Cancer Research AACR Legacy Telomeres
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Retter, M. W.
Right arrow Articles by Rock, K. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Retter, M. W.
Right arrow Articles by Rock, K. L.
[Cancer Research 65, 6425-6434, July 15, 2005]
© 2005 American Association for Cancer Research

Immunology

Characterization of a Proapoptotic Antiganglioside GM2 Monoclonal Antibody and Evaluation of Its Therapeutic Effect on Melanoma and Small Cell Lung Carcinoma Xenografts

Marc W. Retter1, Jeffrey C. Johnson1, David W. Peckham1, Jeannette E. Bannink1, Chaitanya S. Bangur1, Karen Dresser2, Feng Cai1, Teresa M. Foy1, Neil A. Fanger1, Gary R. Fanger1, Bruce Woda2 and Kenneth L. Rock2

1 Antigen Discovery and Preclinical Biology, Corixa Corporation, Seattle, Washington and 2 Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts

Requests for reprints: Kenneth L. Rock, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. Phone: 508-856-2521; Fax: 508-856-1094; E-mail: kenneth.rock{at}umassmed.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The advent of monoclonal antibodies (mAb) provided a reliable source of well-defined, monospecific antibody (1) and thereby made the practical development of antibody-based tumor immunotherapy feasible. Numerous mAbs have been used to treat cancers successfully in preclinical animal models, and to date, eight have been approved for therapeutic use in man, whereas >50 are currently undergoing evaluation in clinical trials (reviewed in refs. 2–5). Based on this experience, there is a very strong rationale for continued development of new mAbs for immunotherapy.

Potential molecular targets for mAb-mediated immunotherapy must be accessible to the antibody and therefore are typically membrane antigens. Moreover, the ideal target antigen is expressed in a tumor-specific mannr, unless the normal cell is dispensable or not affected by antibody binding. Those antigens that are present on normal cells but are highly overexpressed on tumors, such as certain gangliosides (reviewed in ref. 6), are also possible targets for antibody immunotherapy. Finally, to be effective as an unmodified, or "naked" therapeutic agent, the antibody must directly affect the tumors' growth and/or survival (refs. 7, 8; reviewed in ref. 9) This can occur when the antibody, upon tumor binding, initiates the recruitment and activation of immune effector mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), blocks growth factor receptor activation, or transduces negative signals that impair tumor cell growth or survival (reviewed in ref. 9).

Previously, we described mAb DMF10.62.3 that was raised against a murine T-cell lymphoma and showed its ability to block proliferation and induce apoptosis in this tumor when cultured in vitro (10). This mAb also reacted with a number of other murine and human tumors including T- and B-cell lymphomas, melanomas, and sarcomas. Where examined, the DMF10.62.3 mAb had similar antiproliferative and proapoptotic effects on these tumors in vitro. In contrast, DMF10.62.3 mAb did not react with the nonmalignant counterparts of several of these tumors, such as normal T cells or fibroblasts, or other normal adult cells that were examined. These findings raised the possibility that the epitope recognized by DMF10.62.3 might be selectively expressed on tumors and thus might be useful in immunotherapy. However, at the time, the ligand for DMF10.62.3 mAb remained unknown.

The present studies were initiated to define the antigen recognized by DMF10.62.3 and a clonally related antibody DMF10.167.4 and determine the expression of this epitope on normal tissues and tumors. In addition, we sought to evaluate the potential of DMF10.167.4 antibody for tumor immunotherapy. We found that the ligand recognized by these mAbs is the ganglioside GM2 (reviewed in refs. 6, 11), which is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma (SCLC) but not on normal primary lines or most normal tissues. Extending the previous functional analysis revealed that DMF10.167.4 mAb was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 SCLC tumor cells. No apoptotic activity was observed after culture of this antibody with normal cells. In vivo, DMF10.167.4 mAb was well tolerated in mice and caused no histologically detectable damage to normal tissues. Moreover, this mAb was able to prevent establishment of murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 SCLC tumors and block progression of established CHL-1 and SBC-3 SCLC tumors in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DMF10.167.4 hybridoma and antibody purification. The generation of hybridoma DMF10.62.3 was described by Fernandes et al. (10). Hybridoma DMF10.167.4 was derived from the same fusion that generated DMF10.62.3 and is clonally related to this hybridoma as determined by nucleotide sequence analysis of the rearranged immunoglobulin heavy and light chain variable region genes of both hybridomas (data not shown; ref. 12). The DMF10.167.4 hybridoma was cultured in DMEM containing 10% fetal bovine serum (FBS) with low IgG (Life Technologies Invitrogen, Carlsbad, CA), penicillin/streptomycin, NEAA and L-glutamine (Life Technologies Invitrogen) at 37°C in 5% CO2. DMF10.167.4 mAb was purified from spent culture supernatants using Protein A/G chromatography, followed by elution from the column matrix with 0.5 mol/L glycine (pH 3). Following dialysis of the eluate, the mAb was resuspended in 100 mmol/L sodium citrate buffer (pH 5.5) and stored at –20°C.

Antiganglioside ELISA. To determine ligand specificity of the DMF10.167.4 antibody, gangliosides lyso-GM1, asialoGM1, GM1, GD1b, GD1a, GT1b, GM2, GD2, asialoGM2, GM3, and GD3 (Sigma, St. Louis, MO) were resuspended to a concentration of 1 mg/mL in chloroform/methanol (50:50%) and coated in methanol at 0.5 µg per well onto 96-well ELISA plates overnight (Costar, Corning, NY). Wells were then blocked for 2 hours with PBS + 1% bovine serum albumin (BSA) followed by incubation for 1 hour with primary antibodies DMF10.167.4, DMF10.62.3, or Hamster IgG (PharMingen, San Diego, CA) as an irrelevant control, at a concentration of 1 µg/mL in PBS + 1% BSA. Following five washes in PBS + 1% BSA, plates were incubated for 30 minutes with goat anti-hamster immunoglobulin/horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) diluted 1:1,000 in PBS + 1% BSA. Plates were washed in PBS and developed in TMB substrate for 10 minutes before quenching with 1 mol/L H2SO4. Absorbance was measured at 450 to 570 nm by a microplate reader.

