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Supplementary Figure Legends - PDF file 88K, Supplementary Figure Legends 1-9

Supplementary Methods - PDF file 94K, The supplementary methods section describes the following: Construction, expression and purification of different EGFRvIII deletion mutants, cloning of variable heavy (VH) and variable light (VL) domains of P588 and D2C7 mAb, surface plasmon resonance, stability assay, and immunohistochemistry of frozen D270MG brain tumor tissue

Purpose: The EGF receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583-637 AAs) and EGFRvIII (292-346 AAs) proteins.Experimental Design: The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII were measured by surface-plasmon resonance and flow cytometry. In vitro cytotoxicity of D2C7-(scdsFv)-PE38KDEL was measured by inhibition of protein synthesis in human EGFRwt-transfected NR6 (NR6W), human EGFRvIII-transfected NR6 (NR6M), EGFRwt-overexpressing A431-epidermoid-carcinoma, and glioblastoma xenograft cells (43, D08-0493MG, D2159MG, and D270MG). In vivo antitumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated using 43, NR6M, and D270MG orthotopic tumor models.Results: The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6 × 10−9 mol/L and 1.3 × 10−9 mol/L, respectively. Flow cytometry with NR6W and NR6M cells confirmed the specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII. The D2C7-(scdsFv)-PE38KDEL IC50 was 0.18 to 2.5 ng/mL on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was approximately 0.25 ng/mL on EGFRvIII-expressing cells (NR6M) and on EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells (D2159MG and D270MG). Significantly, in intracranial tumor models of 43, NR6M, and D270MG, treatment with D2C7-(scdsFv)-PE38KDEL by convection-enhanced delivery prolonged survival by 310% (P = 0.006), 28% (P = 0.002), and 166% (P = 0.001), respectively.Conclusions: In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, or both. Clin Cancer Res; 19(17); 4717–27. ©2013 AACR.

Supplementary Figures 1-9 - Supplementary Figures 1-9 - PDF file 516K, Supplementary Figure S1. Deduced amino acid sequence of the D2C7 scFv cloned from D2C7 hybridoma. VH (A) and VL (B) antigen-binding regions of the D2C7 scFv. Amino acid numbering and CDR (underlined) delimitation were determined according to the Kabat database. Supplementary Figure S2. Purified D2C7-(scdsFv)-PE38KDEL immunotoxin. For this analysis, 200 ng of the purified D2C7-(scdsFv)-PE38KDEL immunotoxin was run on NuPAGE 4-12% Bis-Tris gel (Invitrogen), along with a standard molecular weight protein marker. Supplementary Figure S3. Surface plasmon resonance analysis of D2C7-(scdsFv)-PE38KDEL. Binding kinetics and affinity constants of D2C7-(scdsFv)-PE38KDEL for EGFRwt (A) and EGFRvIII (B) were determined by surface plasmon resonance against bacterially expressed recombinant EGFRwt or EGFRvIII extracellular domain (ECD) proteins. The association and dissociation rates from the sensogram were KA = 6.3 �~ 108 M-1 and KD = 1.6 �~ 10-9 M against EGFRwt and KA = 7.8 �~ 108 M-1 and KD = 1.3 �~ 10-9M against EGFRvIII. Supplementary Figure S4. Flow cytometric analysis of CD133 and D2C7 mAb on 43 and D2159MG glioblastoma xenograft cells. FACS analysis demonstrates the double staining of CD133 and D2C7 (percentage of cells in Q2) on 71% (A) and 86% (B) of cells freshly isolated from glioblastoma xenografts 43 and D2159MG, respectively. Q1 represents the percentage of cells positive for D2C7 only; Q4 represents the percentage of cells positive for CD133 only and Q3 represents the percentage of cells negative for both CD133 and D2C7. Supplementary Figure S5. Stability of D2C7-(scdsFv)-PE38KDEL. D2C7-(scdsFv)-PE38KDEL was incubated with 0.2% BSA/PBS at 40 ��g/mL (A) and 7.5 ��g/mL (B), over a 7 day period at 37{degree sign}C and then assayed for cytotoxic activity on NR6M cells. Cytotoxicity data are given as IC50 value versus radioactive leucine incorporation. Supplementary Figure S6. Flow cytometric analysis of D2C7 mAb and mouse EGFR antibody on normal mousekidney fibroblast cells (499) or avian sarcoma virus transformed mouse glioma cells (539). FACS analysis demonstrates the reactivity of mouse EGFR (msEGFR) antibody (pink peaks) to EGFR expressing 499 (A) and 539 (C) cells and the absence of D2C7 (pink peaks) reactivity to both 499 (B) and 539 (D) cells, confirming that D2C7 is human specific and does not react with murine EGFR. Isotype-specific antibody (IgG1) was used as control (filled purple peaks). Supplementary Figure S7. Survival of mice injected intracranially with 43 xenograft cells (A), NR6M cells (B), or D270MG xenograft cells (C). Mice were injected intracranially with 1x105 43, NR6M, or D270MG cells and were checked daily for survival. Data are presented as the survival time in days versus percentage of mice surviving. Supplementary Figure S8. Toxicity of D2C7-(scdsFv)-PE38KDEL immunotoxin administered to NSG mice. Different doses of D2C7-(scdsFv)-PE38KDEL were delivered intracranially over a 7 day period to NSG mice (5 mice/group). Animals were monitored for toxicity related death. Data are expressed as percentage of mice surviving versus time. Supplementary Figure S9. Immunohistochemical detection of D2C7-(scdsFv)-PE38KDEL distribution in D270MG orthotopic model after immunotoxin treatment. Acetone-fixed frozen D270MG-sham (negative control) (a) and D270MG-D2C7-(scdsFv)-PE38KDEL group (test) (c) sections were stained with 1.0 ��g of mouse-anti-PE38KDEL antibodies. Tumor sections from D270MG-D2C7-(scdsFv)-PE38KDEL group, pre-stained with D2C7-(scdsFv)-PE38KDEL was used as positive control (b)

