Keywordsbrain metastases, non-small cell lung cancer, epidermal growth factor receptor, urokinase-type plasminogen activator receptor, diphtheria toxin, DTATEGF, bioluminescence, in vivo , in vitro
Diphtheria Toxin (DT) 159
DTATEGF Construction 160
NSCLC Cell Preparation 161
In vitro DTATEGF Efficacy 161
Intracranial NSCLC Tumor Model 161
Pump Implantation and DTATEGF Delivery 162
Bioluminescent Imaging (BLI) 162
Body Weights, Histology, and Statistical Analysis 162
DTATEGF in vitro and in vivo Results 163
Targeted Toxin Limitations 166
Brain metastases affect 25% of patients with systemic cancer and are at least four times more common than primary brain tumors ( ). Nearly half of brain metastases arise from the lung and involve the cerebral hemispheres 80% of the time ( ). Non-small cell lung cancer (NSCLC) constitutes 80% of lung cancer in patients that usually have advanced systemic disease or metastatic spread ( ). Despite treatment with corticosteroids and whole brain radiation therapy, the median survival for patients with NSCLC brain metastases is only 6 months leading clinicians to seek new innovative treatments ( ). Targeting intracellular pathways that are responsible for tumor growth and progression is one direction being pursued by some investigators. The epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptor (VEGF) pathways can be blocked in patients with NSCLC metastases ( ). A high therapeutic response rate and poor prognosis have been associated with EGFR expression ( ). The EGFR tyrosine kinase inhibitors erlotinib and gefitinib have been used clinically ( ). High levels of urokinase plasminogen activator receptor (uPAR) expression in NSCLC and other human cancers are associated with poor prognosis. Because uPAR is expressed on tumor cells and the endothelial cells of tumor blood vessels, it is suitable for targeting when attempting to interfere with pathogenic plasminogen activation ( ).
Radiotherapy and chemotherapy represent the primary treatment modalities for metastatic NSCLC, although both therapies are of limited benefit because they are not selective for cancer cells and they cause serious side effects due to toxicity to normal cells. Surgical resection and stereotactic radiosurgery represent focal therapies that do not address the micrometastatic disease that is present outside the treatment field. In order to address this problem, one option is to combine a targeting ligand that will recognize tumor cells with a toxin that kills the tumor cells. Targeted toxins or immunotoxins are extremely potent compounds that bind to antigens on the surface of tumor cells that block protein synthesis after cellular entry ( ). Targeted toxins are effective in vitro against numerous cell lines and in vivo in flank tumor models in athymic mice where complete tumor regression was seen ( ). The systemic delivery of these compounds is impractical because of the large size of these compounds and their inability to cross the blood–brain barrier (BBB) into the central nervous system (CNS) and the innate toxicity of these agents to some normal cells. Another factor that has diminished the efficacy of targeted toxins includes the immunogenicity of the toxin component of the molecule. In order to gain entry to the CNS by bypassing the BBB, a delivery technique that was introduced in the 1990s was convection enhanced delivery (CED) whereby the drug was infused into the brain parenchyma at a slow continuous rate. By inducing a fluid wave through positive pressure, the drug is administered into the brain. The potential application of this technique was recognized and confirmed in preclinical studies and in early phase clinical trials ( , 2006, 2009).
A bispecific immunotoxin DTATEGF was constructed with two different carrier ligands, epidermal growth factor (EGF) and the amino-terminal fragment (ATF) of urokinase plasminogen activator (uPA) that targeted the EGF receptor (EGFR) and uPA receptor (uPAR), respectively, linked to a truncated diphtheria toxin (DT). Compared to the monospecific targeted toxins, DTATEGF was more potent against some cell lines ( ). DTATEGF was tested against a human metastatic NSCLC PC9-BrM3 cell line in vitro and in vivo ( ). In the human NSCLC mouse xenograft brain tumor model, the cells were genetically labeled with the firefly luciferase reporter gene to allow for real-time bioluminescent imaging (BLI). DTATEGF was administered directly into the intracerebral PC9-BrM3 NSCLC tumor cell via CED over 7 days using an implanted osmotic minipump.