Cell line culture and flow cytometry. The following tumor cell lines described in Table 1 were obtained from the American Type Culture Collection (Rockville, MD): SCLC (HTB 175, HTB-171, HTB-120, NCI-H187, DMS79, HTB-180, NCI-H69, SHP-77, HTB173, HTB-172), melanoma (CHL-1, Mel S, Mel D, and MTL450-5), kidney (HEK293 and HICK 10-4), leukemia (K562, Jurkat, HL-60, THP-1, and 721), pancreas (PCT391-34, CRL-1687, and CRL-1837), breast (MDA-MB-415, SK-BR-3, and BT-474), prostate (Du145, PC3, and LnCap), and ovarian (OV1036, ES-2, OTL298-95, and SK-OV-3). LU-134B, Lu135, and Lu139 were obtained from JCRB (Osaka, Japan). The SBC-3 line was obtained from the Japan Health Sciences Foundation (Tokyo, Japan). All lines were cultured according to provided instructions, typically in either DMEM or RPMI 1640 (Life Technologies Invitrogen) supplemented with 10% FBS (Hyclone, Logan, UT), 2-mercaptoethanol, penicillin/streptomycin, and L-glutamine (Life Technologies Invitrogen). Primary human lung epithelium was obtained from Clonetics (Rockland, MD) and cultured according to provided instructions.


View this table:
[in this window]
[in a new window]

  		Table 1. Summary of GM2 expression on human tumor cell lines

 
For flow cytometric analysis, single cell suspensions were generated. Cells were then washed thrice with ice-cold staining buffer (PBS + 1% BSA + 10 µg/mL human IgG + Azide). Next, cells were incubated for 30 minutes on ice with 10 µg/mL of purified DMF10.167.4 mAb or purified hamster IgG (Jackson ImmunoResearch). Cells were washed thrice with staining buffer and incubated with a 1:100 dilution of an anti-hamster IgG/FITC reagent (PharMingen) for 30 minutes on ice. Following three buffer washes, cells were resuspended in staining buffer containing propidium iodide, a vital stain that allows for identification of permeable cells, and analyzed by flow cytometry using a FACSCalibur instrument (Becton Dickinson, Franklin Lakes, NJ) with CellQuest Pro software (Becton Dickinson).

Analysis of apoptosis induction by Annexin V or caspase staining. For analysis of apoptosis induction, 2 x 105 cells were incubated for 16 to 20 hours with 10 to 20 µg/mL of DMF10.167.4 mAb or control mAbs and assayed for annexin positivity and active caspase content by incubating the cells with an Annexin V-Alexa488 conjugate (Molecular Probes, Eugene, OR) or with the CaspaTag reagent (Intergen, Norcross, GA) following the manufacturer's protocols. Cells were analyzed by flow cytometry using a FACSCalibur instrument (Becton Dickinson) with CellQuest Pro software (Becton Dickinson) to determine the amount of annexin or CaspaTag binding, respectively, as measures of induced apoptotic activity.

Immunohistochemistry and histopathology. Tissues were obtained from surgical resections on humans or were harvested from untreated mice or mice injected i.p. 1 or 5 days earlier with 0.5 mg of either DMF10.167.4, normal polyclonal hamster IgG or monoclonal hamster anti-GST IgG mAb (a kind gift of Dr. Robert Schreiber, Washington University, St. Louis, MO). For immunohistochemistry, 5-µm sections were prepared from snap-frozen tissues or cell pellets, transferred to charged slides, and allowed to dry for 1 hour. The slides were then treated with cold acetone for 10 minutes, dried for >20 minutes, and sequentially treated with cold acetone for 10 minutes, 70% acetone for 1 minute, 50% acetone for 1 minute, and several changes of water. Staining was done on a DaKoCytomation autostainer (DAKO, Carpinteria, CA) as follows. Slides were rinsed with buffer and treated with DAKO Protein Block for 5 minutes. Slides made with tissues from untreated mice were then incubated with 4 to 12 µg/mL of DMF10.167.4 or a control hamster IgG (polyclonal normal IgG or an anti-GST monoclonal antibody) in DAKO antibody diluent for 45 minute and rinsed with buffer; this antibody incubation step was skipped in those experiments where tissues were harvested from mice injected with antibodies. Slides were then incubated with a 1:300 dilution of biotinylated-goat anti-hamster IgG (Vector Labs, Burlingame, CA) for 30 minutes and rinsed with buffer. Endogenous peroxidase was blocked with 3% hydrogen peroxide (Ventana, Tucson, AZ) for 5 minutes followed by rinsing. Slides were then incubated sequentially with avidin/biotin/peroxidase complex (DAKO) for 30 minutes, rinsed, treated with 3,3'-diaminobenzidine (DAKO) for 10 minutes and then DAKO Enhancer for 2 minutes followed by counter staining with hematoxylin (Ventana) for 1 minute, dehydration, and clearing.

For histopathology, tissues were fixed in neutral buffered formalin and embedded in paraffin. Tissue sections were prepared and stained with H&E.

Analysis of in vivo antitumor efficacy. AKR mice were purchased from Jackson Labs (Bar Harbor, ME) and CB17.SCID mice were obtained from Charles River Laboratories (Wilmington, MA) and used according to approved Institutional Animal Care and Use Committee guidelines.

To examine the effect of DMF10.l67.4 mAb on a transplantable mouse tumor, AKR mice were injected i.p. with 5 x 106 syngeneic E710.2.3 cells. These mice were also injected i.p. with 0.5 mg of DMF10.167.4 or control hamster IgG on the day of tumor implantation and again 10 days later. Untreated, E710.2.3 cells grow as disseminated lymphomas; therefore, the health and survival of the mice was monitored; animals were sacrificed when they were moribund.

For prophylactic (mAb treatment at the time of tumor implantation) in vivo human tumor efficacy models, 5 x 106 CHL-1 melanoma or SBC-3 SCLC tumor cells were implanted s.c. into female CB17.SCID mice followed by i.v. injection of various doses of DMF10.167.4 mAb twice per week for 3 weeks. For therapeutic (established tumor) models, 5 x 106 CHL-1 or SBC-3 SCLC tumor cells were implanted s.c. into female CB17.SCID mice and allowed to grow until tumors measured ~10 to 20 mm2. At that time, mice were then injected i.v. with various doses of DMF10.167.4 mAb twice per week for 3 weeks. Tumors were then measured biweekly.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of hamster DMF10.167.4 monoclonal antibody and characterization of its specificity: identification of ganglioside GM2 as its cognate antigen. A hamster hybridoma, termed DMF10.62.3, was raised against murine E710.2.3 thymic lymphoma cells as described by Fernandes et al. (10). The IgG{kappa} antibody produced by this hybridoma bound to a surface antigen expressed on numerous murine and human tumor cell lines, blocked proliferation of the E710.2.3 line in vitro, and induced apoptosis in several types of tumor cell lines, including the human Jurkat T-cell leukemia line.