Monoclonal antibodies have become essential tools for diagnostic and therapeutic purposes. Antibody affinity is one of the critical factors influencing the therapeutic success of tumor targeting antibodies. Therefore, developing an accurate and reliable method for determining antibody affinity is crucial. In this study, we describe a fluorescent cell-based immunosorbent assay that can accurately measure antibody affinity (KD) in the nanomolar range. This method involves the addition of fluorescently labelled antibodies to antigen-positive and antigen-negative cell lines fixed on 96-well plates. The fluorescent signals from nonspecific binding to negative control cell lines is subtracted from the specific binding to the antigen-positive cell lines. The KD values obtained by this method were comparable with values obtained by the flow cytometry and radioactive (I125) scatchard assay. Our results demonstrate that this modified cell-based fluorescent method allows for a convenient and efficient identification of therapeutically relevant leads.

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel recombinant Pseudomonas exotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. Initial pre-clinical testing demonstrated the anti-tumor efficacy of D2C7-IT against orthotopic glioblastoma xenograft models expressing EGFRwt, EGFRvIII, or both EGFRwt and EGFRvIII. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter in Escherichia coli BLR (λ DE3). D2C7-IT was produced by a 10-L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300 mg of purified protein. The final vialed batch of D2C7-IT for clinical testing was at a concentration of 0.12 ± 0.1 mg/mL, the pH was at 7.4 ± 0.4, and endotoxin levels were below the detection limit of 10 EU/mL (1.26 EU/mL). The stability of the vialed D2C7-IT has been monitored over a period of 42 months through protein concentration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, size exclusion chromatography, cytotoxicity, sterility, and pH measurements. The vialed D2C7-IT is currently being tested in a phase I/II clinical trial by intratumoral convection-enhanced delivery for 72 h in patients with recurrent glioblastoma (NCT02303678, D2C7 for Adult Patients with Recurrent Malignant Glioma; clinicaltrials.gov ).

The lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1 have been identified as tumor-associated antigens whose formation is initiated by the Lc3-synthase. Until now, high-affinity IgG monoclonal antibodies (mAbs) against 3'-isoLM1 and 3',6'-isoLD1, which are highly expressed in gliomas, have not been developed, although mAbs against lacto-series gangliosides are powerful tools for functional studies. We previously produced the Lc3-synthase gene beta3Gn-T5 knockout mice. In this study, we immunized beta3Gn-T5 knockout mice with 3'-isoLM1/3',6'-isoLD1 and produced the anti-3'-isoLM1/3',6'-isoLD1 mAb GMab-1, of the IgG(3) subclass, which should be useful for functional analysis of lacto-series gangliosides and for antibody-based therapy of gliomas.

Adults with primary malignant glioma have an unacceptably poor outcome. Although temozolomide plus radiotherapy improves survival of newly diagnosed glioblastoma multiforme (GBM) patients,1 most patients develop tumor progression within 1 – 2 years. Outcome following recurrence is poor.2 Most tumors recur at or adjacent to the site of origin, indicating that failure to eradicate local tumor growth is a major factor contributing to poor outcome.3 For this reason, we have focused on augmenting local control to improve overall outcome by administering tumor-associated radiolabeled monoclonal antibodies (mAbs) directly into spontaneous tumor cysts, surgically created resection cavities (SCRCs), the intrathecal space, and solid tumors.4–8 Tenascin, an extracellular matrix hexabrachion glycoprotein, is expressed ubiquitously in several cancers, including high-grade gliomas, but not in normal brain.9,10 mAb 81C6, a murine isotype 2b immunoglobulin G (IgG2b) that binds to an alternatively spliced region of tenascin,9–12 reacts specifically with tenascin-expressing tumors.13 When labeled with 131I, 81C6 delays tumor growth and prolongs survival in flank and intracranial human xenograft models.14,15 An initial human experience demonstrated the specificity of 131I-labeled murine 81C6 (131I-81C6) mAb compared to 125I-labeled nonspecific IgG2b mAb but also revealed limited intratumor penetration following intravenous or intraarterial administration.16 Hence, subsequent studies incorporated administration into an SCRC, tumor cyst, or intrathecal space. Prior phase I studies established the maximum tolerated dose (MTD) of 131I-81C6 injected into the SCRC of patients with newly diagnosed and recurrent malignant brain tumors to be 120 mCi and 100 mCi, respectively.7,8 Phase II studies demonstrated that patients treated with 131I-81C6 had favorable overall survival compared to established historical controls.4,5 Based on constraints of our U.S. Food and Drug Administration (FDA)-approved phase I and II studies, patients received a “fixed” dose of 131I-81C6 in which the administered level of radioactivity was the same among groups of patients. As a result of this design, 131I dosages were not adjusted to compensate for patient-specific variables such as SCRC volume and SCRC residence time. Dosimetry of patients treated on these studies revealed a wide range of radiation absorbed doses at the SCRC margin. Moreover, outcome correlated with delivered radiation dose to the SCRC. Specifically, patients who received less than 44 Gy were more likely to develop recurrent tumor, whereas those who received significantly more than 44 Gy were more likely to develop radionecrosis. Therefore, a 44-Gy boost to the SCRC margin was considered optimal.17 We now report a pilot study using a novel, patient-specific dosing strategy of 131I-81C6 designed to achieve a 44-Gy boost to the 2-cm SCRC margin in adults with newly diagnosed malignant gliomas.

