monoclonal antibody t-199 directed against human ......(t-199) was selected showing the desired...

(CANCER RESEARCH 49, 4338-4343. August I, 1989]

Monoclonal Antibody T-199 Directed against Human Medu Iloblastoma:

Characterization of a New Antigenic System Expressed onNeuroectodermal Tumors and Natural Killer Cells1

Hans-Joachim Feickert,2 Torsten Pietsch, Martin R. HadanÂ»,Hermann Mildenberger, and HansjÃ¶rgRiehm

Abteilung Paediatrische Haematologie/Onkologie [H. F., T. P., H. R.], Ableitung Kinderchirurgie [M. K. H., H. M.], Zentrum Kinderheilkunde und Humangenetik,Medizinische Hochschule Hannover, Konstanty-Gutschow-Str. 8, 3000 Hannover 61, Federal Republic of Germany

ABSTRACT

A monoclonal antibody (niAh) (T-199) of IgGl isotype was raisedagainst medulloblastoma by immunizations of mice with the medullo-blastoma cell line TE-671. Studies of the specificity of mAb T-199 oncell lines as well as fresh frozen sections of normal and malignant tissuesrevealed the antigen in high amounts on the cell surface of neuroectoder-mally derived tumors such as medulloblastoma, neuroblastoma, retino-blastoma, and astrocytoma. Some melanomas and a subgroup of rhah-domyosarcomas also expressed the antigen. In contrast, mesenchymaltumors, osteosarcomas, and Ewing's sarcomas did not bear the T-199

antigen. Reactivity of T-199 with normal tissues has not been found withfew exceptions; in certain areas of the brain, especially in the cerebellumand part of the hypothalamus, in the adrenal glands, and in the pancreaticislet cells small amounts of antigen were detectable. Natural killer cellscould also be demonstrated to express the T-199 antigen similar to theNKH-1 antigen. However, despite some striking similarities, the antigensor antigen epitopes recognized by mAbs T-199 and NKH-1 are notidentical. Therefore, mAb T-199 seems to detect a unique differentiationantigen on neuroectodermal tumors, coexpressed in low amounts onnormal neuroectodermally derived cells and natural killer cells. Thepattern of reactivity and the biochemical properties of the T-199 antigenare different from other cell surface markers for neuroectodermal cellscoexpressed on natural killer cells or T-cells (HNK-1, NKH-1, orThy-1). Biochemical analysis of the T-199 antigen showed that it is aheat-labile protein.

INTRODUCTION

Brain tumors constitute the most common group of solidtumors in children, medulloblastomas accounting for aboutone-fourth among them (1). Medulloblastomas are the mostmalignant neoplasms in childhood, arising from embryonalneuroectodermal tissue. Usually, they occur within the firstdecade of life. In contrast to other intracranial malignancies,medulloblastomas frequently metastasize via the cerebrospinalfluid, and occasionally to the skeleton forming sclerotic lesions.

Despite recent advances in the treatment of childhood malignancies by combined modality chemotherapy, long term prognosis for patients with medulloblastoma is still poor with ratesof less than 50% disease-free survival after 5 years (1). Reasonsfor treatment failure are that the tumors develop drug resistance(2) or are primarily insensitive to radiation and chemotherapy.Therefore, the hybridoma technique may offer new therapeuticapproaches such as autologous bone marrow transplantationwith purged bone marrow or application of monoclonal antibodies in the treatment of disseminated solid tumors which arecurrently under investigation (3-5).

To date, only four niAbs1 against medulloblastomas have

Received 5/20/88; revised 3/28/89; accepted 4/20/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by Deutsche Forschungsgemeinschaft (Fel81/3-l).2 Fellow of the Kind-Phillip-Stiftung, Federal Republic of Germany. To whom

requests for reprints should be addressed.3The abbreviations used are: mAb, monoclonal antibody; FACS, fluorescence-

activated cell sorter, MHA, mixed hemadsorption assay; NK cell, natural killercell.

been described (6-8). These antibodies also show reactivity witha broad range of other tumor types and normal tissues. Therefore, their use for in vivo treatment will be questionable.

