Monoclonal Antibody R24 Research Articles

E-45. Small cell lung cancer: Targeting minimal residual disease

The outcome for patients with small cell lung cancer has improved marginally over the last 20 years. Standard chemotherapy regimens yield high response rates and some cures when combined with thoracic irradiation in limited stage patients. Nonetheless, SCLC has a high relapse rate due to drug resistance. This has resulted in continued poor survival for most patients. Developing ways to maintain response after induction chemotherapy or chemoradiotherapy has been a major research focus. The lack of an assay capable of measuring residual disease limits our ability to gauge the risk for relapse or to assess the efficacy of therapy in this setting. At this time all patients are considered at high risk and the challenge is to maintain their remission status. The chemosensitivity of SCLC logically supports the use of maintenance chemotherapy or high dose treatments as a way to attack residual metastatic disease. However, trials using more chemotherapy have generally resulted in more toxicity with no improvement in survival. In 13 randomized trials of maintenance chemotherapy, only one showed an overall survival advantage, five showed improvements only in subgroups of patients, and one showed a shorter survival with continued treatment [ 11. At least 36 studies of high dose chemotherapy with autologous bone marrow or stem cell rescue have been reported [2,3]. In these trials, only a small fraction of patients can complete the high dose chemotherapy due to co-morbid illnesses or toxicity, treatment related mortality is significant, and survival remains poor. In order to change outcomes in SCLC substantially, new approaches are needed. The matrix metalloproteinase (MMP) inhibitors are a novel class of drugs that were tested in SCLC patients as a potential therapy aimed at maintaining remission, but unfortunately yielded disappointing results in phase III trials. MMPs are a class of over 30 extracellular enzymes that degrade connective tissue and play an important role in bone development, wound healing, and vascular remodeling. Several inhibitors of these enzymes have been developed to decrease the cell migration responsible for metastatic spread. Dr. Shepherd reported the results of a phase III NCICEORTC trial in which 532 patients with small cell lung cancer who completed initial therapy were randomized to receive marimastat, a broad-spectrum MMP inhibitor, or placebo [4]. There was no difference between the marimastat and placebo groups with regards to overall survival or progression free survival. Furthermore, myalgias and arthralgias necessitated dose reduction or drug discontinuation in 33% and 32% of the patients receiving marimastat. The results of this trial, as well as the early discontinuation of a similar trial with BAY 12-9566, raise doubts as to the efficacy of this class of agents in SCLC, though specific inhibition of other MMPs could be explored. At Memorial Sloan-Kettering, we are attempting to take advantage of unique epitopes present on SCLC to develop vaccines. One potential target identified is the ganglioside GD3 expressed on cells of neuroectodermal origin. Using an anti-idiotype approach, the mouse monoclonal antibody BEC2 was developed against the binding region of the mouse monoclonal antibody R24 which binds to GD3. A pilot study was performed in which 1.5 patients with SCLC who had a major response to induction chemotherapy were immunized with BEC2. BCG was also added as an immunologic adjuvant [5]. Of the 13 evaluable patients, all developed anti-BEC2 antibodies (HAMA). Five patients had measurable anti-GD3 IgM antibody and 3 of those patients developed IgG antibodies. Median overall survival was 21 months; median time to relapse was 11 months in the extensive stage group and had not been reached in the limited stage group (>47 months). These results were a significant improvement when compared to two historical control groups treated at our center, and prompted an international phase III trial in SCLC patients with limited disease and a complete or partial response after initial therapy (the SILVA trial, lead by the EORTC). This trial completed accrual in October, 2002. Currently, we are also testing the immunogenicity of vaccines against other targets in SCLC such as GM2, Fucosyl GM1 [6,7], Globo H, and polysialic acid [S]. In order to optimize the likelihood of immunologic effect, we plan to combine the vaccines into a polyvalent vaccine. Only new strategies will improve outcomes for SCLC patients. Eligible individuals should enroll in clinical trials after chemotherapy and irradiation.

Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti-melanoma mouse IgG3 monoclonal antibody R24.

R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acalpha8Neu5Acalpha3Gal beta4Glcbeta1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma. The IgG molecule basically contains an asparagine-linked biantennary complex type oligosaccharide on the C(H)2 domain of each heavy chain, which is necessary for its in vivo effector function. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO(2)/HCO(3) (-) (pH 7.4, 7.2, and 6.9) and HEPES buffered serum-free medium. Growth, metabolism, and IgG production of hybridoma cells (ATCC HB-8445) were analyzed on a 2-L bioreactor scale using fed-batch mode. Specific growth rates (mu) and MAb production rates (q(IgG)) varied significantly with maximum product yields at pH 6.9 (q(IgG) = 42.9 microg 10(-6) cells d(-1), mu = 0.30 d(-1)) and lowest yields in pH 7.4 adjusted batches (q(IgG) = 10.8 microg 10(-6) cells d(-1), mu = 0.40 d(-1)). N-glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS). The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation. In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained. The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411. Different culture buffering gave rise to altered glycosylation pattern of the MAb R24. Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide-dependent biological activity.

Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.

Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.

Enrichment and localization of ganglioside G D3 and caveolin-1 in shed tumor cell membrane vesicles

Tumor cell ganglioside shedding has been implicated in the process of tumor formation. Previously, we identified three forms of tumor ganglioside shedding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified human ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G D3. Immunostaining using the monoclonal antibody R24 confirmed G D3 expression and its presence in the plasma membrane of these cells. Cellular gangliosides were detected in the culture supernatant by HPTLC autoradiography, confirming an active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell membranes also express caveolin-1, a characteristic protein marker for caveolae, which was detected by flow cytometric analysis and by Western blotting in both the cell membranes and the isolated membrane vesicles. To further define the expression of G D3 and caveolin-1, we used immunogold electron microscopy. This revealed localization of G D3 in small clusters in the plasma membrane as well as enrichment and localization of ganglioside G D3 and caveolin-1 in shed membrane vesicles, with 58–78% of vesicles carrying both G D3 and caveolin-1. Together, these results suggest that membrane vesicle shedding originates in plasma membrane domains enriched in gangliosides and caveolin-1.

Analysis of therapeutic and immunologic effects of R24 anti‐GD3 monoclonal antibody in 37 patients with metastatic melanoma

BACKGROUND Antitumor effects of antibodies against ganglioside antigens of melanoma have been reported, but neither optimal doses nor mechanisms have been established. METHODS This Phase IB trial of the murine immunoglobulin IgG3 monoclonal antibody R24 against disialoganglioside GD3 was conducted with 37 patients to define better the dose-response relation and mechanism of action of R24 in patients with metastatic melanoma. RESULTS Dose-limiting toxicity consisted of a pulmonary capillary leak syndrome in 3 of 5 patients in the 80 mg/M2/day dosage tier. Serial blood and tumor biopsy samples were obtained prior to therapy and on Days 5, 9, and 22 following R24 infusion. Tumor biopsy-infiltrating lymphocytes were enumerated in peritumoral, endotumoral, and perivascular compartments: endotumoral CD4+ and CD8+ T cells and HLA-DR+ T cells increased over time on R24 antibody. Endotumoral CD4 lymphoid infiltrate activation (DR expression) and antibody-dependent cytotoxicity were the greatest in the one patient who achieved a complete response. CONCLUSIONS Clinical response was associated with depression in natural killer (CD56+ and CD56+DR+) blood cells (P = 0.03) and was associated with R24 dosage (P = 0.01). A complete response that lasted 2 years and a partial response that lasted 2 months occurred at a dose of 1 mg/M2/day. The limited number of clinical responses observed in this trial hampered the correlation of antitumor and immune parameters but provided a rational foundation for the future evaluation of antiganglioside antibodies. Cancer 2000;88:2693–702. © 2000 American Cancer Society.

