Immunotoxin therapy for primary malignant brain tumors

International Congress Series 1247 (2002) 185 – 197

Immunotoxin therapy for primary malignant brain tumors Ki-Uk Kim a,*, Daniel A. Vallera b, Hsiao-Tzu Ni c, Kwan H. Cho b, Stephen R. Spellman c, Walter C. Low c, Walter A. Hall c a

Department of Neurosurgery, College of Medicine, Dong-A University, 3Ga-1, Dongdaeshin-Dong, Seo-Gu, Pusan 602-715, South Korea b Department of Therapeutic Radiology – Radiation Oncology, University of Minnesota School of Medicine, Minneapolis, MN, USA c Department of Neurosurgery, University of Minnesota School of Medicine, Minneapolis, MN, USA

Abstract The prognosis of malignant brain tumors is poor in spite of aggressive treatment. Immunotoxin therapy is a novel approach for the treatment of tumors. Targeted fusion toxins, chimeric protein consisting of the cytotoxic domain of the natural toxin and carrier ligands such as a growth factor or an antibody, have been introduced in the treatment of malignant central nervous system (CNS) tumors, with promising results. The toxin is internalized into the cytosol of the target cell via cellsurface receptors and it is essential for these receptors on the tumor cell to be highly expressed in order for the immunotoxin to have specific anti-tumor activity. Studies on expression of cell-surface receptor for immunotoxin targeting was performed on transferring (TR), insulin-like growth factor-1 (IGF-1R), and interleukin-4 (IL-4R) receptors of human glioblastoma (U373-MG and T98-G) and medulloblastoma (Daoy) cell lines, and the effect of irradiation on expressibility of these receptors was studied. Recombinant diphtheria toxin – murine interleukin-4 conjugate (DT390 – mIL4) was developed and its cytotoxic efficacy against murine glioblastoma (SMA-560) and neuroblastoma (Neuro-2a and NB41-A3) cell lines was examined, and the combined effect with irradiation was tested. Receptors were expressed in all cases except IGF-1R on T98-G. After irradiation, TR expression was increased for Daoy, IGF-1R expression was increased on Daoy and U373-MG, and IL-4R expression was decreased on Daoy and U373-MG. DT390 – mIL4 exhibited dose-dependent, specific cytotoxic effects on all cell lines tested with IC50 (concentration of DT390 – mIL4 that inhibit 50% of protein synthesis) value of 0.56  10 9 M in SMA-560, 1.28  10 9 M in Neuro-2a and 0.95  10 10 M in NB41-A3. Cytotoxicity was additive when DT390 – mIL4 at 10 9 M was administered with irradiation.

*

Corresponding author. Tel.: +82-51-240-5243; fax: +82-51-248-2204. E-mail address: [email protected] (K.-U. Kim).

0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 1 0 5 4 - 3

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Targeted fusion toxins are characterized by great potency and high specificity with minimal damage to normal neuronal tissue, and interleukin-4 receptor is one of the proper targets for fusion toxin. The cytotoxicity of the immunotoxin is additive when it is administrated with irradiation. Targeted toxin therapy is a promising adjuvant treatment modality for the malignant brain tumors. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Absolute expression index; Glioblastoma; Immunotoxin; Insulin-like growth factor receptor; Interleukin-4 receptor; Transferrin receptor

1. Introduction The improvement in survival of patients with malignant glioma has been achieved with the advent of adjuvant therapeutic modalities, but the prognosis for these patients is still poor [4,26,29]. In addition to radiation therapy, that has improved patient survival [26,30,35], chemotherapy is included in multidisciplinary treatment protocols and offers some beneficial effect [5,8,19,27,34]. Targeted fusion toxins, combining the cytotoxic domain of the natural toxin with the carrier ligand, are being developed for the treatment of cancers, including malignant central nervous system (CNS) tumors [10,11,32]. The toxins bind to receptors on the surface of the target cell via the ligand specific to it and are internalized by endocytosis [22]. The cytotoxic activity of the fusion toxin is highly specific to the tumor cells and damage to the normal neuronal cells is minimal. In these studies, authors studied expression of several growth factor receptors on brain tumor cell lines and effect of irradiation on expressibility of these receptors. We developed a recombinant DT390 – mIL4 fusion protein, and examined its specific cytotoxic activity against murine glioblastoma and neuroblastoma cell lines. We also determined the effect of combining DT390 – mIL4 with irradiation and discuss the characteristic features of this immunotoxin when it was combined with other therapeutic modalities.