A number of mAbs produced against neuroectodermal tissuessuch as fetal brain, neuroblastomas, or the T-cell line HUT 78have been reported to show also cross-reactivity with medulloblastomas (9-23). However, the broad reactivity of most ofthese monoclonal antibodies precludes their clinical applicability, except for the mAbs UJ 13A (10, 24) and FMG25 (16)which seem to react predominantly with neuroectodermal tumors. These mAbs also react with normal brain, thyroid, adrenals, and nerve tissue.

Therefore, in the present study we tried to raise monoclonalantibodies with specificity for medulloblastomas. Assumingthat any monoclonal antibody will have some kind of cross-reactivity to normal tissues, we selected mAbs which did notreact with any vital tissue, thus allowing possible clinical application in the future.

MATERIALS AND METHODS

Cell Lines. Continuously cultured human cell lines were establishedfrom medulloblastomas, neuroblastomas, and sarcomas.4 Additional

cell lines were obtained from our cell bank and from the collection ofDr. J. Fogh and Dr. L. J. Old (Sloan-Kettering Institute, New York,NY). All cell lines used were free of Mycoplasma and bacterial infections.

Serological Procedures. The MHA was performed using human Otype erythrocytes conjugated with rabbit anti-mouse or goat anti-mouseIgM (25, 26) and sheep RBC coated with mouse anti-sheep RBC andgoat anti-mouse immunoglobulin (25). The immunoglobulin class ofmAbs was determined by Ouchterlony analysis using class-specificantibodies (Nordic Immunology, Tilburg, The Netherlands). Absorption analysis and tests for heat stability of antigens have been described(27). Immunofluorescence and immunoperoxidase analysis were carriedout on 5-fim cryostat sections of fresh frozen tissues after fixation with3.7% formaldehyde in phosphate-buffered saline for 5 min or acetonefor 10s. Antibody binding was compared by two methods: with fluo-rescein-conjugated goat anti-mouse immunoglobulin (Cappel Laboratories, Cochranville, PA); and/or peroxidase-antiperoxidase as de

scribed (25, 27).Generation of Monoclonal Antibodies. Female BALB/c X C57BL/6

FI mice were immunized six to ten times at intervals of 2 to 4 weeksby inoculation of approximately 1 x IO7cells of the established humanmedulloblastoma cell line TE-671 (28, 29). The initial injection wasgiven s.c. with 100 UKcomplete Freund's adjuvant and subsequent

immunizations were carried out by i.p. injections. Three days after thelast injection one mouse was sacrificed and the spleen cells were fusedto NS/1 myeloma cells as described (30). Superna tan ts of wells withgrowing clones were tested by enzyme-linked immunosorbent assay(31) for immunoglobulin production as well as for specific antibodiesby MHA assays for reactivity with cell surface antigens using a screeningpanel of tissue culture cells consisting of tumor cell lines, normalfibroblasts, and normal kidney cells. Selected clones were subcloned atleast four times by limiting dilution. Specificity analysis was performed

4 Manuscript in preparation.

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MONOCLONAL ANTIBODY DIRECTED AGAINST MEDULLOBLASTOMA

with purified and concentrated culture supernatants from hybridomasgrowing in serum-free medium.

Flow Cytometry. Peripheral blood mononuclear cells were isolatedby standard Ficoll-Hypaque density gradient centrifugation, washedtwice, and distributed to round-bottomed microtiter plates (Becton-Dickinson; No. 3103). Immunofluorescence labeling was performed asdescribed (32) using commercial immunoglobulin (Gammonativ; Kabi)to competitively block Fc receptors and fluoresceinated goat immunoglobulin I-'(ab');. fragments directed against mouse IgG and IgM, preab-

sorbed with human serum proteins (Jackson; Dianova, Hamburg, Germany) as developing reagent. All incubations and washes were performed in phosphate-buffered saline containing 0.1% bovine serumalbumin and 0.02% NaN,. Blocking studies were set up similarly usingphycoerythrin-labeled mAb NKH-1 (NKH-l-RD-1; Coulter) togetherwith different amounts of mAb T-199 in the same incubation.