Purpose To assess the efficacy and toxicity of intraresection cavity iodine-131–labeled murine antitenascin monoclonal antibody 81C6 (131I-m81C6) among recurrent malignant brain tumor patients. Patients and Methods In this phase II trial, 100 mCi of 131I-m81C6 was injected directly into the surgically created resection cavity (SCRC) of 43 patients with recurrent malignant glioma (glioblastoma multiforme [GBM], n = 33; anaplastic astrocytoma [AA], n = 6; anaplastic oligodendroglioma [AO], n = 2; gliosarcoma [GS], n = 1; and metastatic adenocarcinoma, n = 1) followed by chemotherapy. Results With a median follow-up of 172 weeks, 63% and 59% of patients with GBM/GS and AA/AO tumors were alive at 1 year. Median overall survival for patients with GBM/GS and AA/AO tumors was 64 and 99 weeks, respectively. Ten patients (23%) developed acute hematologic toxicity. Five patients (12%) developed acute reversible neurotoxicity. One patient (2%) developed irreversible neurotoxicity. No patients required reoperation for radionecrosis. Conclusion In this single-institution phase II study, administration of 100 mCi of 131I-m81C6 to recurrent malignant glioma patients followed by chemotherapy is associated with a median survival that is greater than that of historical controls treated with surgery plus iodine-125 brachytherapy. Furthermore, toxicity was acceptable. Administration of a fixed millicurie dose resulted in a wide range of absorbed radiation doses to the SCRC. We are now conducting a phase II trial, approved by the US Food and Drug Administration, using patient-specific 131I-m81C6 dosing, to deliver 44 Gy to the SCRC followed by standardized chemotherapy. A phase III multicenter trial with patient-specific dosing is planned.

PURPOSE: To assess the efficacy and toxicity of intraresection cavity 131I-labeled murine antitenascin monoclonal antibody 81C6 and determine its true response rate among patients with newly diagnosed malignant glioma. PATIENTS AND METHODS: In this phase II trial, 120 mCi of 131I-labeled murine 81C6 was injected directly into the surgically created resection cavity of 33 patients with previously untreated malignant glioma (glioblastoma multiforme [GBM], n = 27; anaplastic astrocytoma, n = 4; anaplastic oligodendroglioma, n = 2). Patients then received conventional external-beam radiotherapy followed by a year of alkylator-based chemotherapy. RESULTS: Median survival for all patients and those with GBM was 86.7 and 79.4 weeks, respectively. Eleven patients remain alive at a median follow-up of 93 weeks (range, 49 to 220 weeks). Nine patients (27%) developed reversible hematologic toxicity, and histologically confirmed, treatment-related neurologic toxicity occurred in five patients (15%). One patient (3%) required reoperation for radionecrosis. CONCLUSION: Median survival achieved with 131I-labeled 81C6 exceeds that of historical controls treated with conventional radiotherapy and chemotherapy, even after accounting for established prognostic factors including age and Karnofsky performance status. The median survival achieved with 131I-labeled 81C6 compares favorably with either 125I interstitial brachy-therapy or stereotactic radiosurgery and is associated with a significantly lower rate of reoperation for radionecrosis. Our results confirm the efficacy of 131I-labeled 81C6 for patients with newly diagnosed malignant glioma and suggest that a randomized phase III study is indicated.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine-131 (131I)–labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment. PATIENTS AND METHODS: In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of 131I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment. RESULTS: We treated 42 patients with 131I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively. CONCLUSION: The MTD for administration of 131I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.

About 60 percent of glioblastomas highly express the gangliosides 3'-isoLM1 and 3',6'-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3'-isoLM1 and 3',6'-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3'-isoLM1 and 3',6'-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3'-isoLM1 and 3',6'-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the KD of DmAb14m-IT for gangliosides 3'-isoLM1 and 3',6'-isoLD1 was 2.6 × 10(-9)M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3'-isoLM1 and 3',6'-isoLD1. The DmAb14m-IT IC 50 was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3'-isoLM1 and 3',6'-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3'-isoLM1 and 3',6'-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and eventually in the clinical setting.

These studies evaluated the efficacy of sequential pretreatment with L-amino acid oxidase (LOX) and LOX antiserum in the modulation of melphalan activity against intracranial glioma in athymic nude mice. LOX produced statistically significant (P0.01) depletion of the large neutral amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine, and valine in murine plasma at doses of 100 and 200 micrograms administered intravenously. Polyclonal anti-LOX antibody was successfully produced in mice, rabbits, and goats subsequent to immunization with LOX. Staphylococcal protein A-purified rabbit anti-LOX serum inhibited approximately 50% of LOX activity in vitro relative to control samples. This antiserum was used in vivo to inactivate LOX after it had depleted the large neutral amino acids, thereby preventing LOX-mediated catabolism of melphalan. Inoculation of three mice with rabbit anti-LOX serum after the treatment with LOX (100 micrograms) reduced LOX activity by 100%, 89%, and 100% at 6 h compared with reductions of 80%, 59%, and 52% over the same period in animals receiving LOX alone. In three separate studies using groups of eight to ten mice bearing intracranial human glioma xenografts, pretreatment with LOX followed by anti-LOX serum increased the antitumor activity of melphalan as compared with treatments with melphalan plus LOX, melphalan plus anti-LOX serum, or melphalan alone.