Analysis of therapeutic and immunologic effects of R24 anti-GD3 monoclonal antibody in 37 patients with metastatic melanoma

Antitumor effects of antibodies against ganglioside antigens of melanoma have been reported, but neither optimal doses nor mechanisms have been established. This Phase IB trial of the murine immunoglobulin IgG(3) monoclonal antibody R(24) against disialoganglioside GD3 was conducted with 37 patients to define better the dose-response relation and mechanism of action of R(24) in patients with metastatic melanoma. Dose-limiting toxicity consisted of a pulmonary capillary leak syndrome in 3 of 5 patients in the 80 mg/M(2)/day dosage tier. Serial blood and tumor biopsy samples were obtained prior to therapy and on Days 5, 9, and 22 following R(24) infusion. Tumor biopsy-infiltrating lymphocytes were enumerated in peritumoral, endotumoral, and perivascular compartments: endotumoral CD4(+) and CD8(+) T cells and HLA-DR(+) T cells increased over time on R(24) antibody. Endotumoral CD4 lymphoid infiltrate activation (DR expression) and antibody-dependent cytotoxicity were the greatest in the one patient who achieved a complete response. Clinical response was associated with depression in natural killer (CD56(+) and CD56(+)DR(+)) blood cells (P = 0.03) and was associated with R(24) dosage (P = 0.01). A complete response that lasted 2 years and a partial response that lasted 2 months occurred at a dose of 1 mg/M(2)/day. The limited number of clinical responses observed in this trial hampered the correlation of antitumor and immune parameters but provided a rational foundation for the future evaluation of antiganglioside antibodies.

Glycolipid Sialyltransferases Are Enhanced during Neural Differentiation of Mouse Embryonic Carcinoma Cells, P19

We have studied ganglioside alterations and their enzymatic basis during the course of neural differentiation of mouse embryonic carcinoma cell line P19. This cell line can differentiate into neurons and astrocytes on cell aggregation after treatment with retinoic acid (RA) or into muscle cells on dimethyl sulfoxide (DMSO) treatment. GD3, detected on immunostaining after thin-layer chromatography (TLC) with monoclonal antibody (MAb) R24, was markedly present in aggregates treated with RA. GM3 synthase (α2,3-sialyltransferase, SAT-I) in neurons was found to exhibit the highest activity. GD3 synthase (α2,8-sialyltransferase, SAT-II) and GD3 synthase mRNA, as analyzed by Northern blotting, were also markedly present in aggregates and neurons induced by RA. However, on treatment with DMSO, which induces muscle cells, there was no change in the level of GD3 synthase activity, and its transcript was hardly detected during the course of muscle differentiation. GT1b synthase (α2,3-sialyltransferase, SAT-IV) was present at similar levels in undifferentiated cells and aggregates treated with RA, but a higher level was observed in neurons. On the other hand, the level of GQ1b synthase (α2,8-sialyltransferase, SAT-V) in RA-induced aggregates was significantly higher than that in neurons. These results show that RA but not DMSO induces the expression of GM3, GD3, GT1b and GQ1b synthases, and particularly GD3 synthase mRNA, in the ganglioside biosynthetic pathway during the neural differentiation of embryonic carcinoma P19 cells.

Detection of medulloblastoma and astrocytoma-associated ganglioside G D3 in cerebrospinal fluid

Shedding of gangliosides by tumor cells may enhance tumor development. We recently showed that cells of the human brain tumor, medulloblastoma, shed gangliosides in vitro and have therefore examined ganglioside shedding by pediatric brain tumors into the cerebrospinal fluid (CSF). G D3, a major ganglioside in medulloblastoma and astrocytoma, was the target for detection in the CSF by immunostaining using the monoclonal antibody R24 and enhanced chemiluminescence detection. Mean CSF G D3 levels in patients with medulloblastomas ( n=9) and astrocytomas ( n=10) were significantly higher than those of controls (mean±SD 44.7±8.4 versus 18.2±1.9 pmol/ml, n=20, P<0.0002). Mass spectrometric analysis showed that tumor-derived ganglioside G D3 contained heterogeneous ceramide structures and, interestingly, the ceramide subspecies with shorter fatty acyl chains were selectively shed. The elevated CSF G D3 concentrations in patients with medulloblastoma and astrocytoma support the concept that ganglioside shedding, which may have significant biological consequences, is characteristic of human brain tumors.

