J Neurosurg 76:838-844, 1992
In vitro efficacy of transferrin-toxin conjugates against
glioblastoma multiforme WALTER A. HALL, M.D., ASLAK GODAL, PH.D., Stm JUELL, AND ~u FODSTAD~ M.D., PH.D. Department of Tumorbiolog~; Institule for Cancer Research, The Norwegian Radium Hospital, and Hafslund Nycomed A/S, Oslo, Norway; and Department of Neurosurgery, University of Minnesota Hospital and Clinic, Minneapolis, Minnesota
L,- The cytotoxic activity of immunotoxins constructed with human diferric transferrin (Tfn) as the carrier ligand and an abrin variant Pseudomonas exotoxin A (PE) and the diphtheria toxin mutant cross-reacting material (CRM) 107 as the toxin moieties were studied in vitro. Three malignant human cell lines, the glioblastomas multiforme SNB19 and SF295 and the LOX melanoma, and a nonhuman control routine melanoma cell line B16 were assessed. The presence of transferrin receptors on the cell lines was confirmed by direct 12~I-Tfnbinding assays. The 50% protein synthesis inhibitory concentration (IC5o) values for all cell lines demonstrated that Tfn-abrin variant and Tfn-PE had comparable potency and were both more effective than Tfn-CRM 107. Monensin, a carboxylic ionophore, potentiated the effect of Tfn-abrin variant against glioma cells approximately 35-fold with IC50values of 4.0 • 10 -t3 M and 4.7 • 10-~2 M for SNBI9 and SF295, respectively. Cytotoxic activity of Tfn-abrin variant (with or without monensin) and Tfn-PE was correlated with the degree of Tfn receptor expression measured on the cell lines. The exquisite in vitro cytotoxicity of Tfn-abrin variant and Tfn-PE immunotoxins against glioma and melanoma cells warrants further in vivo evaluation and future consideration of these agents for potential clinical application against glioblastoma multiforme and leptomeningeal neoplasia. KEY WORDS brain neoplasm transferrin receptor
9
URRENX treatment of primary malignant tumors of the central nervous system (CNS), including surgery, radiation therapy, and systemic chemotherapy, has not improved the prognosis for patients with this disease. 33"39"51'52"54"63'64"66 The 2-year survival rate for glioblastoma multiforme is less than 20%. 12"64 The lack of specificity of conventional chemotherapy for malignant cells with severe dose-limiting side effects has led investigators to pursue the development of innovative treatment modalities? 4 Not until the advent of monoclonal antibody technology was interest renewed in the "magic bullet" concept of Paul Ehrlich: toxic compounds linked to carrier molecules specific for a target cell.4-j~ This interest has generated a new class of compounds called immunotoxins, tumor-specific monoclonal antibodies, or other ligands covalently linked to toxic proteins that combine exquisite cell-type selectivity with extraordinary potency. 4"13"24'31'33"45"62"66'67 To obtain a selective effect using immunotoxins, the target for this directed
C
838
brain-neoplasm antigen
9 immunotoxin
9
therapy should be preferentially expressed by neoplastic cells, compared to normal cells. 5~ Despite raising antibodies to human glioma-associated antigens, polyclonal and monoclonal serological methodologies have not identified antigens specific for this tumor type. 6~2 Explanations for the inability to identify tumor-specific antigens include the biological heterogeneity of malignant gliomas and the variable expression of antigens among and within tumors. 3'4'65 Although there are no antigens exclusively expressed by malignant glial cells, the transferrin receptor (TR) and the epidermal growth factor receptor (EGFR) or an amplified rearranged EGFR are present in higher numbers on surgical glioblastoma multiforme tissue samples and glioblastoma-derived tissue-culture cells than on normal CNS tissue, a9~8"23262735~50 The high requirement of rapidly dividing tumors, such as the glioblastoma multiforme, for iron should prevent antigenic modulation, 43 antigenic heterogeneity, 7 or genetic loss 28 of the TR, leaving it intact for immunotoxin therapy. J. Neurosurg. / Volume 76/May, 1992
Transferrin-toxin conjugates against glioblastoma Efficient internalization of TR's by cancer cells makes them an ideal target for immunotoxins/~~s Techniques for determining the TR presence and the degree of expression on glioblastoma multiforme have included radioimmunoassay, immunohistochemistry, flow cytofluorometry,, radioreceptor assay, slot blot ribonucleic acid (RNA) analysis, and in vitro cytotoxicity assays?"~'-'-':3'-~~These previous studies provide additive support for the TR as an appropriate target for transferrin (Tfn)-toxin conjugates until a more specific cellsurface antigen is identified for this tumor type]-' Here we describe the construction of three Tfn-toxin conjugates and demonstrate in three different malignant tissue-culture cell lines an efficacy that correlates with TR expression.
Materials and Methods
Chemicals Human Tfn was saturated with iron according to the method of Shindelman, et al? ~ Monensin* was added to the appropriate eytotoxicity bioassay wells to yield a final concentration of 10-7 M. Free sulfhydryl groups were generated on Tfn with 2-iminothiolane.t A stable thioether bond between Tfn and toxin was created using the bifunctional cross-linking agent, sulfo-N-succinimidyl-4-(N-malimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC).:]: 37 Toxins Whole abrin was extracted from Abrus precatorius and purified as previously described/4 A whole-abrin variant was used in the synthesis of abrin-based immunotoxins. Cross-reacting material (CRM) 107, a diphtheria toxin mutant,w was isolated by Laird and Groman 34 and purified by a previously described method. 32 Pseudomonas exotoxin A (PE)II is made by the bacterium Pseudomonas aeruginosa. Cell Lines Glioblastoma-derived tissue-culture cell lines SNB 19 and SF295 were used.** At the time of this study, SNBI9 was in its 237th passage and SF295 was in its 101st passage. The LOX melanoma cell line was established in our laboratory from a patient biopsy specimen. '4 Murine melanoma cell line BI6 was used as a negative control for human TR level expression experiments * Monensin obtained from Calbiochem, Lucerne, Switzerland. t 2-1minothiolane obtained from Pierce Chemical Co., Rockford, Illinois. SuIfo-SMCC obtained from Pierce Chemical Co., Rockford, Illinois. wCross-reacting material 107 obtained by courtesy of Dr. R. Youle, Surgical Neurology Branch, National Institutes of Health. [1Pseudomonas exotoxin A obtained from Swiss Serum and Vaccine Institute, Berne, Switzerland. ** SNBl9 and SF295 obtained from the National Cancer Institute Frederick Cancer Research Facility, Frederick, Maryland, by courtesy of Dr. R. Shoemaker.
J. Neurosurg. / Volume 76/May, 1992
and in cytotoxicity assays to Tfn-toxin conjugates. All cell lines were maintained at 37~ as monolayer cultures in RPMI 1640 mediumt containing 10% fetal calf serum (FCS), 2 mM glutamine, 1000 IU/ml of penicillin, and 100 ug/ml of streptomycin.