Introduction Malignant glioma remains a significant therapeutic challenge, and immunotherapeutics might be a beneficial approach for these patients. A monoclonal antibody (MAb) specific for multiple molecular targets could expand the treatable patient population and the fraction of tumor cells targeted, with potentially increased efficacy. This motivated the generation of MAb D2C7, which recognizes both wild-type epidermal growth factor receptor (EGFRwt) and a tumor-specific mutant, EGFRvIII. Methods D2C7 binding affinity was determined by surface plasmon resonance and its specificity characterized through comparison to EGFRwt-specific EGFR.1 and EGFRvIII-specific L8A4 MAbs by flow cytometry and immunohistochemical analysis. The three MAbs were labeled with 125 I or 131 I using Iodogen, and paired-label internalization assays and biodistribution experiments in athymic mice with human tumor xenografts were performed. Results The affinity of D2C7 for EGFRwt and EGFRvIII was 5.2×10 9 M −1 and 3.6×10 9 M −1 , and cell-surface reactivity with both receptors was documented by flow cytometry. Immunohistochemical analyses revealed D2C7 reactivity with malignant glioma tissue from 90 of 101 patients. Internalization assays performed on EGFRwt-expressing WTT cells and EGFRvIII-expressing NR6M cells indicated a threefold lower degradation of 125 I-labeled D2C7 compared with 131 I-labeled EGFR.1. Uptake of 125 I-labeled D2C7 in NR6M xenografts (52.45±13.97 %ID g −1 on Day 3) was more than twice that of 131 I-labeled L8A4; a threefold to fivefold tumor delivery advantage was seen when compared to 131 I-labeled EGFR.1 in mice with WTT xenografts. Conclusions These results suggest that D2C7 warrants further evaluation for the development of MAb-based therapeutics against cancers expressing EGFRwt and EGFRvIII.

Background Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. Results B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. Conclusions These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.

Introduction: Podoplanin/aggrus is a mucin-like sialoglycoprotein that is highly expressed in malignant gliomas. Podoplanin has been reported to be a novel marker to enrich tumor-initiating cells, which are thought to resist conventional therapies and to be responsible for cancer relapse. The purpose of this study was to determine whether an anti-podoplanin antibody is suitable to target radionuclides to malignant gliomas. Methods: The binding affinity of an anti-podoplanin antibody, NZ-1 (rat IgG2a), was determined by surface plasmon resonance and Scatchard analysis. NZ-1 was radioiodinated with 125 I using Iodogen [ 125 I-NZ-1(Iodogen)] or N-succinimidyl 4-guanidinomethyl 3-[ 131 I] iodobenzoate ([ 131 I]SGMIB-NZ-1), and paired-label internalization assays of NZ-1 were performed. The tissue distribution of 125 I-NZ-1 (Iodogen) and that of [ 131 I]SGMIB-NZ-1 were then compared in athymic mice bearing glioblastoma xenografts. Results: The dissociation constant (KD) of NZ-1 was determined to be 1.2×10 −10 M by surface plasmon resonance and 9.8×10 −10 M for D397MG glioblastoma cells by Scatchard analysis. Paired-label internalization assays in LN319 glioblastoma cells indicated that [ 131 I] SGMIB-NZ-1 resulted in higher intracellular retention of radioactivity (26.3±0.8% of initially bound radioactivity at 8 h) compared to that from the 125 I-NZ-1(Iodogen) (10.0±0.1% of initially bound radioactivity at 8 h). Likewise, tumor uptake of [ 131 I]SGMIB-NZ-1 (39.9±8.8 % ID/g at 24 h) in athymic mice bearing D2159MG xenografts in vivo was significantly higher than that of 125 I-NZ-1(Iodogen) (29.7±6.1 %ID/g at 24 h). Conclusions: The overall results suggest that an anti-podoplanin antibody NZ-1 warrants further evaluation for antibody-based therapy against glioblastoma.

Background Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). Methods We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. Results Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). Conclusions Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.

Epidermal growth factor receptor (EGFR) is overexpressed or mutated in a high percentage of tumors. EGFR has long been considered a promising target for cancer diagnostic and therapeutic applications. However, monoclonal antibodies and other large antibody constructs diffuse into tumors slowly, limiting their efficacy. To develop lower molecular weight probes for EGFR and other tumor cell receptors, the authors immunized a llama with the extracellular domains (ECDs) of EGFR and an oncogenic mutant receptor, EGFRvIII, and with extracts of tumor cell lines. From the immune repertoire of the llama, the authors constructed a heavy chain variable domain (VHH domain)—phage library. At ~16 kDa, the VHH domain is a tenth of the size of a monoclonal antibody and is the smallest antibody fragment that retains specificity. By affinity selection from this library, the authors isolated many VHH domains with specificity for EGFR. The VHH domains bind to whole cells expressing the receptor but not to control cells lacking the receptor and can immunoprecipitate EGFR from cell lysates. Some VHH domains have cross-specificity with existing anti-EGFR monoclonal antibodies and have reasonably high (nM) affinities. The llama-VHH domain library is also potentially a rich source of targeting agents directed toward other tumor cell receptors. ( Journal of Biomolecular Screening 2009:77-85)

