A murine monoclonal antibody that recognizes an extracellular domain of the human c-erbB-2 protooncogene product

A murine IgM monoclonal antibody, designated SV2-61, was generated against human c-erbB-2 gene-transfected NIH-3T3 (SV11) cells. SV2-61 defined a 185-kDa molecule present on the surface of SV11 cells, another line of c-erbB-2 gene-transfected NIH-3T3 (A4-15) cells, and MKN-7 human gastric cancer cell line carrying an amplified human c-erbB-2 gene. The SV2-61-defined antigen was found to show protein kinase activity in vitro. The SV2-61 was reactive with human c-erbB-2 gene-transfected NIH-3T3 cell lines but not with transfectants carrying c-erbB-2 gene mutants which lack a coding region for the extracellular domain. It was reactive with a portion of human epithelial cell lines but not with native NIH-3T3, TGF-alpha-coding gene-, activated c-raf gene- or Ha-ras gene-transfected NIH-3T3 cells, or non-epithelial human cells. These results indicate that the SV2-61 is an antibody which recognizes an extracellular domain of the c-erbB-2 gene product, 185-kDa protein.     

Therapy of a murine sarcoma using syngeneic monoclonal antibody

Syngeneic monoclonal antibodies (MoAb) to Moloney sarcoma cells (MSC) were produced by fusion of spleen cells from MSC regressor mice to myeloma SP2/0. MoAb 244-19A, an immunoglobulin G2b, bound to MSC cells and did not bind to two other sarcomas (K-BALB and Ha2), a carcinoma (Line 1), a fibroblast (A31) or a fibroblast infected with C-type virus (A31-Moloney leukemia virus). In contrast, MoAb 271-1A bound to the MSC and Ha2 sarcoma and line 1 carcinoma as well as to the normal and infected fibroblast cultures. Antibodies were tested for therapeutic effect using three schedules of antibody injection. Injection i.p. of ascites fluid containing 244-19A MoAb given on Days-1, 0, and +1 relative to tumor cell injection increased life span significantly over that of control animals given injections (P3, immunoglobulin G, or MoAb 271-1A) and produced some seven of 19, one of five, and one of five long-term survivors in three separate experiments. Antibody given to animals with established tumors (4 days after implantation) also prolonged life span significantly and produced three of nine long-term survivors. Antibody given to animals with very large tumor burdens (10 days after implantation) did not prolong life span significantly. Optimal dose, schedule, and mechanism studies concerning this therapy are in progress.     

Characterization and reactivity of a monoclonal antibody that recognizes a 76 kilodalton human colon tumor antigen

A monoclonal antibody (MAb 76) was produced by immunizing mice with a soluble cytoplasmic protein fraction from a human adenocarcinoma of the colon. MAb 76 showed specific immunoreactivity against a 76 kDa protein in immunoblot studies using total colon tumor cytosol proteins. Immunoprecipitation of phosphorylated cytosolic protein products with MAb 76 and subsequent analysis on SDS containing polyacrylamide gels revealed a single 38 kDa band, indicating that the 76 kDa antigen is associated with a 38 kDa phosphoprotein species. Indirect immunofluorescence analysis of primary tumor specimens and human colon tumor cell lines showed positive immunoreactivity with 6/7 human colon adenocarcinoma tissues and 15/18 human colon tumor cell lines. MAb 76 was unreactive with normal colon, liver and lung specimens from human, mouse and hamster. The epitope-bearing monomer detected by MAb 76 is immunologically conserved in a high percentage of colon tumor cells and tissues and may represent a cellular product that is characteristic of the transformed colon cell phenotype.     