Tran,sjbrrin Receptor Determination on TissueCulture CWI Lines The amount of TR expressed on the surface of the cells was determined by incubating a suspension of i x 10~'cells in 0.5 ml of phosphate-buffered saline (PBS; 7 mM phosphate buffer, pH 7.4, with 0.14 M NaC1) containing human serum albumin (1 mg/ml) with ~251labeled human Tfn,~ 1 x 105 cpm at 4~ for 4 hours. The reaction was stopped by centrifugation, the cells were washed, and the cell-bound radioactivity was counted in a gamma counter.w Synthesis and Pm4fication ~/ lmmunotoxins Transferrin was conjugated to CRM 107 after generating tree sulfhydryl groups on Tfn with 2-iminothiolane dissolved in 0.1 M sodium borate (pH 8.5), by incubating at room temperature for 1 hour with Tfn in an 8:1 molar ratio. Free 2-iminothiolane was separated from the modified Tfn by gel filtration on a Sephadex G-25 column equilibrated with PBS. After dissolution in H20, sulfo-SMCC was added to the CRM 107 in a 5:1 molar excess for 30 minutes at room temperature. The abfin variant and PE conjugates were prepared in the same manner with a molar excess of toxin in a 10:1 ratio. 37 The sulfo-SMCC-conjugated toxin was separated on a Sephadex G-25 column, mixed with thiolated Yfn in a 1:1.3 molar ratio, and incubated overnight at room temperature, Purification was performed by gel filtration on Sephacryl S-200 with 0.1 M sodium phosphate buffer (pH 7.0) to remove excess toxin. The pooled peak fractions of the immunotoxin were used for all cytotoxicity experiments. To remove abrin variant conjugates with exposed binding sites, the immunotoxin was passed through a Sepharose 4B column as described by Thorpe, el al. 57 The final concentrations of the immunotoxins, determined in the protein assay, ll were 0.18 mg/ml for Tfn-abrin variant, 0.1 mg/ml for Tth-CRM 107, and 0.06 mg/ml for Tfn-PE. Qvtotoxicity Assa)z~ The cytotoxic effect of the immunotoxins on the tissue-culture cell lines was assessed by measuring their ability to inhibit protein synthesis. Cells growing in monolayer culture were treated for 5 minutes with 10 mM ethylenediamine tetra-acetic acid in PBS containing t RPMI 1640 medium manufactured by G1BCO Biocult, Glasgow, Scotland. Human transfcrrin obtained from Amersham International, Ltd., Amersham, Buckinghamshire, England. wGamma counter manufactured by Beckman Instruments, Irvine, California. 1IProtein assay system manufactured by Bio-Rad Laboratories, Munich, Germany. 839
W. A. Hall, et al. 0.05% KCI to obtain a suspension. After washing, the cells were diluted to a concentration of 5 • 104 cells/ ml with RPMI medium containing 10% FCS and 20 mM HEPES, pH 7.2. One ml of cell suspension was added to each well of a 24-well tissue-culture tray, and the cells were incubated for 3 hours at 37~ to obtain a confluent monolayer. Transferrin, immunoconjugate, or control solution was added to the cells in duplicate wells and incubated for 20 hours at 37~ Monensin was added to the appropriate wells in a final concentration of 10-7 M before overnight incubation. The cells were washed once with PBS and leucine-free medium before 1 uCi [3H]-leucine/ml ( 131 mCi/mmol) was added. After incubation for 30 minutes at 37~ the cells were processed and the trichloroacetic acid-precipitable radioactivity was measured and compared to untreated control cell cultures, as previously described. ~9.53The concentration required to inhibit 50% of protein synthesis (ICs0) was derived from dose-effect curves. All cytotoxicity assays were performed two to five times in duplicate. Results
Binding of r25I-Labeled Transferrin to TissueCulture Cell Lines A noncompetitive radioreceptor assay (as described above) was used to detect the presence of TR on the tissue-culture cell lines. The results are presented in Table 1, with cell lines arranged in decreasing order of antigen expression as measured by the binding of ~25ITfn. Both glioblastoma-derived cell lines SNB19 and SF295 had significant levels of TR expression at 4.9% and 2.7%, respectively, whereas the LOX melanoma cell line had a level of 3.1%. As expected, murine melanoma cell line BI6 bound very low amounts of ~25I-Tfn; levels of TR expression under 1% may represent either nonspecific binding or homology of the TR between species.
Cytotoxic Activity of Transferrin- Toxin Conjugates on Tissue-Culture Cell Lines The plant toxin abrin inhibits protein synthesis by inactivating ribosomes in a manner similar to that of ricin. 24'44 Alternatively, toxins derived from bacteria such as PE and CRM 107 block protein synthesis by catalyzing the transfer of adenosine diphosphate-ribose to elongation factor-2, resulting in discontinuation of the addition of amino acids to the growing polypeptide chain. 33 The action of these toxins is ultimately lethal to cells susceptible to toxin entry. A measurement of [3H]-leucine incorporation into the acid-insoluble fraction of cells exposed to different concentrations of Tfn-toxin conjugates determines their effect on protein synthesis and allows for the calculation of inhibitory concentration (IC) values. The ICs0 value represents the molar concentration of immunotoxin that inhibits 50% of protein synthesis. 840
TABLE 1
Binding ~?f~25I-tranfferrin to tissue-culture cell lines* Cell Line
Origin
% Bound
SNB 19 LOX SF295 B 16
human glioblastoma human melanoma human glioblastoma routine melanoma
4.9 3.1 2.7 0.8
* Expressed as % bound (cpm)/total (cpm) of ~2sI-transferrinincubated with differentcell lines for 4 hours at 4~
Iron-loaded Tfn, in the same concentrations as the Tfn-toxin conjugates, did not inhibit protein synthesis (data not shown), and concentrations of Tfn greater than 1 mg/ml were necessary to obtain toxicity in all cell lines. The least effective immunotoxin in the cell lines tested was Tfn-CRM 107. The cell line most susceptible to this conjugate was SNBI9 (ICs0 3.5 x 10-~~ M). Transferrin-PE was 20 times more potent than Tfn-CRM 107 in SNBI9 cells, which were 27 times more sensitive to Tfn-abrin variant than to TfnCRM 107. Immunoconjugates made with PE and abrin variant toxins had very similar ICso values (Table 2) and were very effective against SNBI9 cells, with ICs0 values of 1.7 • 10-t~M and 1.3 • 10-~ M, respectively. A concentration of 1.7 • 10-~~ M for Tfn-PE and for Tfn-abrin variant was equally potent for the SF295 cell line. The LOX melanoma cell line demonstrated almost identical sensitivites to Tfn-PE (ICso 4.1 • 10-~ M) and Tfn-abrin variant (ICs0 4.7 • 10-~j M) immunotoxins. A 20- to 250-fold difference in cytotoxieity to Tfnabrin variant and a 12- to 120-fold difference to TfnPE was seen between B16 cells and the other cell lines when exposed to the Tfn-abrin variant conjugate alone. The fact that the immunotoxins made with human Tfn were cytotoxic to the murine melanoma cell line B16 may represent either antigenic cross-reactivity of the TR between species or nonspecific binding. Adding monensin to the cytotoxicity bioassays enhanced the activity of the Tfn-abrin variant immunotoxin in all cell lines. The degree of potentiation is indicated in Table 2 as the potentiation factor. For LOX melanoma and both SNB 19 and SF295 glioma cell lines, the potentiation factor ranged from 33 to 36. In contrast to previous reports, 6,7 monensin had virtually the same effect on each human cell line we studied. In SNB 19 cells, monensin improved the cytoxicity of Tfnabrin variant 43-fold over Tfn-PE and 875-fold over Tfn-CRM 107. The cytotoxic effect of Tfn-abrin variant immunotoxins in the SNB19 and SF295 glioma cell lines, with and without monensin, is demonstrated in Fig. 1. Cytotoxic effects of the Tfn-abrin variant, with or without monensin, and Tfn-PE immunotoxins correlated directly with the degree of TR expression on LOX, SNBI9, SF295, and B16 cell lines as demonstrated in Tables 1 and 2. Transferrin receptor expression was greatest on the cell line most sensitive to abrin variant and PE immunotoxins, namely SNB19. The mouse
J. Neurosurg. / Volume 76/May, 1992
Transferrin-toxin conjugates against glioblastoma TABLE 2 7?zwcay (lCs,~ value,s? (?[lran.~[errin-toxitt cr Tumor Cells
Tfn-CRM 107
SNBI9 LOX SF295 B16
3.5 • >7.0X > 7,0 • a.9 x
10 -~i' 10 ~ 10-t~ 10-~~
ffn-PE 1.7 4.1 1.7 2.0
• x x x
10-~t 10-~t 10 lo 10 9
on li.~,sue-cu/ture cell lines*
Tfn-Abrin Variant t.3 • 4.7X 1.7 • 3.3 x
10 ii I0 -H 10 1o I0 9
Tfn-Abrin Variant + Mo 4.0 • 1.3X 4.7 x 2.0 x
10 ~3 10 -~2 10 12 10-~t
Potentiation Factor 33 35 36 167
* lCso is the molar concentration of immunotoxin inhibiting 50% of protein synthesis. Tfn = transferrin; PE = Pseudomonas exotoxin A; Mo = monensin (10 -7 M); potentiation factor = the increase in cytotoxicily with added monensin (IC5o Tfn-abrin variant/ICs0 Tfn-abfin variant plus monensin).
