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Ann Thorac Surg 1996;62:1454-1459
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, University of Iowa Hospitals and Clinics, Iowa City, Iowa
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Abstract |
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Methods. 4D5 and gelonin were covalently modified and linked. Purification was confirmed by SDS–polyacrylamide gel electrophoresis. Dose-dependent cytotoxicity was quantified using 3H-thymidine uptake by CALU-3 cells after incubation with 4D5-gelonin conjugate or with control substances (4D5, gelonin, unconjugated 4D5 + gelonin, or control antibody MOPC-21).
Results. The 4D5-gelonin conjugate showed a 50% inhibitory concentration of 3.5 x 10-10 mol/L, but 4D5 alone demonstrated no cytotoxic effect. Gelonin and the unconjugated 4D5-gelonin mixture had one tenth the cytotoxicity of the 4D5-gelonin conjugate (inhibitory concentration = 6.5 x 10-9 mol/L and 8.5 x 10-9 mol/L, respectively). The conjugate exhibited minimal toxicity against a p185-negative cell line (NIH3T3).
Conclusions. Selective and efficient killing of human lung adenocarcinoma cells can be achieved in vitro using c-erbB-2/p185–directed therapy.
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Introduction |
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Lung cancer is the leading cause of deaths due to cancer in men and women in the United States, with nearly 180,000 new cases estimated for 1996 [1]. Most patients are not candidates for surgical resection at the time of diagnosis. Efforts to improve long-term survival using chemotherapy or radiotherapy, or both, have met with disappointment. Systemic therapy directed at tumor-specific molecular targets should demonstrate enhanced potency and reduced collateral toxicity.
The c-erbB-2 gene is overexpressed in numerous human cancers, including up to one third of all non–small cell lung cancers [2]. Its overexpression in lung cancer has been associated with decreased survival [3]. The protein product of c-erbB-2 is p185, a transmembrane glycoprotein with a cystine-rich extracellular domain [4, 5]. The intracellular domain is known to possess tyrosine kinase activity and to participate in signal transduction [6]. Its background expression in normal human tissues is low and known to occur infrequently [7]. As such, p185 potentially represents a viable candidate for targeted therapy in the treatment of lung cancer.
For many years, naturally occurring toxins have formed the backbone of our therapeutic arrmamentarium. Type 1 plant toxins appear ideally suited for targeted therapy because of their defined molecular structure, powerful enzymatic mode of action, and relative lack of collateral toxicity [8]. Gelonin, a well-characterized type 1 ribosomal inhibitory protein found in the seeds of the plant Gelonium multiflorum, potentially fulfills these criteria well [9].
This set of experiments was designed to determine whether c-erbB-2/p185 can be therapeutically targeted in human lung adenocarcinoma using an anti-p185–gelonin conjugate.
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Methods |
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Antibodies
Purified muMoAb-4D5, an antihuman p185 (extracellular domain) IgG1 antibody from a murine hybridoma, was the kind gift of Genentech (South San Francisco, CA). MOPC-21, a mouse myeloma IgG1, was obtained from Sigma Chemical Company (St. Louis, MO).
Gelonin Preparation
Seeds of Gelonium multiflorum were purchased from United Chemical & Allied Products (Calcutta, India). Gelonin was extracted from these seeds by a micromodification of the method described by Stirpe and associates [10]. Briefly, shelled seeds were blended to a fine powder, then homogenized and dialyzed against 5-mmol/L sodium phosphate (pH, 6.5). The dialyzed extract was then applied to a sizing column and the adsorbed material eluted with a NaCl gradient. A bimodal peak was collected in fractions, and purity was confirmed using SDS–polyacrylamide gel electrophoresis (SDS-PAGE analysis). The biologic activity of this fraction was confirmed using a rabbit reticulocyte lysate assay (Amersham International, Buckinghamshire, England) [11]. Uptake of 3H-methionine into synthesized protein was determined using standard liquid scintillation–counting techniques. Saline controls were compared with 50-ng/mL concentrations of control gelonin (Pierce, Inc, Rockford, IL) and two 50-ng/mL aliquots of experimental gelonin extract.
Preparation of Antibody-Gelonin Conjugate
Gelonin in 10-mmol/L potassium phosphate (pH, 7.2) containing sodium chloride (145 mmol/L) was mixed with 0.5-mol/L triethanolamine/HCl buffer (pH, 8.0) and 0.1-mol/L EDTA (ethylene diaminetetraacetic acid), so that the final concentrations of triethanolamine and EDTA were 60 mmol/L and 1 mmol/L, respectively. The solution was degassed and kept under nitrogen until being treated with 2-iminothiolane/HCl (1 mmol/L) at 0°C for 90 minutes. The excess reagent was removed by gel filtration on a column of cross-linked dextran equilibrated with 5-mmol/L bis-tris/acetate buffer (pH, 5.8) containing 50-mmol/L NaCl and 1-mmol/L EDTA.
