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[Cancer Research 61, 4048-4054, May 15, 2001]
© 2001 American Association for Cancer Research

Immunology

Monoclonal Antibody 8H9 Targets a Novel Cell Surface Antigen Expressed by a Wide Spectrum of Human Solid Tumors1

Shakeel Modak, Kim Kramer, S. Humayun Gultekin, Hong Fen Guo and Nai-Kong V. Cheung2

Departments of Pediatrics [S. M., K. K., H. F. G., N-K. V. C.] and Pathology [S. H. G.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor-restricted surface antigens may be targets for diagnosis and immune-based therapies. Monoclonal antibody 8H9 is a murine IgG1 hybridoma derived from the fusion of mouse myeloma SP2/0 cells and splenic lymphocytes from BALB/c mice immunized with human neuroblastoma. By immunohistochemistry, 8H9 was highly reactive with human brain tumors, childhood sarcomas, and neuroblastomas, and less so with adenocarcinomas. Among primary brain tumors, 15 of 17 glioblastomas, 3 of 4 mixed gliomas, 4 of 11 oligodendrogliomas, 6 of 8 astrocytomas, 2 of 2 meningiomas, 3 of 3 schwannomas, 2 of 2 medulloblastomas, 1 of 1 neurofibroma, 1 of 2 neuronoglial tumors, 2 of 3 ependymomas, and 1 of 1 pineoblastoma tested positive. Among sarcomas, 21 of 21 Ewing’s/primitive neuroectodermal tumor, 28 of 29 rhabdomyosarcomas, 28 of 29 osteosarcomas, 35 of 37 desmoplastic small round cell tumors, 2 of 3 synovial sarcomas, 4 of 4 leiomyosarcomas, 1 of 1 malignant fibrous histiocytoma, and 2 of 2 undifferentiated sarcomas tested positive with 8H9. Eighty-seven of 90 neuroblastomas, 12 of 16 melanomas, 3 of 4 hepatoblastomas, 7 of 8 Wilms’ tumors, 3 of 3 rhabdoid tumors, and 12 of 27 adenocarcinomas also tested positive. In contrast, 8H9 was nonreactive with normal human tissues including bone marrow, colon, stomach, heart, lung, muscle, thyroid, testes, pancreas, and human brain (frontal lobe, cerebellum, pons, and spinal cord). Reactivity with normal cynomolgus monkey tissue was restricted similarly. Indirect immunofluorescence localized the antigen recognized by 8H9 to the cell membrane. The antigen is proteinase sensitive and is not easily modulated off the cell surface. 8H9 immunoprecipitated a Mr 58,000 band after N-glycanase treatment, most likely a protein with a heterogeneous degree of glycosylation. This novel antibody-antigen system may have potential for tumor targeting.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
mAbs3 such as 3F8 (1) and 14.18 (2) against GD2 in neuroblastoma, M195 against CD33 in acute leukemia (3) , anti-HER2 antibodies in breast cancer (4) , and anti-CD20 antibodies in lymphoma (5) have shown efficacy in recent clinical trials. The prognosis in glial brain tumors and metastatic mesenchymal and neuroectodermal tumors remains dismal despite innovations in chemotherapy and radiation therapy. Immunotherapy may offer new possibilities for improving the outcome in these patients.

Tumor antigens expressed on cell membranes are potential targets in immunotherapy. Examples of tumor antigens expressed on glial tumors include NCAMs (6) , gangliosides such as GD2 and GM2 (7) , and neurohematopoietic antigens (8) . Recent investigations have focused on growth factor receptors as immune targets, in particular EGFRvIII, which has been shown to be expressed on 50% of glial brain tumors (9) . Notwithstanding the universal expression of NCAM by neuronal cells, two clinical studies have used anti-NCAM antibodies in patients. mAb UJ13A was shown to accumulate in gliomas by virtue of disruption of the blood brain barrier locally (10) , and another antibody, ERIC-1, was used in a therapeutic setting in resected glioma cavities with some clinical benefit (11) .

