Glioma-associated antigens defined by monoclonal antibodies against an avian sarcoma virus-induced rat astrocytoma

Journal of Neuroimmunologv, 13 (1986) 183-202

183

Elsevier JNI 00428

Glioma-Associated Antigens Defined by Monoclonal Antibodies against an Avian Sarcoma Virus-Induced Rat Astrocytoma Yi-sheng Lee, Carol J. Wikstrand and Darell D. Bigner Department of Pathology, Duke University Medical Center, Durham, NC 27710 (U.S.A.)

(Received27 March, 1986) (Revised, received23 June, 1986) (Accepted 23 June. 1986)

Summaff $69-c15 is a highly immunogenic cell line derived from an avian sarcoma virus (ASV)-induced astrocytoma in F-344 rats. Monoclonal antibody (Mab) production was attempted by fusing F-344 rat splenocytes and mouse P3 × 63/Ag8.653 myeloma cells after a syngeneic immunization protocol. 336 fusion clones were screened by cell surface radioimmunoassay (CS-RIA) against the immunizing line $69-c15, rat kidney fibroblast line $203-cll and Walker rat carcinoma line. Mabs 7G4, 9F1, 10E3 and 10E7 which reacted only with $69-c15 were chosen. Further analysis demonstrated that these Mabs reacted only with rat (13/23 astrocytomas, 2 / 4 gliomas, 1/11 neurinomas) or mouse ( 2 / 1 0 astrocytomas) neurogenic tumor cells induced by both viral and chemical agents. Reciprocal competition assays suggested that 7G4, 9F1 and 10E3 recognized the same epitope and that 10E7 reacted with a spatially close determinant. Antigen activity could not be found in adult rat tissues (brain, heart, lung, liver, kidney, spleen, thymus, intestine, muscle and peripheral nerve) and fetal brain (8, 12, 20 days gestation) by either absorption analysis or tissue staining. Preliminary characterization indicated that the epitope may be polypeptide-associated. Further antigen purification and tumor localization can be attempted with these Mabs. This work was supported by NIH Grants 1P0 NS/CA 20023-02, P01 CA 32672, and 2R01 CA 1189814. All correspondenceto: Dr. Darell D. Bigner,M.D., Ph.D., Department of Pathology,Box 3156, Duke Unviersity Medical Center, Durham, NC 27710, U.S.A. 0165-5728/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)

184

Key words: A s t r o c y t o m a - A u i a n s a r c o m a virus - Cell s u r f a c e r a d i o i m m u n o a s s a y Monoclonal antibody

Introduction

The existence of human glioma-associated antigens has been suggested by earlier studies demonstrating glioma-specific cellular and humoral immune responses (reviewed in de Tribolet and Carrel 1980; Wikstrand and Bigner 1980). Such responses have been observed not only in autologous and allogeneic systems (Kornblith et al. 1974; Coakham et al. 1980), but also in xenogeneic hyperimmune antisera (Wikstrand et al. 1977). Extensive absorption analysis of reactive antisera and analysis of monoclonal antibodies has categorized four classes of glioma-associated antigens: (1) those idiotypic to autologous tumors; (2) those common to gliomas; (3) those cross-reactive between tumor cells of neural-crest origin including gliomas, neuroblastomas, ependymomas and melanomas; and (4) oncofetal antigens (Coakham et al. 1980; Schnegg et al. 1981; Carrel et al. 1982; Bourdon et al. 1983). However, antigens that are truly glioma-specific in a structural sense are still to be identified. Molecular characterization of these glioma-associated antigens must be achieved in order to learn how each class of antigen contributes to tumor rejection or growth in vivo, to obtain more sensitive and specific diagnostic reagents, and to provide immune modulation therapy for patients as an alternative treatment regimen. Development of syngeneic monoclonal antibodies against brain tumors in a rodent model was contemplated based upon two considerations. First, by syngeneic immunization, potentially weakly immunogenic tumor-specific antigens (Herberman 1977) might be detected as the immune response against irrelevant but highly immunogenic species and tissue antigens seen in most xenogeneic immunization regimens could be avoided. Second, in vivo use of syngeneic Mabs for diagnostic and therapeutic purposes could be performed without the induction of antibodies against xenogeneic immunoglobulins, which might otherwise inhibit antigen-antibody binding or even provoke adverse reactions. The $69-c15 cell line was derived from an astrocytoma induced by intracerebra! injection of B-77 (Bratislava-77) ASV in F-344 rats. Cultured $69-c15 (Copeland et al. 1976; Harwood et al. 1977) cells contain nervous system-associated S-100 protein and are highly tumorigenic in syngeneic neonatal rats and in nude mice. However, $69-c15 cells were rejected when transplanted to adult rats either intraperitoneally or subcutaneously, and the majority of invasive tumors produced in newborns regressed as the young rats reached maturity. Cellular immunity can be observed in rats with primary brain tumors induced by ASV (Adams et al. 1976), and rats actively immunized with ASV-induced glioma cells developed a strong humoral antibody response as detected by complement-dependent cytotoxicity assays. This evidence suggested that $69-c15 cells carry operational tumor-specific moieties

185 responsible for the rejection phenomenon and thus provided us with a possible model for developing Mabs in syngeneic animal systems. We have produced four Mabs using a syngeneic immunization protocol. These four Mabs recognize either a common epitope or nearby or overlapping antigenic determinants. The antigenic specificity detected is restricted to neurogenic tumors induced in rats and mice by viral or chemical induction protocols. Heterogeneity of antigen expression in cell lines was also observed.