Ganglioside expression in lung cancer cell lines

Gangliosides are complex glycolipid constituents of cell membranes. They are involved in many biological functions including cell–cell recognition, cell–matrix attachment, cell growth and cell differentiation. Analysis of tumour associated gangliosides may aid in the characterisation of tumour cells and their degree of malignant transformation. We have characterised a total of eight lung cancer cell lines (four small cell and four non-small cell lung cancer) with respect to ganglioside and αv integrin receptor expression. Ganglioside GD3 was detected using the monoclonal antibody R24. Ganglioside GM1 was detected using the β-subunit of cholera toxin. Ganglioside 9-O-acetyl GD3 and the αv integrin receptor were measured using commercially available monoclonal antibodies. Our results indicate that small cell lung cancer cell lines express significant levels of GD3 and 9-O-acetyl GD3. Ganglioside GM1 and αv integrin receptor were not specific to any histological subtype. The expression of ganglioside GM1 and GD3 was independent of cell–cycle phase. We conclude that GD3 and 9-O-acetyl GD3 expression may be additional markers of the Small Cell Lung Cancer phenotype, but their significance is unknown. Therefore a characteristic ganglioside pattern cannot be defined according to histological subtype. αv integrin receptor expression is not unique to cells expressing GD3.

Human Melanoma Cell Lines Deficient in GD3 Ganglioside Expression Exhibit Altered Growth and Tumorigenic Characteristics

We have selected GD3-deficient human melanoma cell lines, in order to investigate the function of GD3 ganglioside. This was done by treating SK-MEL-28 cells with anti-GD3 antibody (R24) and rabbit complement and subsequent subcloning of the surviving cells, resulting in the derivation of two cell lines deficient in the cell surface expression of GD3. Neither cell line (designated SK-MEL-28-N1 and SK-MEL-28-N2) had detectable cell surface expression of GD3 as analyzed with monoclonal antibody R24, and no GD3 was detectable in either cell line by glycolipid isolation, thin-layer chromatography, or resorcinol-HC1 spray, but thin-layer chromatography immunostaining with monoclonal antibody R24 showed the presence of low amounts of GD3 in both N1 and N2 (1/40 of the amount in the parent cell line in N1 and 1/500 in N2). In SK-MEL-28-N1, the residual GD3 was shown by immunofluorescence assays on permeabilized cells to be present in discrete intracellular organelles, suggesting that these cells have a defect in the transport of GD3 as well as in its synthesis. Both SK-MEL-28-N1 and -N2 had an increase in detectable GM3 expression. The mutant cell lines had altered cell morphology in comparison to the parent cell line and both had slower growth rates in vitro and lower tumorgenicity in nu/nu mice. These results indicate that GD3 ganglioside plays an important role in proliferation and growth of melanoma cells.

T cell activation via the disialoganglioside GD3: analysis of signal transduction.