melanoma B I6 had the lowest TR expression and the least susceptibility to Tfn-abrin variant and Tfn-PE. No correlation was seen between TR expression and cytotoxicity in any cell line assayed with Tfn-CRM 107. Discussion
Expression of TR, a transmembrane glycoprotein that mediates cellular iron uptake, is regulated by replication. ~9'6~ Thus, proliferating cells express greater numbers of TR's than do nondividing cells? 1.59.66Using immunohistochemistry, Gatter, et al., ~7 demonstrated TR expression on human surgical tissue samples of adenocarcinomas, squamous-cell carcinomas, germ-cell tumors, sarcomas, and Hodgkin's and non-Hodgkin's lymphomas. The basal epidermis, endocrine pancreas, hepatocytes, Kupffer cells, testis, and pituita~' gland also expressed TR's. iv Others have found ve~' high TR levels on certain tumors.l ~,~6.55 Glioblastoma multiforme, a rapidly dividing primary, malignant brain tumor, has been shown by many investigators to express high numbers of TR's in vitro and in vivo. 9"22"23"335~ Transferrin receptors have
FIG. [. Dose-response curves showing cytotoxic effects of transferrin-abrin variant immunotoxins on glioblastoma-derived tissue-culture cell lines SNBt9 (squares) and SF295 (circles), with (open symbols') and without (closed symbols) monensin. Protein synthesis was estimated by measuring the incorporation of I uCi [SH]-leucine. J. Neurosurg. / Volume 7 6 / M a y , 1992
been identified on glioblastoma multiforme tissue samples by immunohistochemistry, a radioreceptor assay, and solid-phase indirect radioimmunoassay) z5~ In addition to other techniques that have detected TR's on glioma cells in vitro, 95~'-~6 the direct binding of radiolabeled Tfn to the SNB19 and SF295 glioma cell lines in this study indicates TR expression on the cell surface. The differential expression of TR on glioblastoma compared to normal brain tissue suggests that TR's may be a suitable target for Tfn-toxin therapy. 22~23'66 Moreover, the binding of 12~l-Tfn to LOX melanoma provides suggestive evidence for the presence of TR's on this tumor. The propensity for melanoma to metastasize to the CNS as discrete mass lesions or as carcinomatous meningitis provides the rationale for evaluating this tumor type as a potential candidate for immunotoxin therapy. 538 In prior in vitro studies against glioblastoma-defived cell lines, immunotoxins targeted to TR's were constructed with either Tfn or a murine monoclonal antibody as the binding ligand, linked to the toxin component. 9.33's~'reToxins used previously to create effective immunotoxins against glioma cells have included intact ricin, ricin A chain, and a point-mutated diphtheria toxin CRM 107.9'21"33"51 Zovickian, el al., 66 reported that an anti-TR-ricin immunoconjugate killed more than 50% of glioma cells after 18 hours at a concentration of 5.6 x 10 - ~ M , in the presence of the earboxylic ionophore monensin. Against five glioma cell lines, Recht, et al., 51 demonstrated potent cytotoxicity of an anti-TR monoclonal antibody-ricin A chain conjugate at concentrations as low as 7.0 x 10-'2 M, with monensin. Monensin decreased 16- to 842-fold the IC~0 of the ricin A chain immunotoxin against glioma cells. As reported by Johnson, et al., 32 immunotoxins composed of the anti-TR monoclonal antibody 454A12 and ricin A chain or CRM 107 demonstrated comparable cytotoxicity against the most sensitive glioma cells tested at concentrations of 3.6 x 10-~ M and 1.6 x 10-j~ M, respectively. The toxin CRM 107 coupled to Tfn was more effective against glioma ceils than either antibody-toxin conjugate with an IC5o value of 2.6 x 10-12 M. According to Colombatti, el al., g Tfn linked to ricin A chain showed high cytotoxic activity, being 5000 times more toxic than the A chain alone, in the presence of monensin. 841
W. A. Hall, el al. Of interest in the present study is the difference in efficacy of our Tfn-CRM 107 in SNB19 and SF295 compared to that reported by Johnson, el al., ~2 in the SNB75, SNB101, and U251 glioma-derived celt lines. This difference in sensitivity to Tfn-CRM 107 between glioma cell lines may be due to a difference in TR expression. Conjugates made with PE and abrin variant, without monensin, as the toxin components had similar cytotoxic activities against malignant melanoma- and glioma-derived cell lines. Our Tfn-PE conjugate demonstrated comparable cytotoxicity in our glioma cell lines when compared to other reported immunotoxins in different glioma cells. 9"32"51 In the presence of monensin, SNB19 and SF295 cells were more susceptible to Tfn-abrin variant than were other glioma lines tested by Zovickian, et al., 66 Recht, el al., 5~ Johnson, e t a l . , or Colombatti, et al., 9 where Tfn and anti-TR monoclonal antibodies were used as the cartier ligands and ricin, ricin A chain, and CRM 107 were the toxin moieties. Although the mechanism by which monensin potentiates plant-derived abrin and ricin immunotoxins is unclear, disruption of vesicle movement in cells and alteration of endosomal pH have been implicated. T M Other lysosomotropic agents that allow easier access of immunoconjugates to the cytosol include chloroquine4s and the calcium channel-blocking agents verapamil and diltiazem. Abrin immunotoxins have been shown to be more toxic to melanoma cell lines than the corresponding ricin conjugate despite similarities between structure and mechanism of action? 9 Before and after the addition of monensin, our Tfn-abrin variant immunotoxin was approximately 100-fold more toxic to glioma cells than was the Tfn-ricin A chain conjugate reported by Colombatti, et al? Abrin- and ricin-based immunotoxins have the advantage over PE- and CRM 107-based immunotoxins against glioblastoma in that their activity can be potentiated by monensin, as we and others have illustrated. Furthermore, prior immunization of the adult population may result in neutralizing antibodies that would inhibit immunotoxins made with diphtheria toxin-derived CRM 107. Many studies demonstrating the efficacy of immunotoxins against glioblastoma-derived tissue-culture cells have used a murine anti-human TR monoclonal antibody as the carrier ligand. 32-5L66As previously done by Johnson, et al.,32 and Colombatti, e t a l . , 9 we have constructed our conjugates with a Tfn carrier to presumably increase transvascular and intratumoral diffusion by decreasing molecular weight and size. A reduction in size may facilitate penetration of toxin conjugates into malignant brain-tumor tissue, after either intrathecal, intratumoral, or intra-arterial delivery, resulting in improved clinical efficacy. Blood-brain barrier modification with the osmotic agent mannitol, as reported in the treatment of primary malignant brain tumors with chemotherapeutic drugs and metastatic melanoma to the CNS with monoclonal antibodies, may improve the distribution of immunotoxins into tu842
mot after intra-arterial administration? ~176 Agents such as monensin that enhance immunotoxin entry into the cytosol can be administered simultaneously into the cerebrospinal fluid (CSF) with conjugates utilizing abrin or ricin toxin moieties to enhance the cytotoxic effect. 8"49 Although there are few examples of the in vivo treatment of CNS malignancy, early results against leptomeningeal neoplasia are encouraging. Extended survival was reported by Zovickian and Youle ~7 in guinea pigs treated with an anti-idiotype monoclonal antibody (M6)-intact ricin immunotoxin delivered intrathecally 24 hours after delivery of L2C leukemia cells into the cisterna magna. Fuchs, et al., 15 established an animal model of human neoplastic meningitis in the athymic nude rat using a glioma cell line. Intrathecal instillation of 4-hydroperoxycyclophosphamide 5 days after tumor cell inoculation prolonged median survival by 23%. ~5 Dissemination of glioblastoma multiforme throughout the neuraxis, although an infrequent occurrence, has been demonstrated in younger patients and in patients with extended survival. 6j Toxicity trials in guinea pigs have shown a maximum tolerated intrathecal Tfn-CRM 107 dose of 2 x 10-9 M. 32 This same dose of immunotoxin, administered intrathecally to rhesus monkeys, was nontoxic and represented a CSF concentration 20- to 5000-fold higher than the effective in vitro dose in glioma cells, suggesting that a significant therapeutic window may exist for immunotoxin treatment of leptomeningeal neoplasia. Recombinant c~-interferon enhances the action of immunotoxins both in vitro and in v i v o 46 and could be coinstilled with immunotoxins into the intrathecal space of patients with carcinomatous meningitis. Intratumoral administration of a-interferon and immunotoxins into glioblastoma multiforme represents a potential form of immunotherapy, similar to that already reported with interleukin-2 and lymphokine-activated killer cellsY 9'3~Previously mentioned lysosomotropic agents may be used in combination with immunotoxins if intrathecal or intratumoral delivery is considered. Future directions for determining the in vivo efficacy of our Tfn-toxin conjugates include the treatment of human neoplastic meningitis, established in animal models. ~s Preliminary results from our laboratory in a nude rat model of meningitis due to LOX melanoma suggest that Tfn-PE given intrathecally can delay the onset of paraplegia to a statistically significant degree. The demonstration of a therapeutic effect in animals will add support for the clinical application of these agents in Phase I and II clinical trials against CNS neoplasia, in the form of carcinomatous meningitis or primary malignant brain tumors, where present therapeutic options are limited and alternatives to the conventional treatment of these diseases are clearly needed.