Recent studies have targeted immunotherapy to the extracellular matrix around tumor cells. Tenascin has been reported to be expressed in 50–95% of glial brain tumors, as well as on mesenchymal tumors, carcinomas, and normal human glial, liver, and kidney cells (12) . Antitenascin mAbs 81C6 (13) , BC-2, and BC-4 (14) , administered intracavity, have been reported recently to show efficacy in the treatment of patients with malignant gliomas. However, because these antigens are also present to varying degrees on normal human neural and non-neural cells, their clinical utility would depend on their overexpression by brain tumors when compared with normal tissues. With the exception of EGFRvIII, the glial tumor antigens described to date are generally found on normal brain tissue or are restricted to intracellular compartments, thus with limited clinical utility for antibody targeting.

Membrane antigens that have been targeted on osteosarcoma include GD2 (15) , CD55 (16) , and an as-yet-undefined osteosarcoma-associated antigen recognized by the mAbs TP-1 and TP-3 (17) . However, these antigens are present to varying degrees on normal tissues. Similarly, the glycoprotein p30/32 coded by the MIC2 oncogene and recognized by the mAb O13 in the Ewing’s family of tumors is expressed on normal tissues (18) . In rhabdomyosarcoma, the MyoD family of oncofetal proteins is nuclear in localization (19) and therefore inaccessible to antibody-targeted immunotherapy.

An ideal tumor antigen for targeted immunotherapy should be absent on normal tissues and abundantly expressed on tumor cell surfaces. Such tumor-specific antigens, e.g., idiotypes in B-cell lymphoma, are rare (20) . Moreover, a generic tumor-specific antigen expressed on tumor cells of varying lineage recognized by mAbs may have broader utility in antibody-based strategies. We describe here a novel tumor-associated antigen, recognized by a murine mAb, 8H9, expressed on cell membranes of a broad spectrum of tumors of neuroectodermal, mesenchymal, and epithelial origin, with restricted distribution on normal tissues.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor and Normal Tissue Samples.
Frozen tumors from 330 patients with neuroectodermal, mesenchymal, and epithelial neoplasia were analyzed. All of the diagnoses of tumor samples were confirmed by H&E assessment of paraffin-embedded specimens. Fifteen normal human tissue samples and eight normal cynomolgus monkey tissue samples obtained at autopsy were also analyzed.

Cell Lines.
The human neuroblastoma cell line LAN-1 was provided by Dr. Robert Seeger (Children’s Hospital of Los Angeles, Los Angeles, CA). Human neuroblastoma cell lines LA-1-15-N, LA-1-66-N, LA-1-5-S, LA-1-19-S, and LA-1-19-N were provided by Dr. Robert Ross (Fordham University, New York, NY), and IMR32 and NMB7 by Dr. Shuen-Kuei Liao (McMaster University, Hamilton, Ontario, Canada). Breast carcinoma cell lines SW480 and ZR75-1 were provided by Dr. S. Welt (Memorial Sloan-Kettering Cancer Center) and the melanoma line SKMel28 by Dr. P. Chapman (Memorial Sloan-Kettering Cancer Center). Neuroblastoma cell lines SKNHM, SKNHB, SKNJD, SKNLP, SKNER, SKNMM, SKNCH, and SKNSH; rhabdomyosarcoma cell line SKRJC; and Ewing’s/PNET cell lines SKPPR, SKPRT, and SKNMC were derived from patients with metastatic disease treated at Memorial Sloan-Kettering Cancer Center. The following cell lines were purchased from American Type Culture Collection (Bethesda, MD): melanoma cell lines HTB63 and HTB67; rhabdomyosarcoma cell line HTB82; small cell lung cancer cell line HTB 119; acute T-leukemia cell line Jurkat; glioblastoma multiforme cell line Glio72; breast cancer cell line HTB22; colon carcinoma cell line SK Co-1; HeLa; embryonal kidney 293; and osteosarcoma cell lines CRL1427, HTB86, and HU20596. All of the cell lines were grown at 37°C in a 5% CO2 incubator using standard culture medium, which consisted of RPMI 1640 supplemented with 10% bovine calf serum, 2 mM glutamine, 100 IU/ml of penicillin, and 100 µg/ml of streptomycin. Normal human hepatocytes were purchased from Clonetics (San Diego, CA) and processed immediately upon delivery. Normal human mononuclear cells were prepared from heparinized bone marrow samples by centrifugation across a Ficoll-Hypaque density separation gradient. EBV lymphoblastoid cell lines were derived from human mononuclear cells.