The antibody to be conjugated to gelonin (4D5 or MOPC-21) was placed in 100-mmol/L sodium phosphate buffer (pH, 7.0) containing EDTA (0.5 mmol/L) and then mixed with a tenfold molal excess of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). The mixture was incubated at 30°C for 30 minutes. The excess reagent was removed by gel filtration on a column of cross-linked dextran equilibrated with 5-mmol/L bis-tris/acetate buffer (pH, 5.8) containing 50-mmol/L NaCl and 1-mmol/L EDTA.
Modified antibody (4D5 or MOPC-21) in 100-mmol/L sodium phosphate buffer (pH, 7.0) containing EDTA (0.5 mmol/L) was mixed with an equimolal amount of modified gelonin in 5-mmol/L bis-tris/acetate buffer (pH, 5.8) containing NaCl (5 mmol/L) and EDTA (1 mmol/L). The pH of the mixture was adjusted to 7.0 by the addition of 0.5-mmol/L triethanolamine/HCl buffer (pH, 8.0) and then kept under nitrogen at 4°C for 20 hours. The conjugation was halted by a 1-hour incubation with of iodoacetamide (2 mmol/L).
Purification of Antibody-Gelonin Conjugates
The conjugation mixture underwent buffer exchange into 10-mmol/L sodium phosphate (pH, 7.0) containing NaCl (0.15 mmol/L). To remove unreacted gelonin, the unpurified conjugate was then applied to a Sephacryl S-200 column (1 x 30 cm) previously equilibrated with the just-mentioned buffer. The initial peak was collected and concentrated to approximately 1 mg/dL. This semipure conjugate was applied to a blue sepharose CL-6B column (2 x 10 cm) equilibrated with 10-mmol/L sodium phosphate (pH, 7.0) containing NaCl (0.15 mmol/L). The column was then washed with three column volumes of the starting buffer. The pure conjugate was then eluted with 10-mmol/L sodium phosphate (pH, 7.0) containing 1-mmol/L NaCl and fractionally collected.
Flow Cytometric Analysis
CALU-3 cells underwent flow cytometry against the 4D5-gelonin conjugate, 4D5, MOPC-21, and sterile phosphate-buffered saline solution as a control, using antimouse immunoglobulin G-FITC (fluorescein isothiocyanate isomer) conjugate as a secondary antibody-marker.
Cytotoxicity Assays
Cells in log-phase growth were plated onto 96-well plates (2.5 x 103 cells/well). Each plate contained a control group of ten wells treated only with sterile phosphate-buffered saline solution. Experimental groups were set up in quadruplicate wells and received either antibody-gelonin conjugate, a mixture of antibody and gelonin, gelonin alone, antibody alone, or an irrelevant antibody (MOPC-21) in doses ranging from 10-11 to 10-7 mol/L.
These cells were incubated at 37°C (90% humidity/5% CO2) until controls were 50% to 75% confluent, at which time they were pulsed with 3H-thymidine (0.001 µCurie/well) and allowed to incubate for an additional 48 hours. Cells were then incubated with trypsin-EDTA and harvested using a PHD Cell Harvester (Cambridge Technologies, Watertown, MA). The incorporation of 3H-thymidine was determined by liquid-scintillation counting.
Statistical Analysis
Results from each experiment were examined with a log-dose response model, using a nested analysis of covariance to determine statistical significance.
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Results |
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Purity of the 4D5-gelonin conjugate was demonstrated by SDS-PAGE analysis (Fig 1
). Image analysis of this gel revealed 94% to 99% purity (determined by NIH Image, version 1.53). Purified conjugate consisted of forms containing one, two, or three gelonin molecules per antibody. The yield averaged approximately 10% of starting products.
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The potency of the 4D5-gelonin conjugate against CALU-3 cells is demonstrated by its IC50 (concentration resulting in a 50% inhibition of growth compared with the growth in controls) of 3.5 x 10-10 mol/L. The mixture of unconjugated gelonin and 4D5 as well as gelonin alone demonstrated much less toxicity, with an IC50 of 8.5 x 10-9 and 6.5 x 10-9 mol/L, respectively, and 4D5 and MOPC-21 had no effect (Fig 4).