Acknowledgment We express our thanks to Dr. Richard J. Youle, Chief, Biochemistry Section, Surgical Neurology Branch, National J. Neurosurg. / Volume 7 6 / M a y , 1992
Transferrin-toxin conjugates against glioblastoma Institutes of Health, Bethesda, Maryland, for providing the CRM 107 for immunotoxin synthesis. References 1. Akiyama S, Seth P, Pirker R, et al: Potentiation of cytotoxic activity of immunotoxins on cultured human cells. Cancer Res 45:1005-1007, 1985 2. Barba D, Saris SC, Holder C, el al: Intratumoral LAK cell and interleukin-2 therapy of human gliomas. J Neurosurg 70:t75-182, 1989 3. Bigner DD: Biology ef gliomas: potential clinical implications of glioma cellular heterogeneity. Neurosurgery 9: 320-326, 1981 4. Blythman HE, Casellas P, Gros O, etal: Immunotoxins: hybrid molecules of monoclonal antibodies and a toxin subunit specifically kill tumour cells. Nature 290:145-146, 1981 5. Brega K, Robinson WA, Winston K, etal: Surgical treatment of brain metastases in malignant melanoma. Cancer 66:2105-2110, 1990 6. Bullard DE, Bigner DD: Applications of monoclonal antibodies in the diagnosis and treatment of primary brain tumors. J Neurosurl~ 63:2-16, 1985 7. Capone PM, Papsidero LD, Chu TM: Relationship between antigen density and immunotherapeutic response elicited by monoclonal antibodies against solid tumors. JNCI 72:673-677, 1984 8. Casellas P, Bourrie BJP, Gros P, et at: Kinetics of cytotoxicity induced by immunotoxins. Enhancement by lysosomotropic amines and carboxylic ionophores. J Biol (;hem 259:9359-9364, 1984 9. Colombatti M, Bisconti M, Dell'Arciprete L, et al: Sensitivity of human glioma cells to cytotoxic heteroconjugates, lnt J Cancer 42:441-448, 1988 10. Ehrlich P: The relationship existing between chemical constitution, distribution, and pharmacological action, in Himmelweite F, Marquardt M, Dale H (eds): The Collected Papers of Paul Ehrlich. Elmsford, NY: Pergamon, 1956, Vol 1, pp 596-618 11. Faulk WP, HsJ BL, Stevens PJ: Transferrin and transferrin receptors in carcinoma of the breast. Lancet 2:390-392, 1980 12. Fischer DK, Chen TL, Narayan RK: Immunological and biochemical strategies for the identification of brain tumor-associated antigens. J Nenrosurg 68:165-180, 1988 13. FitzGerald D, Pastan I: Targeted toxin therapy for the treatment of cancer. JNCI 81:1455-1463, 1989 14. Fodstad D, Aamdal S, McMenamin M, etal: A new experimental metastasis model in athymic nude mice, the human malignant melanoma LOX. Int J Cancer 41: 442-449, 1988 15. Fuchs HE, Archer GE, Colvin OM, et ah Activity of inlrathecal 4-hydroperoxycyclophospbamidein a nude rat model of human neoplastic meningitis. Cancer Res 50: 1954-1959, 1990 16. Galbraith GMP, Galbraith RM, Faulk WP: Transferrin binding by human lymphoblastoid cell lines and other transformed cells, Cell Immunol 49:215-222, 1980 17. Gatter KC, Brown G, Trowbridge IS, et al: Transferrin receptors in human tissues: their distribution and possible clinical relevance. J Clin Pathol 36:539-545, 1983 18. Gerosa MA, Talarico D, Fognani C, etal: Overexpression of N-ras oncogene and epidermal growth factor receptor gene in human glioblastomas. JNCI 81:63-67, 1989 19. Godal A, Fodstad ~, Morgan AC, et al: Human melanoma cell lines showing striking inherent differences in sensitivity to immunotoxins containing holotoxins. JNCI 77:1247-1253, 1986 Y. Neurosurg. / Volume 76 / M a y , 1992
20. Greenberg HS, Ensminger WD, Chandler WF, et al: lntraarterial BCNU chemotherapy for treatment of malignant gliomas of the central nervous system. J Neurosurg 61: 423-429, 1984 21. Greenfield L, Johnson VG, Youle RJ: Mutations in diphtheria toxin separate binding from entry and ampli~ immunotoxin selectivity. Science 238:536-539, 1987 22. Hall WA: Transferrin receptor on glioblastoma multiforme. J Neurosurg 74:313-314, 1991 (Letter) 23. Hall WA, Merrill MJ, Walbridge S, et al: Epidermal growth factor receptors on ependymomas and other brain tumors. J Neurosarg 72:641-646, 1990 24. Hertler AA, Frankel AE: Immunotoxins: a clinical review of their use in the treatment of malignancies. J Clin Oncol 7:1932-1942, 1989 25. Hochberg FH, Pruitt AA, Beck DO, et al: The rationale and methodology for intra-arterial chemotherapy with BCNU as treatment for glioblastoma. J Neurosurg 63: 876-880, 1985 26. Humphrey PA, Wong AJ, Vogelstein B, et al: Amplification and expression of the epidermal growth factor receptor gone in human glioma xenografts. Cancer Res 48: 2231-2238, 1988 27. Humphrey PA, Wong AJ, Vogelstein B, et al: Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma. Proc Natl Acad Set USA 87: 4207-42l 1, 1990 28. Hyman R, Cunningham K, Stallings V: Evidence for a genetic basis for the class A thy- 1- defect, lmmunogenetics 10:261-271, 1980 29. Jacobs SK, Wilson DJ, Kornblith PL, et al: Interleukin-2 and autologous lymphokine-activated killer cells in the treatment of malignant glioma. Prelimina~, report. J Neurosarg 64:743-749, 1986 30. Jacobs SK, Wilson DJ, Kornblith PL, et al: Inlerleukin-2 or autologous lymphokine-activated killer cell treatment of malignant glioma: phase I trial. Cancer Res 46: 2101-2104, 1986 31. Jansen FK, Blythman HE, Carri~re D, et al: lmmunotoxins: hybrid molecules combining high specificity and potent cytotoxicity. Immunol Rev 62:185-216, 1982 32. Johnson VG, Wilson D, Greenfield L, et al: The role of the diphtheria toxin receptor in cytosol translocation. J Biol Chem 263:1295-1300, 1988 33. Johnson VG, Wrobel C, Wilson D, et al: Improved tumorspecific immunotoxins in the treatment of CNS and leptomeningeal neoplasia. J Neurosurg 70:240-248, 1989 34, Laird W, Groman N: Isolation and characterization of tox mutants of corynebacteriophage beta. J Virol 19: 220-227, 1976 35. Libermann TA, Nusbaum HR, Razon N, etal: Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 313:144-147, 1985 (Letter) 36. Libermann TA, Razon N, Bartal AD, et ah Expression of epidermal growth factor receptors in human brain tumors. Cancer Res 44:753-760, 1984 37. Morgan AC Jr, Sivam G, Beaumier P, et al: Immunotoxins of Pseudomonas exotoxin A (PE): effect of linkage on conjugate yield, potency, selectivity and toxicity. Mol lmmunol 27:273-282, 1990 38. Moseley RP, Davies AG, Richardson RB, et al: lntrathecal administration of ~321radiolabelled monoclona[ antibody as a treatment for neoplastic meningitis. Br J Cancer 62: 637-642, 1990 39. Nazzaro JM, Neuwelt EA: The role of surgery in the management of supratentorial intermediate and high-grade astrocytomas in adults. J Neurosurg 73:331-344, 1990 843