mAb.
Female BALB/c mice were hyperimmunized with human neuroblastoma according to methods outlined previously (21) . Lymphocytes derived from these mice were fused with SP2/0 mouse myeloma cell line. Clones were selected for specific binding on ELISA. The 8H9 hybridoma secreting an IgG1 mAb was selected for further characterization after subcloning.

Immunohistochemical Studies.
Eight-µm sections from fresh frozen tumor were fixed in acetone and washed in PBS. Immunohistochemical studies were performed as described previously (22) . Endogenous peroxidases were blocked in 0.3% H2O2 in PBS. Sections were incubated in 10% horse serum (Life Technologies, Inc., Gaithersburg, MD) after blocking with avidin and biotin. Incubation with purified 8H9 (2 µg/ml) in PBS was carried out at room temperature for 1 h. An IgG1 myeloma was used as a control (Sigma Chemical Co., St. Louis, MO). Sections were incubated with a secondary horse antimouse biotinylated antibody (Vector Laboratories, Burlingame, CA), followed by incubation with avidin-biotin complex (Vector), and developed with Vector VIP peroxidase substrate or 3,3'-diaminobenzidine peroxidase substrate kit (Vector). A 10% hematoxylin counterstain was used for 4 min. Staining was graded as positive or negative, and homogeneous or heterogeneous reactivity was noted.

Indirect Immunofluorescence.
One million target cells were washed in PBS and then spun at 180 x g for 5 min. The pellets were then reacted with 100 µl of 15 µg/ml of 8H9 at 4°C for 1 h. After washing the cells with PBS, they were allowed to react with 100 µl of FITC-conjugated goat antimouse IgG + IgM F (ab')2 (Biosource International, Camarillo, CA) at 4°C. Flow cytometric analysis was performed using FACSCalibur Immunocytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).

To study loss of antigen after binding to 8H9, 106 NMB7 and U2OS cell pellets were prepared as above and reacted with 100 µl each of 15 µg/ml of 8H9 or the anti-HLA-A,B,C antibody, HB-95 (American Type Culture Collection), at 4°C for 1 h. NMB7 cells were also similarly reacted with the anti-GD2 mAb 3F8. After washing with PBS, cells were cultured at 37°C in standard culture medium for 0, 1, 2, 4, 8, 12, 24, 36, and 48 h. They were then reacted with FITC-conjugated secondary antibody goat antimouse IgG + IgM F (ab')2 (Biosource International) at 4°C. Flow cytometric analysis was performed. Geometric mean immunofluorescence was compared with that of control cells incubated for similar time intervals in standard culture medium in the absence of mAbs and then immunostained with HB-95 (U2OS) or 3F8 (NMB7).

Antigen sensitivity to proteinase was tested by incubating 0.5 x 106 of HTB82, U2OS, and NMB7 cells at 37°C for 30 min in RPMI 1640 with increasing concentrations of neutral proteinase, Pronase E from streptomyces griseus (E. Merck, Darmstadt, Germany). After washing, cells were stained with 8H9 or 3F8 and studied by indirect immunofluorescence.