Materials and Methods Cultured cell lines

The majority of the 88 established cell lines used in this study (Tables 1-4) were derived from experimental tumors of viral or chemical induction protocols. Many of these cell lines have been described (Copeland et al. 1976; Cloyd and Bigner 1977; Harwood et al. 1977). Neurogenic tumors, with the exception of six spontaneous mouse astrocytomas and one spontaneous mouse neuroblastoma, were induced either by intracranial injection of Bratislava-77-C (B-77) or Schmidt-Ruppin-D (CSR-D) avian sarcoma virus (ASV) or by oral or intravenous administration of nitrosourea. Details of these tumor induction protocols have been published (Bigner et al. 1975; Bigner and Swenberg 1977). $69-c15, the cell line used for syngeneic immunization in this study, was derived from a B-77 ASV-induced F-344 rat astrocytoma (Bigner and Swenberg 1977). The nervous system marker protein S-100 can be found in most of the astroglial tumor cell lines including $69-c15 and neurinomas induced in rats by nitrosourea. Neuron-specific enolase (14-3-2) can be found in the neuroblastoma cell lines. The inducing agents used for non-neurogenic tumors were more diverse; these cell lines were induced in vivo as well as in vitro. Chemically induced tumor cell lines and kidney and embryonal fibroblast lines were established in this laboratory. The rat Morris hepatoma MH1C1, Leydig cell carcinoma line R2C, Walker carcinoma line LLC-WRC 256, Buffalo rat pituitary tumor line P500 and rat anti-mouse Lyt-1 hybridoma cell line TIB-104 (53-7-313) were from the American Type Culture Collection, Rockville, MD. The murine myeloma cell line P3 × 63/Ag8.653 was obtained from Dr. H. Koprowski of the Wistar Institute. All cell lines were grown in zinc option medium/10% fetal calf serum (ZO/10% FCS) (Gibco Laboratories, Grand Island, NY) in 5% CO 2 at 37°C. For cell line freezing, the cells were frozen in 10% DMSO, ZO/10% FCS. Cultures were considered free of mycoplasma contamination by the inability of conditioned medium to cause the conversion of 2'-deoxyadenosine to adenine or thymidine to thymine (Kurtzberg and Hershfield 1985). Normal and tumor tissues

For absorption analysis, normal rat tissues were derived from 12-week-old F-344 rats (Charles River). The rats were anesthetized with ether and perfused with 5% glucose solution through right ventricle infusion. Tissues were frozen in DPBS

186 (Dulbecco's phosphate-buffered saline) at - 7 0 ° C until used. No cryoprotectant was used. For the induction of tumors, 1 × 10 6 $69-c15 cells in serum-free ZO medium were injected subcutaneously in newborn F-344 rats in both flanks. Growing tumors were excised between 25 and 30 days after injection. Tumors were also frozen in DPBS at - 7 0 ° C until used. For immunohistologic analysis, tissues were snap-frozen with freon cooled by liquid nitrogen and sectioned (4/~m). The sections were immediately fixed in cold acetone ( - 2 0 ° C ) for 30 s and stored in a desiccator. Tissues examined included tumors, normal adult tissues (cerebrum, cerebellum, heart, lung, thymus, spleen, testis, epididymis, ovary, intestine, peripheral nerve, kidney, liver) and fetal brains (8, 12, 20 days in gestation). All tissues were examined at least twice. Primary hepatocyte culture was prepared as described (Seglen 1976). Briefly, a single-cell suspension of hepatocytes derived from F-344 rat liver which had been perfused with buffer containing collagenase was plated into 96-well, flat-bottomed plates (Flow Laboratories, Hamden, CT) at 2 × 104 cells/well. Assays were done after overnight incubation. Testes cell suspension and epididymal sperm were prepared as described (Bechtol et al. 1979). Capsule-removed testes were minced in phosphate-buffered saline, pH 7.2. Single-cell suspensions were obtained by gently pipetting the minced preparation with a pasteur pipette. Epididymides were cut into small sections and the spermatids were allowed to swim out in 30 min during room temperature incubation. Spleen, lymph node and thymus were disrupted with needles and passed through a gauge 30 mesh wire. Spleen and peripheral blood lymphocytes were further prepared by Ficoll-Hypaque separation. For red blood cells, heparinized rat blood was centrifuged and washed in DPBS three times, carefully removing the buffy coat after each wash.

Immunization Six 12-week-old F-344 rats were immunized with rat astrocytoma cell line $69-c15 cell suspensions prepared from confluent monolayers. The cells were scraped from tissue plates with a rubber policeman, washed twice with DPBS, and a single-cell suspension obtained. Each rat was injected i.p. with 1 × 10 6 cells on day 0 and was boosted i.p. on days 28, 56 and 72 with 1 × 10 7 cells. Individual rat serum titers against the immunizing cell line $69-c15 were determined 7 days after each booster by cell surface radioimmunoassay (CS-RIA). The rat with the highest titer was given an i.v. injection of 1 × 10 6 cells on day 97. Three days later, the rat was killed by cervical dislocation, and spleen removed aseptically. Cell fusion and cloning Cell fusion of immunized F-344 rat spleen cells and the P3 x 63/Ag8.653 mouse myeloma cell line was performed by the protocol of Gefter et al. (1977). Briefly, 1.5 x 108 rat spleen cells and 3 × 107 mouse myeloma cells were fused with 0.5 ml 35% polyethylene glycol (MW 1500) in serum-free medium. The fused cells were plated into two 100-mm tissue culture dishes in complete medium. After overnight incubation, non-adherent cells were collected and plated into 96-well, flat-bottomed plates at 5 x 104 cells/well in 200 /~1 HAT-selective medium plus 20% FCS-309

187 (Gibco Laboratories, Grand Island, NY). Cells were fed every 3 days by half-volume medium replacement. Supernatants of the outgrowing hybridomas were tested against the immunizing cell line $69-c15, rat LLC-WRC 256 Walker carcinoma line and $203-cll rat kidney fibroblast cell line monolayers by CS-RIA. Hybridomas that reacted with $69-c15 but not with LLC-WRC 256 were chosen and expanded to 1 ml culture and cloned in methylcellulose semisolid media (Wikstrand and Bigner 1982). The cloning procedure was performed at least twice until the percentage of positive reacting clones reached 100%.

Cell surface radioimmunoassay (CS-RIA) Binding of hybridoma culture supernatant to cell surface antigens was detected by an indirect radioimmunoassay described previously (Wikstrand and Bigner 1982). Briefly, confluent cell monolayers in 96-well plates coated with rat tail collagen extract (Strom and Michalopoulos 1982) were incubated with 50 /~1 of hybridoma cell supernatant for 1 h at 37°C. Following three washes with HBSS (Hanks' buffered saline solution), cultures received 50/~1 containing 100 000 cpm of 125I-labeled affinity-purified goat anti-rat IgG antibodies (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and further incubated for 1 h at 37°C. The cultures were then washed seven times with HBSS, dissolved in 2 N NaOH, and sampled with cotton-tipped swabs which were counted in a Packard gamma spectrometer. Non-adherent cell assays were carried out in 96-well, V-bottomed plates (Cooke Engineering) at 2 X 105 cells/well. All assays were done in triplicate. Each assay was performed with P3 x 63/Ag8 (mouse IgG1) supernatant, medium, and a rat anti-mouse Lyt-1 hybridoma TIB-104 (rat IgG2a) supernatant as negative controls. Data was expressed as a binding ratio (B.R.) obtained by dividing the experimental cpm by the negative control cpm. B.R. >~3 was considered positive, as it exceeded the control mean by three standard deviations. Collagen-coated plates prevented cell loss during washing without increasing observed background binding.