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Comment |
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Developments in the field of molecular biology have revealed the identity of cellular genes that may play a role in the neoplastic process. Although this has begun to shed light on the mechanisms of neoplastic transformation, their therapeutic utility has yet to be realized. One such gene is the c-erbB-2/p185 protooncogene system. Amplification and overexpression of this gene in patients with non–small cell lung cancer has been correlated with disease recurrence and shorter survival [3]. The oncogene codes for a transmembrane protein (p185), which possesses tyrosine kinase activity and may act as a growth factor receptor. In other tumor systems, antibodies directed against the extracellular domain of p185 have been shown to suppress tumor growth in a murine model in a cytostatic fashion [12]. It may be possible to target cytotoxic agents to this protein because of its well-characterized nature and its unique pattern of distribution in vivo [13, 14].
Phase I clinical trials in humans have been completed for immunotoxins directed at hematologic cancers and solid tumors. Most of these trials have used native or modified ricin as the toxic moiety. The first immunoconjugate tried in patients was an anti–CD5-ricin A chain (RTA) construct. Transient clearance of CD5-positive cells was observed, but no significant clinical responses occurred [15]. Lack of efficacy was attributed to the presence of circulating free CD5. In another phase I trial [16], partial responses, with remission periods lasting 2 to 8 months, were achieved.
In the management of solid tumors, ricin A chain immunotoxins have been investigated in phase I trials against breast cancer, ovarian cancer, melanoma, colorectal cancer, and small cell lung cancer. On the basis of on data from a murine xenograft model, Z60F9-RTA conjugate has been examined in clinical trials performed in breast cancer patients. Preclinical studies showed minimal toxicity in animals, and the conjugate prepared with recombinant ricin A chain was highly effective in inhibiting MX1 human breast cancer cell growth in athymic nude mice. Phase I evaluation of the conjugate was carried out in metastatic breast cancer patients at two centers [17, 18].
Using a phase I protocol, an anti–CD56-monoclonal antibody conjugated to chemically blocked ricin has been used to treat recurrent or refractory disease in patients with small cell lung cancer [19]. One partial response was observed. Based on the phase I results, a phase II trial with this conjugate is planned for patients with small cell lung cancer after chemotherapy induction.
Judging from the limited data available, it is clear that immunoconjugates may demonstrate biologic activity in vivo in selected patients with advanced cancers, including solid tumors. However, nonspecific toxicity has limited dosing in current constructs. Ricin A chain may not be optimal for use in vivo because of its nonspecific glycoprotein cell surface binding. To date, chemical modification has failed to eliminate collateral toxicity. It is likely that other cytotoxic agents with similar potency but reduced toxicity will produce improved results in vivo.
Gelonin is a small protein (molecular weight, 30 kD) extracted from the seeds of the plant Gelonium multiflorum, which belongs to the same family of proteins as ricin, known as ribosomal inhibitory proteins [10]. Like ricin, it possesses the capacity to enzymatically inhibit protein synthesis by inactivating cellular ribosomal systems. Also like ricin, gelonin catalytically hydrolyzes the glycosidic bond of adenosine residue 4324 in the 28S-RNA. Depurination of adenine residue from 28S-RNA results in irreversible inactivation of ribosomes and inhibition of protein synthesis, leading to cell death [20]. However, unlike ricin, which is a glycosylated heterodimer, the protein is a single-chain, nonglycosylated polypeptide. It is nontoxic to intact cells unless internalized, and cells lack the surface receptors needed to allow for internalization. When conjugated to a monoclonal antibody directed at a cell surface target, this protein has been found to be a highly potent cellular toxin. It may represent an ideal agent for in vivo targeting because of its high efficiency and minimal nonspecific toxicity. Although in animal models gelonin has shown significant antitumor activity when conjugated to targeting antibodies [21], to date there have been no human trials with constructs using this toxin.
Advances in the treatment of lung cancer will very likely result from a clearer understanding of the molecular biology of these tumors. The neu oncogene was first observed in neuroblastomas induced in a rat model through exposure to ethylnitrosourea [22]. Subsequently, three groups independently identified the human homologue of this gene [23–25]. The clinical value of c-erbB-2 and other molecular targets may be in their future exploitation for directed therapies.
In this set of experiments, we have shown that the c-erbB-2/p185 system can serve as a therapeutic target in human lung adenocarcinoma. It is likely that, because of its relatively large size and immunologically active components, the current conjugate will require significant structural modification before it can be used in vivo. Ideal constructs likely will be composed only of the antigen-recognizing region of the targeting molecule, along with the chemically active portion of the toxin, synthesized as a single biologically active molecule. It should be possible to synthesize these constructs using currently available technology once appropriate molecular targets are defined.
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Footnotes |
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Address reprint requests to Dr Lanza, Mayo Clinic Scottsdale, 13400 E Shea Blvd, Scottsdale, AZ 85259.
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