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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3909-3914
NEOPLASIA
From the Medizinische Klinik I der Universität zu Koeln,
Cologne, Germany.
The human lymphocyte activation marker CD30 is highly overexpressed
on Hodgkin/Reed-Sternberg cells and represents an ideal target for
selective immunotherapy. We used the murine anti-CD30 hybridoma Ki-4 to
construct a new recombinant immunotoxin (rIT) for possible clinical use
in patients with CD30+ lymphoma. Hybridoma V genes were
polymerase chain reaction-amplified, assembled, cloned, and expressed
as a mini-library for display on filamentous phage. Functional Ki-4
scFv obtained by selection of binding phage on the CD30-expressing
Hodgkin lymphoma cell line L540cy was inserted into the bacterial
expression vector pBM1.1 and fused to a deletion mutant of
Pseudomonas exotoxin A (ETA'). Periplasmically expressed
Ki-4(scFv)-ETA' demonstrated specific activity against a variety
of CD30+ lymphoma cells as assessed by different in vitro
assays. To evaluate in vivo antitumor activity, severe combined
immunodeficient mice challenged with human lymphoma cell lines were
treated with the immunotoxin. The blood distribution time
t1/2
Although patients with Hodgkin lymphoma or anaplastic
large-cell lymphoma can be cured by polychemotherapy and extended field radiotherapy, less than 30% of those whose lymphoma relapses remain disease-free after second-line treatment. The major reason for this
poor outcome is the persistence of residual tumor cells surviving standard therapy.1-3 Thus, the selective elimination of
minimal residual disease using monoclonal antibody-based
immunoconjugates after standard treatment might improve the outcome in
malignant lymphoma.
One of the most promising target antigens for immunotherapy of
malignant lymphoma such as Hodgkin lymphoma or anaplastic large-cell lymphoma is the CD30 receptor. This antigen was originally discovered on cultured Hodgkin/Reed-Sternberg (H-RS) cells using the monoclonal antibody (mAb) Ki-1.4 The gene encoding for the CD30
receptor molecule5 is located on chromosome 1p36. The
naturally occurring CD30 ligand also has been identified and
cloned.6 The CD30/CD30 ligand system triggers cytolytic
cell death in malignant lymphoma cell lines and induces proliferation
and cytokine production in T cells or neutrophils.7
Monoclonal antibodies against CD30 have been explored as vehicles for
cytostatic drugs8 or plant toxins.9
Immunotoxins constructed with anti-CD30 mAbs chemically linked to
catalytically active toxins demonstrated specificity and potent
anti-tumor activity against Hodgkin lymphoma cells in vitro and in
mouse models.10-12 Twelve patients with refractory relapsed
Hodgkin lymphoma were treated with an immunotoxin constructed by
conjugating the anti-CD30 mAb BerH2 to Saporin-6
(Ber-H2-S6).13 Five patients exhibited a rapid
reduction in tumor mass, underlining the validity of CD30 as a target
antigen in this malignancy. The dose-limiting toxicity in this and
other trials was related to vascular leak syndrome (VLS) consisting of
decreased serum albumin level, weight gain, edema, pulmonary edema, and
aphasia.14,15 Recently, it was reported that vascular
epithelial cells are damaged by IL-2 and the catalytic proportions of
applied toxic enzymes, which share a common (x)D(y)
amino acid sequence motif where x = L, I, G, or V and
y = V, L, or S and that this damage to epithelial cells might
initiate VLS.16
To circumvent VLS, new therapeutics such as recombinant immunotoxin
(rIT) with deleted (x)D(y) amino acid sequence motifs might be developed by DNA technology. Chimeric proteins composed of a
truncated, binding-deficient toxin fused to a single-chain antibody
fragment (scFv), rITs are defined, compact molecules that can be
modified more easily and produced more economically than their chemical counterparts.
Our group has developed and evaluated a number of chemically linked
immunotoxins for clinical application.14,15 One of the
constructs, Ki-4.dgA,12 is based on the anti-CD30 mAb Ki-4, which has been demonstrated to inhibit the shedding of the
extracellular part of the CD30 molecule.17 We subsequently
used phage display technology for the selection of a specific,
high-affinity Ki-4 scFv that was then genetically fused to a modified
Pseudomonas exotoxin A gene (ETA').11
Recombinant immunotoxins expressed so far under standard conditions
remained primarily associated with inclusion bodies18 and
were purified after careful denaturation and renaturation
procedures.19 Under optimized conditions, only 5% to 10%
of the input protein was properly folded and active. Thus, we developed
a new protocol for the periplasmic expression of recombinant proteins
under osmotic stress conditions in the presence of compatible solutes
resulting in more than 95% functional activity in terms of specific in
vitro binding and cytotoxic activity.20 In the current
study we demonstrate the specific effects of the first recombinant
anti-CD30 immunotoxin against disseminated human Hodgkin lymphoma in
severe combined immunodeficiency (SCID) mice.
