Tumor Cells Of Neuroectodermal Origin Research Articles

Abstract 4650: Quantitative assessment of the intracellular uptake of chlorotoxin in a U87 human glioma mouse model for the targeted drug delivery system

Abstract This study quantitatively evaluates the tumor cell-targeting capabilities and intracellular uptake pattern of chlorotoxin (CTX) when delivered intratumorally (IT) and intravenously (IV) using subcutaneous (SC) U87 human glioblastoma mouse model. CTX is a scorpion-derived polypeptide that selectively binds to tumor cells of neuroectodermal origin, such as glioma and a wide range of other tumor cells, but not normal cells. The quantification of CTX cellular uptake presented here will assist in the further development of our tumor-targeting drug delivery system (DDS) consisting of a chemotherapeutic encapsulated in nonionic surfactant vesicles embedded in a thermosensitive cross-linked chitosan hydrogel, which has affinity for MUC1 receptor overexpressed on many tumor cells, including low-differentiated glioma. CTX addition to the DDS will serve as a second tumor-targeting molecule. In this study, U87 human glioma cells were implanted SC in the flank of nude athymic/ncr/nus mice. When tumors reached approximately the volume of 0.25 cm3, mice were injected either IT or IV (tail vein) with 50 μl of 100 μg CTX or phosphate buffer saline (PBS) as a control. The tumors were harvested at 5 minutes post IT injection or 7 hours post IV injection, fixed, sectioned at 5 μm, and followed by either immunofluorescence or immunocytochemistry using anti-CTX primary antibody and AlexaFluor-594 conjugated secondary antibody or immunoperoxidase- AEC kit, and evaluated by confocal or scanning microscopy, respectively. CTX levels were compared between three different tumor regions (A, B, and C; A = injection site marked by methylene blue dye, C = most distant from injection site, B = equidistant between these two) after direct IT injection and compared to CTX levels in tumor tissues following IV injection. Quantitative evaluation of CTX levels was performed with ImageJ software, while further intracellular analysis of CTX presence utilized MATLAB system. CTX was found to be evenly distributed between cells within each region post IT injection, but decreased in intensity from the injection site (A) toward the tumor edge. With region A set at 100% CTX intensity, the levels decreased to 23.7% (in region B), and 2.1% (in region C), compared to 1.8% post IV injection. Our previous in vitro studies showed CTX localization near the nucleus in U87 glioma cells but not in normal human astrocytes. Current evaluation of tumor z-sections quantified levels of CTX inside U87 glioma cells in vivo. These results confirm the specificity of CTX uptake by U87 human glioma cells in vitro and in vivo with CTX tissue levels post IV injection, which is comparable to those post IT injection in the distant C tumor region. This proves CTX is an important second tumor-targeting molecule for our DDS system. Citation Format: Alan D. Roberts, Joseph O. Johnson, Aleksandra Karolak, Norma Alcantar, Katarzyna A. Rejniak, Marzenna Wiranowska. Quantitative assessment of the intracellular uptake of chlorotoxin in a U87 human glioma mouse model for the targeted drug delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4650.