2. Materials and methods 2.1. Brain tumor cell lines and irradiation One medulloblastoma cell line (Daoy) and two glioblastoma cell lines (U373-MG and T98-G) were obtained from ATCC (Rockville, MD), and used for the detection of TR, IGF-1R, and IL-4R. The acute effects of irradiation on receptor expression was studied for TR, IGF-1R, and IL-4R on Daoy cells, for IGF-1R and IL-4R on U373-MG, and for TR on T98-G after irradiating 500 cGy. Each cell line was divided into five groups with three samples for each group—the no-irradiation group, 1-hour group (stain receptors 1 h after irradiation), 3-hour group (stain receptors 3 h after irradiation), 6-hour group (stain receptors 6 h after irradiation), and 24-hour group (stain receptors 24 h after irradiation).

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2.2. Immunological staining and flow cytometric analysis TR and IGF-1R were detected by flow cytometric analysis by two-color immunofluorescence staining technique, and IL-4R was detected using ‘Fluorokinek Biotinylated Human IL-4’ kit (R&D Systems, Minneapolis, MN). FACScan (Becton Dickinson, San Jose, CA) was used and data were analyzed using CELLQuest software and histograms were obtained. The cells were gated, and total fluorescein intensity was calculated. Absolute expression index (AEI) of the receptor was obtained by subtracting the total fluorescein intensity of background staining from that of receptor staining. 2.3. Construction of modified diphtheria toxin – murine interleukin-4 conjugate A 1549-bp NcoI/XhoI DNA fragment was prepared by splice overlap extension encoding amino acids 1 –389 of the DT gene positioned upstream of a fragment encoding amino acids 25– 144 of murine IL-4. This DT390 –mIL4 hybrid gene was ligated into the NcoI/XhoI cloning sites in the pET21d plasmid (Novagen, Madison, WI), which was transformed into the Escherichia coli strain BL21 (DE3) (Novagen). The fusion protein was expressed and purified by ion exchange chromatography. 2.4. Cytotoxicity assays of DT390 – mIL4 The spontaneous murine astrocytoma cell line, SMA-560 [28] and two neuroblastoma cell lines, Neuro-2a [15] and NB41A3 [3] were used. The cytotoxicity assays of DT390 – mIL4 were performed at different concentrations. The cells were divided into a control group treated with phosphate-buffered saline (PBS), a 10 8 M group (treated with DT390 – mIL4 at the concentration of 10 8 M), a 10 9 M group, a 10 10 M group, and a 10 11 M group. Each group was tested in triplicate. The tumor cells were treated with DT390 – mIL4, and the viable cells were counted after 24, 48, and 72 h of incubation by Trypan blue exclusion. Percent viability (percentage of the number of viable cells of each group compared to that of the control group) was calculated. To determine the specificity of DT390 – mIL4, the tumor cells were pretreated with 11B11, which is antibody of IL-4R, and treated with DT390 – mIL4 at 10 8 M 1 h after treatment with 11B11. Another group of cells was pretreated with A20-1.7 antibody, irrelevant control antibody. The viable cells were counted after 24, 48, and 72 h of incubation, and percent viability was calculated. 2.5. Combined effect of irradiation and DT390 – mIL4 The combined effect of irradiation and DT390 – mIL4 was studied on the SMA-560 and Neuro-2a cell lines at two different DT390 – mIL4 concentrations, 10 9 and 10 10 M. The tumor cells were divided into a PBS-treated group, a DT390 – mIL4-treated group, an irradiated group (500 cGy), and a combined group that was treated with DT390 – mIL4 3 h after irradiation. The viable cells were counted on the second, third, and fourth days after incubation, and the percent viability was calculated.