Another hybridoma, termed DMF10.167.4, was derived from the same fusion that yielded DMF10.62.3. Using molecular analysis, it was determined that the two hybrids were clonally related, or derived from the same parental B-cell clone, as their nucleotide sequences were identical across the immunoglobulin heavy chain CDR3 (data not shown). Overall, the nucleotide and predicted amino acid sequences of the rearranged immunoglobulin variable region genes were nearly identical, as the two hybridomas differed by only one conservative amino acid replacement in each of their heavy and light chains (data not shown). Because the DMF10.167.4 hybridoma grew more rapidly and produced more mAb in culture than the DMF10.62.3 hybridoma, the IgG mAb product of the DMF10.167.4 hybridoma was selected for additional characterization.

At this time, the specificity of the DMF10.167.4 mAb was unknown. After failing to identify its cognate antigen by Western blot experiments, we speculated that the mAb's ligand may not be on a polypeptide. Based on the antibody's tumor-binding profile by flow cytometry, we investigated whether the antigen might be a ganglioside. Purified gangliosides were solubilized in methanol/chloroform and coated onto ELISA plates and probed with DMF10.167.4 antibody. As shown in Fig. 1, of the 11 gangliosides tested, only GM2 was specifically recognized by DMF10.167.4 mAb. The structurally related ganglioside GM3, lacking one terminal galactose residue compared with GM2, and GM1, possessing one additional galactose residue, were not recognized, nor was the sialic acid–deficient ganglioside asialoGM2 (AsGM2). These results suggest that the epitope recognized by DMF10.167.4 mAb consists of a combination of the terminal galactose sugar and the sialic acid residue. Treatment of GM2 with neuraminidase A, which cleaves off the sialic acid residue, led to a loss of DMF10.167.4 binding as measured by ELISA (data not shown), further validating the epitope specificity of this mAb. We conclude that DMF10.167.4 mAb binds GM2.



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Ganglioside GM2 is recognized by clonally related mAbs DMF10.62.3 and DMF10.167.4. Purified gangliosides were solubilized in chloroform/methanol and coated in methanol onto microplate wells. The DMF10.62.3 and DMF10.167.4 mAbs were incubated with the wells and determined to be reactive specifically with GM2. Samples were run in duplicate. Bars, SE. Abbreviations describing the modifications of the individual gangliosides are as follows: As, asialo; Ls, Lyso.

 
Numerous types of tumor cell lines express cell surface GM2 as measured by reactivity with DMF10.167.4 monoclonal antibody. Several groups using different anti-GM2 antibodies have found that numerous human melanoma and lung tumor cell lines (13) and primary tumors (14, 15) express ganglioside GM2 as assessed by flow cytometry and immunohistochemistry. The extent to which DMF10.167.4 mAb reacted with several types of human tumor cell lines was determined by flow cytometric analysis. As summarized in Table 1, 14 SCLC lines were tested for surface expression of GM2. Eight lines, including LU-135, LU-134B, HTB 175, SBC-3, LU-139, HTB 171, HTB-120, and NCI-H187, showed strong positive staining for GM2 expression with DMF10.167.4 mAb compared with an irrelevant hamster IgG control mAb. Five lines, including HTB-180, NCI-H69, DMS-79, SHP-77, and HTB173, showed weak binding, and the SCLC line HTB 172 was negative for ganglioside GM2 expression For the melanoma cell lines tested with DMF10.167.4 mAb, CHL-1, Mel S, and Mel D had strong surface GM2 expression, whereas MTL450-5 cells were negative. In terms of DMF10.167.4 reactivity with other types of tumor cell lines, two kidney carcinoma lines, HEK293 and HICK 10-4, were strongly positive, as were the Jurkat T-cell and K562 B-cell leukemia lines, whereas the HL-60 and THP-1 leukemia lines and the 721 B-cell leukemia line showed no detectable GM2 surface expression. Representative tumor cell lines derived from pancreas, breast, prostate, and ovarian carcinomas were also analyzed. Overall, a low frequency (≤1 of 3) of pancreas (PCT391-94), breast (MDA-MB-415), prostate (Du145), and ovarian (OV1063) tumor cell lines were found to be positive for GM2 surface expression as measured by DMF10.167.4 mAb reactivity. Overall, these data confirm the overexpression of ganglioside GM2 on numerous tumor cell lines.

DMF10.167.4 monoclonal antibody induces apoptosis of several tumor cell lines in vitro. Previously, in vitro cell culture experiments revealed that the hamster DMF10.62.3 mAb induced apoptosis, as measured by Annexin V binding, in several murine and human tumor lines, including the Jurkat T-cell leukemia line (10). To validate the apoptotic activity of DMF10.167.4 mAb and extend the analysis to additional tumor cell lines, Jurkat, CHL-1 melanoma, and SBC-3 SCLC cells were assayed for apoptosis induction by DMF10.167.4 mAb using an Annexin V assay as well as an activated caspase assay. In this latter assay system, a fluorescently labeled DEVD peptide that binds irreversibly to the active site of activated caspases is used for flow cytometric analysis. After overnight incubation, DMF10.167.4 mAb induced apoptosis in all three of these cell lines, with a good correlation between the annexin and caspase assay systems (Table 2). In both the CHL-1 and SBC-3 lines, there was ~2.5-fold increase in the amount of apoptosis when DMF10.167.4 mAb is compared with control IgG treatment, whereas with Jurkat cells, there was a >4-fold increase in apoptosis when comparing DMF10.167.4 mAb with irrelevant control IgG. Collectively, these data show that the DMF10.167.4 mAb induces apoptosis upon cell surface GM2 binding.