Introduction When labeled with iodine-131, the antitenascin monoclonal antibody (mAb) 81C6 has shown promise as a targeted radiotherapeutic in patients with brain tumors. Because of its more favorable γ-ray properties, lutetium-177 might be a better low-energy β-emitter for this type of therapy. Materials and Methods Chimeric 81C6 (ch81C6) was labeled with 177 Lu using the acyclic 1B4M ligand and the macrocyclic ligands NHS-DOTA and MeO-DOTA and evaluated for binding to tenascin. Three paired-label tissue distribution experiments were performed in normal mice receiving one of the 177 Lu-labeled immunoconjugates plus 125 I-labeled ch81C6 labeled using Iodogen. Paired-label experiments in athymic mice bearing subcutaneous D54 MG human glioma xenografts were done to directly compare the biodistribution of ch81C6–1B4M– 177 Lu and 125 I-labeled ch81C6, and ch81C6–MeO-DOTA– 177 Lu and 125 I-labeled ch81C6. Similar comparisons were done using murine (mu) instead of ch81C6. The primary parameter utilized for evaluation was the 177 Lu/ 125 I uptake ratio in each tissue. Results In the studies performed in normal mice, the NHS-DOTA ligand yielded the highest 177 Lu/ 125 I uptake ratios in tissues indicative of loss of label from the chelate; for this reason, only 1B4M and MeO-DOTA were evaluated further. The 177 Lu/ 125 I ratio in bone increased gradually with time for the chimeric conjugates; however, there were no significant differences between ch81C6–1B4M–DTPA– 177 Lu and ch81C6–MeO-DOTA– 177 Lu. In contrast, mu81C6–1B4M-DTPA– 177 Lu and mu81C6–MeO-DOTA– 177 Lu showed a more dramatic increase in the 177 Lu/ 125 I ratio in bone — from 2.4±0.3 and 1.7±0.2 at Day 1 to 8.5±1.1 and 4.2±0.5 at Day 7, respectively. Conclusion With these antitenascin constructs, the nature of the mAb had a profound influence on the relative degree of loss of 177 Lu from these immunoconjugates. MeO-DOTA shows promise as a bifunctional chelate for labeling 81C6 mAbs with 177 Lu.

Results from animal experiments have shown that human IgG2/mouse chimeric antitenascin 81C6 (ch81C6) monoclonal antibody exhibited higher tumor accumulation and enhanced stability compared with its murine parent. Our objective was to determine the effect of these differences on the maximum tolerated dose (MTD), pharmacokinetics, dosimetry, and antitumor activity of (131)I-ch81C6 administered into the surgically created resection cavity (SCRC) of malignant glioma patients.In this phase I trial, eligible patients received a single injection of (131)I-ch81C6 administered through a Rickham catheter into the SCRC. Patients were stratified as newly diagnosed and untreated (stratum A), newly diagnosed after external beam radiotherapy (XRT) (stratum B), and recurrent (stratum C). (131)I-ch81C6 was administered either before (stratum A) or after (stratum B) conventional XRT for newly diagnosed patients. In addition, chemotherapy was prescribed for all patients after (131)I-ch81C6 administration. Dose escalation was performed independently for each stratum. Patients were observed for toxicity and response until death or progressive disease.We treated 47 patients with (131)I-ch81C6 doses up to 4.44 GBq (120 mCi), including 35 with newly diagnosed tumors (strata A and B) and 12 with recurrent disease (stratum C). Dose-limiting hematologic toxicity defined the MTD to be 2.96 GBq (80 mCi) for all patients, regardless of treatment strata. Neurologic dose-limiting toxicity developed in 3 patients; however, none required further surgery to debulk radiation necrosis. Median survival was 88.6 wk and 65.0 wk for newly diagnosed and recurrent patients, respectively.The MTD of (131)I-ch81C6 is 2.96 GBq (80 mCi) because of dose-limiting hematologic toxicity. Although encouraging survival was observed, (131)I-ch81C6 was associated with greater hematologic toxicity, probably due to the enhanced stability of the IgG2 construct, than previously observed with (131)I-murine 81C6.

The objective was to perform dosimetry and evaluate dose-response relationships in newly diagnosed patients with malignant brain tumors treated with direct injections of (131)I-labeled anti-tenascin murine 81C6 monoclonal antibody (mAb) into surgically created resection cavities (SCRCs) followed by conventional external-beam radiotherapy and chemotherapy.Absorbed doses to the 2-cm-thick shell, measured from the margins of the resection cavity interface, were estimated for 33 patients with primary brain tumors. MRI/SPECT registrations were used to assess the distribution of the radiolabeled mAb in brain parenchyma. Results from biopsies obtained from 15 patients were classified as tumor, radionecrosis, or tumor and radionecrosis, and these were correlated with absorbed dose and dose rate. Also, MRI/PET registrations were used to assess radiographic progression among patients.This therapeutic strategy yielded a median survival of 86 and 79 wk for all patients and glioblastoma multiforme (GBM) patients, respectively. The average SCRC residence time of (131)I-mu81C6 mAb was 76 h (range, 34-169 h). The average absorbed dose to the 2-cm cavity margins was 48 Gy (range, 25-116 Gy) for all patients and 51 Gy (range, 27-116 Gy) for GBM patients. In MRI/SPECT registrations, we observed a preferential distribution of (131)I-mu81C6 mAb through regions of vasogenic edema. An analysis of the relationship between the absorbed dose and dose rate and the first biopsy results yielded a most favorable absorbed dose of 44 Gy. A correlation between decreased survival and irreversible neurotoxicity was noted. A comparative analysis, in terms of median survival, was performed with previous brachytherapy clinical studies, which showed a proportional relationship between the average boost absorbed dose and the median survival.This study shows that (131)I-mu81C6 mAb increases the median survival of GBM patients. An optimal absorbed dose of 44 Gy to the 2-cm cavity margins is suggested to reduce the incidence of neurologic toxicity. Further clinical studies are warranted to determine the effectiveness of (131)I-mu81C6 mAb based on a target dose of 44 Gy rather than a fixed administered activity.