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3. Results TR and IL-4R were expressed on all cell lines. IGF-1R was expressed on Daoy and U373-MG, but was not detectable on T98-G (Fig. 1). 3.1. Effect of irradiation on transferrin receptors of Daoy and T98-G In Daoy, the average AEI was increased 62.7% in the 1-hour group, 45.9% in the 3hour group, and 94% in the 6-hour group, but decreased 11.4% in the 24-hour group (Fig. 2). Results showed significant differences between the no-irradiation group and the 1-hour, 3-hour, and 6-hour groups, but no significant difference between the no-irradiation group and 24-hour group ( P < 0.05). In T98-G, the average AEI was decreased by 3.1%, 2.4%, 2.6% and 1% in 1-hour, 3hour, 6-hour, and 24-hour groups, respectively. Results showed no significant differences between the no-irradiation group and the 1-hour, 3-hour, 6-hour, 24-hour groups ( P < 0.05). 3.2. Effect of irradiation on type-1 insulin-like growth factor receptors of Daoy and U373MG In Daoy, the average AEI was increased 22.1%, 30.1%, 57.6%, and 8.6% in 1-hour group, 3-hour group, 6-hour group, and 24-hour group, respectively. Results showed

Fig. 1. Expression of transferrin receptor on Daoy. Solid: isotype control for background staining. Blank: receptor staining.

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Fig. 2. Expression of transferrin receptor on Daoy was increased after irradiation.

significant differences between the no-irradiation group and 3-hour, 6-hour groups, but no significant differences between the no-irradiation group and the 1-hour and 24-hour groups ( P < 0.05). In U373-MG, The average AEI was increased 7.6% in the 1-hour group, 37.5% in the 3-hour group, and 37.1% in the 6-hour group, but decreased 24.2% in the 24-hour group. Results showed significant differences between the no-irradiation group and the 3-hour and 6-hour groups, but no significant differences between the no-irradiation group and the 1-hour and 24-hour groups ( P < 0.05). 3.3. Effect of irradiation on interleukin-4 receptors of Daoy and U373-M In Daoy, the average AEI was decreased 61.4% in the 1-hour group, 36.7% in the 3hour group, and 36.6% in the 6-hour group, but increased 4.4% in the 24-hour group (Fig. 3). Results showed significant differences between the no-irradiation group and the 1-hour, 3-hour, and 6-hour groups, but no significant difference between the no-irradiation group and the 24-hour group ( P < 0.05).

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Fig. 3. Expression of interleukin-4 receptor on Daoy was decreased after irradiation.

In U373-MG, The average AEI was decreased 49.6% in the 1-hour group, 24.7% in the 3-hour group, 23.7% in the 6-hour group, and 15.3% in the 24-hour group. Results showed no significant differences between the no-irradiation group and each irradiation group ( P < 0.05). 3.4. Cytotoxicity of DT390 – mIL4 on SMA-560, Neuro-2a, and NB41A3 The percent viability of SMA-560 cells on the fourth day after incubation was 10.4% at 10 8 M, 46.7% at 10 9 M, 60.2% at 10 10 M, and 113.4% at 10 11 M. The survival curve shows that DT390 – mIL4 inhibited SMA-560 in a dose-dependent manner (Fig. 4). The cytotoxicity of DT390 – mIL4 on SMA-560 was neutralized by an excess of 11B11 antibody, which reacted with mouse interleukin-4 (Fig. 5). In Neuro-2a, the percent viability on the fourth day was 6.2%, 63.3%, and 95% at 10 8, 10 9, and 10 10 M of DT390 –mIL4 concentration, respectively. The survival curve shows dose-dependent cytotoxic activity of DT390 –mIL4 and a tendency for the percent viability to decrease over time. The cytotoxic activity of DT390 – mIL4 on Neuro2a was blocked by an excess of 11B11. In NB41A3, the percent viability on the fourth day was zero at 10 8 M, 2.3% at 10 9 M, and 56.2% at 10 10 M. The cytotoxicity shown in the survival curve was dose-

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Fig. 4. SMA-560 was treated with DT390 – mIL4 at the concentration range 10 8 – 10 11 M after the first day of incubation and the viable cells were counted on the second, third, and fourth days. DT390 – mIL4 showed dosedependent cytotoxicity.