NK cells were isolated by two-color cell sorting on a FACS using amarker combination to select for CD3-CD4-CD8weak+ cells and further expanded in vitro with irradiated feeder cells and conditionedmedium as described (32).

Flow cytometry was performed on a FACS 440 cell sorter with 488nm excitation (300 mW). Gating was performed on both forward andright-angle scatter and resulting histograms (containing 20,000 cellseach) recorded on a three decade log scale. Evaluation was done on aPDP-11/73-based computer system (Consort 40; Becton-Dickinson).

Immunoprecipitation Procedures. TE-671 medulloblastoma cells werebiosynthetically labeled with [35S]methionine, [l4C]leucine, and [14C]-glucosamine and extracted with Nonidet P-40 in Tris buffer as described(25). Alternatively, cellular proteins were iodinated with 125Ias described (33). Indirect immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis have also been described (25). Fordirect immunoprecipitation, mAb T-199 was purified from serum-freeculture supernatants and coupled to cyanobromide-activated Sepharose4B (Sigma, St. Louis, MO). Molecular weights of immunoprecipitatedproteins were determined by comparison with marker proteins: myosin(M, 200,000); phosphorylase b (M, 97,400); bovine serum albumin (M,68,000); ovalbumin (M, 43,000); a-chymotrypsinogen (M, 25,700);0-lactoglobulin (M, 18,400); cytochrome c (M, 12,300).

Western Blot Analysis. TE-671 cells were grown in serum-free RPMI1640 supplemented with 1% nonessential amino acids and 1% penicillin/streptomycin solution. Approximately 1 x IO7cells were lysed with0.5% Nonidet P-40 in Tris buffer as described (25) and the cellularproteins were separated on a 5-15% polyacrylamide gel under reducingand nonreducing conditions. The separated proteins were transferredto nitrocellulose and reactivity with mAb T-199 was probed accordingto standard procedures utilizing the alkaline phosphatase anti-alkalinephosphatase and peroxidase techniques (34).

RESULTS

Monoclonal antibodies were produced with specificity forneuroectodermally derived tumors and/or medulloblastomas.

From one fusion experiment one of 463 generated clones(T-199) was selected showing the desired restricted specificity

in initial screenings, cloned, and further characterized. Theimmunoglobulin subclass of this monoclonal antibody T-199was shown by Ouchterlony analysis to be IgGl.

Specificity of T-199 on Tissue Culture Cell Lines. The expression of the T-199 antigen was tested on 84 established humantumor cell lines by the MHA assay, including 6 new permanentcell lines established in our laboratory. mAb T-199 reactedstrongly with 1 of 2 medulloblastoma cell lines, 7 of 9 neuroblastoma cell lines, 1 retinoblastoma cell line, and 2 of 9melanoma cell lines. All other cell lines tested, including 12gliomas and all epithelial cancers, showed no reactivity withmAb T-199. The results are summarized in Table 1.

Reactivity of mAB T-199 with Normal Human Tissues. Specificity analysis on fresh frozen tissue sections by indirect im-munofluorescence revealed that small (but significant) amountsof T-199 antigen were detectable within certain areas of thebrain, notably in the cerebellum (Fig. 1), hypothalamus, hippocampus, substantia nigra, and spinal cord. In other areas ofthe brain as well as on peripheral nerve tissue, no antigen couldbe detected by indirect immunofluorescence.

Other normal tissues were T-199 antigen negative. However,a small subpopulation of cells in bone marrow smears stainedpositive with mAb T-199. Characterization of these cells wascarried out further by FACS analysis described below. Theseresults were confirmed by a specificity analysis applying themore sensitive indirect immunoperoxidase technique. However,with the latter detection system, T-199 antigen expression couldbe demonstrated in addition on pancreatic islet cells and adrenalcells. The data are summarized in Table 2.