Radioimmunotherapy of human synovial sarcoma using a monoclonal antibody against FZD10

We previously reported Frizzled homolog 10 ( FZD10 ), a member of the Frizzled family, to be a promising therapeutic target for synovial sarcomas. In this report, we established a murine monoclonal antibody (MAb), namely, MAb 92-13 that had specific binding activity against native FZD10 product expressed in synovial sarcoma cell lines. Subsequent immunohistochemical analyses with the MAb 92-13 confirmed an absence or hardly detectable level of FZD10 protein in any normal human organs. We confirmed the specific binding activity of this MAb in vivo after injection of fluorescent-labeled MAb i.p. or i.v. into the mice carrying synovial sarcoma xenografts by the use of the in vivo fluorescent imaging system as well as radioisotopes. Moreover, MAb 92-13 was effectively internalized into the synovial sarcoma cells after its binding to FZD10 on the cell surface. A single i.v. injection of the Yttrium-90 (⁹⁰Y)-MAb 92-13 drastically suppressed tumor growth of synovial sarcoma in mice without any severe toxicity. Median time to tumor progression was 58 days for mice treated with ⁹⁰Y-MAb 92-13 and 9 days for mice treated with non-labeled antibody control or untreated mice (difference = 49 days; P  = 7 × 10⁻⁵). This result indicates that MAb 92-13 could be utilized as the novel treatment modality for synovial sarcoma and other FZD10-positive tumors. ( Cancer Sci 2008; 99: 432–440)     

Possible differential diagnosis of neuroblastoma from rhabdomyosarcoma and Ewing's sarcoma by using a panel of monoclonal antibodies

The accurate diagnosis of malignant tumor type is essential to enable the correct therapeutic regimen to be followed and to predict a patient's prognosis. However, the differential diagnosis of "small-round-cell" tumors, represented by neuroblastoma, rhabdomyosarcoma, lymphoma/leukemia and Ewing's sarcoma, can occasionally be difficult by conventional morphological and biochemical methods. If tumor membrane markers were available, these could provide rapid and accurate diagnostic aids. In the present work, a panel of 9 monoclonal antibodies raised against hematopoietic cells (BA-1, BA-2, J-5 and B7/21), brain cells (UJ-13A, UJ-127-11 and anti-Thy-1), and neuroblastoma cells (HSAN1.2 and PI153/3) was used to analyze the membrane phenotypes of 12 neuroblastoma, 4 rhabdomyosarcoma and 3 Ewing's sarcoma cell lines and cells of 3 fresh bone marrow tumors. BA-1, UJ-127-11 and PI153/3 antibodies may be useful for the differential diagnosis of neuroblastoma from rhabdomyosarcoma and Ewing's sarcoma.     

Radiotherapy as a component of treatment for Ewing's sarcoma

The main stages of development of such conservative modalities as radio- and chemoradiotherapy for localized Ewing's sarcoma are discussed. Factors affecting five-year overall and recurrence-free survival of patients treated with chemoradiotherapy are presented.     

Inhibition of growth of mouse leukemic cell lines in vitro and in vivo by a monoclonal antibody that recognizes an interleukin 3 receptor-associated protein

Myeloid cell lines that have achieved leukemic transformation may also have acquired the ability to produce hematopoietic growth factors. In certain instances, neutralizing antibodies directed against the growth factor have inhibited growth, supporting an autocrine mechanism in their transformation. The effect of anti-receptor antibodies on their growth and transformed phenotype has not been reported. We have developed a monoclonal antibody, 4G8, directed against a protein that is associated with the IL-3 receptor complex; 4G8 inhibits IL-3 binding and signal transduction in nonleukemic IL-3-dependent cell lines. In this study, we examined the effect of 4G8 on the growth in vitro and in vivo of leukemic cell lines, including WEHI-3B, which spontaneously produces IL-3, and NFS-60, an IL-3-dependent cell line. Our results demonstrate that the 4G8 antigen can be detected in both WEHI-3B and NFS-60 cells by flow cytometry and Western blotting; 4G8 inhibits the autonomous growth of WEHI-3B and the IL-3-dependent growth of both WEHI-3B and NFS-60. In addition, s.c. administration of 4G8 prolonged the survival of syngeneic mice given s.c. injections of WEHI-3B. These results support the conclusion that an autocrine mechanism involving IL-3 and its receptor plays a critical role in the growth and transformed phenotype of WEHI-3B and raises the possibility that anti-IL-3 receptor antibodies may be useful in the treatment of some leukemias.     