We report a phase 1 study of pharmacokinetics, dosimetry, toxicity, and response of 131I anti-tenascin chimeric 81C6 for the treatment of lymphoma. Nine patients received a dosimetric dose of 370 MBq (10 mCi). Three patients received an administered activity of 1480 MBq (40 mCi), and 2 developed hematologic toxicity that required stem cell infusion. Six patients received an administered activity of 1110 MBq (30 mCi), and 2 developed toxicity that required stem cell infusion. The clearance of whole-body activity was monoexponential with a mean effective half-life of 110 hours (range, 90-136 hours) and a mean effective whole-body residence time of 159 hours (range, 130-196 hours). There was rapid uptake within the viscera; however, tumor uptake was slower. Activity in normal viscera decreased proportional to the whole body; however, tumor sites presented a slow clearance (T1/2, 86-191 hours). The mean absorbed dose to whole-body was 67 cGy (range, 51-89 hours), whereas the dose to tumor sites was 963 cGy (range, 363-1517 cGy). Despite lack of a “blocking” antibody, 1 of 9 patients attained a complete remission and 1 a partial remission. These data demonstrate this radiopharmaceutical to be an encouraging agent for the treatment of lymphoma particularly if methods to protect the normal viscera are developed.

We have obtained encouraging responses in recent Phase I studies evaluating (131)I-labeled human/murine chimeric 81C6 anti-tenascin monoclonal antibody (ch81C6) administered into surgically-created tumor resection cavities in brain tumor patients. However, because the blood clearance is slow, hematologic toxicity has been higher than seen with murine 81C6 (mu81C6). In the current study, a series of paired-label experiments were performed in athymic mice bearing subcutaneous D-245 MG human glioma xenografts to compare the biodistribution of the fragment ch81C6 F(ab')(2) labeled using Iodogen to a) intact ch81C6, b) mu81C6, and c) ch81C6 F(ab')(2) labeled using N-succinimidyl 3-[(131)I]iodobenzoate. Tumor retention of radioiodine activity for the F(ab')(2) fragment was comparable to that for intact ch81C6 for the first 24 h and to that for mu81C6 for the first 48 h; as expected, blood and other normal tissue levels declined faster for ch81C6 F(ab')(2.) Radiation dosimetry calculations suggest that (131)I-labeled ch81C6 F(ab')(2) may warrant further evaluation as a targeted radiotherapeutic for the treatment of brain tumors.

Purpose: The objective of this study was to perform the dosimetry and evaluate the dose–response relationships in newly diagnosed patients with malignant brain tumors treated by direct injections of 131 I-labeled 81C6 monoclonal antibody (MAb) into surgically created resection cavities (SCRCs). Methods and Materials: Absorbed doses to the 2-cm-thick shell as measured from the margins of the resection cavity interface were estimated for 42 patients with primary brain tumors. MR images were used to assess the enhanced-rim volume as a function of time after radiolabeled MAb therapy. Biopsy samples were obtained from 15 patients and 1 autopsy. Results: The average absorbed dose [range] to the 2-cm shell region was 32 [3–59] Gy. For the endpoint of minimal time to MR contrast enhancement, the optimal absorbed dose and initial dose-rate were 43 ± 16 Gy and 0.41 ± 0.10 Gy/h, respectively. There was a correlation between the absorbed dose and dose rate to the shell region and biopsy outcome (tumor recurrence, radionecrosis, and tumor recurrence and/or radionecrosis). In this Phase I study, the maximum tolerated dose (MTD) was 120 mCi. At this MTD, the estimated average absorbed dose and initial dose rate to the 2-cm shell were 41 [9–89] Gy and 0.51 [0.24–1.13] Gy/h, respectively. These values are in agreement with the optimal values based on the time to MR lesion rim enhancement. Conclusions: The average absorbed dose to the 2-cm shell region varied considerably and mainly depended on cavity volume. In future clinical trials, the administered activity of 131 I-labeled 81C6 MAb may be adjusted based on cavity volume in order to deliver the optimal absorbed dose of 43 Gy rather than giving a fixed administered activity.