Expression of T-199 Antigen on Fresh Frozen Tissue Sectionsof Malignant Human Tumors. All medulloblastomas and neu-roblastomas were stained strongly on the cell surface by mAbT-199 (Fig. 2). Metastasis of neuroblastoma to the bone marrowcould be clearly demonstrated in one case (data not shown).One pheochromocytoma and 5 astrocytomas expressed theantigen strongly in contrast to astrocytoma cell lines. Oneganglioneuroma showed weak and more cytoplasmic staining.Two of four rhabdomyosarcomas were weakly stained by mAbT-199, raising questions about the origin of these tumors, sincethey were the only nonectodermally derived tumors stained byT-199 as shown in Table 3.

FACS Analysis of T-199 Antigen Expression. On bone marrow smears only a few cells (less than 3%) were T-199 positive.In order to further characterize this cell population, coexpres-

Table 1 Reactivity of monoclonal antibody T-199 with established human cell lines detected by MHA assay

CelltypeMedulloblastomaNeuroblastomaPrimitive

neuroectodermaltumorRetinoblastomaGliomaMelanomaSarcomaLung

carcinomaRenalcarcinomaBladdercarcinomaBreastcarcinomaCarcinomasLeukemiasNormal

kidneycellsNormalfibroblastsCell

lineTE-671;MHH-MB-3IMR-5;

IMR-32; SK-N-SH; SMS-SAN; SMS-KAN; MHH-NB-1 1; LAN-l(N) and-5(S);SK-N-MCSK-PN-DWY-79U-251-MG;

SK-MG-2, -4, -9, -11, -14, -15, -16. -17, -23; MHH-GB-1,-2MEWO;SK-MEL-13, -23, -28, -29, -37, -1 13, -130,-131RD-2;

MHH-RS-3; 6647; A-4573; SW-594, -684, -872. -981. -1045; KS-165;MHH-ES-1;U-209, -459; TE-85; SK-OS-2, -3B, -5,-10SK-LC-1,

-2, -4, -6, -9;CALU-6SK-RC-1,-26A, -29, -35, -37;RiderRT-4;

J-82; SW-800; 486P; 636P; 647V; VM-CUB-1, -2,-3MDA-MB-157;BT-20; CAMA; ALAB;SK-BR-7SVV;A-10;LNcAPJurkat;

K-562; HL-60No.

positive/no. of celllinestested1/27/90/11/10/122/90/180/60/60/90/50/30/30/10/1

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Table 2 Reactivity of mAb T-199 with normal tissues as detected by indirectimmunofluorescence and indirect peroxidase-antiperoxidase staining

Â«Â£***':$Â£â€¢&â€¢>+'&Fig. l. A, cryostat section of normal cerebellum. Positive staining of neuronal

cells by mAb T-199 demonstrated by indirect immunoperoxidase technique. B,cryostat section of normal cerebellum. Negative staining with control antibody.

sion of T-199 antigen with various cell surface markers ofhematopoietic cells was investigated on buffy coat cells ofperipheral blood as well as on defined lymphocyte populationsand NK cell clones by FACS analysis. Phenotyping peripheralblood cells of a large number of nonselected individuals consistently showed reactivity of mAb T-199 with a small lymphocyte subset. Monocytes, granulocytes, erythrocytes, and platelets were not stained by mAb T-199. The subset of lymphocyteswas shown to have the scatter characteristics of large granularlymphocytes. Comparison with other cell surface markers revealed a virtually complete overlap of this cell population withNK cells as defined by mAb NKH-1 (data not shown). Furthermore, T-199 also displayed the histogram pattern characteristicfor NKH-1 on fresh peripheral blood lymphocytes. The apparent similar reactivity was most evident with cultured NK cells,which were consistently stained by both markers intensively.This is exemplified in Fig. 3 in which a CD2 + CD3 - CD 16+NK cell line is readily stained by both reagents. To test whetherboth antibodies react with the same antigen or epitope, directlylabeled mAb NKH-1 was assessed in competition experimentswith increasing doses of mAb T-199. As shown in Fig. 4 neitherthe standard amount for surface labeling (10 Â¡Aculture supernatant of mAb T-199) nor an at least 1000-fold excess (20 n\ascites of mAB T-199, titer 1:106) were effective at displacingNKH-1 activity. Thus mAb T-199 and mAb NHK-1 seemto react with different epitopes of the same antigen molecule or two independent although closely linked antigens. Thisis further supported by the fact that the NKH-1 antigen isstrongly expressed on RD-2 (rhabdomyosarcoma) cells but notthe T-199 antigen.