W. A. Hall, et al. 40. Neuwelt EA, Balaban E, Diehl J, et al: Successful treatment of primary central nervous system lymphomas with chemotherapy after osmotic blood-brain barrier opening. Neurosurgery 12:662-671, 1983 41. Neuwelt EA, Frenkel EP, Gumerlock MK, et al: Developments in the diagnosis and treatment of primary CNS lymphoma. A prospective series. Cancer 58:1609-1620. 1986 42. Neuwelt EA, Specht HD, Barnett PA, et al: Increased delivery of tumor-specific monoclonal antibodies to brain after osmotic blood-brain harrier modification in patients with melanoma metastatic to the central nervous system. Neurosurgery 20:885-895, 1987 43. Old LJ, Stockert E, Boyse EA, et al: Antigenic modulation. Loss of TL antigen from cells exposed to TL antibody. Study of the phenomenon in vitro. J Exp Med 127:523-539, 1968 44. Olsnes S: Toxic and nontoxic lectins from Abrus precatorius. Methods Enzymol 50:323-330, 1978 45. Pastan I, Willingham MC, FitzGerald DJP: Immunotoxins. Cell 47:641-648, 1986 46. Pearson JW, Hedrick E, Fogler WE, et al: Enhanced therapeutic efficacy against an ovarian tumor xenografi of immunotoxins used in conjunction with recombinant alpha-interferon. Cancer Res 50:6379-6388, 1990 47. Pressman BC: Biological applications of ionophores. Annu Rev Biochem 45:501-530, 1976 48. Ramakrishnan S, Houston LL: Inhibition of human acute lymphoblastic leukemia cells by immunotoxins: potentiation by chloroquine. Science 223:58-61, [984 49. Rasp V, Lawrence J: Carboxylic ionophores enhance the cytotoxic potency of ligand- and antibody-delivered ricin A chain. J Exp Med 160:1234-1240, 1984 50. Recht L, Tortes CO, Smith TW, etal: Transferrin receptor in normal and neoplastic brain tissue: implications for brain-tumor immunotherapy. J Neurosurg 72:941-945, 1990 51. Recht LD, Griffin TW, Rasp V, et al: Potent cytotoxicity of an antihuman transferrin receptor-ricin A-chain immunotoxin on human glioma cells in vitro. Cancer Res 50:6696-6700, 1990 52. Salcman M: The morbidity and mortality of brain tumors. A perspective on recent advances in therapy. Neurol Clin 3:229-257, 1985 53. Sandvig K, Olsnes S: Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. I. Requirement for calcium, d Biol Chem 257:7495-7503, 1982 54. Shapiro WR: Treatment of neuroectodermal brain tumors. Ann Neurol 12:231-237, 1982 55. Shindelman dE, Ortmeyer AE, Sussman HH: Demonstration of the transferrin receptor in human breast cancer tissue. Potential marker for identifying dividing cells. Int J Cancer 2"/:329-334, 1981 56. Shitara N, Kitamura K, Wada T, et al: Transferrin recep-
844
57.
58.
59. 60. 61. 62. 63.
64.
65.
66.
67.
tor molecules in human cell lines derived from CNS malignant gliomas: immunohistochemical and flow-cytometric study. Acta Histochem Cytochem 22:275-288, 1989 Thorpe PE, Ross WCJ, Brown ANF, et al: Blockade of the galactose-binding sites of ricin by its linkage to antibody. Specific cytotoxic effects of the conjugates. Eur J Biochem 140:63-71, 1984 Trowbridge IS, Domingo DL: Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumour cells. Nature 294: 171-173, 1981 Trowbridge IS, Newman RA, Domingo DL, et al: Transferrin receptors: structure and function. Biochem Pharmacol 33:925-932, 1984 Trowbridge IS, Shackelford DA: Structure and function of transferrin receptors and their relationship to cell growth. Biochem Soc Syrup 51:117-129, 1986 Vertosick FT Jr, Selker RG: Brain stem and spinal metastases of supratentorial glioblastoma multiforme: a clinical series. Neurosurgery 27:516-522, t990 Vitetta ES, Fulton RJ, May RD, etal: Redesigning nature's poisons to create anti-tumor reagents. Science 238: 1098-I 104, 1987 Walker MD, Alexander E Jr, Hunt WE, et at: Evaluation of BCNU and/or radiotherapy in the treatment of ariaplastic gliomas. A cooperative clinical trial. J Neurosurg 49:333-343, 1978 Walker MD, Green SB, Byar DP, et al: Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. N Engl J Med 303:1323-1329, 1980 Wikstrand CJ, Bigner SH, Bigner DD: Demonstration of complex antigenic heterogeneity in a human glioma cell line and eight derived clones by specific monoclonal antibodies. Cancer Res 43:3327-3334, 1983 Zovickian J, Johnson VG, Youle RJ: Potent and specific killing of human malignant brain tumor cells by an antitransferrin receptor antibody-ricin immunotoxin. J Neurosurg 66:850-86 l, 1987 Zovickian J, Youle RJ: Efficacy of intrathecal immunotoxin therapy in an animal model of leptomeningeal neoplasia. J Neurosurg 68:767-774, 1988
Manuscript received July 11, 1991. This work was supported by the Van Wagenen Fellowship of the American Association of Neurological Surgeons and the Norwegian Cancer Society. Address reprint requests to." Walter A. Hall, M.D., Department of Neurosurgery, University of Minnesota, Box 96 UMHC, 420 Delaware Street SE, Minneapolis, Minnesota 55455.