Abstract 3909: Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study

Abstract Chlorotoxin is a scorpion-derived peptide that preferentially binds to tumor cells of neuroectodermal origin, like glioma, but not to normal non-transformed cells. We have previously reported the development of a targeted nanodelivery system using chlorotoxin. Chlorotoxin has been studied as an imaging and targeting agent for drug and radioisotope delivery, but the mechanism of interaction of chlorotoxin with cancer cells has not been well understood and it needs to be further evaluated in order to optimize its use as a targeting compound for cancer cells. In this study, we used U87 human glioma cell line to evaluate the binding kinetics of chlorotoxin by Attenuated Total Reflection Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. As a sensitive and label free technique, ATR-FTIR is a powerful diagnostic tool for the comparison of cancer cells with normal cells. First, we characterized the signature spectra of chlorotoxin and U87 cells by assigning and evaluating the infrared absorption bands and their second derivatives. Next, we studied the spectral differences between U87 cells incubated with and without chlorotoxin for incremental time periods ranging from 15 minutes to 24 hours. We also examined the metabolic changes in U87 cells induced with chlorotoxin at different incubation time points by measuring the ratios of integrated areas corresponding to different chemical conformations of proteins, lipids, carbohydrates, and nucleic acids. Our results revealed spectral changes in U87 cells at different stages of incubation with chlorotoxin. The most notable change occurred after 30 minutes of incubation, where the band assigned to CH3 bending of lipid membranes shifted from 1456 cm-1 to 1450 cm-1. Consequently, a new shoulder appeared at wavenumber of 1466 cm-1 assigned to CH2 bending of lipid membranes for U87 cells treated with chlorotoxin. These changes indicate a direct interaction due to the presence of a molecular binding site between chlorotoxin and the lipids of the cell membrane. Another significant alternation occurred after 1 hour of incubation of U87 cells with chlorotoxin in the spectral region assigned for nucleic acids. This interaction included a large upshift of the band at 1036 cm-1 to 1051 cm-1 assigned to C-O stretching vibrations of ribose ring in RNA due to the hydrogen bonding with chlorotoxin. These findings suggest at least two different interactions of chlorotoxin with U87 human glioma cells that can be further explored for improving chlorotoxin containing drug delivery systems. We are currently using the ATR-FTIR method to conduct control experiments by measuring possible interactions of chlorotoxin with normal human astrocytes. Citation Format: Rana Falahat, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3909.

Abstract 3677: Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system

Abstract We report a development of a novel localized nanodelivery system featuring an enhanced targeted and controlled paclitaxel (PTX) delivery to epithelial and neuroectodermal origin tumor cells. The presented drug delivery system consists of a chitosan hydrogel embedded with PTX loaded non-ionic surfactant vesicles (PTX-niosomes) and chlorotoxin (CTX). We found a higher accumulation of chitosan on the surface of ovarian epithelial carcinoma cells known to overexpress MUC1 antigen as compared to normal ovarian epithelial cells. We therefore hypothesized that besides serving as a localized drug delivery platform, chitosan hydrogel can actively target MUC1 overexpressing tumor cells. To further improve the tumor-specific delivery of PTX, we have incorporated CTX, a 36-amino acid peptide, which binds to tumor cells of neuroectodermal origin, but not to non-transformed cells. Using a High Performance Liquid Chromatography (HPLC) assay, we have measured the in-vitro release profiles of PTX from chitosan hydrogel embedded with PTX-niosomes and CTX. We have assessed the morphology of CTX and PTX-nisomes as well as chitosan hydrogel embedded with PTX-niosomes and CTX using Transmission Electron Microscopy (TEM). We have used Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy to investigate the possible molecular interactions between chitosan hydrogel and various tumor cells. To better understand the mechanism involved in interactions between chitosan and specific tumor cells, we have developed a new Cell based Enzyme Linked Immunosorbent Assay (Cell ELISA) allowing us to quantify the expression level of MUC1 in epithelial and neuroectodermal origin tumor cells. Our Cell ELISA results revealed higher expression level of MUC1 (around 1.5 fold) in ovarian epithelial carcinoma cell line (OV2008) when compared to normal ovarian epithelial cell line (MCC3). The ATR-FTIR results showed a specific interaction of chitosan with OV2008 cells implying the mucoadhesive property of chitosan and its potential in targeting of MUC1 overexpressing tumor cells. We observed a sustained PTX release from chitosan hydrogel embedded with PTX-niosomes (< 40% of cumulative release occurred within two weeks). Release studies in cell-free system also indicated that embedding CTX along with the niosomes does not have any adverse effect on the controlled release of PTX from the chitosan network. In fact, attachment of CTX to the surface of PTX-niosomes shown by TEM imaging improves the stability of PTX-niosomes resulting in extended release rates. Together these findings indicate that the proposed drug delivery system with the distinctive tumor targeting features and extended release profiles would improve the specific delivery of PTX to epithelial and neuroectodermal origin tumor cells. Citation Format: Rana Falahat, Eva Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2015-3677

Molecular mechanisms of fenofibrate-induced metabolic catastrophe and glioblastoma cell death.