The monoclonal antibody (mAb) R24 is a murine immunoglobulin G3 (IgG3) that reacts with the GD3 disialoganglioside present on melanoma cells as well as a subset of T cells. R24 mAb has induced antitumor responses both alone and in combination with interleukin-2 (IL-2) in clinical trials. We have reported T cell activation via GD3 as measured by the induction of tyrosine phosphorylation. In this study a more detailed analysis of signal transduction after ligation of GD3 was performed in an attempt to understand the mechanism of in vivo therapeutic benefits observed. Analysis of subsequent events indicated that GD3 engagement resulted in phospholipase C(gamma) phosphorylation and calcium flux. When ras-associated events were examined, GD3 signaling resulted in ras activation as determined by GDP/GTP conversion as well as dose-and time-dependent IP3 activation. In addition, the majority of the IP3 activation by GD3 was inhibited by herbimycin A pretreatment. Elucidation of the nature and potential role of this moiety in GD3 signal transduction should be useful. Collectively, these data suggest a novel mechanism of T cell activation via a single, non-protein, surface moiety. This novel form of T cell-mediated activation may permit the delivery and local activation of effector cells at the tumor resulting in site-specific activation of the immune system.

Glycosphingolipid composition of murine neuroblastoma cells: O-acetylesterase gene downregulates the expression of O-acetylated GD3.

We have studied the glycosphingolipid composition in an F-11 neuroblastoma cell line originated from hybridization of a mouse neuroblastoma cell line (N18TG-2) with rat dorsal root ganglion cells. The total lipid-bound glucose of F-11 cells was estimated to be 0.28 micrograms/mg of protein and the total lipid-bound sialic acid was 0.82 micrograms/mg of protein. The major neutral glycosphingolipids were Gb4 (37% of the total neutral glycosphingolipids), Gb3 (15%), LacCer (21%), and GlcCer (15%). The major gangliosides were found to be GM3 (37% of the total gangliosides), GD3 (27%), O-acetylated GD3 (18%), and GD1a (4%), with trace amounts of GD2. The unusually high concentration of O-acetylated GD3 is consistent with its putative role as a tumor marker. Immunocytochemical localization studies of GD3 and O-acetylated GD3, examined by mouse monoclonal antibodies R24 and D1.1, respectively, revealed that the cell bodies and processes were all positively stained. To elucidate the role of O-acetylated GD3 in tumorigenesis, we transfected F-11 cells with the O-acetylesterase gene from influenza C virus. Compared with the original cell line, the transfected cells showed a dramatic increase in the level of GD3 (150% of that in the control cells) and a significant decrease of the concentration of O-acetylated GD3 (27% of control cells). In addition, the transfected F-11 cells exhibited a morphology different from the parental cells with enlarged cell bodies and elongated neurites. We conclude that alteration of ganglioside composition, particularly the expression of GD3 and O-acetylated GD3, may be associated with the morphological changes observed in this cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase I Trial of Cisplatin, WR-2721, and the Murine Monoclonal Antibody R24 in Patients with Metastatic Melanoma

The therapeutic and biologic effects of murine monoclonal antibodies in patients with malignancies have been widely investigated. Attempts to enhance results by combining these agents with cytotoxic drugs are now under study. A Phase I trial was performed to assess the toxicity and biologic effects of escalating doses of R24 (0-40 mg/m2/day 1-5, 8-12), an antibody that binds to the ganglioside GD3 present on melanoma cells, administered in combination with cisplatin (120 mg/m2) and WR-2721 (740 mg/m2) on day 1. Twenty-three patients with metastatic malignant melanoma were treated and are evaluable. The true maximum tolerated dose of R24 given as part of this combination was not reached. The toxicity of the regimen was moderate and included fever and urticaria, which were attributed to R24. Severe but reversible renal failure was noted in six patients in subsequent (two or more) treatment cycles, but when cisplatin was administered in 3% saline, this toxicity was not seen. Responses were seen in 2 of 19 patients receiving all three agents and in 1 of 4 patients receiving only cisplatin and WR-2721. No significant enhancement of natural killer, lymphokine-activated killer, and antibody-dependent cellular cytotoxicity lytic activity or significant changes from baseline in lymphocyte subsets secondary to R24 were seen. In 4 of 10 patients tumor localization of mouse monoclonal antibody was found and appeared greatest at higher R24 doses and during week 1 of therapy. Human anti-mouse antibody responses developed by day 22 in 17 of 19 patients treated with R24, and the coadministration of cisplatin did not appear to abrogate this response. Finally, the half-life and Cmax of cisplatin were not affected by R24. In summary, the combination was well tolerated, responses were few, and significant biologic interactions or immunomodulation were not observed.