Hemagglutination assay The hemagglutination assay was performed as described (Misra et al. 1982) with slight modification. Briefly, the assay was performed by combining 25 ~1 2% dextran, 25 /~1 hybridoma supernatant and 25 ffl of a 0.4% solution of F-344 rat erythrocytes in DPBS. The mixture was incubated at 37°C for 1 h and the results were determined microscopically. For the indirect hemagglutination assay, 50/~1 of a 1 : 100 dilution of rabbit anti-rat IgG antiserum (Pel-Freeze Biol., Rogers, AR) was added to the mixtures described above after 30 min; this was followed by a further incubation of 1 h.

Antibody absorption Tissue specimens from F-344 rats were mechanically disrupted and homogenized on ice with a glass-Teflon homogenizer in a 5-fold volume of cold PBS with 1 mM PMSF and aprotinin at the concentration of 2 trypsin inhibitor units/ml (Sigma Chemical Co., St. Louis, MO). The entire homogenate was frozen and lyophilized immediately. For homogenate washing, the homogenates were washed three times

188 with homogenizing buffer, with 100 000 x g centrifugation for 30 min between each wash. The pellets were resuspended and aliquoted. $69-c15 cell pellets and tumors were treated in the same way. Sequential absorption analysis was performed by incubating 500/~1 of monoclonal antibodies at 0.3/~g/ml with 5 mg of lyophilized material for 1 h at room temperature. Protein concentration was determined by the Lowry method (Lowry et al. 1951). The mixture was then centrifuged at 100 000 × g for 15 min in a Beckman L2-65B ultracentrifuge; the unbound supernatant was collected. Each absorption consisted of five samples, each absorbed differently, from once only to five times sequentially. The remaining binding activity in the supernatant to $69-c15 monolayers detected with CS-RIA was determined at each absorption step.

Reciprocal competitive inhibition assay Monoclonal antibodies were purified by a bovine anti-rat affinity column prepared in this laboratory and were labeled with 125I by the chloramine-T method (Greenwood et al. 1963). Briefly, 200/~g of affinity-purified monoclonal antibodies in 100 t~l 0.05 PBS, pH 7, were labeled with 0.4 mCi carrier-free 125I (New England Nuclear, Boston, MA). The radiolabeled monoclonal antibody was titrated against $69-c15 cell monolayers in 96-well, flat-bottomed plates at serial 1 : 2 dilutions. In a competitive inhibition assay, 50 ~1 of unlabeled antibody at 50/~g/ml in ZO/10% FCS were added and incubated for 1.5 h at room temperature. Without further washing, radiolabeled antibody at the concentration of 12.5 t~g/ml, which gave 50% maximal binding activity in direct titrations, was added and incubated for 1 h at room temperature. The plates were washed and the contents of each well dissolved in 2 N NaOH and counted. The results were expressed as percentage binding of 125I-labeled antibody following preincubation with each specified unlabeled competing antibody versus negative control in HBSS with 0.5% BSA. Enzyme digestion and heat treatment For enzyme digestion experiments, $69-c15 cell monolayers (1 × 105 cells/well) grown on 96-well plates were treated separately with trypsin (Gibco Laboratories, Grand Island, NY), protease (Gibco Laboratories, 6 units/mg protein), pronase (Gibco Laboratories, 45000 P U K / g protein, at a concentration of 100 ~g/250 ~l/well neuraminidase (Sigma Chemical Co., St. Louis, MO, 5 units/mg protein using NAN-lactose) at 1 unit/well, or DNAse (Sigma Co., 1520 K U / m g protein) at 6/~g/well for 30 min at 37°C. All of these enzymes except neuraminidase were in HBSS, pH 7.0 with Ca 2+ and Mg 2+ supplementation. Neuraminidase digestion was done in citrate-phosphate buffer, pH 6.0. Antigen activity was determined by CS-RIA with each monoclonal antibody. For protease-treated cells, centrifugation at 200 × g for 5 min between washings was needed since cells detached from the wells. All assays were performed in triplicate. For the heat-sensitivity study, either $69-c15 monolayers or whole-tissue homogenates (5 mg homogenate/250 /~1 DPBS) were heated at 100°C for 1 min. The monolayers were examined with CS-RIA and tissue homogenates were assayed using sequential absorption analysis. Tissue homogenates were also treated at 60 ° C for 3 min.

189

Glycolipid isolation and assay Glycolipids were isolated as described (Pukel et al. 1982). Briefly, $69-c15 cells were homogenized in chloroform-methanol (c/m, 2 : 1) at a 1 : 10 cell/solvent ratio. After filtration, the homogenate was re-extracted with c / m (1 : 1). The amount of cells used was equivalent to that which gave positive antigen activity in the aqueous extraction described previously. The pooled glycolipid extract was aliquoted into tubes and dried by low vacuum evaporation. Sequential absorption analysis was performed as before. Tissue staining Both Mabs 7G4 and TIB-104 were biotinylated with N-hydroxy-succinimide/biotin (Pel-Freeze) at 1:100 molecular ratio. The tissue sections were first blocked with normal horse serum for 30 min and then incubated with biotinylated antibodies at 10/~g/ml for 1 h. Tissues with high endogenous biotin content such as liver and kidney were also first blocked with sequential incubation of free avidin and biotin. After washing, the sections were further incubated with avidin-biotinperoxidase complex (ABC kit, Vector Laboratory, Burlingame, CA) for 1 h (Wikstrand et al. 1983). The sections were developed with H202 and diaminobenzidine (Sigma, St. Louis, MO). A buffer control was also included during each staining. Results