J. Neurosurg. / Volume 76 /May, 1992
In vitro efficacy of transferrin-toxin conjugates against
glioblastoma multiforme WALTER A. HALL, M.D., ASLAK GODAL, PH.D., Stm JUELL, AND ~u FODSTAD~ M.D., PH.D. Department of Tumorbiolog~; Institule for Cancer Research, The Norwegian Radium Hospital, and Hafslund Nycomed A/S, Oslo, Norway; and Department of Neurosurgery, University of Minnesota Hospital and Clinic, Minneapolis, Minnesota
L,- The cytotoxic activity of immunotoxins constructed with human diferric transferrin (Tfn) as the carrier ligand and an abrin variant Pseudomonas exotoxin A (PE) and the diphtheria toxin mutant cross-reacting material (CRM) 107 as the toxin moieties were studied in vitro. Three malignant human cell lines, the glioblastomas multiforme SNB19 and SF295 and the LOX melanoma, and a nonhuman control routine melanoma cell line B16 were assessed. The presence of transferrin receptors on the cell lines was confirmed by direct 12~I-Tfnbinding assays. The 50% protein synthesis inhibitory concentration (IC5o) values for all cell lines demonstrated that Tfn-abrin variant and Tfn-PE had comparable potency and were both more effective than Tfn-CRM 107. Monensin, a carboxylic ionophore, potentiated the effect of Tfn-abrin variant against glioma cells approximately 35-fold with IC50values of 4.0 • 10 -t3 M and 4.7 • 10-~2 M for SNBI9 and SF295, respectively. Cytotoxic activity of Tfn-abrin variant (with or without monensin) and Tfn-PE was correlated with the degree of Tfn receptor expression measured on the cell lines. The exquisite in vitro cytotoxicity of Tfn-abrin variant and Tfn-PE immunotoxins against glioma and melanoma cells warrants further in vivo evaluation and future consideration of these agents for potential clinical application against glioblastoma multiforme and leptomeningeal neoplasia. KEY WORDS brain neoplasm transferrin receptor
9
URRENX treatment of primary malignant tumors of the central nervous system (CNS), including surgery, radiation therapy, and systemic chemotherapy, has not improved the prognosis for patients with this disease. 33"39"51'52"54"63'64"66 The 2-year survival rate for glioblastoma multiforme is less than 20%. 12"64 The lack of specificity of conventional chemotherapy for malignant cells with severe dose-limiting side effects has led investigators to pursue the development of innovative treatment modalities? 4 Not until the advent of monoclonal antibody technology was interest renewed in the "magic bullet" concept of Paul Ehrlich: toxic compounds linked to carrier molecules specific for a target cell.4-j~ This interest has generated a new class of compounds called immunotoxins, tumor-specific monoclonal antibodies, or other ligands covalently linked to toxic proteins that combine exquisite cell-type selectivity with extraordinary potency. 4"13"24'31'33"45"62"66'67 To obtain a selective effect using immunotoxins, the target for this directed
C
838
brain-neoplasm antigen
9 immunotoxin
9
therapy should be preferentially expressed by neoplastic cells, compared to normal cells. 5~ Despite raising antibodies to human glioma-associated antigens, polyclonal and monoclonal serological methodologies have not identified antigens specific for this tumor type. 6~2 Explanations for the inability to identify tumor-specific antigens include the biological heterogeneity of malignant gliomas and the variable expression of antigens among and within tumors. 3'4'65 Although there are no antigens exclusively expressed by malignant glial cells, the transferrin receptor (TR) and the epidermal growth factor receptor (EGFR) or an amplified rearranged EGFR are present in higher numbers on surgical glioblastoma multiforme tissue samples and glioblastoma-derived tissue-culture cells than on normal CNS tissue, a9~8"23262735~50 The high requirement of rapidly dividing tumors, such as the glioblastoma multiforme, for iron should prevent antigenic modulation, 43 antigenic heterogeneity, 7 or genetic loss 28 of the TR, leaving it intact for immunotoxin therapy. J. Neurosurg. / Volume 76/May, 1992
Transferrin-toxin conjugates against glioblastoma Efficient internalization of TR's by cancer cells makes them an ideal target for immunotoxins/~~s Techniques for determining the TR presence and the degree of expression on glioblastoma multiforme have included radioimmunoassay, immunohistochemistry, flow cytofluorometry,, radioreceptor assay, slot blot ribonucleic acid (RNA) analysis, and in vitro cytotoxicity assays?"~'-'-':3'-~~These previous studies provide additive support for the TR as an appropriate target for transferrin (Tfn)-toxin conjugates until a more specific cellsurface antigen is identified for this tumor type]-' Here we describe the construction of three Tfn-toxin conjugates and demonstrate in three different malignant tissue-culture cell lines an efficacy that correlates with TR expression.
Materials and Methods
Chemicals Human Tfn was saturated with iron according to the method of Shindelman, et al? ~ Monensin* was added to the appropriate eytotoxicity bioassay wells to yield a final concentration of 10-7 M. Free sulfhydryl groups were generated on Tfn with 2-iminothiolane.t A stable thioether bond between Tfn and toxin was created using the bifunctional cross-linking agent, sulfo-N-succinimidyl-4-(N-malimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC).:]: 37 Toxins Whole abrin was extracted from Abrus precatorius and purified as previously described/4 A whole-abrin variant was used in the synthesis of abrin-based immunotoxins. Cross-reacting material (CRM) 107, a diphtheria toxin mutant,w was isolated by Laird and Groman 34 and purified by a previously described method. 32 Pseudomonas exotoxin A (PE)II is made by the bacterium Pseudomonas aeruginosa. Cell Lines Glioblastoma-derived tissue-culture cell lines SNB 19 and SF295 were used.** At the time of this study, SNBI9 was in its 237th passage and SF295 was in its 101st passage. The LOX melanoma cell line was established in our laboratory from a patient biopsy specimen. '4 Murine melanoma cell line BI6 was used as a negative control for human TR level expression experiments * Monensin obtained from Calbiochem, Lucerne, Switzerland. t 2-1minothiolane obtained from Pierce Chemical Co., Rockford, Illinois. SuIfo-SMCC obtained from Pierce Chemical Co., Rockford, Illinois. wCross-reacting material 107 obtained by courtesy of Dr. R. Youle, Surgical Neurology Branch, National Institutes of Health. [1Pseudomonas exotoxin A obtained from Swiss Serum and Vaccine Institute, Berne, Switzerland. ** SNBl9 and SF295 obtained from the National Cancer Institute Frederick Cancer Research Facility, Frederick, Maryland, by courtesy of Dr. R. Shoemaker.
J. Neurosurg. / Volume 76/May, 1992
and in cytotoxicity assays to Tfn-toxin conjugates. All cell lines were maintained at 37~ as monolayer cultures in RPMI 1640 mediumt containing 10% fetal calf serum (FCS), 2 mM glutamine, 1000 IU/ml of penicillin, and 100 ug/ml of streptomycin.
Tran,sjbrrin Receptor Determination on TissueCulture CWI Lines The amount of TR expressed on the surface of the cells was determined by incubating a suspension of i x 10~'cells in 0.5 ml of phosphate-buffered saline (PBS; 7 mM phosphate buffer, pH 7.4, with 0.14 M NaC1) containing human serum albumin (1 mg/ml) with ~251labeled human Tfn,~ 1 x 105 cpm at 4~ for 4 hours. The reaction was stopped by centrifugation, the cells were washed, and the cell-bound radioactivity was counted in a gamma counter.w Synthesis and Pm4fication ~/ lmmunotoxins Transferrin was conjugated to CRM 107 after generating tree sulfhydryl groups on Tfn with 2-iminothiolane dissolved in 0.1 M sodium borate (pH 8.5), by incubating at room temperature for 1 hour with Tfn in an 8:1 molar ratio. Free 2-iminothiolane was separated from the modified Tfn by gel filtration on a Sephadex G-25 column equilibrated with PBS. After dissolution in H20, sulfo-SMCC was added to the CRM 107 in a 5:1 molar excess for 30 minutes at room temperature. The abfin variant and PE conjugates were prepared in the same manner with a molar excess of toxin in a 10:1 ratio. 37 The sulfo-SMCC-conjugated toxin was separated on a Sephadex G-25 column, mixed with thiolated Yfn in a 1:1.3 molar ratio, and incubated overnight at room temperature, Purification was performed by gel filtration on Sephacryl S-200 with 0.1 M sodium phosphate buffer (pH 7.0) to remove excess toxin. The pooled peak fractions of the immunotoxin were used for all cytotoxicity experiments. To remove abrin variant conjugates with exposed binding sites, the immunotoxin was passed through a Sepharose 4B column as described by Thorpe, el al. 57 The final concentrations of the immunotoxins, determined in the protein assay, ll were 0.18 mg/ml for Tfn-abrin variant, 0.1 mg/ml for Tth-CRM 107, and 0.06 mg/ml for Tfn-PE. Qvtotoxicity Assa)z~ The cytotoxic effect of the immunotoxins on the tissue-culture cell lines was assessed by measuring their ability to inhibit protein synthesis. Cells growing in monolayer culture were treated for 5 minutes with 10 mM ethylenediamine tetra-acetic acid in PBS containing t RPMI 1640 medium manufactured by G1BCO Biocult, Glasgow, Scotland. Human transfcrrin obtained from Amersham International, Ltd., Amersham, Buckinghamshire, England. wGamma counter manufactured by Beckman Instruments, Irvine, California. 1IProtein assay system manufactured by Bio-Rad Laboratories, Munich, Germany. 839
W. A. Hall, et al. 0.05% KCI to obtain a suspension. After washing, the cells were diluted to a concentration of 5 • 104 cells/ ml with RPMI medium containing 10% FCS and 20 mM HEPES, pH 7.2. One ml of cell suspension was added to each well of a 24-well tissue-culture tray, and the cells were incubated for 3 hours at 37~ to obtain a confluent monolayer. Transferrin, immunoconjugate, or control solution was added to the cells in duplicate wells and incubated for 20 hours at 37~ Monensin was added to the appropriate wells in a final concentration of 10-7 M before overnight incubation. The cells were washed once with PBS and leucine-free medium before 1 uCi [3H]-leucine/ml ( 131 mCi/mmol) was added. After incubation for 30 minutes at 37~ the cells were processed and the trichloroacetic acid-precipitable radioactivity was measured and compared to untreated control cell cultures, as previously described. ~9.53The concentration required to inhibit 50% of protein synthesis (ICs0) was derived from dose-effect curves. All cytotoxicity assays were performed two to five times in duplicate. Results
Binding of r25I-Labeled Transferrin to TissueCulture Cell Lines A noncompetitive radioreceptor assay (as described above) was used to detect the presence of TR on the tissue-culture cell lines. The results are presented in Table 1, with cell lines arranged in decreasing order of antigen expression as measured by the binding of ~25ITfn. Both glioblastoma-derived cell lines SNB19 and SF295 had significant levels of TR expression at 4.9% and 2.7%, respectively, whereas the LOX melanoma cell line had a level of 3.1%. As expected, murine melanoma cell line BI6 bound very low amounts of ~25I-Tfn; levels of TR expression under 1% may represent either nonspecific binding or homology of the TR between species.