Bacterial strains, oligonucleotides, and plasmids
Cell culture
Cloning Ki-4 VH and VL genes were amplified, assembled, and cloned into pCANTAB6 as previously described.11 Plasmids were transfected into 50 µL E coli TG-1 by electroporation as described elsewhere.29 The cells were grown for 1 hour in 2 × TY medium at 37°C before plating on 2 × TY agar medium containing 100 µg ampicillin/mL and 2% (wt/vol) glucose (2 × TY-Amp-Glu). Binding Ki-4(scFv) molecules were enriched on L540cy cells as reported.11 Their genes were released from phagemid vector pCANTAB6 by SfiI/NotI digestion and inserted into SfiI/NotI-restricted expression vector pBM1.1 containing the ETA' gene.23 The resultant plasmids were transfected into E coli BL21(DE3).Periplasmic expression and purification of the recombinant immunotoxins As described recently,20 rITs were expressed under the control of the isopropyl -thiogalactoside-inducible
Taq promotor in the E coli strain
BL21(DE3).30 Briefly, bacteria were grown overnight at
26°C in Terrific broth containing 0.5 mmol/L ZnCl2 and
50 µg/mL kanamycin. The culture was diluted 30-fold in 200 mL of the
same medium, at an OD600 of 2, and was supplemented with
0.5 mmol/L sorbitol, 4% NaCl, and 10 mmol/L betaine. Then it was
incubated at 26°C for additional 30 to 60 minutes. Thereafter, immunotoxin production was induced by the addition of 2 mmol/L isopropyl-thiogalactoside at 26°C. Fifteen hours later cells were harvested by centrifugation at 3700g for 10 minutes at
4°C. For all the following steps, tubes were kept on ice. The
bacterial pellet was centrifuged, and its wet weight was determined.
Cells were frozen at  196°C. After they thawed, the cells
were resuspended in 75 mmol/L Tris HCl, pH 8, 300 mmol/L NaCl, 1 capsule protease inhibitors/50 mL NaCl (Complete; Roche
Diagnostics, Mannheim, Germany), 5 mmol/L dithiothreitol, 10 mmol/L
EDTA, and 10% glycerol, and they were sonicated 6 times for 30 seconds
at 200 W. The periplasmic fraction was recovered as supernatant after
centrifugation at 21 000g for 30 minutes at 4°C and
transferred to 75 mmol/L Tris HCl, pH 8, 1 mol/L NaCl, and 10%
glycerol using Hitrap desalting columns (Pharmacia). Recombinant
immunotoxin was partially purified by immobilized IMAC using
nickel-nitriloacetic acid (Ni2+-NTA) chelating Sepharose
(Qiagen) on a BioLogic workstation (BioRad, München, Germany).
Bound protein was eluted with 250 mmol/L imidazole in 75 mmol/L Tris
HCl, pH 8, 1 mol/L NaCl, and 10% glycerol. Fractions containing
RFT5(scFv)-ETA' or Ki-4(scFv)-ETA' were pooled and concentrated by ultrafiltration. Functional rIT was finally purified through size exclusion chromatography (SEC) using Bio-Prep SE-100/17 (BioRad) on the BioLogic workstation by separation in
phosphate-buffered saline (PBS), pH 7.4, and 1 mol/L NaCl. Purified
protein was analyzed by SDS-PAGE and quantified by densitometry (GS-700
Imaging Densitometer; BioRad) after Coomassie staining according to
bovine serum albumin standards.