Production and preliminary screening of rat-mouse hybridomas Sera from syngeneically immunized adult F-344 rats were tested for antibody activity against the immunizing cell fine $69-c15 by CS-RIA analysis. Binding activity was observed after three monthly immunizations and increased significantly following further boosts. There was no tumor growth in any rat as observed at elective autopsy 8 months after initial immunization. Spleen cells from the rat that gave the highest titer were fused with mouse myeloma cells P3 × 63/Ag8.653. Culture supernatants from wells with growing hybrids (336 of 576) were tested against $69-c15 by CS-RIA and 63 wells were initially found to be positive. However, only 38 hybrids continued to produce antibodies following screening on two negative control cell lines: the LLC-WRC 256 rat carcinoma cell line and $203-cll rat kidney fibroblast cell lines. Four hybridomas (7G4, 9F1, 10E3 and 10E7) were selected on the basis of positive reactivity against $69-c15 but not for LLC-WRC 256 and $203-c11. Mab 12G8, produced in the same fusion, was chosen as positive control. It reacted with both $69-c15 and LLC-WRC 256. Each hybridoma was cloned by Methocel at least twice until the percentage of positive clones against $69-c15 reached 100%. The stability of these cloned hybridomas was good, but recloning every 6 months was needed to maintain high titer antibody production if the hybridoma was to be carried in culture continuously (data not shown). Monoclonal antibodies 7G4, 9F1, 10E3 and 12G8 are of IgG2a isotype and 10E7 is of IgG2b isotype as determined by immunodiffusion analysis with immunoglobulin isotype-specific antisera (Miles Laboratories, Elkart, IN).

190

Cell line specificity analysis by CS-RIA The four selected hybridomas (7G4, 9F1, 10E3 and 10E7) were further tested by CS-RIA analysis against a large panel of rat and mouse cell lines derived from spontaneous or experimentally induced tumors and normal fibroblasts (Tables 1-4). All four monoclonal antibodies showed the same pattern of binding activity on cell lines tested, with 7G4, 9F1 and 10E3 giving similar binding ratios, and 10E7 a consistently higher value. Of rat neurogenic tumors tested (Tables 1 and 2),

TABLE 1 CS-RIA REACTIVITY PATTERN OF ANTI-RAT ASTROCYTOMA HYBRIDOMAS A G A I N S T S Y N G E N E I C (F-344) RAT N E U R O G E N I C T U M O R CELL LINES Cell line

Strain

Inducing

origin

agent

F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344 F-344

B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV B-77 ASV SR-D ASV ENU ENU ENU ENU ENU

F-344 F-344 F-344 F-344 F-344

Passage

B.R.

BKG b

a

7G4

9F1

10E3

10E7

cpm

16 19 17 18 19 23 23 25 20 41 17 14 15 50 16 22 25 25 24

3 6 5 1 6 11 1 1 3 1 6 4 4 1 1 2 1 1 1

3 6 6 1 6 13 1 1 3 1 6 4 4 1 1 2 1 1 1

3 6 5 1 5 12 1 1 3 1 7 4 4 1 1 2 1 1 1

4 8 5 1 7 17 1 1 4 1 6 4 4 1 1 2 1 1 1

208 318 248 271 330 198 235 290 236 296 216 219 275 262 267 284 384 280 200

ENU ENU ENU ENU Unknown

41 6 15 17 40

1 1 1 1 1

1 1 1 1 1

1 1 1 1 1

1 1 1 1 1

276 266 282 310 260

Unknown

44

2

2

2

3

320

Unknown Unknown

13 42

1 5

1 4

1 4

1 8

461 206

A strocytoma $69 $69cll $69c12 $69c13 $69c14 $69c15 $70 $70cll $70c12 $70c13 $83 $83cll $83c12 $83c15 P379 P480 P481 P482 P494

Neurinoma P476 P477 P478 P479 P519

Anaplastic glioma P502

F-344

Differentiated glioma P503 P506

F-344 F-344

a B.R.: Binding ratio is calculated as Exp. ( c p m ) / B K G (cpm). B.R./> 3 is considered significant. b BKG: Background cpm represents the counts bound of P3 mouse Ig or TIB-104 rat anti-mouse Lyt-1 Mab in control wells.

191 TABLE 2 CS-RIA REACTIVITY PATTERN OF ANTI-RAT ASTROCYTOMA HYBRIDOMAS AGAINST ALLOGENEIC RAT NEUROGENIC TUMOR CELL LINES Cellline

Strmn orion

Inducing agent

Passage

BDIX BDIx Random-bred Wistar-Furth Random bred Wistar-Furth

Unknown SR-D ASV MNU

BDIX BDIX BDIX BDIX BDIX BDIX

B.R.a

BKG b cpm

7G4

9F1

10E3

10E7

21 4 Unknown

2 1 4

2 1 4

2 1 4

3 1 4

261 336 241

MNU

47

5

5

5

6

230

ENU ENU ENU ENU ENU Unknown

35 24 15 12 15 36

4 1 1 1 1 1

3 1 1 1 1 1

3 1 1 1 1 1

6 1 1 1 1 1

301 414 318 318 366 232

ENU ENU ENU ENU

16 47 38 41

1 1 1 1

1 1 1 1

1 1 1 1

1 1 1 1

303 303 438 427

6

1

1

1

1

227

Aslrocvtoma P522 $240 C-6 C-6c12

Neurinoma $349 $349cl I P416 P417 P475 P505

Neuroblastoma P471 P472 P473 P474

BIDX BDIX BDIX BDIX

Mixed glioma P520 a

b

BDIX

Unknown

B.R.: Binding ratio. B.R./> 3 is considered significant. B K G : Background cpm represents the counts bound of P3 mouse Ig or TIB-104 rat anti-mouse Lyt-1 hybridoma in control wells.

significant b i n d i n g (B.R. >/3) was d e t e c t e d on 1 3 / 2 3 astrocytomas, 1 / 1 1 neurinomas, a n d 2 / 4 gliomas. N o relationship was f o u n d b e t w e e n positivity an d strain of origin o f cell lines or agents used for t u m o r induction. D i f f e r e n c e s b e t w e e n several p a r e n t a l cell lines a n d their cloned sublines were also o b s e r v e d in b o t h virally i n d u c e d ($69 and clones, $70 a n d clones) and c h e m i c a l l y i n d u c e d ($349 an d c11) cell lines. E x a m i n a t i o n of the reactivity of these f o u r m o n o c l o n a l a n t i b o d i e s o n n o n - n e u r o g e n i c cell fines (Table 3) p r o v e d negative. W h e n m o u s e cell lines were tested (Table 4), two c h e m i c a l l y i n d u c e d a s t r o c y t o m a cell lines were f o u n d to be positive. F u r t h e r , t w o h u m a n g l i o m a cell lines and o n e osteogenic s a r c o m a cell line tested (data n o t shown) were negative.