Hemorrhagic tumor necrosis during a pilot trial of tumor necrosis factor-alpha and anti-GD3 ganglioside monoclonal antibody in patients with metastatic melanoma

Hemorrhagic tumor necrosis is an inflammatory event that leads to selective destruction of malignant tissues, with both potentially toxic and beneficial consequences. A pilot clinical trial was undertaken combining tumor necrosis factor-alpha (TNF-alpha) with the monoclonal antibody R24 (MoAb R24) against GD3 ganglioside in patients with metastatic melanoma. Patients received MoAb R24 to recruit leukocytes to the tumor followed by low doses of recombinant TNF-alpha to activate leukocytes. Eight patients were treated and seven patients had mild toxicity. One patient with extensive metastatic melanoma developed tumor lysis syndrome within hours after treatment with almost complete necrosis of bulky tumors in multiple visceral sites. To our knowledge, this is the first documented case of hemorrhagic tumor necrosis in a patient with metastatic cancer in multiple visceral sites.

Hemorrhagic Tumor Necrosis During a Pilot Trial of Tumor Necrosis Factor-α and Anti-GD3 Ganglioside Monoclonal Antibody in Patients With Metastatic Melanoma

Hemorrhagic tumor necrosis is an inflammatory event that leads to selective destruction of malignant tissues, with both potentially toxic and beneficial consequences. A pilot clinical trial was undertaken combining tumor necrosis factor-alpha (TNF-alpha) with the monoclonal antibody R24 (MoAb R24) against GD3 ganglioside in patients with metastatic melanoma. Patients received MoAb R24 to recruit leukocytes to the tumor followed by low doses of recombinant TNF-alpha to activate leukocytes. Eight patients were treated and seven patients had mild toxicity. One patient with extensive metastatic melanoma developed tumor lysis syndrome within hours after treatment with almost complete necrosis of bulky tumors in multiple visceral sites. To our knowledge, this is the first documented case of hemorrhagic tumor necrosis in a patient with metastatic cancer in multiple visceral sites.

Treatment with high dose mouse monoclonal (anti-GD3) antibody R24 in patients with metastatic melanoma.

R24 is a mouse IgG3 monoclonal antibody that reacts with the ganglioside GD3 expressed by melanoma cells and other cells of neuroectodermal origin (e.g. adrenal medulla). Antitumour activity of R24 was demonstrated in initial phase I and pilot trials, but treatment was limited by urticaria at cumulative doses of 400 mg/m2. A trial exploring intensification of the dose of R24 was conducted in eight patients. Planned doses of R24 antibody were 800 and 1200 mg/m2 over 6-8 days by continuous i.v. infusion. All patients received concomitant therapy with hydroxyzine hydrochloride and cimetidine to minimize urticaria. One patient developed anaphylaxis, after which no further therapy was given. All patients developed peripheral blood lymphopenia and marked decreases in serum complement values during treatment, suggesting depletion of two possible effector mechanisms of the antitumour effects of R24. A vascular leak syndrome, manifested by weight gain, oedema and hypotension, was evident in seven patients during the initial 24-36 h of treatment. Serum sickness syndrome was observed in six of seven evaluable patients between days 5 and 8, coincident with the onset of the human anti-globulin response to R24. One patient given 1200 mg/m2 had a minor response (38% reduction in pelvic nodes) lasting 12 months. There was no detectable increase (by immunohistochemical staining) in deposition of R24 within tumour sites at doses used in this trial compared to that observed at doses of 240 and 400 mg/m2. The maximum tolerated dose was 800 mg/m2. Dose-limiting toxicity was manifest as reversible hypertension with end-organ symptoms (chest pain or visual field defects) in patients treated with a dose of 1200 mg/m2.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of disialogangliosides GD2 and GD3 on human soft tissue sarcomas.