Immunoprecipitation.
Immunoprecipitation was carried out using a modification of the standard technique (23) . 8H9-positive cell lines (NMB7, LAN-1, HTB82, U2OS, HELA, and 293) and 8H9-negative cell lines (Jurkat and HTB119) were used. Two x 107 viable cells were washed in Tris-buffered saline [TBS; 0.05 M Tris-HCl (pH 8) with 0.15 M NaCl] and incubated with 10 units of lactoperoxidase (Sigma Chemical Co.), 100 µl of 100 units/ml in TBS, 1 mCi of 125I (2.7 µl), and 1/6000 dilution of 30% hydrogen peroxide for 5 min at 20°C. Five units of lactoperoxidase (50 µl) and the same dilution of hydrogen peroxide (50 µl) were added every 3 min with mixing for a total of three times. The cells were washed extensively in TBS containing 2 mg/ml of NaI. The iodinated cells were washed three times in TBS and lysed on ice (30 min) in 500 µl of modified RIPA buffer [0.01 M Tris-HCl (pH 7.2), 0.15 M NaCl, 1% sodium deoxycholate, 1% NP40, 0.1% SDS, and 0.01 M EDTA] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 µg/ml bestatin, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 10 µg/ml of E-64). The lysates were clarified by centrifugation at 15,000 rpm for 5 min at 4°C and then incubated with 1 mg of 8H9 or IgG1 control antibody for 16 h at 4°C with mixing. The antigen-antibody complex was collected by adsorption onto 100 µl of protein G-Sepharose beads (Sigma Chemical Co.) for 6 h at 4°C. The immune complex immobilized on protein G was washed three times with modified RIPA buffer, washed once with RIPA buffer containing 1 M NaCl, and then washed twice with modified TNN buffer [0.05 M Tris-HCl (pH 8), 0.15 M NaCl, and 0.05% NP40]. Bound proteins were removed by elution with SDS-sample buffer [0.03 M Tris-HCl (pH 6.8), 5% Glycerol, 1% SDS, 2.5% 2ß-Mercaptoethanol, 0.001% Bromophenol blue] and analyzed by 7.5% SDS-PAGE, followed by autoradiography. Deglycosylation of the radiolabeled antigen was carried out on the protein G-Sepharose using N-glycanase (Glyco, Novato, CA) and O-glycanase (Glyco) according to manufacturer’s instructions. Molecular weight was estimated using Quantity One software from Bio-Rad, Inc. (Hercules, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical Studies.
Frozen sections from 330 tumors with histologically confirmed diagnoses of cancer were analyzed for immunoreactivity with mAb 8H9 (Tables 1Citation and 2Citation ). Fifteen histologically normal human tissues and eight normal monkey tissues were also analyzed (Table 3)Citation .


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  		Table 1 8H9 reactivity: Neuroectodermal tumors

 

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  		Table 2 8H9 reactivity: Mesenchymal, epithelial and other tumors

 

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  		Table 3 8H9 reactivity in normal human and cynomolgus monkey tissues

 
Heterogeneous, nonspecific cytoplasmic staining was noticed in normal human pancreas, stomach, liver, and adrenal cortex, which was diminished when 8H9 F(ab')2 fragments were used instead of the whole antibody for immunostaining (data not shown). None of the other human tissues showed reactivity with 8H9. In particular, normal human brain tissue sections including frontal lobe, spinal cord, pons, and cerebellum were completely negative. Normal tissues from cynomolgus monkey also demonstrated similarly restricted reactivity with nonspecific staining observed in stomach and liver (Table 3)Citation .

The majority of neuroectodermal and mesenchymal tumors tested showed positive reactivity with 8H9, and epithelial tumors showed positive reactivity to a lesser extent. 8H9 immunoreactivity was seen in a characteristic, homogeneous, cell membrane distribution in 286 of the 330 (87%) tumor samples examined (Fig. 1)Citation . Eighty-eight % of neuroectodermal tumors, 94% of mesenchymal tumors, and 44% of epithelial tumors tested positive with 8H9 (Tables 2Citation and 3Citation ).