dependent and time-dependent at 10 8 and 10 9 M. An excess of neutralizing anti-IL4 antibody abolished the cytotoxic activity of DT390 –mIL4. 3.5. Additive effect of DT390 – mIL4 and irradiation on SMA-560 At a concentration of 10 9 M, the percent viability of the DT390 – mIL4-treated group was 78.8% on the second day, 66.7% on the third day, and 59.5% on the fourth day (Table 1). The percent viabilities on the third and fourth days represented statistically significant cytotoxicity. On the third day, the percent viability was 70.2% in the irradiated group and 26.3% in the combined group with statistical significance. To evaluate the combined cytotoxic activity of DT390 –mIL4 and irradiation, the relative percent viability of the combined group (percentage of the number of viable cells in the combined group compared to that of the DT390 – mIL4-treated group and the irradiated group) was calculated. The relative percent viability of the combined group was 39.4% for the DT390 –mIL4treated group and 37.5% for the irradiated group. Both had statistical significance. On the fourth day of cell incubation, the percent viability was 53.8% in the irradiated group and 24.8% in the combined group, and the relative percent viability of the combined group was

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Fig. 5. An excess of 11B11 and A20-1.7 antibodies were added 1 h before treating DT390 – mIL4 at 10 cytotoxicity of DT390 – mIL4 was specific for cells expressing interleukin-4 receptor.

8

M. The

41.7% for the DT390 – mIL4-treated group and 46.1% for the irradiated group with statistical significance (Fig. 6) ( P < 0.05). At a concentration of 10 10 M, the percent viabilities of the DT390 – mIL4-treated group were 106.4% on the second day, 89.6% on the third day, and 94.6% on the fourth day. The cytotoxicity did not have statistical significance. Taken together, the combination of 10 9 M DT390 –mIL4 and irradiation with 500 cGy showed an additive cytotoxic effect on SMA-560.

Table 1 Percent viability of SMA-560 Txa

10

DT390 – mIL4 Irradiation Combination a b c

9

Mb

10

10

Mb

2c ( P)

3c ( P)

4c ( P)

2c ( P)

3c ( P)

4c ( P)

78.8 71.5 42.2 ( < 0.05)

66.7 ( < 0.05) 70.2 ( < 0.05) 26.3 ( < 0.05)

59.5 ( < 0.05) 53.8 ( < 0.05) 24.8 ( < 0.05)

106.4 71.5 69.7

89.6 70.2 ( < 0.05) 58.0 ( < 0.05)

94.6 53.8 ( < 0.05) 48.8 ( < 0.05)

Mode of treatment. Concentration of DT390-mIL4. Days after treatment.

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Fig. 6. The cytotoxic activity of DT390 – mIL4, irradiation, and combination of both on SMA-560 (PBS: PBStreated group, IR: irradiated group, DT: DT390 – mIL4-treated group, CB: combined group). The dosage of irradiation was 500 cGy and tumor cells of the combined group were irradiated 3 h before treating DT390 – mIL4. The concentration of DT390 – mIL4 was 10 9 M.

3.6. Additive effect of DT390 – mIL4 and irradiation on Neuro-2a At a concentration of 10 9 M, the percent viability of the DT390 – mIL4-treated group was 74.1% on the second day, 89.6% on the third day, and 63.3% on the fourth day (Table 2). The percent viability on the fourth day represented statistically significant cytotoxicity. On the fourth day after cell incubation, the percent viability was 32.5% in the irradiated group and 16.2% in the combined group, with the relative percent viability of the Table 2 Percent viability of Neuro-2a Txa

10

DT390 – mIL4 Irradiation Combination a b c

9

Mb

10

10

Mb

2c ( P)

3c ( P)

4c ( P)

2c ( P)

3c ( P)

4c ( P)

74.1 58.0 ( < 0.05) 39.7 ( < 0.05)

89.6 63.7 ( < 0.05) 54.3 ( < 0.05)

63.3 ( < 0.05) 32.5 ( < 0.05) 16.2 ( < 0.05)

83.8 58.0 ( < 0.05) 50.5 ( < 0.05)

77.5 63.7 ( < 0.05) 52.5 ( < 0.05)

94.7 32.5 ( < 0.05) 19.1 ( < 0.05)

Mode of treatment. Concentration of DT390-mIL4. Days after treatment.