TissueBrainPosterior

lobeHypothalamusStriatumNucleus

nigerCerebellumMedulla

oblongataSpinalcordPituitary

glandRespiratory

systemLungBronchusGastrointestinal

systemEsophagusStomachIleumColonSigmaLiverUrogenital

systemKidneyBladderUreterProstate

glandUterusOvary,

testisMammary

glandSkinPlacentaEndocrine

systemThyroidglandParathyroidglandAdrenal

glandPancreasHematopoietic

systemThymusTonsilSpleenLymph

nodeBonemarrowPeripheral

bloodHeart

muscleSmooth

muscleBlood

vesselsT-199e*oe0o0o00000000o000000o00000"0e0000eo0oo" â€¢¿�,strong staining; 9, weak/inhomogeneous staining; O, no staining.* Positive by peroxidase-antiperoxidase staining.f Islet cells positive by peroxidase-antiperoxidase staining.

Biochemical Analysis of the T-199 Antigen. The T-199 antigen is trypsin and heat labile, shown by absorption analysis(Fig. 5). Immunoprecipitation of the T-199 antigen failed despite various attempts of biosynthetic radiolabeling as well asradioiodination of cellular antigens. Binding of radioiodinatedmAB T-199 to TE-671 medulloblastoma cells could be displaced by excess unlabeled mAb T-199 but not by excess unla-beled mAb NKH-1, indicating that the recognized respectiveantigens or antigen epitopes are not identical (Fig. 6).

Western Blot Analysis of the T-199 Antigen. The T-199antigen could not be identified by Western blot analysis byeither reducing or nonreducing conditions for the separation ofcellular proteins.

DISCUSSION

In our present study, a monoclonal antibody (T-199) wasraised against the established medulloblastoma cell line TE-

4340




PPSTTe.SÂ«! * 5778 / SUM*RY

ALGG

CD3

LGG

NKH1

Counts= aeeee.HS= 256 VS= ieZ

CD2

T133

Fig. 3. FACS analysis performed on cultured NK cells. The tested cells wereCD3 (A) and mostly CD2 (B) positive. The staining pattern for mAbs NKH-1(C) and T-199 (D) were very similar. HS. horizontal scale; VS. vertical scale;LGG green fluorescence.

Fig. 2. A, cryostat section of medulloblastoma cell line TE-671. Positivestaining of all tumor cells by mAb T-199 detected by indirect immunoperoxidasetechnique. B, cryostat section of medulloblastoma cell line TE-671. Negativestaining with control antibody.

Table 3 Reactivity of mAb T-199 with tumors as detected by indirectimmunofluorescence and indirect peroxidase-antiperoxidase technique

TumorsNo. of T-199 posi

tive/no, tested

MedulloblastomaNeuroblastomaGanglioneuroblastomaAstrocytomaPituitary adenomaPheochromocytomaMeningiomaRhabdomyosarcomaChondrosarcomaEwing's sarcoma

OsteosarcomaNephroblastomaHepatoblastomaBrain metastasis (carcinoma)Leukemias (T-cell acute lymphocytic

leukemia, acute myeloblastic leukemia, common acute lymphocytic leukemia)

6/612/12iVi5/5iVil/l0/22-/S

0/10/70/20/30/10/10/3

t 5875 / H<H1 - T199 BLOCK

LGR A

NEC CTRL

LGR

NKH1 + T199

10 ul Spnte

Counts=HS= 256 VS= 1024.