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  		Fig. 1. A, desmoplastic small round cell tumor (x40) immunostained with 8H9 showing strong membrane positivity and typical histology. B, glioblastoma multiforme (x40) stained with 8H9 showing binding to cell membranes and fibrillary stroma. C, embryonal rhabdomyosarcoma (x40) stained with 8H9 showing cell membrane reactivity. D, negative staining of embryonal rhabdomyosarcoma (x40) with MOPC21, an irrelevant IgG1 control antibody

 
Indirect Immunofluorescence.
8H9 immunoreactivity in 35 neuroblastoma, melanoma, rhabdomyosarcoma, small cell lung cancer, osteosarcoma, glioblastoma, leukemia, breast cancer, and colon cancer cell lines was tested using indirect immunofluorescence. Moderate to strong cell membrane reactivity (Fig. 2)Citation with 8H9 was detected in 16 of 16 neuroblastoma cell lines, 3 of 3 melanoma cell lines, 2 of 2 rhabdomyosarcoma cell lines, 1 of 1 glioblastoma multiforme cell line, 3 of 3 breast cancer cell lines, and 1 of 1 colon cancer cell line studied. Two of three Ewing’s/PNET cell lines and 2 of 3 osteosarcoma cell lines were strongly positive, whereas the others showed weak positivity. The small cell lung cancer cell line tested negative with 8H9, as did the Jurkat T-cell acute lymphocytic leukemia cell line and EBV-transformed lymphoblastoid cells. Normal human bone marrow mononuclear cells and hepatocytes had no reactivity with 8H9 (Table 4)Citation . In the neuroblastoma cell lines studied, indirect immunofluorescence with 8H9 was weaker (mean fluorescence, 73.73; negative control, 3.95) when compared with the anti-GD2 antibody 3F8 (mean fluorescence, 249.95).



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  		Fig. 2. Indirect immunofluorescence staining of osteosarcoma cell line CRL 1427 with 8H9. A, confocal microscope image (x2000) showing cell membrane immunoreactivity. B, histogram comparing 8H9 (bold line) mean geometric fluorescence (Y axis) with nonspecific IgG1 mAb MOPC21.

 

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  		Table 4 8H9 reactivity with cell lines by indirect immunofluorescence

 
8H9 binding to U2OS as detected by indirect immunofluorescence did not diminish significantly after 48 h of incubation at 37°C. During the same period, binding to the anti-HLA antibody HB-95 diminished by 89%. Similarly, there was no significant loss of 8H9 binding to NMB7 cells, whereas 3F8 binding diminished by 61% (Fig. 3)Citation .



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  		Fig. 3. Persistence of 8H9 binding to U2OS cells (A) and NMB7 cells (B) as studied by indirect immunofluorescence. X axis, relative immunofluorescence; Y axis, h of incubation. U2OS cells were reacted with 8H9 and HB95, and NMB7 cells were reacted with 8H9 and 3F8. After washing, cells were recultured, and the persistence of immunoreactivity of the primary antibodies was evaluated by indirect immunofluorescence using FITC-conjugated secondary antibody. Relative immunofluorescence of 8H9 on U2OS cells dropped to 80% after 48 h (HB95 to 11%), whereas that on NMB7 cells showed no significant drop off at 36 h (3F8 dropped to 39%).

 
There was a Pronase dose-dependent reduction in reactivity with 8H9, with 75–85% loss of immunofluorescence achieved at a final Pronase concentration of 0.3 mg/ml (Fig. 4)Citation . There was no appreciable loss of reactivity with 3F8 on NMB7 cells. Furthermore, the 8H9 antigen was not sensitive to neuraminidase or phosphatidylinositol-specific phospholipase C (data not shown).



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  		Fig. 4. Effect of Pronase E on 8H9 immunoreactivity with HTB82, U2OS, and NMB7 cells and on 3F8 immunoreactivity with NMB7 cells as studied by indirect immunofluorescence. X axis, concentration of Pronase E (mg/ml); Y axis, relative immunofluorescence.