A new monoclonal antibody that specifically recognises the MDR‐3‐encoded gene product

The MDR‐3‐encoded P‐glycoprotein (Pgp) is highly expressed in liver and is thought to function as a hepatic transporter of phospholipids into bile. However its role, if any, in other tissues remains undefined. Although transfection experiments have indicated that it may be unable to confer drug resistance, there is evidence that it may be involved in drug resistance in certain B‐cell leukaemias. To date, most work on clinical samples has been performed at the mRNA level; limited work has been performed using polyclonal antibodies raised to MDR‐3 and mdr‐2 (the murine equivalent of MDR‐3). We have generated a new monoclonal antibody, termed 6/1G, which specifically recognises the human MDR‐3 gene‐encoded product. Antibody 6/1G was produced by in vitro immunisation of spleen cells from BALB/c mice with a synthetic 12‐amino acid peptide. Cells from MDR‐3 transgenic mice showed consistent membranous staining with antibody 6/1G. Immunoblotting with 6/1G identifed a band at 170 kDa on lysates of MDR‐3 transgenic cells. Preliminary results with a range of B‐cell leukaemias suggest that MDR‐3 Pgp positivity may be a marker for a more malignant phenotype in B‐CLL. Antibody 6/1G may be useful in defining a role for MDR‐3 in malignancy and drug resistance, as well as in certain liver diseases such as progressive familial intracholeostasis. Int. J. Cancer 80:265–271, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody

We have isolated a monoclonal antibody, designated HMB-50, that is highly specific for melanomas and melanocyte derived lesions. The antibody recognizes melanomas, neonatal melanocytes, and junctional nevi but does not react with adult melanocytes, dermal nevi, or a variety of non-melanocyte derived neoplasms. In tissue culture, the antibody reacts with five of seven human melanoma lines and neonatal foreskin melanocytes but fails to recognize fibroblasts and a number of different carcinomas. HMB-50 identifies a Mr 95,000 glycoprotein that is released into the growth medium by melanoma cells and neonatal melanocytes in vitro. This molecule is unrelated to antigens recognized by a variety of antimelanoma monoclonal antibodies isolated in other laboratories. The Mr 95,000 glycoprotein has been purified by antibody affinity chromatography and a polyclonal rabbit antiserum raised that exhibits identical specificity to the monoclonal antibody. The Mr 95,000 glycoprotein is rapidly released by melanoma cells (within 60 min) and one line produces relatively large quantities of the molecule (1 microgram/10(6) cells/24 h). The molecule in normal melanocytes differs slightly in electrophoretic mobility compared to its counterpart in melanomas and this difference appears to result from posttranslational modification.     

Preparation and characterization of a specific monoclonal antibody against a new gene product: URG11

As a new gene, the monoclonal antibody against URG11 protein is not currently available. In this study, one monoclonal antibody (MAb) against URG11 was obtained with standard cell fusion technique and enzymelinked immunosorbent assay (ELISA) screening. The peptide of URG11 used in making the MAb in this study was synthesized as described. One of the newly developed MAbs is named MAb 3D2, the isotype of which is IgG2a. Immunohistochemistry and Western blot showed that MAb 3D2 could recognize URG11 protein in both native and denatured forms. MAb 3D2 will be a useful tool for the functional research of URG11 in future studies.     

Novel monoclonal antibody specific for the de2-7 epidermal growth factor receptor (EGFR) that also recognizes the EGFR expressed in cells containing amplification of the EGFR gene