Fenofibrate (FF) is a common lipid-lowering drug and a potent agonist of the peroxisome proliferator-activated receptor alpha (PPARα). FF and several other agonists of PPARα have interesting anticancer properties, and our recent studies demonstrate that FF is very effective against tumor cells of neuroectodermal origin. In spite of these promising anticancer effects, the molecular mechanism(s) of FF-induced tumor cell toxicity remains to be elucidated. Here we report a novel PPARα-independent mechanism explaining FF's cytotoxicity in vitro and in an intracranial mouse model of glioblastoma. The mechanism involves accumulation of FF in the mitochondrial fraction, followed by immediate impairment of mitochondrial respiration at the level of complex I of the electron transport chain. This mitochondrial action sensitizes tested glioblastoma cells to the PPARα-dependent metabolic switch from glycolysis to fatty acid β-oxidation. As a consequence, prolonged exposure to FF depletes intracellular ATP, activates the AMP-activated protein kinase-mammalian target of rapamycin-autophagy pathway, and results in extensive tumor cell death. Interestingly, autophagy activators attenuate and autophagy inhibitors enhance FF-induced glioblastoma cytotoxicity. Our results explain the molecular basis of FF-induced glioblastoma cytotoxicity and reveal a new supplemental therapeutic approach in which intracranial infusion of FF could selectively trigger metabolic catastrophe in glioblastoma cells.

Abstract 5410: Enhanced targeting delivery to tumor cells using mucoadhesive chitosan and chlorotoxin

Abstract Specific drug delivery to tumor cells without affecting normal cells remains a major challenge in cancer treatment. We present a localized drug delivery system with enhanced targeting ability consisting of non-ionic surfactant vesicles (niosomes) with chlorotoxin (CTX) embedded in a chitosan hydrogel. This system represents a novel approach in cancer therapy through the controlled and targeted delivery of drugs to tumor cells. We have found the specific accumulation of chitosan on the surface of ovarian epithelial carcinoma cells known to have high expression of mucin antigen MUC1, but not on the normal ovarian epithelial cells. This finding demonstrates the capability of chitosan to target tumor cells expressing MUC1. Similarly, the incorporation of CTX (a 36-amino acid peptide capable of binding preferentially to tumor cells of neuroectodermal origin but not to normal cells) along with niosomes in the chitosan hydrogel has been used as the second targeting strategy to further improve the specific delivery of drugs to tumor cells such as glioma. To investigate the possible molecular interaction between the chitosan hydrogel and various cell lines, Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy has been used. The expression level of MUC1 in different cell lines was quantitatively evaluated using Cell based Enzyme Linked Immunosorbent Assay (Cell-ELISA) to better understand the mechanism involved in interactions between chitosan and specific tumor cells. In vitro release studies were performed to examine the effect of CTX on the release rates. Transmission Electron Microscopy (TEM) is being used for morphology assessments of CTX and nisomes as well as chitosan hydrogel embedded with niosomes and CTX. Cell-ELISA data revealed high expression level of MUC1 in ovarian epithelial carcinoma cell line (OV2008) and a moderate expression level in normal ovarian epithelial cell line (MCC3) implicating higher affinity of chitosan for OV2008 than MCC3 cell lines. This is consistent with ATR-FTIR results as well as the previously observed higher chitosan accumulation on the surface of OV2008 compared to MCC3, confirming the mucoadhesive property of chitosan and indicating its specificity in targeting MUC1 overexpressing tumor cells. In vitro release studies show that embedding CTX along with the niosomes does not disturb the controlled release from the chitosan network. In fact, attachment of CTX to the surface of niosomes shown by TEM imaging improves the stability of niosomes resulting in extended release rates. Based on these findings, we anticipate that the drug delivery system described here would improve tumor cell uptake of drugs due to the enhanced tumor targeting and its controlled, sustained and localized drug delivery to cancer cells. Citation Format: Rana Falahat, Eva Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Enhanced targeting delivery to tumor cells using mucoadhesive chitosan and chlorotoxin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5410. doi:10.1158/1538-7445.AM2014-5410