Binding analyses The binding activity of Ki-4(scFv)-ETA' was determined by CD30 receptor enzyme-linked immunosorbent assay (ELISA) as previously documented.11 Samples were incubated with anti-ETA' mAb TC-1 for 1 hour. After washing, 100 µL (Fab' 2) fragments of peroxidase-coupled goat-antimouse-IgG (Roche Diagnostics) 1:5000 in Tris-buffered saline (TBS) containing 0.5% bovine serum albumin (BSA) and 0.05% Tween 20 were added, and samples were incubated for 1 hour at room temperature. Bound Ki-4(scFv)-ETA' was detected by the addition of 100 µL o-Phenylenediamine-dihydrochloride (Sigma, Deisenhofen, Germany) after incubation for 1 hour. Absorbance at 405 nm was measured with an ELISA reader (MWG Biotech, Ebersberg, Germany). Native mAbs were used as controls.Cytotoxicity assays The effect of Ki-4(scFv)-ETA' on the protein synthesis of the target cell lines was determined by measurement of [3H]-leucine incorporation as published recently.31 Incorporated [3H]-leucine was measured by liquid scintillation (LS/801; Beckman, Munich, Germany). The concentration required to achieve a 50% reduction in protein synthesis compared with untreated control cultures (IC50) was calculated. All measurements were made in triplicate. Competition experiments were performed with L540cy cells in the presence or absence of 10 µg/mL Ki-4 mAb as a competitor for Ki-4(scFv)-ETA'.Animals The SCID mice were obtained from our own colony and were maintained under sterile conditions. No antibiotic prophylaxis was provided. ELISA was performed regularly to detect antibodies that might have leaked into the sera of the animals.Antitumor experiments in mice To evaluate the effects of immunotoxin treatment on the survival of SCID mice challenged with human Hodgkin lymphoma cells, animals were randomly divided into groups of 5. One day after inoculation of L540rec Hodgkin cells, mice received either a single intravenous injection of Ki-4(scFv)-ETA', ETA', or PBS in a volume of 400 µL PBS. The dose of immunotoxin (40 µg functional rIT) represented the maximum tolerated dose in SCID mice, determined in a dose-escalation study of groups of 3 mice.Immunohistochemical staining Gross examination of various tissues of each animal was performed within 24 hours of death, as described recently.27 The tissues were removed, fixed in 4% formaldehyde buffered in TBS, dehydrated through a graded series of ethanol concentrations, and embedded in paraffin. Sections 4-µm thick were stained immunohistochemically with the monoclonal antibody Ber-H2 (anti-CD30) purchased from DAKO Diagnostica (Hamburg, Germany) and visualized by the biotin-streptavidin method using fast red as a chromogen.32 For detection of the CD25 receptor, an antigen-retrieval technique was applied33 using a commercially available antibody, 2A3 (Becton Dickinson, San Jose, CA). Tissue sections were counterstained with hematoxylin, mounted for light microscopy, and digitally documented using the Imaging System KS300 (Kontron Elektronik, München, Germany).Detection of soluble CD30 CD30 receptor assays were performed in triplicate using clarified sera (1 µL/well) and photometric ELISA for quantitative determination of soluble human CD30 antigen, according to the manufacturer's instructions (DAKO Diagnostica).Blood clearance study SCID mice weighing approximately 20 g were injected intravenously through the tail vein with 40 µg rIT. At 20, 40, 60, 120, 240, and 360 minutes after injection, blood was drawn from the orbital sinus. For each time point, blood from 3 mice was drawn. Serum was stored at80°C before immunotoxin levels were determined.
Stability in mouse blood serum The stability of 40 µg/mL rIT was evaluated by incubation in mouse serum at 37°C. Active immunotoxin remaining at different times was determined by cell proliferation and ELISA assays on L540rec cells.Statistical analysis Blood clearance was determined in the 2-compartment model after nonlinear regression of the obtained data with SPSS 8.0. Survival analyses were performed by the Kaplan-Meier method, and statistical significance was compared by the log-rank test. Using the t test, the highest sCD30 concentrations of the PBS groups were correlated with those of the rIT-treated animals.
In vitro characterization of Ki-4(scFv)-ETA' After cloning the RFT5 scFv into the bacterial expression vector pBM1.1 and the transformation of E coli BL21(DE3), bacteria were grown under osmotic stress in the presence of betaine. The 70-kd Ki-4(scFv)-ETA' was functionally accumulated in the periplasmic space and subsequently purified by combinations of IMAC and size-exclusion chromatography. The rIT was substantially stabilized during purification by 1 mol/L NaCl, eluted from Ni2+-NTA columns by 250 mmol/L imidazole, and separated by size-exclusion chromatography between 20 and 100 kd. Only intact Ki-4(scFv)-ETA', but no degradation products or rIT aggregates, was collected. The purity of the prepared protein was determined by densitometry and by receptor ELISA. Functional Ki-4(scFv)-ETA' was enriched to more than 95%. The rIT was purified by combinations of IMAC and size exclusion chromatography to a final concentration of approximately 1 mg/4 g cell paste from bacterial shaking cultures.20 In contrast to our former standard periplasmic expression and extraction protocol34 that resulted in some µg/4 g cell paste, the functionality of Ki-4(scFv)-ETA' was preserved after the extraction procedure.