 
Immunoprecipitation.
8H9 immunoprecipitated a broad band centered around Mr 90,000 from all of the 8H9-positive cell lines (HTB82, NMB7, LAN-1, U20S, HeLa, and 293), whether using native or reducing (2 mercaptoethanol) conditions (data not shown). Neither control IgG1 antibody nor 8H9-negative cell lines (Jurkat or HTB119) showed the Mr 90,000 antigen. After N-glycanase treatment, a single Mr 58,000 band was found. O-Glycanase had no effect. We interpreted this band to be a protein with a heterogeneous glycosylation pattern without disulfide-linked subunits.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We describe a novel Mr 58,000 surface tumor antigen that is detected by the mAb 8H9. This antigen is expressed on a broad spectrum of human neuroectodermal, mesenchymal, and epithelial tumors and appears to be immunohistochemically tumor specific, i.e., it is expressed on cell membranes of tumor cells with no/low membrane reactivity noted on normal human tissues. The antigen is present on 88% of neuroectodermal tumors, 96% of mesenchymal tumors, and 44% of epithelial cancers tested. The specific tissue distribution suggests a unique tumor antigen that has not been reported previously.

The expression of the 8H9 antigen on several glial and nonglial brain tumors and the complete absence on normal brain tissue are unusual. This property contrasts with most of the glial tumor antigens with a cell membrane distribution described previously (Table 5)Citation . Neuroectodermal-oncofetal antigens, e.g., NCAMs, are present to varying degrees on normal adult and fetal tissues (6) . Neurohematopoietic antigens including Thy-1 determinants (24) , CD-44 (8) , and its splice variants (25) are present on normal and neoplastic brain tissue, as well as hematopoietic tissues, principally of the lymphoid lineage. Gangliosides, such as GD2 and GM2, although expressed on tumors of neuroectodermal origin, are also present on normal brain tissue (7) . The lactotetraose series ganglioside 3',6'-iso-LD1 is widely expressed on brain tumors, epithelial cancers, and germ cell tumors, as well as on normal brain tissue (26) .


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  		Table 5 Antigens expressed on glial tumors

 
Another remarkable property of the 8H9 antigen is its expression on tumors of diverse lineage, neuroectodermal and mesenchymal tumors, and to a lesser degree, epithelial tumors. No mAb to date has the binding spectrum described with 8H9. This broad distribution provides mAb 8H9 the potential of being a generic tumor antigen for targeted therapy. Of particular interest is its expression on 28 of 29 rhabdomyosarcoma tumors and the rhabdomyosarcoma cell lines tested by indirect immunofluorescence. Disseminated and high-risk rhabdomyosarcomas have a very poor prognosis with a <40% long-term survival rate (27) . Although the MyoD family of oncofetal proteins is specific to rhabdomyosarcoma, oncofetal proteins are nuclear antigens and therefore unlikely candidates for antibody-based therapy (19) . In a preliminary report, cross-reactivity of the mAb BW575 raised against small cell lung carcinoma with rhabdomyosarcoma cell lines and 2 of 2 rhabdomyosarcoma sections was described. However, this antibody showed cross-reactivity with normal tissues (28) .

Two additional groups of tumors studied were the Ewing’s family of tumors and osteosarcoma. The Ewing’s family of tumors can be differentiated from other small, blue, round cell tumors of childhood by mAbs recognizing glycoprotein p30/32 coded by the MIC2 oncogene. However, this protein is also expressed on normal tissues, severely limiting its utility in radioimaging and therapy (18) . One hundred percent (21 of 21) of Ewing’s family tumors tested showed immunoreactivity with mAb 8H9. Besides GD2 (15) , the osteosarcoma-associated antigen recognized by the mAbs TP-1 and TP-3 (17) , and the decay-accelerating factor CD55 (16) , few tumor-associated antigens have been defined for osteosarcoma. In our study, 28 of 29 (95%) osteosarcomas tested immunohistochemically positive with mAb 8H9. The latter may therefore have clinical utility in the Ewing’s family of tumors and osteosarcomas.

