a 15-fold increased binding to purified recombinant erbB2 using the Biacore method (11)
.
Pharmacokinetics of e23 (dsFv)2-PE38 in Mouse Circulation.
Because of their small size, the t1/2s for both scFv- and dsFv-containing immunotoxins in mice serum were short (20 min; Ref. 18
). Because e23 (dsFv)2-PE38 is larger than the monovalent dsFv, we thought it would have a longer half-life, and we determined its pharmacokinetics in mice. To do this, we injected BALB/c mice i.v. with a single dose of 10 µg of e23 (dsFv)2-PE38 or e23 dsFv-PE38. Blood was drawn at different times after the injection and assayed for immunotoxin activity by measuring its ability to inhibit protein synthesis on A431 cells, as described in "Materials and Methods." In Fig. 2
, the plasma clearance curves of e23 (dsFv)2-PE38 and dsFv-PE38 are shown. The elimination of e23 (dsFv)2-PE38 from mouse blood is much slower than that of the dsFv-PE38 molecule, with a t1/2 = 20 min and t1/2ß = 325 min for e23 (dsFv)2-PE38 and t1/2 = 6 min and t1/2ß = 52 min for e23 dsFv-PE38.
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Fig. 2. Pharmacokinetics of mono- and bivalent e23 dsFv immunotoxin in mice serum. Female BALB/c mice were injected i.v. with a single 10-µg dose of dsFv or (dsFv)2 immunotoxin. Blood samples were drawn at specific time points. The level of immunotoxin was measured by bioassay in which triplicates of diluted serum samples were incubated with A431 cells. The ability of the serum sample to inhibit protein synthesis was measured by incorporation of tritiated leucine. Data points, means from three animals in two independent assays performed on each immunotoxin. A, blood levels for dsFv-PE38. B, blood levels of (dsFv)2-PE38.
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Animal Toxicity Studies.
Toxicity studies were carried out by injecting different doses of each immunotoxin i.v. into BALB/c mice. The LD50 of (dsFv)2-PE38 was found to be 0.5 mg/kg, which is very similar to that of dsFv-PE38 (Table 4
; Ref. 19
).
Antitumor Activity.
On A431 cells, the activity of the e23 (dsFv)2-PE38 was 13-fold higher than the corresponding monovalent dsFv-PE38 molecule (Table 1)
. To determine whether the cytotoxicity data could be translated as antitumor activity in vivo, we assayed the mono- and bivalent e23 dsFv-PE38 immunotoxins for their ability to cause regressions of s.c. tumor xenografts of A431 cells in athymic nude mice. To produce tumors, we injected A431 cells s.c. into immunodeficient mice on day 0. On day 5, when the tumors reached 50 mm3, the animals were injected i.v. with 4 µg (45 pmol) of e23 (dsFv)2-PE38 or 2.9 µg (45 pmol) of e23 dsFv-PE38 every other day x 3. The dose of e23 dsFv-PE38 was chosen (45 pmol) that would produce regression and regrowth of tumors so that a difference in activity between it and the bivalent immunotoxin could be assessed. The control groups received PBS containing 0.2% HSA. The antitumor activity of e23 dsFv-PE38 and e23 (dsFv)2-PE38 are shown in Fig. 3
. Control animals treated with 0.2% PBS-HSA developed large tumors and were sacrificed on day 18, when the tumors had grown to 1.5 x 2.0 cm in size. The tumors for both the e23 dsFv-PE38 and e23 (dsFv)2-PE38 groups decreased to an unmeasurable size after three injections, but the tumors begin to reappear on day 20. Animals treated with 4 µg (45 pmol) of e23 (dsFv)2-PE38 showed tumor regressions that lasted somewhat longer than the corresponding monovalent immunotoxin at equimolar dose of 2.9 µg (45 pmol) of e23 dsFv-PE38. When the dose for the monovalent immunotoxin was increased almost 2-fold (77 pmol), the antitumor activity for both the mono and bivalent immunotoxins were very similar (Fig. 3)
, indicating that the antitumor activity of the bivalent immunotoxin was 2-fold better than the corresponding monovalent immunotoxin.
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DISCUSSION
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We recently developed a new type of recombinant bivalent dsFv immunotoxin using the anti erbB2 antibody e23. The bivalent immunotoxin is 10–20 times more active on two erbB2-expressing cell lines tested than the corresponding monovalent immunotoxin, e23 dsFv-PE38. Here, we examined the cytotoxicity of this bivalent dsFv immunotoxin on 12 other erbB2-expressing cancer cell lines. In addition, we evaluated the antitumor activity and pharmacokinetic behavior of e23 (dsFv)2-PE38 in mice.