View this table:
[in this window]
[in a new window]

  		Table 2. In vitro apoptosis induction by DMF10.167.4 mAb in Jurkat, CHL-1, and SBC-3 cell lines

 
Analysis of reactivity of DMF10.167.4 monoclonal antibody with normal mouse tissues ex vivo. Various anti-GM2 mAbs have been reported by others to show different patterns of reactivity with normal tissues, either showing no reactivity or binding to a large number of epithelial tissues (14, 15). Because reactivity with normal tissues is a critical issue for immunotherapy, we characterized the reactivity of DMF10.167.4 mAb with normal cells. As a positive control, sections were prepared from pellets of cultured E710.2.3 lymphoma cells that were previously shown to be reactive with DMF10.167.4 by immunofluorescence and flow cytometry (10). These sections were incubated with the antibody, and the bound immunoglobulin was visualized using an immunoperoxidase assay. DMF10.167.4 mAb failed to stain these sections when the cells had been fixed and embedded in paraffin, presumably because the processing procedure destroyed the glycolipid epitope. In contrast, DMF10.167.4 mAb, but not control hamster IgG, stained frozen sections of E710.2.3 cell pellets (Fig. 2A and B). Similarly, the antibody stained frozen sections of other DMF10.167.4-positive cell line (e.g., RMA) with a staining pattern that was granular and cytoplasmic (data not shown). Moreover, staining was not observed in frozen sections of the DMF10.167.4-negative cell line RF33.70 that were incubated with DMF10.167.4 antibody or control hamster IgG (Fig. 2C and D). Therefore, it was feasible to use immunoperoxidase assays on frozen sections to determine whether DMF10.167.4 reacted with normal mouse tissues.



View larger version (89K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Immunohistochemistry analysis of E710.3.2 (A and B) and RF33.70 (C and D) cell lines using DMF10.167.4 mAb and control hamster immunoglobulin.

 
A panel of normal mouse tissues was snap frozen, sectioned, and assayed for reactivity with DMF10.167.4 mAb and control hamster immunoglobulin. In several tissues, such as lung and liver, tissue mononuclear cells (macrophages/dendritic cells) stained equivalently with DMF10.167.4 and control hamster immunoglobulin, presumably due to the antibodies' binding to Fc receptors. However, no DMF10.167.4-specific staining was observed in parenchymal cells of any tissue except for small (Fig. 3A) and large (Fig. 3B) intestine. In the intestines, there was strong staining of only the apical region of the surface epithelium cells (Table 3, left column; Fig. 3A and B, d). There was no staining of the intestinal epithelium with control hamster immunoglobulin (Fig. 3A and B, a).



View larger version (142K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. A, analysis of DMF10.176.4 expression and effects on small intestine. a, intestine from untreated animals incubated ex vivo with control hamster IgG and stained for bound hamster immunoglobulin. b, intestine from animals 24 hours after injection of control hamster IgG in vivo stained for bound hamster immunoglobulin. c, histopathology of intestine from animals 24 hours after injection of control hamster IgG in vivo and stained with H&E; histopathology was identical in animals injected 5 days earlier with antibody (data not shown). d, same as (a) except incubated ex vivo with DMF10.167.4 instead of hamster immunoglobulin. e, same as (b) except animals were injected with DMF10.167.4 in instead of hamster immunoglobulin. f, same as (c) except animals were injected with DMF10.167.4 instead of hamster immunoglobulin; histopathology was identical in animals injected 5 days earlier with antibody (data not shown). B, analysis of DMF10.176.4 expression and effects on colon in. (a-f) are the same conditions as in (a-f) in (A), except analyzing colon instead of small intestine.

 

View this table:
[in this window]
[in a new window]

  		Table 3. Analysis of DMF10.167.4 mAb reactivity with mouse tissues in vitro and in vivo

 
Analysis of reactivity of DMF10.167.4 monoclonal antibody with mouse tissues in vivo. From the immunoperoxidase analysis of tissue sections, it was not clear whether DMF10.167.4 mAb was reacting with a cell surface or an internal antigen on intestinal epithelium. This is an important distinction because a therapeutic antibody would be unable to access and bind to internal antigens of intact cells. In addition, it was unclear whether in vivo the antibody would be able to penetrate and bind to the apical surface of intestinal epithelium, which is on an external surface of the body and bounded by a basement membrane. Thus, it was of interest to determine whether systemically given antibody would bind to intestinal epithelium or other tissues in vivo.

To address this question, mice were injected with 0.5 mg of DMF10.167.4 mAb or control hamster immunoglobulin (the same amounts used in immunotherapy studies described below) and 24 hours later, tissues were harvested and probed for the presence of bound hamster immunoglobulin using a labeled anti-hamster immunoglobulin antibody in an immunoperoxidase assay (Table 3, middle column). As a positive control, E710.2.3 cells were incubated for 24 hours with these same antibodies and then processed and analyzed in an identical manner. The DMF10.167.4 mAb–coated E710.2.3 cells stained intensely, whereas those incubated with control hamster immunoglobulin were negative (data not shown). Therefore, this technique can detect DMF10.167.4 mAb that is prebound to cells. As shown in Fig. 3, no DMF10.167.4 mAb was detectably bound to the intestinal epithelium of mice that were injected with this antibody. Thus, DMF10.167.4 mAb injected in vivo does not bind to small intestinal or colonic epithelium at detectable levels.

This analysis was also extended to other tissues (Table 3). In the spleen, a dendritic cell network, presumably follicular dendritic cells, was stained in the white pulp and scattered dendritic cells were stained in the red pulp. Similarly, kidney intraglomerular and thymic cortical dendritic cells were stained, as were scattered dendritic cells in the lamina propria of the small and large intestine. The staining of dendritic cells was surprising because these cells did not stain when frozen sections were probed with DMF10.167.4 mAb and analyzed by immunoperoxidase reaction. Moreover, dendritic cells and macrophages stained ex vivo did not react detectably with DMF10.167.4 mAb even when analyzed by a more sensitive flow cytometry assay. Given these discrepancies it is possible that the injected DMF10.167.4 mAb is binding to Fc receptors on macrophages and immature dendritic cells in vivo. The parenchymal cells of all other tissues examined did not bind detectable amounts of the injected DMF10.167.4 mAb.