Reciprocal competitive inhibition analysis of monoclonal antibodies As o b s e r v e d f r o m the a b o v e cell line panel analysis, 7G4, 9F1, 10E3 an d 10E7 p r o d u c e d the same b i n d i n g p a t t e r n on the 89 cell lines tested; the first three

192 TABLE 3 CS-RIA REACTIVITY PATTERN OF ANTI-RAT ASTROCYTOMA HYBRIDOMAS AGAINST RAT NON-NEUROGENIC CELL LINES Cell line

Strain

Inducing

origin

agent

Fibroblast kidney $205 F-344 $203cll F-344 $188cll BDIX Emb~,o $90c13 F-344 Sarcoma $115cll $202c17 $204cll $278c12 $306cll $326 P483 H1240 HI241 B8617 $292c14 $293c15 P507

F-344 F-344 F-344 F-344 (embryo) F-344 (embryo) F-344 F-344 F-344 (embryo) F-344 F-344 (embryo) BDIX BDIX BDIX

Hepatoma MH1C1 Buffalo Spontaneous tumor LLC-WRC Harlan256 Wistar R2C Inbred WistarFurth P500

Buffalo

Passage

B.R.

BKG b

a

7G4

9F1

10E3

10E7

cpm

-

8

1

1

1

1

41 79

1 1

1 1

1 1

1 1

264 239 273

-

47

1

1

1

1

225

B-77ASV B-77ASV B-77ASV B-77ASV

34 5 17 12

1 1 1 1

1 1 1 1

1 1 1 1

1 1 1 1

220 201 287 246

B-77 ASV

16

1

1

1

1

222

MCA MNU MCA

14 32 26

1

1

1

1

1 1

1 1

1 1

1 1

381 235 347

DBA Human adenovirus 2

14 16

1 1

1 1

1 1

1 1

268 220

B-77 ASV SR-D ASV MCA

25 15 10

1 1 1

1 1 1

1 1 1

1 1 1

362 214 323

FPA

58

1

1

1

1

277

Carcinoma

38

1

1

1

1

230

Carcinoma Leydig cell Pituitary

44

1

1

1

1

275

157

1

1

1

1

280

a B.R.: Binding ratio. B.R./> 3 is considered significant. b B K G : Background cpm represents the counts bound of P3 mouse Ig or TIB-104 rat anti-mouse Lyt-1 Mab in control wells.

a n t i b o d i e s g a v e r e l a t i v e l y t h e s a m e b i n d i n g r a t i o a n d 1 0 E 7 a h i g h e r value. It w a s suspected that these four monoclonal antibodies were actually recognizing the same or a closely related antigenic determinant. Competitive inhibition assay was perf o r m e d w i t h 125I-labeled a f f i n i t y - p u r i f i e d m o n o c l o n a l a n t i b o d i e s a n d v a r i o u s u n l a b e l e d i n h i b i t o r s (Fig, 1). T h e b i n d i n g o f 7 G 4 , 9 F 1

a n d 10E3 w a s c o m p l e t e l y

193 TABLE 4 CS-RIA REACTIVITY PATTERN OF A N T I - R A T ASTROCYTOMA H Y B R I D O M A A G A I N S T MOUSE CELL LINES Cell line

Strain

Inducing

origin

agent

VM/Dk

-

VM/Dk VM/Dk VM/Dk VM/Dk VM/Dk VM/Dk VM/Dk VM/Dk C3He/Fj C3He/Fj

A/Hen DBA C3H

Passage

B.R. a

BKG b

7G4

9F1

10E3

10E7

cpm

48

1

1

1

1

534

Spont. Spont. Spont. Spont. Spont. Spont. B-77 ASV SR-D ASV Unknown c Unknown c

85 40 31 6 13 18 4 3 23 25

1 1 1 1 1 1 1 1 3 3

1 1 1 1 1 1 1 1 3 3

1 1 1 1 1 1 1 1 3 3

1 1 1 1 1 1 1 1 4 3

321 314 267 264 219 262 267 227 286 239

Unknown c Unknown c Unknown ¢

35 48 27

1 1 1

1 1 1

1 1 1

1 1 1

288 216 262

Unknown

Spont.

70

1

1

1

1

260

VM/Dk VM/Dk VM/Dk

MCA MCA MCA

4 3 11

1 1 1

1 1 1

1 1 1

1 1 1

190 218 295

-

32

1

1

1

1

248

Normal glial P534

A stroeytoma P492 P496 P497 P497cl 1 P560cll P560c12 P539 P569 M517 M518

Neurinoma P514 P515 P516

Neuroblastoma P530

Sarcoma P536 P544 P561

Fibroblast kidney P499

VM/Dk

a B.R.: Binding ratio. B.R. 1> 3 is considered significant. b BKG: Background cpm represents the counts bound of P3 mouse Ig or TIB-104 rat anti-mouse Lyt-1 Mab in control wells. c All cell lines were obtained from Dr. W. Wechsler, Max-Planck Institute, F.R.G. Primary tumors were induced with either E N U or MNU, but the exact inducing agent was unknown.

cross-inhibited by each other, but only partial inhibition was observed between these three monoclonal antibodies and 10E7. However, further increase of 7G4, 9F1 or 10E3 concentration to 2 0 0 / ~ g / m l did block lzsI-10E7 binding completely (data now shown). The inhibition was titratable and specific. 10E7 could only block up to 70% of its own binding, even when the concentration was increased to 200 # g / m l . These results suggest that 7G4, 9F1 and 10E3 are recognizing the same epitope and 10E7 either reacts against the same epitope but with a lower affinity, or reacts against a spatially closely related epitope. 7G4 and 10E7 were selected for further studies.

194 125.

-7G4

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-

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Fig. 1. Reciprocal competitive inhibition assay, Binding of 1251-labeled monoclonal antibodies 7G4, 9F1 (A), 10E3 and 10E7 (B) on $69-c15 monolayers following preincubation of the target cells with the designated unlabeled antibody. Results are expressed as percentage binding of ~25I-labeled antibody following preincubation with each specified competing antibody versus preincubation with 0.5% BSA in HBSS. 12G8 is a Mab produced in the same fusion which gave broad cross-reactivity against various rat cell lines including $69-c15.