The advent of monoclonal antibody (MAb) technology has made Ehrlich's postulate of the 'magic bullet' an attainable goal. Although specific localization of polyvalent antibodies to human gliomas was demonstrated in the 1960s, the lack of specific, high affinity antibody populations and of defined target antigens of sufficient density precluded therapeutic applications. Not until the identification of operationally specific tumor-associated antigens (present in tumor tissue but not normal central nervous system tissue); production of homogeneous, high affinity MAbs to such antigens; and the use of compartmental administration (intrathecal or intracystic), has the promise of passive immunotherapy of primary and metastatic central nervous system neoplasms been recognized. We report here preliminary data from Phase I studies of the compartmental administration of the anti-tenascin MAb 81C6 and F(ab2)2 fragments of MAb Me1-14, which recognizes the proteoglycan chondroitin sulfate-associated protein of gliomas and melanomas, to patients with primary central nervous system tumors or tumors metastatic to the central nervous system. Phase I dose escalation studies of intracystically administered 131I-labeled anti-tenascin MAb 81C6 to either spontaneous cysts of recurrent gliomas or surgically created cystic resection cavities have resulted in striking responses. Of five patients with recurrent cystic gliomas treated, four had partial responses, clinically or radiographically. Similarly, in patients with surgically created resection cavities, a partial response at the treatment site and extended stable disease status has been obtained following intracystic administration of 131I-labeled 81C6. No evidence of hematologic or neurologic toxicity has been observed in either patient population, with the exception of transient exacerbation of a pre-existing seizure disorder in a single patient. Dosimetry calculations indicated high intracystic retention for four to six weeks with little or no systemic dissemination; estimated total doses intracystically ranged from 12,700-70,290 rad. Intrathecal administration of labeled MAbs to patients with neoplastic meningitis is more difficult to assess in terms of clinical responsiveness. Of patients so treated with either 131I-labeled 81C6 or 131I-labeled Me1-14 (F(ab)2, cerebrospinal fluid and radiographic responses have been achieved, and survival prolongation through maintenance of stable disease has been observed in several cases. Initial results from pHase I dose escalation trials are encouraging in terms of the proportion of cases of disease stabilization and partial and complete responses obtained. Importantly, neurotoxicity has been virtually nonexistent, and hematologic toxicity rare and rapidly responsive to treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Aim: The aim of this study is firstly to examine if the D2C7-immunotoxin (D2C7-IT) specifically binds to the epidermal growth factor receptor (EGFRwt) and the EGFR-mutant EGFRvIII subsequently inducing cell death. Secondly, to study the internalization of D2C7-IT after binding to EGFRwt/EGFRvIII. Background: Glioblastoma multiforme (GBM) is a malignant primary brain tumor and from diagnose median survival remains around 15 months. Tumor burden can be decreased by operation but eventually the tumor recurs. Thus, novel treatment-strategies for GBM patients are in great demand that would result prolonged median survival and increased quality of life. While EGFRwt and EGFRvIII are not found in normal brain tissue, expression of these are observed in malignant tissue of GBM, and consequently the D2C7-IT has been designed to target both EGFRwt and EGFRvIII. D2C7-IT comprises single chain disulphide stabilized fragment variables (scdsFv) of the bivalent anti-EGFR/EGFRvIII antibody D2C7, a 38 kDa fragment of the pseudomonas exotoxin A (PE38), and finally a C-terminal ‘endoplasmatic reticulum (ER) retention motif’: KDEL. In theory, the D2C7-IT is internalized after binding to EGFR/EGFRvIII and the PE38 moiety is proteolytically cleaved in the endosomal compartment, which releases the C-terminal toxic part of the PE38 into the cytosol. In the cytosol, binding of the KDEL-receptor assures transport to the ER where the toxic moiety inhibits protein synthesis and causes cell death. Methods: Western blotting was applied for examining D2C7-IT-binding to EGFRwt and EGFRvIII in different human malignant cell lines expressing varying levels of EGFRwt or EGFRvIII. MTT assays were used for assessing the impact of D2C7-IT on cell viability in these cell lines. Results: We observed that D2C7-IT binds to cells expressing EGFRwt or EGFRvIII. In addition, the binding on these cells could be inhibited by co-incubating with either EGF or the D2C7 antibody in surplus. Furthermore, D2C7-IT induced a moderate Tyr1173 phosphorylation (marker for internalization) on EGFRwt expressing cells. Concordantly, EGF induced a high level of EGFR-Tyr1173 phosphorylation on EGFRwt expressing cells, which was reduced to a moderate level when co-incubating with D2C7-IT in surplus. D2C7-IT reduced viability in cell cultures expressing EGFR or EGFRvIII while D2C7-IT had no effect on EGFRwt/EGFRvIII negative cells. We are currently trying to visualize the subcellular localizations of D2C7-IT after internalization by fluorescent microscopy. Conclusion: These preliminary results show that D2C7-IT specifically binds to EGFR and EGFRvIII, and reduces viability of EGFRwt/EGFRvIII expressing cells in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2732. doi:1538-7445.AM2012-2732

Murine monoclonal antibody Me1-14, which recognizes an epitope on chondroitin proteoglycan sulfate expressed in malignant glioma and melanoma, has been used for radioimmunolocalization and therapy both in animal models and in patients. Here, we report the generation, characterization, and in vivo biodistribution of mouse/human chimeric Me1-14. Rearranged immunoglobulin genes from the Me1-14 hybridoma were identified by Southern blot analysis. Putative rearranged light- and heavy-chain genes were cloned from Lambda-ZapII Me1-14 genomic libraries and sequenced for nucleotide analysis. One of the putative heavy-chain Eco RI fragments (3.5 kb) had all the features of an intact variable region, including a functional leader sequence, in-frame V-D and D-J junctions, and cysteines 22 and 92. The deduced amino acid sequence from the heavy-chain variable region gene showed considerable homology with the invariant protein sequence of the mouse heavy-chain subgroup IIIB. Like the heavy-chain gene, one of the putative rearranged kappa-chain Hind III fragments (4 kb) had all of the characteristics of the functional variable region, and the deduced amino acid sequence showed homology to the invariant sequence of kappa-chain group V. The variable region genes for heavy- and light-chains were linked to human constant region exons in the expression vectors at the unique sites and cotransfected into mouse SP2/0 cells. The production level of chimeric Me1-14 from ascites in the highest expressing transfectoma was 1.8 mg/ml. The chimeric Me1-14 antibody exhibited the same specificity and similar affinity as that of parent Me1-14. Direct comparison of radioiodinated chimeric and murine Me1-14 in paired-label biodistribution analysis in subcutaneous xenograft-bearing mice showed higher tumor-to-normal organ ratios for chimeric Me1-14 IgG2, suggesting that this chimeric Me1-14 may be potentially useful in vivo for diagnostic and therapeutic purposes in patients.

We have previously demonstrated antibody production to a glioma-associated variant form of the human epidermal growth factor receptor in rabbits that had received a synthetic peptide mimicking the unique primary structure of the variant protein as immunogen. We report here the response of mice, rabbits, goats, and macaques immunized by various protocols to this peptide. Titers to both peptide- and cell-elaborated variant receptor were measured, and the capacity to recognize the variant receptor in human tumor samples was determined. Within the range of species and strains investigated, we demonstrated a variable species-associated response to the peptide (rabbits >mice > goats > rats > macaques). Rabbits and a single goat produced specific, high titer antibody activity to the variant receptor protein following immunization with peptide alone. Murine titers to the parent protein were not appreciable following peptide immunization alone; additional immunization with variant receptor as expressed on cell membranes was used to boost this response.

