Brain Tumor Cell Lines and Pediatric Brain Tumors ...

Efficacy of Transferrin Receptor-targeted Immunotoxins in Brain Tumor Cell Lines and Pediatric Brain Tumors Lori A. Martell, Alka Agrawal, Donald A. Ross, et al. Cancer Res 1993;53:1348-1353. Published online March 1, 1993.

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(CANCER RESEARCH 53. 1348-1353. March 15, 1993]

Efficacy of Transferrin Receptor-targeted Immunotoxins in Brain Iunior Cell Lines and Pediatrie Brain Tbmors1 Lori A. Marteli, Alka Agrawal, Donald A. Ross, and Kann M. Muras/ko2 Department of Surgery. Section of Neurosurgery, the University of Michigan Medical Center, Ann Arbor, Michigan 48109

ABSTRACT

receptor ligand. The directed toxin enters the cytosol of the target cell and inactivates protein synthesis machinery in a catalytic manner (6). ITs containing the ricin A chain or CRM 107 are effective in killing tumor cells in vitro and in cultured cell lines (7-14). The transferrin

The efficacy and cytotoxic properties of Â¡mmunotoxin conjugates di rected against the transferrin receptor were examined in cell lines and operative specimens from pediatrie brain tumors. Dose-response relation ships were assessed for immunotoxin-mediated inhibition of protein syn thesis for two immunotoxins, 454A12-rRA and anti-tfnR-CRM 107. Three

receptor has been utilized as a target for ligand toxin or monoclonal anti-receptor antibody-toxin conjugates (7, 8, 15-20). Transferrin re

target medulloblastoma cell lines (DAOY, D283MED, and D341MED), a glioblastoma (U373), and a neuroblastoma (SH-SY5Y) cell line exhibited similar sensitivity to both immunotoxins with I('sâ€žsin the 10 ''-10 "' M range. The lime course of protein synthesis inhibition by the immunotox ins in DAOY cells showed that inhibition by anti-tfnR-CRM 107 was rapid and apparent by 6 h of incubation. In contrast, a response to 454A 12-rKA was not observed until 16 h. Cell viability was decreased 30-40% by 24 h after removing 454A 12-rRA (l x Kr'' M) and was maximally decreased 70-80% after 3 days. The efficacy of the immunotoxins

on a variety of

ceptors are overexpressed in some cancers, including CNS malignan cies, and thus may be a reasonable target for IT therapy. It has been postulated that the expression of transferrin receptors increases during cell proliferation to fulfill the need of the tumor cell for iron. In vivo studies and clinical trials using ITs to date indicate that they may play an important role in therapy for compartmentalized tumors in the CNS (21-24). This study compares the efficacy of transferrin receptor-targeted ITs in three recently isolated medulloblastoma cell lines (DAOY, D283MED, D341MED) (25), a glioblastoma cell line (U373), and a neuroblastoma (SH-SY5Y) cell line in vitro. In addi tion, we examine the cytotoxic activity of ITs toward primary tumor cell cultures derived from pediatrie brain tumor surgical specimens.

fresh specimens of pediatrie brain tumors was also examined. The more aggressive and malignant tumor types such as glioblastoma multiforme and medulloblastoma had low K 'io-5of 2 Â±0.3"Control9.4 4.5 Â±0.7day1. 32 Â±4IT5.610Â±2Â°7 152Â±7IT48 111Â±7a U3731Control2.5 a P < 0.01 for the IT group compared to the control group at the same lime point.

B

C

F

D

G

H

I

205-

80-

sensitivity is dependent only on the surface expression of receptors. Fig. 5A shows the 90,000 molecular weight immunoreactive protein present in all of the cell lines examined. The intensity of the staining did not appear to differ extensively between cell lines. In contrast, the homogenates from pediatrie brain tumor specimens showed obvious differences in staining intensity of the 90,000 molecular weight im munoreactive product using Western blot analysis (Fig. 55). The M, 90,000 band was much more prominent in specimen numbers 6 (medulloblastoma), 8 (glioblastoma), 10(anaplastic ependymoma), 16 (medulloblastoma), and 18 (medulloblastoma), which are all malig nant tumors. No immunoreactive products were observed in speci mens 4 (choroid plexus papilloma), 5 (craniopharyngioma), and 7