B

NKH1

NKH1 + T193

20 ul Asc

Fig. 4. FACS analysis performed on cultured NK cells. A, reactivity of negativestaining control antibody; B, reactivity of phycoerythrin-labeled mAb NKH-1with NK cells; C, reactivity of phycoerythrin-labeled mAb NKH-1 with NK cellspreincubated with standard amounts of mAb T-199 [culture supernatant(Spnte)]: D, reactivity of FITC-labeled mAb NKH-1 with NK cells preincubated with an at least 1000-fold excess of mAb T-199 [ascites (Asc) fluid]. IIS.horizontal scale; VS, vertical scale; LGR, red fluorescence.

" Weak membrane staining.

671. The initial screening suggested that this antibody did nothave cross-reactivity with most normal tissues, possibly allowing future clinical application.

The specificity analysis of mAb T-199 revealed that therespective antigen is associated with tumors, i.e.. it is stronglyexpressed on medulloblastomas but also on other neuroecto-dermally derived tumors such as neuroblastoma, retinoblas-toma, glioma, pheochromocytoma, and melanoma, but not onmesenchymal or epithelial neoplasms. However, mAb T-199

8 16 256 102432 64 128TITER Â»-

Fig. 5. Study of biochemical properties of the T-199 antigen as demonstratedby absorption analysis on TE-671 medulloblastoma cells. The antibody tiler ofculture supernatant of mAb T-199 was 1:256 (50% rosette formation) using theMHA assay (O). The antibody activity could be absorbed by TE-671 (â€¢)cells.However, treatment of TE-671 cells with trypsin (D) or heat (â€¢)destroyed theirability to bind mAb T-199.

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1 2 3200 -

97 -

68 -

43 -

25 -

18 -

14 ~

Fig. 6. Gradient sodium dodecyl sulfate-polyacrylamide gel; reducing conditions. In Lane 4, heavy and light chains of 125I-mAb T-199 bound to TE-671

medulloblastoma cells were demonstrated after washing and lysis of the cells. InLane 3, preincubation of TE-671 cells with an excess of unlabeled T-199 inhibitsbinding of labeled T-199. Lane 2, preincubation with an excess of unlabeled mAbNKH-1 for 3 h followed by incubation with 125I-T-199 with subsequent lysis ofcells. Lane 1, molecular weight standards; ordinate, molecular weight in thousands.

was also shown to react weakly with a few normal neuroecto-dernial tissues and natural killer cells (NK cells).

The T-199 antigen seems to be a differentiation antigen ofan intermediate maturation stage of neuroectodermal tissueexpressed on the cell surface as indicated by the pattern ofexpression in various tissues and cell lines. Very immatureneuroectodermally derived tumors (like the cell lines SK-PN-DW and SK-N-Mc) do not express the antigen. Also, very well

differentiated tumors like ganglioneuromas show only lowexpression of antigen in contrast to all other neuroectodermaltumors tested with differentiation markers in between the lattergroups. Although the T-199 antigen is expressed in some parts

of normal brain (hypothalamus, cerebellum, spinal cord), thequantity of antigen expression is much lower than in tumortissues as judged by the intensity of immunofluorescence staining. Similar results were reported on tests of melanoma celllines of defined maturation stages (35). The reactivity of mAbT-199 was also observed on 2 (embryonal) of 4 rhabdomyosar-comas. Whether this reactivity with a subgroup of rhabdomyo-

sarcomas can be reproduced on a larger series awaits testing.Unexpectedly, mAb T-199 was found to react also with a

subpopulation of lymphocytes, which were demonstrated to belarge granular lymphocytes (NK cells). FACS analyses on various lymphoid and NK cell populations revealed some overlapin the pattern of reactivity with mAb NKH-1 (36). However,the reactivity of T-199 and NKH-1 on cell lines and tissues