Diphtheria Toxin (DT)
Diphtheria toxin (DT) functions like other toxins by inhibiting protein synthesis either by modifying elongation factor-2 (EF2) or by acting directly on the ribosome ( ). Most toxins are polypeptide chains with several domains that include a cell recognition chain that attaches to cell surface receptors on the target tumor; a translocation domain that enables the toxin to cross the cell membrane and enter the cell; and an inactivation chain that blocks an important intracellular function leading to cell death ( ). After the toxin binds to the overexpressed antigens or receptors on cancer cells, it is endocytosed into the cell and transferred by an endosome to a lysosome or to the Golgi apparatus. The toxin is then separated from the carrier ligand allowing it to inhibit protein synthesis. Toxins can inactivate over 200 EF2s per minute ( Figure 15.1 ). Compared to conventional chemotherapy where 10 5 molecules are needed to kill one cell, a single toxin molecule can kill a cancer cell ( ).
Corynebacterium diphtheriae is a non-encapsulated, non-motile, Gram-positive bacillus that produces the 62-kDa protein DT ( ). The gene for DT is carried beside the promoter and operator sequences, however, the bacterium carries a repressor sequence that regulates gene expression. Gene regulation is dependent on iron concentration. After the repressor binds iron, the resulting complex attaches to the operator to prevent gene transcription ( ). The single polypeptide chain must be enzymatically nicked at an arginine-rich site for the A and B chains to become active against human cells. DT has a cell-binding domain at the C terminus (amino acids 482–539) and the A chain has ADP-ribosylation activity at the N terminus (see Figure 15.1 ). The A chain catalyzes the transfer of adenosine-diphosphate (ADP)-ribose to EF2, preventing the translocation of peptidyl-t-RNA on ribosomes, thereby blocking protein synthesis and subsequently killing the cell ( ). A natural ligand for DT on the cell membrane is the heparin-binding epidermal growth factor (EGF)-like precursor ( ) www.nature.com.ezp-prod1.hul.harvard.edu/nrc/journal/v6/n7/full/nrc1891.html – B6#B6.
Recombinant DT is produced by replacing the C terminal cell-binding domain with a ligand that binds to a growth factor receptor or the Fv fragment of an antibody. The native DT protein consists of 535 amino acids. Variable truncation of the binding segments resulting in 389 and 486 amino acid length toxin conjugates has resulted in the formation of toxins DAB389 and DAB486, respectively ( ). These modified DTs cannot enter a cell without selective binding of their carrier ligand to a receptor. Another modification of DT involves the substitution of two amino acids in the B chain resulting in a new molecule cross-reacting material-107 (CRM-107) ( ). This modification reduced the non-specific binding of DT to human cells by 8000-fold and increased the toxin’s tumor-specificity 10 000-fold.
DNA shuffling and DNA cloning techniques were used to synthesize and assemble the hybrid genes encoding the single-chain DTATEGF ( Figure 15.2 ) ( ). From the 5′ to 3′ end, the assembled fusion gene contained an Nco 1 restriction site, an ATG initiation codon, the first 389 amino acids of the DT molecule (DT390), the seven-amino-acid EASGGPE linker, the genes for human EGF and 135-ATF from uPA linked by 20-amino-acid segment of human muscle aldolase, and a Xho I restriction site ( ). The resulting Nco I/ Xho I fragment gene was spliced into the pET21d expression vector under the control of an isopropyl- h -D-thiogalactopyranoside-inducible T7 promoter. Verification that the gene was correct in sequence and had been cloned in frame was confirmed using DNA sequencing analysis. The monospecific targeted toxins DTAT and DTEGF were created using the same techniques ( ). A Novagen pET expression system was used for protein expression and purification from inclusion bodies. Fast protein liquid chromatography-ion exchange chromatography (Q Sepharose Fast Flow, Sigma) was used to purify refolded proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining was performed to determine protein purity.