In some respects, the EGFR appears to be an attractive target for tumor-targeted antibody therapy: it is overexpressed in many types of epithelial tumor and inhibition of signaling often induces an anti-tumor effect. The use of EGFR specific antibodies, however, may be limited by uptake in organs that have high endogenous levels of the wild type EGFR such as the liver. The de2-7 EGFR (or EGFRvIII) is a naturally occurring extracellular truncation of the EGFR found in a number of tumor types including glioma, breast, lung and prostate. Antibodies directed to this tumor specific variant of the EGFR provide an alternative targeting strategy, although the lower proportion of tumors that express the de2-7 EGFR restricts this approach. We describe a novel monoclonal antibody (MAb 806) that potentially overcomes the difficulties associated with targeting the EGFR expressed on the surface of tumor cells. MAb 806 bound to de2-7 EGFR transfected U87MG glioma cells (U87MG.Delta 2-7) with high affinity (approximately 1 x 10(9) M(-1)), but did not bind parental cells that express the wild type EGFR. Consistent with this observation, MAb 806 was unable to bind a soluble version of the wild type EGFR containing the extracellular domain. In contrast, immobilization of this extracellular domain to ELISA plates induced saturating and dose response binding of MAb 806, suggesting that MAb 806 can bind the wild type EGFR under certain conditions. MAb 806 also bound to the surface of A431 cells, which due to an amplification of the EGFR gene express large amounts of the EGFR. Interestingly, MAb 806 only recognized 10% of the total EGFR molecules expressed by A431 cells and the binding affinity was lower than that determined for the de2-7 EGFR. MAb 806 specifically targeted U87MG.Delta 2-7 and A431 xenografts grown in nude mice with peak levels in U87MG.Delta 2-7 xenografts detected 8 h after injection. No specific targeting of parental U87MG xenografts was observed. Following binding to U87MG.Delta 2-7 cells, MAb 806 was rapidly internalized by macropinocytosis and subsequently transported to lysosomes, a process that probably contributes to the early targeting peak observed in the xenografts. Thus, MAb 806 can be used to target tumor cells containing amplification of the EGFR gene or de2-7 EGFR but does not bind to the wild type EGFR when expressed on the cell surface.     

Ewing's sarcoma of a lower extremity in an infant. A therapeutic dilemma

A 5.5-month-old infant with Ewing's sarcoma of the left femur is described. The clinical and the pathologic features in this infant are presented in detail, and the dilemma faced in diagnosis and therapy of Ewing's sarcoma in infants is discussed. It is suggested that Ewing's sarcoma in an infant with a lower extremity lesion may be adequately managed without primary amputation.     

Characterization of an anti-MUC1 monoclonal antibody with potential as a cancer vaccine

The monoclonal antibody (MAb) AR20.5 is a murine MAb, generated against the tandem repeat protein backbone of the tumor-associated antigen MUC1. MAb AR20.5 reacts strongly with either the soluble form or the cell surface epitope of MUC1 on many human cancer cell lines. It also reacts with a 23-amino acid MUC1 peptide, E23, which includes the core tandem repeat sequence. Epitope mapping confirmed that MAb AR20.5 recognizes a minimum of six residues with the sequence DTRPAP. Inhibition of glycosylation of MUC1 resulted in decreased binding of MAb AR20.5 to cell surface MUC1, suggesting that MAb AR20.5 binding is carbohydrate dependent. The antibody was studied in a human PBL-SCID/beige mouse model to evaluate its effect on progression of NIH:OVARCAR-3 tumors. Tumor reduction was observed in mice injected with MAb AR20.5, but not in mice treated with control murine antibody or PBS (p < 0.001 and p < 0.05, respectively). An anti-tumor effect could also be demonstrated in a CB6F1 mouse model with the MUC1 transfectoma 413BCR.     

Identification of the metastasis-associated, galactoside-binding lectin as a chimeric gene product with homology to an IgE-binding protein

We report the complete primary and secondary structures of a metastasis-associated Mr 34,000 galactoside-binding lectin. The polypeptide sequence (264 amino acids) was derived from the nucleotide sequence of three overlapping complementary DNA clones isolated from lambda gt11 and lambda gt10 phage libraries of UV-induced murine fibrosarcomas. Striking features of the polypeptide sequence are two distinct regions of beta-sheet and globular structures at the amino and carboxy terminals, respectively. Homology search suggests that the polypeptide is a chimeric gene product formed by fusion of the 5'-end of an Mr approximately 14,000 galactoside-binding lectin with an internal domain of the collagen alpha gene. Enzymatic treatment with collagenase confirmed the presence of a collagen-like structure in the polypeptide. Unexpectedly, the entire sequence is greater than 85% homologous to a rat low affinity IgE-binding protein.     