shows clear differences (data not shown). Blocking experiments(Figs. 4 and 6) further support that the detected antigens orantigen epitopes recognized by mAb T-199 and mAb NKH-1are different. NKH-1 has been reported to detect a glycoproteinwith a relative molecule mass of 200,000 Da under reducingand nonreducing conditions (36). While this antigen could be

easily precipitated from TE-671 cells, the T-199 antigen wasunprecipitable when cells labeled by various methods were used(125I,[35S]methionine, [14C]leucine, [14C]glucosamine). This mayalso indicate a different epitope for the T-199 antigen. Heatlability and trypsin sensitivity of the T-199 antigen indicate aprotein rather than a glycolipid or carbohydrate structure. Theantigen instability may also explain why it could not be detectedby Western blot analysis.

MAb T-199 is also different from the NK cell markerHNK-1 (37) which is known to react with neuroectodermaltissues (38). MAb HNK-1 detects a myelin-associated carbohydrate moiety (39) on the cell surface not only on neuroectodermally derived tumors but also on sarcomas, nephroblasto-mas, carcinomas derived from Amine Precursor Uptake andDecarboxylation (APUD) type cells, the normal nervous system, and APUD type cells in lung and adrenals (40).

In comparison to previously described monoclonal antibodiesdirected against neuroectodermal tissues, mAb T-199 shows aunique pattern of reactivity, yet undescribed. However, thereare similarities with some mABs developed against either fetalbrain or neuroectodermally derived tumors indicating the existence of different antigen systems, highly but not exclusivelyrestricted to these tissues (15).

For diagnostic purposes those antibodies may become valuable with respect to therapy and prognosis which allow theprecise determination of a tumor type. Especially in solidchildhood tumors the distinction between neuroectodermallyversus mesenchymal derived tumors is of major clinical importance, since the treatment modalities are very different.

In contrast to all other mAbs described as yet, mAb T-199 isthe only antibody which detects primarily neuroectodermallyderived tumors with the exception of mAbs FMG25 (16) andUJ127:11 (9). Noteworthy is mAb 5A7 (17) which was reportedto react with neuroblastomas only, besides normal brain, colon,and kidney. However, the respective antigen is located intracel-lularly, precluding in vivo application.

Regarding clinical application, only mAb 3F8 which detectsthe glycolipid GD2 has shown promising results thus far (4).Certain glycolipid antigens seem to be expressed in largeamounts on tumor cells in contrast to normal tissues andtherefore may be utilized as tumor markers. GD2has been foundpredominantly in neuroblastomas, melanomas, astrocytomas,an oat cell carcinoma, and an adenocarcinoma of the adrenalcortex (23). In contrast, the ganglioside GD3 is expressed inlarge amounts predominantly in melanomas (25) but not inmedulloblastomas.

Since mAb T-199 reacts primarily with neuroectodermaltumors it may be a valuable tool in the differential diagnosis ofsmall round cell tumors, especially in the diagnosis of neuroblastoma versus Ewing's sarcoma or osteosarcoma. Furthermore, the strong reactivity of mAb T-199 with neuroectodermaltumors, the uniform expression of the antigen on the cellsurface, and the lack of reactivity with most normal tissues,especially heart, liver, and kidney, may allow its clinical application, possibly in combination with other mAbs directedagainst neuroectodermal tumors. In this context the reactivitywith normal brain tissue is irrelevant since the brain is protectedin vivo by the blood-brain barrier (41).

Since mAb T-199 is of the IgGl subtype, clinical applicationof the antibody conjugated to some drug, radionuclide, or toxinmay be necessary and should be feasible.

ACKNOWLEDGMENTS

The authors thank K. Appelhans and B. Joggerst-Thomalla forexcellent technical assistance and Dr. A. Knuth for critical reading ofthe manuscript.

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1989;49:4338-4343. Cancer Res Hans-Joachim Feickert, Torsten Pietsch, Martin R. Hadam, et al. Expressed on Neuroectodermal Tumors and Natural Killer CellsMedulloblastoma: Characterization of a New Antigenic System Monoclonal Antibody T-199 Directed against Human

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