Normal tissue antigen distribution analysis Normal tissue antigen distribution was first examined on isolated normal rat hepatocytes, spermatids, testicular ceils and lymphocytes by CS-RIA. Hemagglutination assays were performed on rat red blood cells which were observed to express a high density of class I transplantation antigens on the cell surface. Monoclonal antibodies showed negative results in both assays (Table 5). Tissue antigen localization was further examined by sequential absorption analysis. Absorption was first performed with washed tumor and tissue homogenates prepared as described (Bourdon et al. 1983). Persistent negative absorptions were obtained in either tumors or normal tissues. Whole-tumor homogenates were later found necessary to remove antibody activity (Fig. 2). Primary ASV-induced tumors were not used for absorption due to long induction time and small amounts of tumor tissue available. Sequential absorption analysis using whole-tissue homogenates showed higher variation than those using washed tissue homogenates. The increased binding activity observed in kidney for 7G4 and heart and muscle for 10E7 could be the result of antibody aggregation during absorption in these tissue homogenates, which would give apparent higher binding specificity on $69-c15 monolayers in CS-RIA. For both antibodies, testis gave persistent partial absorption of antibody activity which was contrary to data obtained in CS-RIA on isolated cells. In all, absorption analysis failed to demonstrate antigen activity in brain, heart, lung, kidney, liver, spleen, thymus and muscle.

195 TABLE 5 A N T I - R A T A S T R O C Y T O M A H Y B R I D O M A R E A C T I V I T Y ON I S O L A T E D N O R M A L R A T CELLS W I T H CS-RIA A N D H E M A G G L U T I N A T I O N ASSAY B.R. a

BKG b

7G4

9F1

10E3

10E7

cpm

1 1 1 1

1

1

1

1

1

1

1 1

1 1

1 1

275 159 260 357

1 1 1 1

1 1 1 1

1 1 1 1

1 1 1 1

397 453 823 345

CR-RIA Primary hepatocytes Spermatids Testicular cells Red blood cells ', Lymphocytes - peripheral blood - l y m p h node - spleen - thymus

Hemagglutination Direct assay Indirect assay a B.R.: Binding ratio. B.R./> 3 is considered significant. b BKG: Background c p m represents the counts b o u n d of P3 mouse Ig or TIB-104 rat anti-mouse Lyt-1 M a b in control wells.

7G4 257

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Fig. 2. Absorption analysis of anti-rat astrocytoma monoclonal antibodies 7G4 and 10E7 with normal rat tissue homogenates and tumor homogenate of rat astrocytoma cell line $69-c15 grown in syngeneic F-344 newborn rats. Each antibody was absorbed sequentially 1 - 5 times with a fixed weight of lyophilized materials. Remaining antibody activity was assayed by CS-RIA on $69-c15 monolayers and is expressed as the percentage of pre-absorbed binding activity.

196

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Fig. 4. Absorption analysis Of anti-rat astrocytoma monoclonal antibody 7G4 with untreated and heated tumor homogenates from rat astrocytoma cell line $69-c15 grown in syngeneic F-344 rats. Antibody was absorbed sequentially 1-5 times with a fixed weight of lyophilized homogenate. Remaining antibody activity was assayed by CS-RIA. W h e n tissue sections were examined with biotinylated antibodies, both subcutaneous and intracranial $69-c15 transplanted tumors were positive with strong perinuclear staining (Fig. 3). TIB104 was positive on B A L B / c mouse spleen. All other tissues tested were negative.

Characterization of antigenic determinants Viable $69-c15 cells were treated with various enzymes, and a n t i b o d y binding capacity determined by C S - R I A . Antigen activity was found to be sensitive to various enzymes such as trypsin, proteases and pronase, but resistant to neuraminidase and D N A s e treatment. Heat sensitivity tests were done with both intact cells using C S - R I A (Dippold et al. 1980) and homogenized antigen preparations using absorption analysis. The antigen activity as determined by both methods was lost after treatment at 1 0 0 ° C for 1 min (Fig. 4). Lipid solubilization failed to d e m o n strate lipid-extractable antigen activity. These data suggested that the antigen recognized by these four monoclonal antibodies was a polypeptide-associated epitope rather than a glycolipid.

Discussion In syngeneic immunization systems, the differences between the host and tumor i m m u n o g e n are minimal; this system also provides the most convincing evidence of Fig. 3. Tissue staining of glioma-associated antigen and mouse Lyt-1 antigen. A and C: $69-c15 tumors, subcutaneous and intracranial, T1B-104 Mab staining. B and D: $69-c15 tumors, 7G4 Mab. E: BALB/c mouse spleen, T1B-104. F: BALB/c mouse spleen, 7G4.

198 the existence of tumor-associated antigens (Law et al. 1980). However, except in certain special cases such as swine melanoma (Berkelhammer et al. 1982) and ultraviolet radiation-induced murine skin tumor systems (Schmitt et al. 1983), syngeneic tumors seem to be only weakly immunogenic and usually produce fatal, progressively growing tumors after transplantation. This is also true for avian sarcoma virus (ASV)-induced mammalian tumors which usually produce tumors that rarely regress (Bauer and Fleischer 1981), and other procedures, such as tumor ligation or transplantation of irradiated tumor cells for immunization (Herberman 1977) must be employed. The availability of the B-77 ASV-induced F-344 rat astrocytoma cell line $69-c15 which was persistently rejected in adult syngeneic rats and produced regressing tumors in newborns provided us the opportunity to investigate the possibility of establishing a well-defined syngeneic animal model of monoclonal antibody production and therapeutic application in an animal glioma system. The fusion between mouse myeloma cell line P3 x 63/Ag8.653 and spleen cells from an adult F-344 rat immunized with $69-c15 cells produced four monoclonal antibodies (Mabs: 7G4, 9F1, 10E3 and 10E7) which gave a highly restrictive reactivity pattern against a large panel of cell lines derived from different species. They reacted with identical cell lines; three of the four (7G4, 9F1 and 10E3) apparently recognized the same antigenic determinants; 10E7 reacted with a spatially close or overlapping epitope as determined by reciprocal competitive inhibition assays. This suggests that this epitope(s) is highly immunogenic and may be responsible for the majority of specific immune reactivity in this syngeneic immunization system. The antigenic specificities recognized by these four Mabs are expressed only on neurogenic tumor cell lines as detected with CS-RIA. More interestingly, these specificities exist not only on rat astrocytomas induced by ASV but are also found on those induced with chemical carcinogens, mainly nitrosourea. Cross-species reactivity is also demonstrated by their activity against two chemically induced murine glioma cell lines. No antigenic activity was found in normal tissues examined either as isolated cells or in tissue sections. Sequential absorption analysis with homogenates of normal rat tissues and tumors also confirmed this data except that testis homogenates partially absorbed antibody activity. This apparent contradiction between CS-RIA, staining and absorption analysis may be explained by the higher sensitivity of absorption analysis (Dippold et al. 1980). Further characterization is needed to clarify this discrepancy. Initial attempts to characterize these antigenic specificities biochemically established that the activity seems to reside on a peptide molecule that can be easily released from the cell during tissue homogenization. This phenomenon has also been observed for common tumor antigen P53 (Law et al. 1980) which is found in both virally and chemically induced tumors (Levine 1982), and is readily released into the supernatant after homogenization. However, P53 exists intranuclearly, and has a distribution (including fibrosarcomas) unlike that recognized by the four Mabs described here.