Effects of administering DMF10.167.4 monoclonal antibody in vivo to normal mice. In vivo, pathology is a paramount issue when evaluating mAbs for use in immunotherapy. Because DMF10.167.4 mAb has antiproliferative and proapoptotic activity, it could potentially cause tissue injury if it bound to normal cells and induced similar effects. Although our studies indicated that DMF10.167.4 mAb was not binding to most cells in vivo, it was possible that some cells were binding small amounts of mAb that were below the limits of detection in our assay, or binding to cells that were not included in our tissue panel (Table 3). Therefore, to further evaluate this issue, mice were injected with DMF10.167.4 mAb or control hamster immunoglobulin and monitored for 1 to 5 days, followed by tissue harvest for histopathologic analysis. No overt signs of distress were evident in either the DMF10.167.4 or control immunoglobulin-treated mice during this time course, or in additional experiments that extended several months.

In animals injected with DMF10.167.4 mAb, tissues were harvested after 24 hours or 5 days. Sections of paraffin-embedded tissues were stained with H&E and examined by microscopy. All tissues examined, including small (Fig. 3A) and large intestine (Fig. 3B) were histologically normal (Table 3, right column) and there were no signs of inflammation or cell death. Therefore, the acute administration of DMF10.167.4 mAb does not cause detectable pathologic changes.

Anti-GM2 monoclonal antibody eliminates a syngeneic mouse lymphoma in vivo. Because mice tolerated administration of DMF10.167.4, we next evaluated whether the antibody could be used to treat a transplantable tumor. To determine if the anti-GM2 mAb would suppress tumor formation, AKR mice were injected with the syngeneic E710.2.3 lymphoma. When left untreated, this tumor disseminates and grows progressively until it is uniformly fatal (16). Mice were treated with 0.5 mg of DMF10.167.4 mAb or control hamster immunoglobulin on the day they were injected with the E710.2.3 tumor and again 10 days later and survival of these mice was monitored over time. As described in Table 4, all mice that were treated with control hamster immunoglobulin developed tumors and survived for a mean time of 35 days. Mice that received DMF10.167.4 mAb remained healthy with no visible signs of tumor for >3 months. These results indicate that the DMF10.167.4 antibody was able to successfully treat a lethal tumor challenge without apparent toxicity.


View this table:
[in this window]
[in a new window]

  		Table 4. Immunotherapy with DMF10.167.4 mAb

 
Anti-GM2 monoclonal antibody prevents the establishment of human tumor xenografts in vivo. To extend the immunotherapy studies to human tumors, CB17.SCID mice were injected s.c. with 5 x 106 human CHL-1 melanoma cells or 4 x 106 SBC-3 SCLC cells. Mice were then injected i.v. with 100 µg of DMF10.167.4 mAb or control hamster immunoglobulin twice a week for 3 weeks, with the first mAb injection occurring on the day they were injected with tumor cells. All five animals that received no treatment or irrelevant IgG treatment developed easily detectable tumors by day 10 that continued to expand throughout the course of the study until their sacrifice at day 30, whereas mice that received DMF10.167.4 mAb showed no evidence of tumor establishment (data not shown). These results show that DMF10.167.4 can prevent the establishment of human tumors in vivo.

Anti-GM2 monoclonal antibody blocks the progression of established human tumor xenografts in vivo. Next, we assessed whether DMF10.167.4 mAb could suppress progression of established tumors in more physiologically stringent models. CB17.SCID mice were injected s.c. with 5 x 106 human CHL-1 melanoma cells, and on day 4 when tumors were ~10 to 15 mm2, mice were randomized into treatment groups of five animals. One group of mice remained untreated; one group received 100 µg of irrelevant IgG; and the other groups received 10, 30, or 100 µg of DMF10.167.4 mAb twice a week for 3 weeks. Tumor areas were measured for >35 days. As shown in Fig. 4A, all five of the animals that received no treatment ({bullet}) or irrelevant IgG treatment (same as no treatment group, thus data not shown) developed easily detectable tumors by day 10 which expanded throughout the course of the study, leading to their sacrifice at day 30. Mice that had received 10 µg ({lozenge}) or 30 µg ({blacktriangleup}) of DMF10.167.4 mAb over time showed a ~50% reduction in tumor size compared with untreated mice, whereas mice that received 100 µg ({square}) of DMF10.167.4 mAb showed a >75% reduction in tumor size versus the control mice.



View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. A, suppression of CHL-1 tumor progression in vivo following treatment with anti-GM2 antibody DMF10.167.4. CB17.SCID mice (five per group) were injected with 5 x 106 CHL-1 cells on day 0. Antibody (10, 30, or 100 µg/injection) was given i.v. starting on day 4 and continued twice per week for 3 weeks. Point, mean tumor area from five animals; bars, SD. B, suppression of SBC-3 tumor progression in vivo following treatment with anti-GM2 antibody DMF10.167.4. CB17.SCID mice (five per group) were injected with 4 x 106 SBC-3 cells on day 0. Antibody (100 µg/injection) was given i.v. starting on day 7 and continued twice per week for 3 weeks. Point, mean tumor area from five animals; bars, SD.

 
Additional studies using established SBC-3 human SCLC tumors were done. CB17.SCID mice were injected s.c. with 4 x 106 SBC-3 SCLC cells and tumors were allowed to establish for 7 days. Mean areas for the SBC-3 tumors were 20 mm2, approximately equal to the CHL-1 tumors in the previous study. At this time, the mice were randomized into groups of five mice, with one group receiving 100 µg of irrelevant IgG and the other receiving 100 µg of DMF10.167.4 twice a week for 3 weeks. Tumor areas were measured for >30 days. As shown in Fig. 4B, SBC-3 tumor growth continued in all of the animals that received irrelevant IgG treatment ({bullet}), leading to their sacrifice at 28 days. Remarkably, mice that received DMF10.167.4 mAb ({square}) showed a total suppression of tumor growth progression. Collectively, these in vivo tumor model studies show the potential therapeutic efficacy of this mAb for treatment of human melanoma and SCLC tumors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A hamster monoclonal antibody, termed DMF10.63.2, was raised against E710.2.3 mouse thymic lymphoma cells and shown to block the tumor's proliferation and induce it to undergo apoptosis in vitro. The mAb also showed reactivity with a number of other mouse tumor cell lines, as well as human T- and B-cell lymphomas, and sarcomas (10). A clonally related and nearly identical mAb termed DMF10.167.4 was also generated and, in this report, we have determined that these two mAbs recognize ganglioside GM2.