Potentiality of Anti-idiotypic Antibodies Mimicking GD2 to Induce Cellular Immunity

Background: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma, small cell lung carcinoma and neuroblastoma. Immunity against GD2 has anti-tumor activities, but GD2 is poorly immunogenic. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In our previous study, we produced anti-idiotypic antibodies mimicking GD2 (3A4 and 3H9), which induced humoral immunity. However, cellular immunity is essential to eradicate tumor cells in vivo as well as humoral immunity. In the present study, we investigated whether these anti-idiotypic antibodies 3A4 and 3H9 could induce cellular immunes responses. Methods: BALB/C mice were immunized with anti-idiotypic antibody 3A4 or 3H9, or normal mouse IgG as a negative control. Lymphoproliferative responses, cytokine production responses, and delayed-type hypersensitivity reactions were measured in mice immunized with the anti-idiotypic antibodies. Results: Both the anti-idiotypic antibody 3A4 and 3H9 induced GD2-specific lymphoproliferative responses and IFN-γ production of lymph node lymphocytes in BALB/C mice. Only anti-idiotypic antibody 3H9 induced significant GD2-specific delayed-type hypersensitivity in the mice. Conclusion: These results show that anti-idiotypic antibodies 3A4 and 3H9 have the potentiality of inducing GD2-specific cellular immune responses that cannot be induced by the native antigen GD2 itself. (Immune Network 2004;4(4):229-236)

N-(4-Hydroxyphenyl)retinamide Inhibits Retinoblastoma Growth through Reactive Oxygen Species-Mediated Cell Death

Retinoblastoma arises from a subset of developing retinal cells lacking the RB-1 gene product pRB, which have lost the ability to respond to apoptotic signals. A better understanding of retinoblastoma biological response to therapeutic agents with low toxicity could improve the development of novel approaches for treatment and prevention of the disease. Naturally occurring retinoids inhibit growth and induce differentiation of Y79 human retinoblastoma cells in vitro. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has been shown to induce apoptosis and/or necrosis of tumor cells of neuroectodermal origin. We examined the sensitivity of Y79 retinoblastoma cells to 4HPR in vitro, and in a xenograft model of tumor growth in nude mice in vivo. 4HPR treatment in the range 2.5 to 10 microM induced a loss of Y79 cell viability, as determined by crystal violet, trypan blue exclusion, and long-term clonogenic assays, and impairment of mitochondrial function detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Reactive oxygen species were elevated in 4HPR-treated cells and antioxidants rescued cell viability, indicating that 4HPR-induced cell death was mediated by oxidative stress. 4HPR inhibited growth of Y79 xenografts in vivo in both chemoprevention and intervention settings. Tumor growth inhibition by 4HPR was also associated with significant inhibition of angiogenesis in vivo. These findings could have an important translational value for chemoprevention or early intervention in the treatment of retinoblastoma.

Characterization of Anti-anti-idiotypic Antibodies (Ab3) Induced by Immunization of Anti-idiotypic Antibodies (Ab2) Mimicking Disialoganglioside GD2