Cytotoxic Activity of Transferrin- Toxin Conjugates on Tissue-Culture Cell Lines The plant toxin abrin inhibits protein synthesis by inactivating ribosomes in a manner similar to that of ricin. 24'44 Alternatively, toxins derived from bacteria such as PE and CRM 107 block protein synthesis by catalyzing the transfer of adenosine diphosphate-ribose to elongation factor-2, resulting in discontinuation of the addition of amino acids to the growing polypeptide chain. 33 The action of these toxins is ultimately lethal to cells susceptible to toxin entry. A measurement of [3H]-leucine incorporation into the acid-insoluble fraction of cells exposed to different concentrations of Tfn-toxin conjugates determines their effect on protein synthesis and allows for the calculation of inhibitory concentration (IC) values. The ICs0 value represents the molar concentration of immunotoxin that inhibits 50% of protein synthesis. 840
TABLE 1
Binding ~?f~25I-tranfferrin to tissue-culture cell lines* Cell Line
Origin
% Bound
SNB 19 LOX SF295 B 16
human glioblastoma human melanoma human glioblastoma routine melanoma
4.9 3.1 2.7 0.8
* Expressed as % bound (cpm)/total (cpm) of ~2sI-transferrinincubated with differentcell lines for 4 hours at 4~
Iron-loaded Tfn, in the same concentrations as the Tfn-toxin conjugates, did not inhibit protein synthesis (data not shown), and concentrations of Tfn greater than 1 mg/ml were necessary to obtain toxicity in all cell lines. The least effective immunotoxin in the cell lines tested was Tfn-CRM 107. The cell line most susceptible to this conjugate was SNBI9 (ICs0 3.5 x 10-~~ M). Transferrin-PE was 20 times more potent than Tfn-CRM 107 in SNBI9 cells, which were 27 times more sensitive to Tfn-abrin variant than to TfnCRM 107. Immunoconjugates made with PE and abrin variant toxins had very similar ICso values (Table 2) and were very effective against SNBI9 cells, with ICs0 values of 1.7 • 10-t~M and 1.3 • 10-~ M, respectively. A concentration of 1.7 • 10-~~ M for Tfn-PE and for Tfn-abrin variant was equally potent for the SF295 cell line. The LOX melanoma cell line demonstrated almost identical sensitivites to Tfn-PE (ICso 4.1 • 10-~ M) and Tfn-abrin variant (ICs0 4.7 • 10-~j M) immunotoxins. A 20- to 250-fold difference in cytotoxieity to Tfnabrin variant and a 12- to 120-fold difference to TfnPE was seen between B16 cells and the other cell lines when exposed to the Tfn-abrin variant conjugate alone. The fact that the immunotoxins made with human Tfn were cytotoxic to the murine melanoma cell line B16 may represent either antigenic cross-reactivity of the TR between species or nonspecific binding. Adding monensin to the cytotoxicity bioassays enhanced the activity of the Tfn-abrin variant immunotoxin in all cell lines. The degree of potentiation is indicated in Table 2 as the potentiation factor. For LOX melanoma and both SNB 19 and SF295 glioma cell lines, the potentiation factor ranged from 33 to 36. In contrast to previous reports, 6,7 monensin had virtually the same effect on each human cell line we studied. In SNB 19 cells, monensin improved the cytoxicity of Tfnabrin variant 43-fold over Tfn-PE and 875-fold over Tfn-CRM 107. The cytotoxic effect of Tfn-abrin variant immunotoxins in the SNB19 and SF295 glioma cell lines, with and without monensin, is demonstrated in Fig. 1. Cytotoxic effects of the Tfn-abrin variant, with or without monensin, and Tfn-PE immunotoxins correlated directly with the degree of TR expression on LOX, SNBI9, SF295, and B16 cell lines as demonstrated in Tables 1 and 2. Transferrin receptor expression was greatest on the cell line most sensitive to abrin variant and PE immunotoxins, namely SNB19. The mouse
J. Neurosurg. / Volume 76/May, 1992
Transferrin-toxin conjugates against glioblastoma TABLE 2 7?zwcay (lCs,~ value,s? (?[lran.~[errin-toxitt cr Tumor Cells
Tfn-CRM 107
SNBI9 LOX SF295 B16
3.5 • >7.0X > 7,0 • a.9 x
10 -~i' 10 ~ 10-t~ 10-~~
ffn-PE 1.7 4.1 1.7 2.0
• x x x
10-~t 10-~t 10 lo 10 9
on li.~,sue-cu/ture cell lines*
Tfn-Abrin Variant t.3 • 4.7X 1.7 • 3.3 x
10 ii I0 -H 10 1o I0 9
Tfn-Abrin Variant + Mo 4.0 • 1.3X 4.7 x 2.0 x
10 ~3 10 -~2 10 12 10-~t
Potentiation Factor 33 35 36 167
* lCso is the molar concentration of immunotoxin inhibiting 50% of protein synthesis. Tfn = transferrin; PE = Pseudomonas exotoxin A; Mo = monensin (10 -7 M); potentiation factor = the increase in cytotoxicily with added monensin (IC5o Tfn-abrin variant/ICs0 Tfn-abfin variant plus monensin).