Gangliosides are cell membrane bound glycosphingolipids composed of sialic acid, carbohydrates, and ceramide. They are highly expressed on the cell surface of most cancers of neuroectodermal and epithelial origin, including glioblastoma, neuroblastoma, melanoma, and SCLC. These gangliosides are minor components of normal tissues, suggesting that they are overexpressed in certain cancers and might play some role in malignancy (6, 11). In fact, ganglioside interactions with extracellular matrix proteins may play a role in metastasis of tumor cells (17, 18). Due to the cell surface overexpression of gangliosides, particularly GD2, GD3, and GM2, an enormous amount of effort has been focused on the preclinical validation of these molecules as targets for immunotherapy (6).

Numerous mAbs have been generated that are specific for gangliosides, including GM2 (13, 14, 19–22). These previously reported anti-GM2 mAbs were found to react with several different tumor types including melanoma, SCLC, lung adenoma, lung squamous, and large cell carcinomas. We also found that DMF10.167.4 mAb reacts with human tumors and show in this report that these tumors include a large percentage of small cell lung carcinoma and melanoma cell lines, as well as a smaller number of other tumor types. Interestingly, DMF10.167.4 mAb does not seem to show an identical pattern of reactivity to the various tumor types reported for other anti-GM2 antibodies.

Other groups have analyzed the reactivity of normal tissues with different anti-GM2 antibodies. One immunoglobulin M (IgM) anti-GM2 mAb showed no detectable binding to a large panel of normal tissues, including brain, heart, liver, and lung (14), whereas a second IgM anti-GM2 mAb was found to react with the secretory borders of normal epithelial cells from several tissues, including lung and prostate (15). DMF10.167.4 mAb has a distinct pattern of reactivity to normal (mouse) tissues, as it reacts with the apical border of intestinal epithelium but not with epithelium of other tissues such as lung. To evaluate whether this differential reactivity between the various anti-GM2 antibodies with several normal tissues was due to a difference between human and mouse tissues, we have examined the reactivity of DMF10.167.4 with human lung. DMF10.167.4 does not stain human lung epithelium by immunohistochemistry, whereas under the same conditions it strongly stains the surface of human intestinal epithelium, as it does in mouse (data not shown). Similarly, we also failed to detect DMF10.167.4 binding to human lung epithelium, using the much more sensitive technique of immunofluorescence and flow cytometry (data not shown). Therefore, the most likely explanation for the differences in tumor and normal tissue binding observed between the various anti-GM2 mAbs is that they recognize distinct epitopes on GM2. DMF10.167.4 therefore seems an anti-GM2 antibody with unique antigen-binding properties.

Several of the previously described anti-GM2 mAbs have been of IgM isotype (14, 19, 20, 22). This is not unexpected for an antigen that likely does not elicit T-cell help, which promotes isotype switching. In any case, IgM antibodies are less amenable to clinical use because of potential stability issues as well as large-scale commercial manufacturing and purification difficulties. In contrast, DMF10.167.4 mAb is of the IgG isotype, which is much more adaptable to clinical use, as shown by the large number of IgG antibodies that have been used successfully to date for immunotherapy (reviewed in refs. 3, 5).

Antitumor antibodies can eliminate tumors through several different mechanisms. The Fc region of both IgM and some IgG antibodies can activate complement proteins to generate products that induce inflammation and create pores in the tumor cell membrane (23). Antibodies can also bind Fc receptors on macrophage, neutrophils, and natural killer cells and thereby target these cells to kill the tumor by ADCC. Other anti-GM2 mAbs have shown both CDC activity or, when chimerized or humanized to IgG isotypes, ADCC function in vitro and in vivo (13, 21, 24, 25). DMF10.167.4 can also trigger CDC and ADCC in vitro (data not shown) but is of particular interest because of its ability to trigger tumor cells to undergo apoptosis. DMF10.167.4 mAb was able to rapidly induce apoptosis and/or block cellular proliferation when cultured with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 SCLC lines. Previously, only one described anti-GM2 mAb has been shown to induce apoptosis in vitro, but this effect only occurred after several days of incubation and was only observed with tumor heterospheroids and not monolayer cultures (26). Thus, DMF10.167.4 seems to have distinct functional properties compared with previously reported anti-GM2 antibodies.

Because DMF10.167.4 seems to bind a distinct epitope (see above), we speculate that the mAbs' proapoptotic activity may be due to its unique binding specificity. Exactly how DMF10.167.4 stimulates apoptosis is not known. However, mAbs to other gangliosides such as GD2 also induce apoptosis in SCLC cells in vitro. Yoshida et al. speculated that antibody-induced GD2 clustering resulted in the modulation of unknown molecules that stimulate apoptosis (27). It has been shown that gangliosides exist on the cell surface in clusters and form microdomains, variously called lipid rafts or caveolae (28). Ganglioside-associated signal transduction may occur in these membrane structures (29–31), possibly through interactions with tetraspanins or other signaling molecules located within these microdomains that form a functional unit termed a glycosynapse (32, 33). Ganglioside GD3 has also been shown to be involved in the induction of CD95 and ceramide-mediated apoptosis (34, 35).

Antibodies, such as DMF10.167.4, that inhibit tumor cell growth and induce apoptosis in vitro are of potential interest for use in immunotherapy. However, it is essential that the antibody does not bind normal cells in ways that cause pathology. Thus, it was important to determine whether DMF10.167.4 mAb would bind to and/or effect normal tissues. No apoptotic activity was observed after culture of this antibody with normal cells (data not shown). The most important experiments to address this issue were done by dosing mice with DMF10.167.4 and evaluating their tissues for pathology. We found the antibody only bound to follicular dendritic cells in the spleen and in scattered mononuclear/dendritic cells primarily in lymphoid tissues, presumably through Fc receptors. We detected no binding to the apical surface of intestinal epithelium, although this region of cells reacted with the antibody in tissue sections. Presumably, this epitope is not expressed on the cell surface of the epithelium or the antibody does not gain access to this site. Most importantly, these mAb treated animals remained healthy and no gross or microscopic pathologic changes could be detected in their tissues, including the small and large bowels. This result indicates that if the antibody is binding to intestinal epithelium or other tissues, it is not affecting their survival or integrity. Therefore, the antibody is well tolerated in vivo.