199 It is a commonly held maxim that chemically induced tumors usually express individually distinctive tumor-associated transplantation antigens that are not shared by other tumors induced with the same carcinogen, or even in the same animal (Baldwin 1973); virally induced tumors produce cross-reactive tumor antigens among tumors induced by the same virus even in different animal species (Bauer and Fleischer 1981). However, this difference is not absolute when more extensive studies were done with conventional immunoassays (Porta and Parmiani 1976; Embleton and Baldwin 1979) and monoclonal antibody technology (Kuzumaki et al. 1982; Gallagher and Burk 1983). Further, cross-reactive antigens expressed in both virally and chemically induced tumors from different species have been demonstrated (Ferracini et al. 1982). Cross-reactive glioma antigens, as found in this study, could be attributed to the activation of endogenous type C virus in chemically induced glioma cell lines (Anzil and Stavrou 1978; Stavrou et al. 1979); for example a subline of the positively reactive MNU-induced rat glioma line C-6 in our study was observed to produce type C virus after steroid treatment (Armelin et al. 1983). A 70 kDa protein was immunoprecipitated with immune serum against rat type C endogenous virus from two transformed fibroblast cell lines induced separately by B-77 ASV and 3,4benzo(a)pyrene, and a similar 70 kDa protein was also detected on B-77 ASV-transformed rat cells by antiserum against Friend murine leukemia virus envelope glycoprotein gp70 (Duraj et al. 1982). In addition to viruses, embryonic antigens have also been suggested as the basis for common antigenicity in chemically induced tumors (Alexander 1976); in virally induced systems, cross-reactive embryonic and transformation-specific non-viral antigens have been demonstrated (Ignjatovic et al. 1978), The expression of these cross-reactive antigens has been correlated with the expression of the ASV s r c gene (Kuzamaki et al. 1984). As all normal cells possess genes homologous to the various oncogenes (Bishop 1983) including the s r c gene, and oncogene activation has also been demonstrated in chemical carcinogenesis (Eva and Aaronson 1983), it is likely that the c - s r c gene or other oncogenes with related protein kinase activity are activated during tumor induction. These activated cellular oncogene products and the transduced v - s r c gene product may interact with a cell surface moiety to give rise to the cross-reactive antigenic determinant observed in these experiments. Until the molecular nature of the antigenic determinant is recognized, we cannot predict the relationship of this antigen with endogenous type C virus-related antigen, embryonic antigens and cellular moieties modified by oncogenic products. Data presented here have confirmed the existence of phenotypic tumor cell heterogeneity (Stravou et al. 1983; Wikstrand et al. 1983); target antigen was not expressed in all subclones of cell lines. Even though most parental cell lines in the positive groups remained antigen positive, the loss of antigen expression in the parental line $70 but not in its clones warrants the use of early-passage cells in tumor studies and the advisability of cloning to retain stable phenotypes. Another implication of tumor antigen heterogeneity is that the use of monoclonal antibodies in immunotherapeutic and diagnostic applications would necessitate use of a mixture of monoclonal antibodies recognizing different epitopes.

200 I n s u m m a r y , t h e results suggest the e x i s t e n c e of a g l i o m a - s p e c i f i c a n t i g e n , o n e w h i c h is c r o s s - r e a c t i v e w i t h b o t h A S V - a n d c h e m i c a l l y i n d u c e d t u m o r s in b o t h rats a n d mice. S i n c e t h e s e g l i o m a - s p e c i f i c a n t i b o d i e s are s y n g e n e i c to rat a n d t h e A S V i n d u c t i o n o f rat g l i o m a s is well c h a r a c t e r i z e d ( B i g n e r et al. 1975), it is p o s s i b l e to u s e t h e s e M a b s w i t h i n a s y n g e n e i c s y s t e m in the s t u d i e s of i n t r a c e r e b r a l a n d s u b c u t a n e o u s t u m o r l o c a l i z a t i o n as w e l l as t u m o r p r e v e n t i o n a n d t h e r a p y . T h i s m a y p r o v i d e us w i t h v a l u a b l e i n f o r m a t i o n for t h e f u t u r e d e v e l o p m e n t a n d a p p l i c a t i o n of human monoclonal antibodies against gliomas.