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Fig. 7. The cytotoxic activity of DT390 – mIL4, irradiation, and combination of both on Neuro-2a (PBS: PBStreated group, IR: irradiated group, DT: DT390 – mIL4-treated group, CB: combined group). The dosage of irradiation was 500 cGy and tumor cells of the combined group were irradiated 3 h before treating DT390 – mIL4. The concentration of DT390 – mIL4 was 10 9 M.

combined group of 25.6% for the DT390 – mIL4-treated group and 50% for the irradiated group. All had statistical significance (Fig. 7) ( P < 0.05). At a concentration of 10 10 M, the percent viabilities of the DT390 – mIL4-treated group were 83.8% on the second day, 77.5% on the third day, and 94.7% on the fourth day. The cytotoxicity did not have statistical significance. Taken together, an additive cytotoxic activity of 10 9 M DT390 – mIL4 and irradiation with 500 cGy on Neuro-2a was demonstrated.

4. Discussion In primary malignant brain tumors, a significant number of activated oncogenes have been identified. Some of these oncogenes are growth factors that result in the stimulation of cell proliferation [1,7]. The receptors on the cell surface bind to their specific growth factors usually with high affinity. It has also been reported that growth factor receptors are overexpressed on many malignant brain tumor cells, allowing the tumor cells to bind growth factors to stimulate cellular division. These receptors include the transferrin

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receptors [25,31,37], IGF receptors [2,6], and IL-4 receptors [24]. Expression of growth factor receptors can be changed by gene mutation or by cellular responses to certain stimuli. In addition to the role in tumorigenesis, overexpression of growth factor receptors is an important aspect in the treatment of tumor with targeted toxins. Because these receptors have preferential expression on the tumor cells compared with normal brain tissue, they are frequently chosen as targets for immunotoxins [11,13,17]. Immunotoxin therapy has developed as an adjuvant therapy which has shown promising results in vitro, in vivo, and in clinical studies [10 –12,32,33,38]. In patients with recurrent malignant brain tumors, regional therapy with the TF-CRM107 immunotoxin exhibited at least a 50% reduction in tumor volume on magnetic resonance imaging in nine of 15 patients, including complete responses in two patients [17]. The interleukin-4 receptor (IL-4R) is commonly expressed on many malignant brain tumor cells but not on normal brain tissue, and fusion proteins directed against IL-4R were reported to have a potent tumor-killing effect in some studies [13,23,24]. Radiation therapy is a well-known cornerstone of most treatment protocols for the management of malignant brain tumors. In addition to the damage of DNA, irradiation affects signal transduction systems [14,20,36] and may result in changes of receptor expression on the cell membrane. Knowledge about changes in receptor expression on target cells after irradiation should help to establish a treatment strategy for targeted toxins. DNA is the main intracellular target for ionizing radiation. Ionizing radiation (X-ray or gray) interacts with water molecules within the cell producing hydroxyl radicals, which break DNA strands. The effect of irradiation is dose-dependent, oxygen-dependent, and cell cycle-dependent. The cells in the mitotic or G2 phase are more sensitive to radiation therapy than those in the synthetic (S) or resting (G0) phase [9,16]. Oxygen tissue tension is also important for predicting the response to irradiation and hypoxic cells are known to be resistant to radiation. Tumor cells located at the periphery of a mass divide actively and receive higher oxygen supply than those located in the necrotic center. These cells, which remain after surgery, can be targets for radiation treatment. Surgery, radiation therapy, chemotherapeutic drugs, and newly developed therapeutic measures such as immunotoxins have limitations, such as the ability of tumor cells to fail to respond to the first line of treatment and grow as a recurrent tumor. Many multidisciplinary treatment plans have been designed that combine surgery, radiation therapy, and chemotherapy [18,21], but protocols which include immunotoxins have not yet been introduced. Clinical trials in which fusion proteins are delivered during irradiation, and additional studies combining fusion toxins and chemotherapeutic drugs are recommended.

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