In contrast to the absence of effects on normal tissues, DMF10.167.4 mAb did affect the growth of tumors in vivo. This was first observed with the transplantable syngenic E710.2.3 murine lymphoma. E710.2.3 normally grows aggressively in mice, but treatment with DMF10.167.4 prevented the establishment of tumor in 100% of the tumor cell–injected animals. We extended these studies to xenogenic models using human tumor cell lines CHL-1 and SBC-3. Importantly, DMF10.167.4 reduced tumor growth even when given to animals bearing established tumors. In all of these situations, there was no antibody toxicity detected in any of the treated animals.

These findings together with earlier ones suggest that the apoptotic functional effects of DMF10.167.4 mAb are tumor specific. We established that it can block tumor growth and extend survival in vivo without detectable toxicity. Antibodies to other gangliosides, such as GD2, GM3, and GD3, have also shown in vivo efficacy for tumor immunotherapy (36–38), yet these gangliosides are immunochemically distinct from GM2 and have different patterns of expression. Moreover, anti-GD3 and anti-GM3 mAbs, respectively, are recent examples of ganglioside-specific mAbs that have undergone phase I clinical trials (refs. 39, 40; reviewed in ref. 6). Potential products that arise from these efforts and an antibody like DMF10.167.4 might complement each other because they target different gangliosides; thus, they could potentially have additive effects and/or target different cancer indications. Considering the unique combination of specificity for GM2, antitumor activity, and reactivity with multiple human tumor types, DMF10.167.4 mAb warrants further evaluation as an immunotherapeutic antibody.


   Acknowledgments
 
Grant support: NIH (K.L. Rock).

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


   Footnotes
 
Note: C.S. Bangur is currently at the Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, C3-168, Seattle, WA 98109-1024.

N.A. Fanger is currently at the Teranode Corporation, 83 South King Street, Suite 800, Seattle, WA 98104.