Background: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma and neuroblastoma. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In this study, to explore the potential of anti-idiotypic antibodies as tumor vaccines, the ability of anti-idiotypic antibodies (Ab2) to induce anti-anti-idiotypic antibodies (Ab3) that bind to the original antigen GD2 was investigated. Methods: Six monoclonal anti-idiotypic antibodies (1A8, 1G5, 2B6, 3A4, 3D6, 3H9) to monoclonal antibody M2058, which is a monoclonal antibody to GD2, were produced in mice. Three (1A8, 3A4, 3H9) of them were selected based on their ability to inhibit the binding of Ab1 to D142.34 (murine melanoma cell expressing GD2). These 3 different Ab2 were injected into rabbits, and rabbit Ab3 induced by each of them were characterized. Results: Ab3-containing sera from two rabbits immunized with 1A8, 3A4, or 3H9 bound significantly (P<0.05) to D142.34 but not to B78.96 (GD2-negative cell), and bound significantly (P<0.05) to isolated GD2 but not to GD1a. Ab3-containing sera from two rabbits immunized with 3A4 or 3H9 inhibited significantly (P<0.05) the binding of Ab1 M2058 to D142.34, and inhibited significantly (P<0.05) the binding of Ab1 M2058 to the Ab2. Conclusion: These results suggest that anti-idiotypic antibodies 3A4 and 3H9 have a potential to be used as vaccines against tumors expressing GD2 by inducing GD2-specific antibodies (Ab3). (Im m un e N etw ork 2003;3(2):118-125)

A monoclonal antibody against Meningococcus group B polysaccharides used to immunocapture and quantify polysialylated NCAM in tissues and biological fluids

Polysialylated isoforms of neural cell adhesion molecule (PSA-NCAM) are transiently expressed in many tissues during development and in discrete areas of the adult central nervous system. In pathological situations, they are expressed by poorly differentiated tumor cells of neuroectodermal origin and by regenerating muscle. An ELISA is introduced here to estimate the relative concentrations of PSA-NCAM expressed by tissues or released into biological fluids. In this double-sandwich assay, an anti-PSA antibody (anti-MenB) was adsorbed onto plastic plates and permitted the immunocapture of PSA-bearing molecules. It is demonstrated that these molecules are major NCAM. The second antibody was directed against an amino acid sequence shared by NCAM isoforms in several species. The standard curves were established using Nonidet P40 extracts of human or mouse embryonic brain known to be rich in PSA-NCAM. The sensitivity of the assay allows for quantitation of PSA-NCAM in muscle during regeneration and in small samples of cerebrospinal fluid from patients with medulloblastoma metastasis.

Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody.

A mouse/human chimeric antibody (ch14.18) was developed that reacts with the disialoganglioside GD2 on the surface of tumor cells of neuroectodermal origin. ch14.18 has the constant regions of a human IgG1 antibody and was expressed in a murine hybridoma. This antibody was produced in tissue culture at concentrations up to 180 mg/liter of spent culture fluid. ch14.18 was characterized and compared to 14.G2a, a murine mAb against GD2 of IgG2a isotype derived from the same parental hybridoma as ch14.18. Scatchard plot analysis of data from saturation binding studies on M21 melanoma cells showed identical binding for ch14.18 and 14.G2a. Indirect immunofluorescence revealed the same staining pattern for ch14.18 and 14.G2a on different melanoma cell lines. Both antibodies were equally capable of targeting M21 xenografts in athymic nude mice. ch14.18- and 14.G2a-activated human C-mediated cytolysis of melanoma cell; however, ch14.18-mediated antibody-dependent cytotoxicity of human effector cells against melanoma cells 50- to 100-fold more efficiently than 14.G2a.

GD3 expression by cultured human tumor cells of neuroectodermal origin.