NSCLC Cell Preparation
Memorial Sloan-Kettering Cancer Center provided the human brain metastasis NSCLC cell line PC9-BrM3 which was then transfected with the luciferase reporter gene. The PC9-BrM3 cells were cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum and 5% streptomycin (Mediatech Inc., Manassas, VA) and maintained at 37°C in a humidified atmosphere of 5% CO 2 . Cells with>95% viability were used for experiments as determined by Trypan blue exclusion.
In vitro DTATEGF Efficacy
An MTT assay was performed to test the in vitro cytotoxicity of DTATEGF against PC9-BrM3 NSCLC tumor cells. Cells were incubated overnight at 37°C with 5% CO 2 in a 96-well flat-bottomed plate containing 10 4 cells/well. The following day control DT, DTATEGF, DTAT, or DTEGF were added in varying concentrations to the wells. After 72 hours of incubation, MTT was added to each well for the last 4 hours. The cells were then washed, dried and counted using standard scintillation techniques. The data from the proliferation assays was reported as a percentage of the control counts. All assays were performed in triplicate and the results were obtained in three independent experiments.
Intracranial NSCLC Tumor Model
The mice used for intracranial tumor experiments were 6-week-old female athymic mice weighing 17–19 grams (Taconic, Hudson, NY). Intraperitoneal ketamine (80 mg/kg) was used to anesthetize the mice for the tumor implantation using a David Kopf stereotactic frame (David Kopf Instruments, Tujunga, CA) for immobilization. Using a midline incision the skull was accessed in order to place a burr hole 0.5 mm anterior to bregma and 2.5 mm lateral to the midline using a drill (Foredom Electric Co., Bethel, CT). A 25-gauge needle was attached to a 10-μL Hamilton syringe in order to deliver the tumor cells stereotactically. The needle was inserted into the brain to a depth of 3.2 mm from the skull surface and kept in place for 2 minutes before injecting tumor cells. Over a period of 5 minutes 4×10 5 PC9-BrM3 cells in 1.4 μL were injected into the brain. After completion of the injection the needle was left in place for 5 minutes before it was withdrawn slowly and the burr hole was covered with sterile bone wax. The skin incision was closed with surgical glue. All surgical procedures were approved by the animal care and use committee of SUNY Upstate Medical University and performed under sterile conditions.
Pump Implantation and DTATEGF Delivery
An implantable osmotic minipump system (ALZET model 1007D, Durect Corporation, Cupertino, CA) was chosen because of its infusion characteristics and size. The system contains a disposable filling tube, flow moderator and an osmotic pump. The PC9-BrM3 cells were implanted intracranially as described above. Four days after the cells were implanted, the first bioluminescent imaging (BLI) was performed to confirm the presence of tumor. The mice were divided into two groups each with six mice; the DTATEGF treatment group and the DT control group. The osmotic minipump system was assembled according to the manufacturer’s instructions and contained 1.5 cm of infusion tubing attached to a 25-gauge 3-mm needle. After the mice were anesthetized and immobilized in the stereotactic frame, the drug-loaded pumps were implanted subcutaneously on the back. The treatment pumps were filled with 1 μg of DTATEGF in 100 μL and the control group pumps received 1 μg DT in 100 μL. The infusion rate was 0.5 μL/hour continuously for 7 days. The animals were again anesthetized on day 8 and all components of the pump system were removed to prevent interference with the BLI or the future potential for hardware exposure over time.
Bioluminescent Imaging (BLI)
Prior to BLI, the animals were anesthetized using isoflurane gas inhalation. BLI was performed with the IVIS 50 imaging system (Xenogen Corporation, Alameda, CA) and the results were analyzed with Living Image 2.5 software ( Figure 15.3 ). An intraperitoneal injection of 150 mg/kg aqueous D-luciferin solution was administered to the mice 10 minutes before BLI. The BLI obtained represents 2 minutes of exposure time and the total number of photons per second per steradian per square centimeter was recorded. BLI was performed on days 4, 14, 30, 60, and 90 after PC9-BrM3 cell implantation.