Received 1/28/05. Revised 4/ 6/05. Accepted 5/ 4/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495–7.[CrossRef][Medline]
  2. von Mehren M, Adams GP, Weiner LM. Monoclonal antibody therapy for cancer. Annu Rev Med 2003;54:343–69.[CrossRef][Medline]
  3. Ross JS, Gray K, Schenkein D, et al. Antibody-based therapeutics in oncology. Expert Rev Anticancer Ther 2003;3:107–21.[CrossRef][Medline]
  4. Reichert JM. Therapeutic monoclonal antibodies: trends in development and approval in the US. Curr Opin Mol Ther 2002;4:110–8.[Medline]
  5. Glennie MJ, van de Winkel JG. Renaissance of cancer therapeutic antibodies. Drug Discov Today 2003;8:503–10.[CrossRef][Medline]
  6. Fredman P, Hedberg K, Brezicka T. Gangliosides as therapeutic targets for cancer. Biodrugs 2003;17:155–67.[CrossRef][Medline]
  7. Sarup JC, Johnson RM, King KL, et al. Characterization of an anti-p185HER2 monoclonal antibody that stimulates receptor function and inhibits tumor cell growth. Growth Regul 1991;1:72–82.[Medline]
  8. Demidem A, Lam T, Alas S, Hariharan K, Hanna N, Bonavida B. Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs. Cancer Biother Radiopharm 1997;12:177–86.[Medline]
  9. Carter P, Smith L, Ryan M. Identification and validation of cell surface antigens for antibody targeting in oncology. Endocr Relat Cancer 2004;:659–87.
  10. Fernandes DM, Baird AM, Berg LJ, Rock KL. A monoclonal antibody reactive with a 40-kDa molecule on fetal thymocytes and tumor cells blocks proliferation and stimulates aggregation and apoptosis. J Immunol 1999;163:1306–14.[Abstract/Free Full Text]
  11. Hakomori S. Aberrent glycosylation in cancer cell membranes as focused on glycolipids: overview and perspectives. Cancer Res 1985;45:2405–14.[Free Full Text]
  12. Litwin S, Shlomchik M. A test for clonal relatedness in a set of lymphocytes. J Exp Med 1990;171:293–7.[Free Full Text]
  13. Nakamura K, Koibe M, Shitara K, et al. Chimeric anti-ganglioside GM2 antibody with antitumor activity. Cancer Res 1994;54:1511–6.[Abstract/Free Full Text]
  14. Nishinaka Y, Ravindranath MH, Irie RF. Development of a human monoclonal antibody to ganglioside GM2 with potential for cancer treatment. Cancer Res 1996;56:5666–71.[Abstract/Free Full Text]
  15. Zhang S, Cordon-Cardo C, Zhang HS, et al. Livingston PO Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides. Int J Cancer 1997;73:42–9.[CrossRef][Medline]
  16. Pinto VB, Rock KL. Characterization of the proliferative response of a CD4–CD8– thymic T cell lymphoma cell line to stimulation by thymic cellular elements. J Immunol 1991;147:42–9.[Abstract]
  17. Nakano J, Yasui H, Lloyd KO, Muto M. Biologic role of gangliosides GM3 and GD3 in the attachment of human melanoma cells to extracellular matrix proteins. J Investig Dermatol Symp Proc 1999;4:173–6.[Medline]
  18. Birkle S, Zeng G, Gao L, Yu RK, Aubry J. Role of tumor-associated gangliosides in cancer progression. Biochemie 2003;85:455–63.[Medline]
  19. Miyake M, Ito M, Hitomi S, et al. Generation of two murine monoclonal antibodies that can discriminate N-glycolyl and N-acetyl neuraminic acid residues of GM2 gangliosides. Cancer Res 1988;48:6154–60.[Abstract/Free Full Text]
  20. Vrionis FD, Wikstrand CJ, Fredman P, Mansson JE, Svennerholm L, Bigner DD. Five new epitope-defined monoclonal antibodies reactive with GM2 and human glioma and medulloblastoma cell lines. Cancer Res 1989;49:6645–51.[Abstract/Free Full Text]
  21. Nakamura K, Tanaka Y, Fujino I, Hirayama N, Shitara K, Hanai N. Dissection and optimization of immune effector functions of humanized anti-ganglioside GM2 monoclonal antibody. Mol Immunol 2000;37:1035–46.[CrossRef][Medline]
  22. Natoli EJ Jr, Livingston PO, Pukel CS, et al. A murine monoclonal antibody detecting N-acetyl and N-glycoyl-GM2: characterization of cell surface reactivity. Cancer Res 1986;46:4116–20.[Abstract/Free Full Text]
  23. LoBuglio AF, Saleh MN. Advances in monoclonal antibody therapy of cancer. Am J Med Sci 1992;304:214–24.[CrossRef][Medline]
  24. Fukumoto H, Nishio K, Ohta S, et al. Effect of a chimeric anti-ganglioside GM2 antibody on ganglioside GM2-expressing human solid tumors in vivo. Int J Cancer 1999;82:759–64.[Medline]
  25. Parajuli P, Yanagawa H, Hanibuchi M, et al. Humanized anti-ganglioside GM2 antibody is effective to induce antibody-dependent cell-mediated cytotoxicity in mononuclear cells from lung cancer patients. Cancer Lett 2001;165:179–84.[Medline]
  26. Nakamura K, Hanibuchi M, Yano S, et al. Apoptosis induction of human lung cancer cell line in multicellular heterospheroids with humanized antiganglioside GM2 monoclonal antibody. Cancer Res 1999;59:5323–30.[Abstract/Free Full Text]
  27. Yoshida S, Fukumoto S, Kawaguchi H, Sato S, Ueda R, Furukawa K. Ganglioside G(D2) in small cell lung cancer cell lines: enhancement of cell proliferation and mediation of apoptosis. Cancer Res 2001;61:4244–52.[Abstract/Free Full Text]
  28. Simons K, Ikonen E. Functional rafts in cell membranes. Nature 1997;387:569–72.[CrossRef][Medline]
  29. Kasahara K, Sanai Y. Functional roles of glycosphingolipids in signal transduction via lipid rafts. Glycoconj J 2000;17:153–62.[CrossRef][Medline]
  30. Anderson RGW. The caveolae membrane system. Annu Rev Biochem 1998;67:199–225.[CrossRef][Medline]
  31. Carver LA, Schnitzer JE. 2003 Caveolae: mining little caves for new cancer targets. Nat Rev Cancer 2003;3:571–81.[CrossRef][Medline]
  32. Hakomori S. The glycosynapse. Proc Natl Acad Sci U S A 2002;99:225–32.[Abstract/Free Full Text]
  33. Hakomori S. Structure, organization and function of glycosphingolipids in membranes. Curr Opin Hematol 2003;10:16–24.[CrossRef][Medline]
  34. De Maria R, Lenti L, Malisan F, et al. Requirement for GD3 ganglioside in CD95- and ceramide-induced apoptosis. Science 1997;277:1652–5.[Abstract/Free Full Text]
  35. Kristal BS, Brown AM. Apoptogenic ganglioside GD3 directly induces the mitochondrial permeability transition. J Biol Chem 1999;274:23169–75.[Abstract/Free Full Text]
  36. Zhang H, Zhang S, Cheung NK, Ragupathi G, Livingston PO. Antibodies against GD2 ganglioside can eradicate syngeneic cancer micrometastases. Cancer Res 1998;58:2844–9.[Abstract/Free Full Text]
  37. Carr A, Mesa C, Del Carmen Arango M, Vasquez AM, Fernandez LE. In vivo and in vitro anti-tumor effect of 14F7 monoclonal antibody. Hybrid Hybridomics 2002;21:463–8.[CrossRef][Medline]
  38. Kanazawa J, Ohta S, Shitara K, et al. Therapeutic potential of chimeric anti-ganglioside GD3 antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis. Cancer Immunol Immunother 2000;49:253–8.[CrossRef][Medline]
  39. Scott AM, Lee FT, Hopkins W, et al. Specific targeting, biodistribution, and lack of immunogenicity of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma: results of a phase I trial. J Clin Oncol 2001;19:3976–87.[Abstract/Free Full Text]
  40. Irie RF, Ollila DW, O'Day S, Morton DL. Phase I pilot clinical trial of human IgM monoclonal antibody to ganglioside GM3 in patients with metastatic melanoma. Cancer Immunol Immunother 2004;53:110–7.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Biswas, K. Biswas, A. Richmond, J. Ko, S. Ghosh, M. Simmons, P. Rayman, B. Rini, I. Gill, C. S. Tannenbaum, et al.
Elevated Levels of Select Gangliosides in T Cells from Renal Cell Carcinoma Patients Is Associated with T Cell Dysfunction
J. Immunol., October 15, 2009; 183(8): 5050 - 5058.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. M. Hernandez, D. Toledo, D. Martinez, T. Grinan, V. Brito, A. Macias, S. Alfonso, T. Rondon, E. Suarez, A. M. Vazquez, et al.
Characterization of the Antibody Response against NeuGcGM3 Ganglioside Elicited in Non-Small Cell Lung Cancer Patients Immunized with an Anti-Idiotype Antibody
J. Immunol., November 1, 2008; 181(9): 6625 - 6634.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
L. Roque-Navarro, K. Chakrabandhu, J. de Leon, S. Rodriguez, C. Toledo, A. Carr, C. M. de Acosta, A.-O. Hueber, and R. Perez
Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity
Mol. Cancer Ther., July 1, 2008; 7(7): 2033 - 2041.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Raval, S. Biswas, P. Rayman, K. Biswas, G. Sa, S. Ghosh, M. Thornton, C. Hilston, T. Das, R. Bukowski, et al.
TNF-{alpha} Induction of GM2 Expression on Renal Cell Carcinomas Promotes T Cell Dysfunction
J. Immunol., May 15, 2007; 178(10): 6642 - 6652.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Biswas, A. Richmond, P. Rayman, S. Biswas, M. Thornton, G. Sa, T. Das, R. Zhang, A. Chahlavi, C. S. Tannenbaum, et al.
GM2 Expression in Renal Cell Carcinoma: Potential Role in Tumor-Induced T-Cell Dysfunction.
Cancer Res., July 1, 2006; 66(13): 6816 - 6825.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Retter, M. W.
Right arrow Articles by Rock, K. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Retter, M. W.
Right arrow Articles by Rock, K. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online