Seven monoclonal antibodies (mAbs) reactive with ganglioside II3(NeuAc)2-LacCer (GD3) were generated; four of these mAbs (DMAb-21, DMAb-22, DMAb-23, and DMAb-24) by immunizing mice with GD3 adsorbed to Salmonella minnesota and the remaining three (DMAb-7, DMAb-8, and DMAb-17) with melanoma line SK-MEL 28, which contains 1.4 nmol sialic acid of GD3 per mg protein. The specificities of the mAbs were defined by high-performance thin-layer chromatography (HPTLC) immunostain and solid-phase radioimmunoassay (SP-RIA) with a panel of purified gangliosides. DMAb-7 and DMAb-8 reacted with GD3, IV3(NeuAc)2nLcOse4Cer(3',8'-LD1), and very weakly with IV3(NeuAc)2II3NeuAcGgOse4Cer (GT1a), but not with II3NeuAc-LacCer (GM3), II3NeuAcGgOse3Cer(GM2), II3NeuAcGgOse4Cer (GM1), II3NeuAc, IV3NeuAcGgOse4Cer (GD1a), II3(NeuAc)2GgOse3(GD2), II3(NeuAc)2GgOse4Cer (GD1b), IV3NeuAcII3(NeuAc)2, GgOse4Cer(GT1b), suggesting the binding epitope to be a terminal tetrasaccharide NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4(Glc or GlcNAc). DMAb-7 and DMAb-8 were used to investigate the expression of GD3 on cultured human tumor cells of neuroectodermal origin. Thirteen of 19 gliomas, 3 of 5 medulloblastomas, 5 of 5 neuroblastomas, 2 of 2 melanomas, and 1 of 3 teratomas were shown to react with DMAb-8 and/or DMAb-7 by cell surface-RIA (CS-RIA) and immunofluorescence (IF) assays. HPTLC and densitometric analysis confirmed these results, as positive immunostains in the GD3 region were obtained with oligoganglioside fractions from 9 glioma, 1 medulloblastoma, 2 neuroblastoma, 1 melanoma, and 1 teratoma cell line. Glioma cell line U-105 MG and medulloblastoma cell line Daoy contain GD3 as shown by HPTLC immunostain analysis of extracts, although GD3 was undetectable on the cell surface as determined by CS-RIA and IF.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphokine-activated killer cells targeted by monoclonal antibodies to the disialogangliosides GD2 and GD3 specifically lyse human tumor cells of neuroectodermal origin.

Monoclonal antibodies 14.18 (IgG3) and 11C64 (IgG3) directed against disialogangliosides GD2 and GD3, respectively, when used in conjunction with human peripheral blood mononuclear cells (PBMCs) stimulated with human recombinant interleukin (rIL-2) lyse both human melanoma and neuroblastoma cells by antibody-dependent cellular cytotoxicity. Such monoclonal antibody-"armed" effector cells are specifically directed to targets expressing the given disialoganglioside without detectable cross-reactivity. In addition, antibody-dependent cellular cytotoxicity as well as the natural killing ability of human PBMCs is augmented by a brief coincubation with rIL-2. PBMCs augmented by rIL-2 and armed with monoclonal antibodies significantly suppressed tumor growth in the xenotransplant nude mouse model. Our results suggest that once a threshold level of activation of PBMCs is achieved, additional rIL-2 (over three orders of magnitude of concentration) does not significantly enhance cytolytic augmentation. Furthermore, anti-GD3 monoclonal antibody 11C64 together with rIL-2-stimulated PBMCs from melanoma patients with widely differing tumor burdens effectively lyse melanoma tumor targets in antibody-dependent cellular cytotoxicity. Our results also suggest that GD2 and GD3 represent distinct and relevant immunotherapeutic target structures on melanoma whereas GD2 does the same for neuroblastoma tumors. Our data suggest that targeting of activated human effector cells may provide a new and effective cancer immunotherapy protocol.

Human antibody to OFA-I, a tumor antigen, produced in vitro by Epstein-Barr virus-transformed human B-lymphoid cell lines.

We established two long-term human B-lymphoblastoid cell lines (L55 and L72) transformed by Epstein-Barr virus that produced IgM kappa antibodies to the human tumor antigen, OFA-I. Periphral blood lymphocytes obtained from melanoma patients were used as the source of the B lymphocytes. Antibody specificity was determined by the immune adherence assay using various human cancer and noncancer tissues as targets. L55 antibody (designated anti-OFA-I-1) reacted with a variety of human tumor types whereas L72 antibody (designated anti-OFA-I-2) reacted only with tumor cells of neuroectodermal origin (melanoma, glioma, and neuroblastoma). The levels of IgM detected in the spent medium of 1 X 10(6) L55 and L72 cells were 4 and 9 micrograms/ml, respectively, by radioimmunoassay.