A unique monoclonal antibody against human plasma fibronectin, which recognizes an epitope on the surface of a subpopulation of polymorphonuclear neutrophils

A new monoclonal antibody (MAb) specific to plasma fibronectin, named MO, which was produced by immunizing a mouse with fragments of fibronectin isolated from human plasma, was found to bind to the surface of a subpopulation of peripheral blood polymorphonuclear neutrophils (PMNs) in the presence of physiological concentration of soluble fibronectin. MAb MO had no reactivity with other blood cells such as lymphocytes, monocytes, or platelets. The percentage of the MAb MO-reactive subpopulation of PMNs from healthy donors varied from 0 to 60%. Ten previously established MAbs against fibronectin tested in this study together with MAb MO had no reactivity with the surface of PMNs at all, when tested simultaneously with MAb MO. These results indicate that MAb MO can be a unique and useful tool for analyzing the structure and function of fibronectin-especially the fibronectin expressed on the surface of PMNs.     

A radioimmunoassay for the detection of a human tumor-associated glycoprotein (TAG-72) using monoclonal antibody B72.3

Monoclonal antibody (MAb) B72.3 was generated against a carcinoma metastasis and has been shown to bind with a high degree of selectivity to a tumor-associated glycoprotein (TAG-72) found in human colon and breast carcinomas, while showing minimal reactivity to any normal adult tissues. Competition radioimmunoassays have been developed for the detection of TAG-72 in tumors and sera from both athymic mice bearing human carcinoma xenografts and patients with colon, breast and other carcinomas. The distribution of TAG-72 in human tumor xenografts was restricted to tumors originating from the LS-174T human colon carcinoma, with no significant reactivity being detected in human tumor xenografts from a different colon carcinoma, a human breast carcinoma, or a human melanoma. The levels of TAG-72 in clinical material obtained from surgery were examined; high levels were found in colon carcinomas and to a lesser extent in breast carcinomas, while no detectable levels were found in normal tissues. Sera from apparently normal patients contained a mean level of 2.2 units per ml of TAG-72. A level of 3 standard deviations above the mean level of TAG-72 found in normals was employed as a cut-off value to indicate a positive test result in subsequent studies. No patient with inflammatory disease or other benign colon disease exhibited elevated levels of TAG-72. Seven out of 20 patients with other carcinomas had elevated serum levels of TAG-72. The serum TAG-72 levels were compared with the serum levels of antigens recognized by the MAbs currently used to screen sera of carcinoma patients (CEA, GICA, and OC125). It was clearly demonstrated that TAG-72 is different from these antigens and can be found in some sera in which no antigen is detected by otherwise available MAb RIAs.     

Identification of an H antigen-like blood group antigen in sera of cancer patients using a novel monoclonal antibody raised against endometrial carcinoma

An IgM class monoclonal antibody (MAb) was derived by immunizing BALB/c mice with a human endometrial carcinoma cell line. This MAb, termed C12, exhibited strong reactivity against endometrial carcinoma, but lesser reactivity against normal endometria. The antigen recognized by MAb C12 (C12 antigen) was detected by radiometric assay in sera from patients with various carcinomas, but not in sera from patients without carcinomas or in sera from normal individuals. MAb C12 was found to agglutinate blood type O erythrocytes, but not A, B, or AB erythrocytes. To clarify the specificity of MAb C12, tissue staining experiments were performed in parallel using MAb C12, Ulex europaeus lectin I (anti-H), and a monoclonal anti-H antibody. In endometrial carcinoma tissues, both H and C12 antigens increased, but the C12 antigen showed a prominent increase, in contrast to the H antigen. Further, the C12 antigen was not found in endothelial cells of blood type O patients. In sera, the level of the H antigen varied according to the host's blood type. The sera from blood type O individuals possessed higher levels of the H antigen than those with blood type A or B. Thus, the H antigen showed no value as a tumor-associated serum marker. In contrast, the presence of the C12 antigen in sera was not determined by ABO blood group status. Thus, MAb C12 was demonstrated to be a unique MAb that reacts with an H-like antigen occurring in the sera of patients with carcinomas irrespective of ABO blood group status. MAb C12 may prove to be a useful marker for cancer patient serum.