Five-day-old Fischer 344 rats were inoculated intracerebrally with BCG cell walls attached to oil (BCG-CW) or with oil emulsion alone. Doses of BCG-CW of 40 μg in 0.01 ml, 200 μg in 0.05 ml, and 400 μg in 0.1 ml were used, with similar volumes of oil emulsion for comparison. Twentyfive days later hydrocephalus, intracerebral granulomas, and arachnoid granulomas were found at autopsy. The frequency and severity of these abnormalities were related to the presence of BCG-CW in the inoculum and the dose administered. There were minimal abnormalities with 40 μg BCG-CW.

The highly reproducible histochemical localization of glial fibrillary acidic protein (GFAP)+ qualifies it as an important marker of astrocytes in both research and clinical applications. The primary objective of this study was to produce monoclonal antibodies having the advantage of invariant specificity, affinity, and titer to GFAP-specific epitopes of wide species distribution. We report here the characterization of four monoclonal antibodies that recognize the same or spatially close epitopes specific to GFAP. The epitope(s) detected has been phylogenetically conserved; human, bovine, ovine, canine, porcine, rabbit, guinea pig, rat, murine, and chicken brain homogenates all specifically absorb monoclonal antibody activity. Of importance to the routine application of these new anti-GFAP monoclonal antibodies is the demonstration here of the stability of the antigen-antibody interaction in normal, reactive, and neoplastic astrocytes of both rat and human origin following various methods of fixation.

Fifteen permanent cell lines derived from human gliomas which are individually distinct by immunologic and biochemical criteria were evaluated to determine if morphologic or cell biologic parameters distinguished the 4 lines which were tumorigenic in athymic nude mice. By subjective morphologic appraisal, the 4 tumorigenic lines were considered "malignant" or "borderline," but 4 of the non-tumorigenic lines were also classified in this way. By objective criteria, these 15 lines varied markedly in percentage of piled-up cells, chromatin pattern, pleomorphism, nuclear to cytoplasmic ratio, number of bizarre multinucleate giant cells, presence of abnormal mitotic figures, percentage of colony formation in soft agar, saturation density, population doubling time, and absolute plating efficiency. Among these criteria, percentage of colony formation in soft agar had the highest correlation coefficiency with tumorigenicity, and when this parameter was held constant the only additional characteristic which correlated significantly (p less than .05) was the number of bizarre multinucleate giant cells. When the 11 non-tumorigenic lines were ranked by these 2 criteria, 1 non-tumorigenic line (U-251 MGsp) had greater than .95 predicted probability of tumorigenicity. Although further tumorigenicity testing may increase the number of tumorigenic lines, the lines with few "malignant" characteristics may correspond to the population resembling cells of low grade astrocytomas seen within glioblastomas. The histologic pleomorphism of human gliomas is reflected in their morphologic and cell biologic diversity in culture.

The intermediate filament, glial fibrillary acidic protein (GFAP) has proven to be an important glial marker in diagnostic neuropathology. We report the histochemical application of three monoclonal antibodies (Mab) produced in this laboratory, 1B4, 2E1, and 4A11, which are monospecific to GFAP by radioimmunoassay, immunoblot electrophoresis, and immunoperoxidase histochemistry. The goal of this study was to compare the specificity and sensitivity of these Mab to GFAP on surgical brain biopsy specimens which had been routinely processed for diagnostic neuropathology with that of a high titer, highly specific, reference polyvalent anti-GFAP antiserum. The Mab stained astrocytes specifically in normal brain. When combined in a "cocktail" preparation, the quality of the immunoperoxidase detection of GFAP by these Mab closely approached that of the reference serum in 71 intracranial and intraspinal neoplasms. As these three Mab represent a continuous supply of a well defined, monospecific reagent, the monoclonal "cocktail" represents a standard reagent for large multi-institutional studies and for studies extending over a period of time.

One of the few spontaneous gliomas in inbred animals, the VM/Dk spontaneous murine astrocytoma (SMA), has seen limited use. Previously restricted to an in vivo system, the SMA was only transplantable intracerebrally (IC) using nonquantifiable suspensions of normal brain and tumor tissue. Prior attempts at establishing permanent tumorigenic SMA cell lines have not succeeded; tumorigenicity was lost during serial in vitro passage. We have established three different cell culture lines from a serially IC-transplanted SMA and two from tumors that arose from intraperitoneal (IP) injection of the IC-transplanted SMA. In contrast to previous cell cultures and transplantable lines of SMA, all five cell lines are not only tumorigenic IC but subcutaneously (SC) as well. Astrocytic feature are present in three of five lines to varying degrees, evidenced by in vitro and in vivo morphology, response to dibutyryl cyclic AMP (db-cAMP), and the presence of neuroglial fibers: None of the lines express CNPase, S-100, or GFA proteins in significant amounts. P560, highly tumorigenic and possessing the most astrocytic features of the five lines, extends the use of the spontaneous astrocytoma system of the inbred VM/Dk mouse strain by allowing quantitative in vivo and in vitro experiments.

Central nervous system (CNS) malignancies represent one ofthe most devastating forms of cancer. Malignant gliomas, the most commonly occurring primary tumor of the CNS, have a particularly bleak prognosis despite surgery and combined radiation and chemotherapy (1, 2). Immunological approaches to diagnosis and treatment have seemed attractive, but in the past have been frustrated by technological limitations (3, 4). Monoclonal antibody methodology offers major advantages over conventional methods of antiserum production: a practically unlimited source of homogeneous, highly specific reagent which may be prepared using impure or complex antigens. This has led to a new age in neuroimmunology and neuro-oncology, with the potential for better understanding of the basic cell biology of CNS neoplasia, and for improvement in diagnosis, imaging, and therapy.