melanoma B I6 had the lowest TR expression and the least susceptibility to Tfn-abrin variant and Tfn-PE. No correlation was seen between TR expression and cytotoxicity in any cell line assayed with Tfn-CRM 107. Discussion
Expression of TR, a transmembrane glycoprotein that mediates cellular iron uptake, is regulated by replication. ~9'6~ Thus, proliferating cells express greater numbers of TR's than do nondividing cells? 1.59.66Using immunohistochemistry, Gatter, et al., ~7 demonstrated TR expression on human surgical tissue samples of adenocarcinomas, squamous-cell carcinomas, germ-cell tumors, sarcomas, and Hodgkin's and non-Hodgkin's lymphomas. The basal epidermis, endocrine pancreas, hepatocytes, Kupffer cells, testis, and pituita~' gland also expressed TR's. iv Others have found ve~' high TR levels on certain tumors.l ~,~6.55 Glioblastoma multiforme, a rapidly dividing primary, malignant brain tumor, has been shown by many investigators to express high numbers of TR's in vitro and in vivo. 9"22"23"335~ Transferrin receptors have
FIG. [. Dose-response curves showing cytotoxic effects of transferrin-abrin variant immunotoxins on glioblastoma-derived tissue-culture cell lines SNBt9 (squares) and SF295 (circles), with (open symbols') and without (closed symbols) monensin. Protein synthesis was estimated by measuring the incorporation of I uCi [SH]-leucine. J. Neurosurg. / Volume 7 6 / M a y , 1992
been identified on glioblastoma multiforme tissue samples by immunohistochemistry, a radioreceptor assay, and solid-phase indirect radioimmunoassay) z5~ In addition to other techniques that have detected TR's on glioma cells in vitro, 95~'-~6 the direct binding of radiolabeled Tfn to the SNB19 and SF295 glioma cell lines in this study indicates TR expression on the cell surface. The differential expression of TR on glioblastoma compared to normal brain tissue suggests that TR's may be a suitable target for Tfn-toxin therapy. 22~23'66 Moreover, the binding of 12~l-Tfn to LOX melanoma provides suggestive evidence for the presence of TR's on this tumor. The propensity for melanoma to metastasize to the CNS as discrete mass lesions or as carcinomatous meningitis provides the rationale for evaluating this tumor type as a potential candidate for immunotoxin therapy. 538 In prior in vitro studies against glioblastoma-defived cell lines, immunotoxins targeted to TR's were constructed with either Tfn or a murine monoclonal antibody as the binding ligand, linked to the toxin component. 9.33's~'reToxins used previously to create effective immunotoxins against glioma cells have included intact ricin, ricin A chain, and a point-mutated diphtheria toxin CRM 107.9'21"33"51 Zovickian, el al., 66 reported that an anti-TR-ricin immunoconjugate killed more than 50% of glioma cells after 18 hours at a concentration of 5.6 x 10 - ~ M , in the presence of the earboxylic ionophore monensin. Against five glioma cell lines, Recht, et al., 51 demonstrated potent cytotoxicity of an anti-TR monoclonal antibody-ricin A chain conjugate at concentrations as low as 7.0 x 10-'2 M, with monensin. Monensin decreased 16- to 842-fold the IC~0 of the ricin A chain immunotoxin against glioma cells. As reported by Johnson, et al., 32 immunotoxins composed of the anti-TR monoclonal antibody 454A12 and ricin A chain or CRM 107 demonstrated comparable cytotoxicity against the most sensitive glioma cells tested at concentrations of 3.6 x 10-~ M and 1.6 x 10-j~ M, respectively. The toxin CRM 107 coupled to Tfn was more effective against glioma ceils than either antibody-toxin conjugate with an IC5o value of 2.6 x 10-12 M. According to Colombatti, el al., g Tfn linked to ricin A chain showed high cytotoxic activity, being 5000 times more toxic than the A chain alone, in the presence of monensin. 841
W. A. Hall, el al. Of interest in the present study is the difference in efficacy of our Tfn-CRM 107 in SNB19 and SF295 compared to that reported by Johnson, el al., ~2 in the SNB75, SNB101, and U251 glioma-derived celt lines. This difference in sensitivity to Tfn-CRM 107 between glioma cell lines may be due to a difference in TR expression. Conjugates made with PE and abrin variant, without monensin, as the toxin components had similar cytotoxic activities against malignant melanoma- and glioma-derived cell lines. Our Tfn-PE conjugate demonstrated comparable cytotoxicity in our glioma cell lines when compared to other reported immunotoxins in different glioma cells. 9"32"51 In the presence of monensin, SNB19 and SF295 cells were more susceptible to Tfn-abrin variant than were other glioma lines tested by Zovickian, et al., 66 Recht, el al., 5~ Johnson, e t a l . , or Colombatti, et al., 9 where Tfn and anti-TR monoclonal antibodies were used as the cartier ligands and ricin, ricin A chain, and CRM 107 were the toxin moieties. Although the mechanism by which monensin potentiates plant-derived abrin and ricin immunotoxins is unclear, disruption of vesicle movement in cells and alteration of endosomal pH have been implicated. T M Other lysosomotropic agents that allow easier access of immunoconjugates to the cytosol include chloroquine4s and the calcium channel-blocking agents verapamil and diltiazem. Abrin immunotoxins have been shown to be more toxic to melanoma cell lines than the corresponding ricin conjugate despite similarities between structure and mechanism of action? 9 Before and after the addition of monensin, our Tfn-abrin variant immunotoxin was approximately 100-fold more toxic to glioma cells than was the Tfn-ricin A chain conjugate reported by Colombatti, et al? Abrin- and ricin-based immunotoxins have the advantage over PE- and CRM 107-based immunotoxins against glioblastoma in that their activity can be potentiated by monensin, as we and others have illustrated. Furthermore, prior immunization of the adult population may result in neutralizing antibodies that would inhibit immunotoxins made with diphtheria toxin-derived CRM 107. Many studies demonstrating the efficacy of immunotoxins against glioblastoma-derived tissue-culture cells have used a murine anti-human TR monoclonal antibody as the carrier ligand. 32-5L66As previously done by Johnson, et al.,32 and Colombatti, e t a l . , 9 we have constructed our conjugates with a Tfn carrier to presumably increase transvascular and intratumoral diffusion by decreasing molecular weight and size. A reduction in size may facilitate penetration of toxin conjugates into malignant brain-tumor tissue, after either intrathecal, intratumoral, or intra-arterial delivery, resulting in improved clinical efficacy. Blood-brain barrier modification with the osmotic agent mannitol, as reported in the treatment of primary malignant brain tumors with chemotherapeutic drugs and metastatic melanoma to the CNS with monoclonal antibodies, may improve the distribution of immunotoxins into tu842
mot after intra-arterial administration? ~176 Agents such as monensin that enhance immunotoxin entry into the cytosol can be administered simultaneously into the cerebrospinal fluid (CSF) with conjugates utilizing abrin or ricin toxin moieties to enhance the cytotoxic effect. 8"49 Although there are few examples of the in vivo treatment of CNS malignancy, early results against leptomeningeal neoplasia are encouraging. Extended survival was reported by Zovickian and Youle ~7 in guinea pigs treated with an anti-idiotype monoclonal antibody (M6)-intact ricin immunotoxin delivered intrathecally 24 hours after delivery of L2C leukemia cells into the cisterna magna. Fuchs, et al., 15 established an animal model of human neoplastic meningitis in the athymic nude rat using a glioma cell line. Intrathecal instillation of 4-hydroperoxycyclophosphamide 5 days after tumor cell inoculation prolonged median survival by 23%. ~5 Dissemination of glioblastoma multiforme throughout the neuraxis, although an infrequent occurrence, has been demonstrated in younger patients and in patients with extended survival. 6j Toxicity trials in guinea pigs have shown a maximum tolerated intrathecal Tfn-CRM 107 dose of 2 x 10-9 M. 32 This same dose of immunotoxin, administered intrathecally to rhesus monkeys, was nontoxic and represented a CSF concentration 20- to 5000-fold higher than the effective in vitro dose in glioma cells, suggesting that a significant therapeutic window may exist for immunotoxin treatment of leptomeningeal neoplasia. Recombinant c~-interferon enhances the action of immunotoxins both in vitro and in v i v o 46 and could be coinstilled with immunotoxins into the intrathecal space of patients with carcinomatous meningitis. Intratumoral administration of a-interferon and immunotoxins into glioblastoma multiforme represents a potential form of immunotherapy, similar to that already reported with interleukin-2 and lymphokine-activated killer cellsY 9'3~Previously mentioned lysosomotropic agents may be used in combination with immunotoxins if intrathecal or intratumoral delivery is considered. Future directions for determining the in vivo efficacy of our Tfn-toxin conjugates include the treatment of human neoplastic meningitis, established in animal models. ~s Preliminary results from our laboratory in a nude rat model of meningitis due to LOX melanoma suggest that Tfn-PE given intrathecally can delay the onset of paraplegia to a statistically significant degree. The demonstration of a therapeutic effect in animals will add support for the clinical application of these agents in Phase I and II clinical trials against CNS neoplasia, in the form of carcinomatous meningitis or primary malignant brain tumors, where present therapeutic options are limited and alternatives to the conventional treatment of these diseases are clearly needed.
Acknowledgment We express our thanks to Dr. Richard J. Youle, Chief, Biochemistry Section, Surgical Neurology Branch, National J. Neurosurg. / Volume 7 6 / M a y , 1992
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Manuscript received July 11, 1991. This work was supported by the Van Wagenen Fellowship of the American Association of Neurological Surgeons and the Norwegian Cancer Society. Address reprint requests to." Walter A. Hall, M.D., Department of Neurosurgery, University of Minnesota, Box 96 UMHC, 420 Delaware Street SE, Minneapolis, Minnesota 55455.
J. Neurosurg. / Volume 76 /May, 1992