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Cytotoxicity of e23 (dsFv)2-PE38 toward Cell Lines Expressing Different Amounts of erbB2.
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Two important factors that are widely accepted to determine the cytotoxicity of an immunotoxin toward a cell line containing a target antigen are the binding affinity and the antigen density on the target cells. The cytotoxicity data described in Table 1
for e23 (dsFv)2-PE38 do not correlate with the erbB2 expression on target cells. The bivalent e23 dsFv immunotoxin is very active on cells expressing relatively low amounts of erbB2 (A431, MCF7, HepG2, and LNCaP); on these cells, it is 9–29-fold more active than the corresponding monovalent dsFv immunotoxin. However, the improved cytotoxicity of the bivalent immunotoxin is not evident on cells overexpressing erbB2 antigen, such as SK-BR-3, BT-474, SK-OV-3, or N87 (20
, 21)
. In fact, the activity of bivalent immunotoxin on those cells is a little less than the corresponding monovalent e23 (dsFv) immunotoxin. Also, in the case of SK-OV-3 cells, in which erbB2 expression is relatively high (21)
, both mono- and bivalent immunotoxins have relatively low activity. There are several possible explanations for this variation in relative cytotoxicity of the mono- and bivalent e23 dsFv immunotoxins on cell lines expressing variable amounts of erbB2. (a) Because of its bivalency, the bivalent immunotoxin may bind better than the monovalent dsFv immunotoxin to cells expressing either high or low antigen-expressing cells, but the rate of internalization for these two immunotoxins might be different on cells with different antigen density. We attempted to measure internalization rates using radiolabeled immunotoxins and found that both the bivalent and monovalent immunotoxins were very slowly internalized; no significant differences could be detected. (b) Perhaps the bivalency of the (dsFv)2 molecule cross-links the antigen in such a way that it slows the internalization of the immunotoxin on erbB2-overexpressing cells. (c) Because of expression of other receptors in the erb family, cross-linking might activate some signaling pathway that protects those cells from toxin-mediated killing. Further studies are clearly needed to understand this unexpected observation.
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Pharmacokinetics of e23 (dsFv)2-PE38.
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The bivalent e23 dsFv-PE38 immunotoxin is larger than the corresponding monovalent dsFv-PE38 immunotoxin. Both monovalent and bivalent molecules are very stable in vitro at 37°C in HSA (11)
. The half-lives of the bivalent immunotoxin in mouse serum were t1/2 = 20 min and t1/2ß = 325 min, which are higher than the corresponding values for e23 dsFv-PE38, t1/2 = 6 min and t1/2ß = 52 min. The larger size of the bivalent molecule probably contributes to its longer retention into the circulation because the smaller molecules are filtered through the kidney glomerulus more efficiently than the larger molecules (22
, 23)
.
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Antitumor Activity of e23 (dsFv)2 Immunotoxin.
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The antitumor potency of the bivalent e23 dsFv-PE38 was only slightly (2-fold) higher compared to the monovalent immunotoxin (Fig. 3)
, although the bivalent immunotoxin was 13-fold more active than the monovalent immunotoxin on A431 cells in vitro (Table 1)
. Thus, the improved cytotoxicity in vitro of the bivalent immunotoxin does not correlate with antitumor activity of the molecule. This is despite the fact that bivalent immunotoxin in the mouse circulation remained for a longer period of time. The most probable explanation for this discrepancy is that the improved affinity of the bivalent immunotoxin retards its penetration into the tumor because of enhanced binding to tumor cells close to the capillaries (24)
.
In summary, our data show that the increase in avidity of an immunotoxin produced by attaching two Fv domains to the toxin does not lead to a dramatic increase in antitumor activity in animals and may not be a very useful strategy for increasing the activity of immunotoxins or other forms of targeted therapy in humans.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be addressed. Phone: (301) 496-4797; Fax: (301) 402-1344; E-mail: pasta{at}helix.nih.gov 
2 The abbreviations used are: MAb, monoclonal antibody; PE, Pseudomonas exotoxin A; IB, inclusion body; HSA, human serum albumin.
Received 12/15/98.
Accepted 6/18/99.
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Top
ABSTRACT
INTRODUCTION
) for (dsFv)2-PE38 and either 77 pmol (
) or 45 pmol (
) for dsFv-PE38 immunotoxin. Control groups received diluent only (
).