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201 Copeland, D.D., M.W. Cloyd, W. Wechsler, P. Ingrain and D.D. Bigner. Surface morphology of avian sarcoma virus and ethyl-nitrosourea transformed rat neuroectodermal ceils and human glioblastoma cells in organ and monolayer culture. In: O. Johari and R.P. Becker (Eds.), The Ninth Scanning Electron Microscopy Symposium, Toronto, Illinois Institute of Technology Research Institute, Chicago, IL, 1976, pp. 93-100. de Tribolet, N. and S. Carrel, Human glioma tumor-associated antigens, Cancer Immunol. Immunother., 9 (1980) 201-211. Dippold, W.G., K.O. Lloyd, T.C: Li, H. Ikeda, H.F. Oettgen and L.J. Old, Cell surface antigens of human malignant melanoma: definition of six antigenic systems with mouse monoclonal antibodies, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 6114-6118. Duraj, J., B. Chorvath and D. Simkovic, Cell surface alterations of avian sarcoma virus B-77- and 3,4-benzo(a)pyrene-transformed rat fibroblast. I. Cell surface proteins characterized by two-dimensional electrophoresis, Neoplasm, 29 (1982) 377-386. Embleton, M.J. and R.W. Baldwin, Tumor-related antigen specificities associated with 3-methylcholanthrene treated rat embryo cells, Int. J. Cancer, 23 (1979) 840-845. Eva, A. and S.A. Aaronson, Frequent activation of C-kis as a transforming gene in fibrosarcoma induced by methylcholanthrene, Science, 220 (1983) 955-956. Ferracini, R., M. Prat and P.M. Comoglio, Dissection of the antigenic determinants expressed on the cell surface of RSV-transformed fibroblasts by monoclonal antibodies, Int. J. Cancer, 29 (1982) 477-481. Gallagher, W.J. and M.W. Burk, Syngeneic monoclonal antibodies directed against chemically induced murine fibrosarcoma, Curt. Surg., 40 (1983) 361-364. Gefter, M.L., D.A. Margulies and M.D. Scharff, A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells, Somatic Cell Mol. Genet., 3 (1977) 231-236. Greenwood, F.C., W.M. Hunter and J.S. Glover, The preparation of 131I labeled human growth hormone of high specificities, Biochem. J., 89 (1963) 114-123. Harwood, S.E., D.D. Bigner, W. Wechsler and E.D. Day, Antigens shared by rat schwannomas, neuroblastomas, and a testicular interstitial cell carcinoma, Cancer Res., 37 (1977) 3379-3384. Herberman, R.B., Immunogenicity of tumor antigens, Biochim. Biophys. Acta, 473 (1977) 93-119. Ignjatovic, J., H. Rubsamen, M. Hayami and H. Bauer, Rous sarcoma virus-transformed avian cells express four different cell surface antigens that are distinguishable by cell mediated cytotoxicityblocking test, J. Immunol., 120 (1978) 1663-1688. Kornblith, P.L., F.C. Donan, W.C. Wood and B.O. Whitman, Human astrocytoma: serum mediated immunologic response, Cancer, 33 (1974) 1512-1519. Kurtzberg, J. and Hershfield, M.S., Determinants of deoxyadenosine toxicity in hybrids between human T and B lymphoblasts as a model for the development of drug resistance in T cell acute lymphoblastic leukemia, Cancer Res., 45 (1985) 1579-1586. Kuzumaki, N., H. Minakawa, T. Miyazaki, S. Haraguchi, T. Matsuo and T.O. Yoshida, Individually distinct tumor-specific cell surface antigens identified by monoclonal antibody on a Rous sarcoma virus-induced mouse tumor, J. Natl. Cancer Inst., 69 (1982) 527-530. Kuzumaki, N., T. Oikawa and T. Yamada, Independent expression of common and private tumor-specific cell surface antigens in somatic cell hybrids between Rous sarcoma virus-transformed and normal cells, J. Natl. Cancer Inst., 72 (1984) 387-393. Law, L.W., M.J. Rogers and E. Appella, Tumor antigens on neoplasms induced by chemical carcinogens and by DNA- and RNA-containing viruses: properties of the solubilized antigens. In: G. Klein and S. Weinhouse (Eds.), Advances in Cancer Research, Academic Press, New York, 1980, pp. 201-235. Levine, A.J., Transformation-associated tumor antigens. In: G. Klein and S. Weinhouse (Eds.), Advances in Cancer Research, Academic Press, New York, 1982, pp. 75-109. Lowry, O.H., N.J. Rosebrough, A . L Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 264-275. Misra, D.N., S.A. Noeman, H.W. Kunz and T.J. Gill, III, Identification of two class I antigens coded by the major histocompatibility complex of the rat by using monoclonal antibodies, J. Immunol., 128 (1982) 1651-1658. Porta, G.D. and G. Parmiani, Antigens of chemically induced tumors, Exp. Cell Biol., 44 (1976) 170-183.

202 Pukel, C.S., K.O. Lloyd, L.R. Travassos, W.G. Dippold, H.F. Oettgen and J.J. Old, GD3, a prominent ganglioside of human melanoma detected and characterized by mouse monoclonal antibody, J. Exp. Med., 155 (1982) 1133-1147. Schmitt, M.K., L.K. Roberts, L.C. Gahring and R.A. Daynes, Enhanced tumorigenicity of cloned UV-regressor tumor lines following selected in vivo and in vitro manipulations, Am. J. Pathol., 113 (1983) 269-278. Schnegg, J.L., A.C. Diserens, S. Carrel, R.S. Accolla and N. de Tribolet, Human gliorna-associated antigens detected by monoclonal antibodies, Cancer Res., 41 (1981) 1209-1213. Seglen, P.O., Preparation of isolated rat liver cells, Methods Cell Biol, 13 (1976) 29-83. Stavrou, D., V. Osterkamp, B. Schroder, A.P. Anzil and K. Zanker, Selected morphological immunocytochemical and growth characteristics of three experimental rat gliomas and of their cell in vitro, Exp. Cell Biol., 47 (1979) 3-21. Stavrou, D., C. Suss, T. Bilzer, U. Kummar and N. de Tribolet, Monoclonal antibodies reactive with glioma cell lines derived from experimental brain tumors, Eur. J. Cancer Clin. Oncol., 19 (1983) 1439-1449. Strom, S.C. and G. Michalopoulas, Collagen as a substrate for cell growth and differentiation, Methods Enzymol., 82 (1982) 544-555. Wikstrand, C.J. and D.D. Bigner, Immunobiologic aspects of the brain and human gliomas, Am..I. Pathol., 98 (1980) 516-567. Wikstrand, C.J. and D.D. Bigner, Expression of human fetal brain antigens by human tumors of neuroectodermal origin as defined by monoclonal antibodies, Cancer Res., 42 (1982) 267-274. Wikstrand, C.J. and D.D. Bigner, Demonstration of complex antigen heterogeneity in a human glioma cell line and eight derived clones by specific monoclonal antibodies, Cancer Res., 43 (1983) 3327-3334. Wikstrand, C.J., M.S. Mahaley and D.D. Bigner, Surface antigenic characteristics of human glial brain tumor cells, Cancer Res., 37 (1977) 4267-4275.