The 8H9 antigen appears to be a novel, previously undescribed antigen. Sensitivity to proteinase suggests that it has a protein component. Conversely, the lack of sensitivity to neuraminidase implies the absence of sialylated epitopes, and the lack of sensitivity to phosphatidylinositol-specific phospholipase C implies that the 8H9 antigen is not glycosylphosphatidylinositol anchored. It is unlikely to be related to the NCAM family because of its unique distribution and restriction of expression among normal tissues (6) . Of the currently described antigens expressed on glial tumors, four have been reported to be restricted to tumor tissues. The mutated EGFRvIII was found to be expressed on 52% of gliomas tested and on breast and lung carcinomas (29) . However, the broad distribution of the 8H9 antigen is different from EGFRvIII. Integrin {alpha}3, a Mr 140,000 protein expressed on gliomas and medulloblastomas, is targeted by the mAb ONS-M21, which does not cross-react with normal brain (30) . However, negative immunoreactivity with neuroblastoma, melanoma, and meningioma has been reported (31) . Similar data on glioma-specific antibodies with no cross-reactivity with normal brain has been published. However, they do not react with other neuroectodermal or mesenchymal tumors, and data regarding reactivity with other tissues are unavailable (32) . A Mr 38,000 antigen has been targeted on glioblastoma cells by the antibody 6DS1. No cross-reactivity with human brain has been reported. Data regarding reactivity with other human tissues are unknown, although a high accumulation of the radiolabeled antibody in mouse kidney has been reported (33) . An ependymoma-specific protein antigen of Mr 81,000, recognized by mAbs that do not cross-react with normal glial cells, has also been described. These antibodies do not react with other glial tumors such as glioblastoma, and cross-reactivity with other tumor tissues is not known (34) .

The homogeneous expression of the 8H9 antigen on cell membrane makes it an attractive candidate for targeted immunotherapy. Furthermore, the persistence of the 8H9 antigen on NMB7 cells after binding to the mAb suggests that the antigen is not easily immunomodulated. To explore its potential for radioimaging, we used 99mTc-conjugated 8H9 to image neuroblastoma xenografts in athymic nude mice. This revealed selective uptake in the xenografts apart from moderate uptake in the liver; the percentage of injected dose/g was 50% of that achieved with the anti-GD2 mAb 3F8 (data not shown). The hydrazino-derivative of 8H9, therefore, retains the immunoreactive properties of the unmodified antibody and may be useful for radioimaging of tumors. We have also demonstrated selective radioimmunolocalization of rhabdomyosarcoma xenografts in athymic mice with no significant uptake in normal tissues using 125I-labeled 8H9 (data not shown).

In summary, the mAb 8H9 recognizes a unique Mr 58,000 tumor-specific antigen with broad distribution across a spectrum of tumors of varying lineage, neuroectodermal, mesenchyma, and epithelial, with restricted expression in normal tissues. 8H9 may have clinical utility in the targeted therapy of these human solid tumors in vitro or in vivo. Further biochemical characterization of the 8H9 antigen is warranted and may be of interest in delineating a possible role in the oncogenic process.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Grant CA61017 from the National Cancer Institute, Grant DE-FG-02-93 ER61658 from the Department of Energy, and grants from the Robert Steel Foundation, Justin Zahn Fund, Katie-Find-a-Cure Fund, JP’s Wish Fund, and Aubrey Fund. Back

2 To whom requests for reprints should be addressed, at Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-8401; Fax: (212) 744-2245; E-mail: cheungn{at}mskcc.org Back

3 The abbreviations used are: mAb, monoclonal antibody; NCAM, neural cell adhesion molecule; EGFRvIII, epidermal growth factor receptor variant III; PNET, primitive neuroectodermal tumor. Back

Received 3/27/00. Accepted 3/19/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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