49.5-

8

10 16 18

Patient Number Fig. 5. Immunoblot analysis of transferrin receptor in brain tumor cell lines and surgical specimens. Homogenates (30 ug protein) were separated on 10%acrylamide gels. The proteins were transferred to lmmobilon-P membranes and incubated with a mono clonal transferrin receptor antibody (1:200). The immunoreactive proteins were detected and visualized using goat anti-mouse IgG horseradish peroxidase and 4-chloro-l-naphthol.

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EFFICACY

IN PEDIATRIC

(recurrent giant cell astrocytoma), which are all relatively slow grow ing and are benign tumors that are not sensitive to ITs. Interestingly, the two pilocytic astrocytomas from patients 2 and 3 demonstrated transferrin receptor expression, even though this type of tumor is generally considered benign. DISCUSSION In this study, we have shown that ITs have potent cytotoxic activity against brain tumor cell lines, including medulloblastoma and glioblastoma, and cultures obtained from various pediatrie brain tumors. Medulloblastomas are poorly differentiated and are among the most common CNS neoplasms of childhood. Other intracranial tumors common in childhood include astrocytomas and ependymomas. Medulloblastomas and ependymomas are often difficult to treat due to their ability to spread through the cerebrospinal fluid pathways. Re cent advances in surgical techniques and radiation therapy have im proved survival in children with these tumors. Chemotherapy regi mens have also been developed to augment radiation therapy (29-31). There are unacceptable long-term sequelae to radiation therapy and chemotherapy. The unacceptable side effects include a decrease in intellectual function, hypothalamic and pituitary dysfunction, a de crease in stature and development of leukomalacia, and radiation necrosis of the brain. ITs have properties different from those of conventional chemotherapeutic agents which render them useful as a complement to or in combination with chemotherapy. ITs have a different mechanism of action, affecting protein synthesis and killing nondividing cells. Most chemotherapeutic drugs, which disrupt DNA synthesis and cell divi sion, require actively proliferating cells to be effective. In addition, those cancer cells expressing resistance to conventional therapies may be susceptible to ITs. The cytotoxicity of ITs is not affected by conditions of hypoxia, an important factor in the resistance observed with radiation and chemotherapy. In addition, because of the high enzymatic activity of the toxins ricin and diphtheria toxin, the entry of one or very few molecules into the cytosol can kill a cell (32). Ricin and diphtheria toxin consist of two functionally distinct polypeptides linked by a disulfide bond (33). The A chain carries the enzymatic activity, whereas the B chain binds to receptors at the cell surface and contains the internalization functions of the toxin. Ricin toxin acts directly on the ribosomes, where it inactivates the large 60S ribosomal subunit (34). Diphtheria toxin inactivates elongation factor 2, an enzyme required for the translocation of the growing peptide chains from the A site to the P site of the ribosome (35). CRM 107 is identical to diphtheria toxin except for a single point mutation in the B chain, which inactivates intrinsic toxin binding (36). Cell lines can provide a useful system for investigating with relative ease the effects of a toxin on a single clonal population of cells. However, a more physiological indication of the usefulness of an IT in the treatment of CNS tumors comes from cells obtained directly from tumor specimens. Data from other laboratories have suggested that anti-transferrin receptor Mab-linked toxins may be effective at killing tumors in vivo. We have shown that many of these primary tumor cells are susceptible to IT cytotoxicity with IC50s similar to those observed with cell lines. In fact, the more aggressive, malignant, and rapidly proliferating tumors such as medulloblastoma and highgrade astrocytomas were killed at a lower concentration of IT than the slower-growing and more benign tumors such as choroid plexus papilloma and craniopharyngioma. These data were further supported by the identification by immunoblot analysis of transferrin receptor pro tein present in the cell lines and surgical specimens. Interestingly, the specimens from pilocytic astrocytomas, common childhood tumors,