Fifty-six freshly frozen human sarcomas were studied for expression of disialogangliosides by avidin-biotin immunohistochemical staining with purified monoclonal antibodies (MoAb) 3F8 (antidisialoganglioside GD2) and R24 (antidisialoganglioside GD3). Ninety-three percent of the tumors tested by the immunohistochemical staining expressed GD2 and 88% expressed GD3. The intensity of expression varied among sarcomas of different histologic types. Liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, and spindle cell sarcoma reacted strongly with 3F8 and R24. Embryonal rhabdomyosarcoma and synovial sarcoma demonstrated substantially weaker staining by either MoAb. Weakly reactive or nonreactive tumors were, in general, high-grade or metastatic sarcomas. Ganglioside extraction and thin-layer chromatography/immunothin-layer chromatography of two liposarcomas confirmed the identities of these gangliosides. GD2 and GD3 may indicate sites for MoAb-targeted imaging and therapy for sarcomas. The association of a diminished stainability by 3F8 and R24 with aggressive sarcomas may indicate prognostic significance.

Anti-ganglioside antibodies reveal subsets of cultured rat dorsal root ganglion neurons

Subsets of cultured dorsal root ganglion neurons were identified by using the anti-ganglioside monoclonal antibodies A2B5, D1.1, R24 and JONES. A2B5 and D1.1 labelled a population of cells that was relatively stable between 2 and 20 days in vitro, while the population of cells labelled with both R24 and JONES decreased with time, suggesting that the gangliosides recognized by Jones and R24 are developmentally regulated. Given the observation that the relative proportions of ganglioside species changes with time in culture, it is very important to carefully define the stability of ganglioside antigens before using them as cell markers.

Tissue distribution of GD3 ganglioside detected by mouse monoclonal antibody R24.

Mouse monoclonal antibody (MoAb) R24 detects the disialoganglioside GD3, a neuroectodermal antigen expressed by normal melanocytes and melanomas. This antibody has been used to define the immunohistochemical distribution of GD3 antigen in normal and malignant human tissues. The present study shows that staining with MoAb R24 in normal organs and tissues where low concentrations of the antigen can be detected. The highest expression of GD3 was detected on the surface of melanoma cells. Therefore, MoAb R24 can be a useful reagent for studying cells of melanocytic lineage, and for imaging and therapeutic studies of melanomas.

Generation and partial characterization of melanoma sublines resistant to lymphokine activated killer (LAK) cells. Relevance to doxorubicin resistance.

To see whether a tumor cell population may contain cells resistant to lymphokine-activated killer (LAK) lymphocytes, cells from a LAK-sensitive melanoma line (Me 665/2) were co-cultured with LAKs. Three sublines were obtained after 1, 2 or 3 immunoselection cycles. Immunoselected (IS) sublines show reduced proliferation, decreased reactivity to the monoclonal antibody (MAb) R24 and appeared morphologically more differentiated in comparison with the parental Me 665/2 line. A progressively reduced sensitivity to LAKs was observed in IS sublines with a more than 8-fold reduction in LAK susceptibility. A reduced complement (C)-mediated lysis was also observed in IS sublines. Since we have previously shown that LAK sensitivity of melanoma cells may be associated with Doxorubicin (Dx) resistance, the sensitivity to Dx was tested in these lines. An augmented sensitivity to Dx was noted in IS sublines as compared with Me 665/2. The differences in LAK susceptibility between the IS sublines and the parental Me 665/2 line remained stable for 2 weeks but declined and disappeared thereafter. These results indicate that (1) a LAK-sensitive tumor line may contain a subpopulation of cells which are significantly less lysed by LAKs; (2) a correlation between LAK sensitivity and susceptibility to C-mediated lysis is also present; and (3) increased sensitivity to Dx is evident in the IS sublines.