Growth of L540rec Hodgkin cells in SCID mice Inoculation of 1 × 107 L540rec cells through the tail vein induced tumor growth in 100% of untreated SCID mice. Untreated animals developed signs of progressive disease approximately 4 weeks after tumor cell challenge. Histologic examination revealed patterns of dissemination of L540rec tumors in various organs of the 32 female PBS-treated SCID mice, as previously published.27 Tumors stained intensively with the mAbs 2A3 (anti-CD25) and Ber-H2 (anti-CD30), as demonstrated by immunohistochemistry. The histology of an involved lymph node is shown in Figure 1.
Toxicity of Ki-4(scFv)-ETA' in SCID mice The MTD of Ki-4(scFv)-ETA' was determined in a dose-escalation study of groups of 3 mice intravenously treated with 20, 30, 40, 50, and 60 µg rIT (Table 1). The MTD was reached at 40 µg per animal.
Pharmacokinetics of Ki-4(scFv)-ETA' To determine the blood clearance of the rIT in SCID mice, animals were treated with a single intravenous dose of 40 µg Ki-4(scFv)-ETA'. Blood was drawn at different time points, and rIT presence was measured by ELISA. Kinetics of the 2-compartment model was determined after nonlinear regression. The dose/time relationship was calculated as: c = 13.95e 0.0371t + 26.05e0.0036t, where
c = concentration (µg), and t = time
(minutes). Thus, the distribution time
t1/2 was 19 minutes, and the serum elimination
time t1/2 was 193 minutes (Figure
2).
Stability of Ki-4(scFv)-ETA' in mouse serum We examined the stability of Ki-4(scFv)-ETA' by incubating 0.25 µg/mL purified protein at 37°C for different periods of time in mouse serum. The biologic activity of incubated rIT was determined by ELISA using rhCD30 and cell proliferation assays with CD30+ L540rec cells. Ki-4(scFv)-ETA' was highly stable under these conditions, retaining 85% to 90% of initial binding activity for at least 24 hours (Table 2). The IC50 dropped between 8.5 and 13 ng/mL after incubation in mouse serum (Table 3). Mouse serum by itself was not cytotoxic to the cells. Thus, Ki-4(scFv)-ETA' was relatively stable in mouse serum at physiological temperatures exceeding the time range calculated for the serum elimination time t1/2.
Effect of rIT treatment on the survival of tumor-bearing SCID mice Female SCID mice were treated with the MTD for Ki-4(scFv)-ETA', corresponding to 40 µg 1 day after tumor challenge by a singular intravenous injection. As documented by Kaplan-Meier analysis (Figure 3), the MST of the PBS- and ETA'-treated control groups was 38.1 ± 1.77 days and 39.8 ± 2.56 days, respectively. There was no difference between animals treated with PBS or ETA' (P = .5804). Treatment of L540cy-challenged SCID mice with 40 µg Ki-4 mAb alone did not lead to tumor-free survival of all the animals tested.12 In contrast, 9 of 10 mice treated with Ki-4(scFv)-ETA' did not show any tumor growth more than 200 days after treatment, extending the MST by a factor of 5 (201.6 ± 17.46 days). This result is statistically significant for both control groups (P < .001). None of the animals treated with 40 µg Ki-4(scFv)-ETA' died of toxicity at this dose level. One of the treated animals died of unknown causes on day 36 after tumor challenge and showed no detectable L540rec cells on examination. The overall results are summarized in Table 4.
In this study, we report the antitumor activity of a recombinant
anti-CD30 immunotoxin against disseminated, growing Hodgkin tumors in
SCID mice. The major findings to emerge from our study are that (1)
Ki-4(scFv)-ETA' isolated from the periplasmic space of E
coli, cultured under osmotic stress in the presence of compatible solutes, can easily be purified in large amounts of functionally active
protein by a combination of immobilized metal affinity and molecular
size chromatography; (2) in vitro characteristics of the recombinant
protein were documented by ELISA, flow cytometry, and cytotoxic
activity against a variety of CD30+ lymphoma cells; (3)
purified protein was stable to 85% to 90% of its biologic activity in
mouse serum for more than 24 hours; (4) Ki4(scFv)-ETA' showed a
blood distribution time t1/2 We thank Gisela Schön for assistance with cloning and Silke
Drillich for performing the cytotoxicity assays.
Submitted November 10, 1999; accepted February 3, 2000.
Supported in part by Deutsche Forschungsgemeinschaft grants SFB502 and
TFB6-98.
S.B. and M.H. contributed equally to this work.
Reprints: S. Barth, Labor fuer Immuntherapie,
Medizinische Klinik I der Universitaet zu Koeln,
Joseph-Stelzmann-Strasse 9, D-50931 Koeln, Germany; e-mail:
stefan.barth{at}uni-koeln.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
of Ki-4(scFv)-ETA' was 19 minutes,
and its serum elimination time t1/2