BRAIN TUMORS

expressed transferrin receptor protein and yet are considered relatively benign tumors. These tumors were also susceptible to the ITs. We have demonstrated that ITs are effective in killing 3 medullo blastoma cell lines, as well as other cell lines and pediatrie brain tumor cultures from surgical specimens. The ITs are potent and specific agents, with IC50s generally in the 10~9-10~" Mrange. The intrathecal dose of IT that is known to cause severe toxic effects is 2 X IO"6 M in monkeys, 2 X IO"6 M in rats, 5 X 10~7 M in guinea pigs, and 5 X 10~7 Min humans (37).4 Thus, the concentrations required to kill cells in vitro are at least 1-2 log units smaller than those concentrations that produce adverse effects in vivo. The specificity of the antibody portion of the ITs was demonstrated in that antibody-toxin conjugates were 1000 times more potent than the unconjugated toxin alone. While the IC50s for the two ITs were similar in the different cell lines, the kinetics of cell killing differed, with the anti-transferrin receptor MabCRM 107 killing at a faster rate than 454A12-rRA. This result has been described previously by others in other cell lines (38) and may suggest a greater utility for the CRM 107 conjugate over the ricin A chain conjugate. The two Mabs directed against the human transferrin receptor, 454A12 and anti-tfnR, do not compete with transferrin for binding to the receptor and are thought to be directed against different domains on the same receptor. Tumor heterogeneity remains a significant obstacle with regard to implications for the therapeutic sensitivity of a tumor to ITs and traditional chemotherapy, as well as metastatic potential (39^12). An ideal target antigen for IT therapy would be expressed only on the tumor cells. However, since this level of specificity may not be at tainable, a more realistic approach is to seek antigens expressed at high levels on target cells and low levels on normal cells. The ex pression of transferrin receptors is related to cellular iron requirements and is thought to represent the proliferative potential of the cell. In normal brain, the most prominent immunostaining for transferrin re ceptors was observed in the capillary endothelial cells (43). In addi tion, Recht et al. (20) have shown that immunostaining for transferrin receptors is much more abundant in high-grade astrocytic tumors and glioblastomas than normal brain and that the staining pattern and intensity roughly correlated with increasing tumor grade. The use of transferrin receptors as a target for toxins, first proposed by Trowbridge and Domingo (7), has been shown to be an effective means of killing tumor cells. Transferrin receptors are effectively internalized upon binding of antibody or ligand and are linked with the rate of DNA synthesis (44). It has been suggested that IT therapy targeted to the transferrin receptor in glioblastomas and medulloblastomas would not be affected by antigenic heterogeneity, antigenic modulation, or genetic loss since the receptor is up-regulated by higher iron require ments by the rapidly dividing cells (24). We have examined the effect of the simple addition of iron-loaded transferrin to cultures of DAOY and U373 cells and found a 50% increase in the rate of DNA synthesis.5 A number of requirements must be fulfilled for an immunoconjugate to be of therapeutic value in vivo. The conjugate must remain intact in the circulation for a sufficient period of time, it must be able to penetrate through capillary walls and into tumors, and it must be specific for tumor tissue over normal tissue. In the treatment of brain tumors and other "compartmentalized" tumors, these issues may not be so critical, since conjugates are administered locally in a compart mentalized space, where high concentrations can be reached without great risk to the systemic circulation. ITs may play an important role in the treatment of residual disease, which commonly results in tumor 4 K. M. Muraszko, C. Sung, S. Wallbridge, L. Greenfield, R. Dedrich, E. Oldfield, and R. Youle. Pharmacokinetics and toxicology of immunotoxins into the subarachnoid space in non-human primates and rodents, submitted for publication. 5 L. A. Marteli and K. M. Muraszko, unpublished observation.

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recurrence. In the case of tumors which spread through cerebrospinal fluid pathways, ITs may provide a means of controlling dissemination and improving morbidity as well as mortality. Thus, the results sug gest that ITs are potent and efficacious in killing aggressive tumors common in childhood and that these agents should be examined in an in vivo model of neoplastic disease. ACKNOWLEDGMENTS We thank Dr. Richard J. Youle (Surgical Neurology Branch, NIH) for immunotoxins, scientific contributions, and his careful review of the manu script.

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