Monoclonal mouse antibodies raised against human lung carcinoma

We have evaluated approximately 10,000 monoclonal antibodies (MoAb) resulting from 25 hybridizations of spleen cells from mice immunized with cells from human non-small cell lung carcinoma or fetal lung. The spleen cells were hybridized with NS-1 myeloma cells, and the resulting hybridomas were screened for production of MoAb to non-small cell lung carcinoma by binding assays with either cell extracts or cells growing in culture, followed by immunohistology on frozen sections. Fourteen MoAb had relative specificity for non-small cell lung carcinoma versus normal tissues. Three of these MoAb (L3, L6, L17) also reacted with most carcinomas of the breast and colon, and two MoAb (L20 and L22) reacted with the four samples of small cell lung carcinoma tested. No MoAb defined an antigen of absolute tumor specificity, and no MoAb reacted substantially more with adenocarcinoma than squamous cell carcinoma of the lung (or vice versa). Five MoAb were Ig G1, two were Ig G2a, and the remaining seven were Ig M. Seven MoAb (L5, L6, L15, L17, L20, L22, L23) could bind to the cell surface. Three MoAb (L6, L15, L17) defined carbohydrate antigens, and three (L3, L5, L20) were to protein antigens, while the antigens to which the remaining MoAb are directed have not been identified. Six MoAb could bind to tumor cells in Carnoy-fixed paraffin-embedded sections. An intercellular variability in antigen expression was detected with all 14 MoAb. At least two of the MoAb, L6 and L20, are good candidates for preclinical testing in view of their high level of tumor selectivity, as shown by both immunohistology and binding assays with living cells.     

Identification of transformation-related antigen by monoclonal antibody on Swiss 3T3 cells induced by transfection with murine cultured colon 36 tumor DNA

The cell surface antigen associated with the transformed state of cells that could grow in an anchorage-independent manner was analyzed by use of techniques of DNA transfection and hybridomas secreting the monoclonal antibody (MoAb). Spleen cells of C57BL/6 mice immunized with a highly tumorigenic, chemically induced murine cultured colon 36 tumor (C-C36) of BALB/c origin were hybridized with NS-1, a hypoxanthine phosphoribosyltransferase-deficient myeloma line of BALB/c mice. Screening of hybridomas revealed an antibody that reacted with C-C36 and transformed Swiss 3T3 cells growing in soft agar after transfection of 3T3 cells with C-C36 DNA. The hybridomas that did not react with nontransformed 3T3 and the less tumorigenic BALB/c hemangioendothelioma line D10 were then selected. An MoAb was designated "#71295." This MoAb immunoprecipitated the antigen that consisted of 65,000- and 14,000-molecular-weight components with soluble C-C36 membrane antigens. It also reacted with 2 other chemically induced syngeneic colon tumor lines, cultured colon 26 tumor line and cultured colon 51 tumor line, and with fibrosarcoma Meth A. However, #71295 was not found in NS-1, D14, and BALB/c normal thymus, liver, colon, and kidney tissues. In addition, this MoAb could not inhibit the anchorage-independent growth of C-C36 and transformed 3T3 cells. These results suggest that although the molecule defined by #71295 might not be associated with the anchorage independence of cell growth, it could be a newly expressed determinant on the cell surface that is related to the events of cell transformation.     

Tissue distribution, molecular profile, and shedding of a cytoplasmic antigen identified by the monoclonal antibody 465.12S to human melanoma cells

The mouse immunoglobulin G2 monoclonal antibody (MoAb) 465.12S reacts with a cytoplasmic antigen present in human melanoma cells but not detectable in melanocytes. Indirect immunofluorescent staining of a large number of surgically removed normal adult and fetal tissues with the MoAb 465.12S detected the cytoplasmic antigen in epithelial cells from several organs. The intensity of staining was greater in adult tissues than in the corresponding fetal tissues. Furthermore, the MoAb 465.12S stained nearly all of the surgically removed tumors tested but did not stain many of the normal tissues from which they originated. In almost all cases, the intensity and frequency of staining wa greater for tumor cells than for corresponding normal tissues. From cultured carcinoma and melanoma cells, the MoAb 465.12S immunoprecipitated four glycopolypeptides with molecular weights of 94,000, 75,000, 70,000, and 25,000. Incorporation of 3H-labeled sugars into the various components of the cytoplasmic antigen revealed that the M.W. 75,000 component was unusual in that it contained only glucosamine and mannose. The antigenic determinant defined by the MoAb 465.12S appears to be protein rather than carbohydrate in nature since it is heat sensitive and is expressed on the antigens synthesized by cells in presence of tunicamycin. Analysis of the spent culture medium of carcinoma and melanoma cell lines revealed that the cytoplasmic antigen is readily shed by these cells and consists of a major M.W. 94,000 and a minor M.W. 72,000 component. Treatment of cultured melanoma cells with the antibiotic tunicamycin showed that glycosylation of the cytoplasmic antigen is required for its shedding and/or stability in the spent culture medium.     

Monoclonal antibody defining fibroblasts appearing in fetal and neoplastic tissues

VIF3 is a hybridoma-derived mouse IgG monoclonal antibody (MoAb) generated in a fusion with the use of a malignant fibrous histiocytoma as the immunizing agent and shown to recognize a 70-kilodalton antigen expressed within connective tissues. Of 55 human tissue culture cell lines tested by indirect immunofluorescence, VIF3 was shown to bind to 20 of 35 (57%) sarcomas, 4 of 9 (44%) normal fibroblasts, and none of 11 carcinomas and other neoplasm-derived cell lines. A panel of over 259 human frozen tissue sections obtained from surgical pathology specimens, postmortem studies, and elective abortions was used to further determine the histopathologic specificity of VIF3. MoAb VIF3 was found to bind to 15 of 19 (79%) human sarcoma tissue sections tested. It was also shown to recognize an antigen expressed on a subset of fibroblasts dispersed within the stroma of carcinomas obtained from all 46 patients tested, as well as a subset of cells within 3 of 10 benign hyperplastic tissues (30%). VIF3-positive cells were detected in all 60 fetal tissues tested including amniotic membranes, placentas, and umbilical cords. In contrast, fibroblasts of normal adult tissues tested stained infrequently (22/97 or 23%) with this reagent. The results confirm that this MoAb is directed against a human connective tissue-specific marker. VIF3 detects a marker appearing on fetal fibroblasts that is typically not present in normal adult tissues, but reappears in association with neoplastic diseases. MoAb VIF3 therefore defines a fibroblast-oncofetal antigen and as such may serve as a probe for immunopathologic studies.     

Differential extraction of tumor-specific transplantation antigen and embryonic antigen from simian virus 40- and adenovirus 7-induced sarcoma cells of hamsters with 1-butanol and 3 M potassium chloride

Simian virus 40 (SV40)-induced sarcomas and adenovirus 7-induced sarcomas (Adv-7) exhibit both specific tumor-specific transplantation antigens (TSTA) and cross-protective embryonic antigens at the cell surface in the LAK:LVG(SYR) strain of Syrian golden hamsters. Specific SV40 TSTA could be released from the surfaces of living hamster sarcoma cells in a 2.5% crude 1-butanol extract (CBE) and served as immunogen to protect syngeneic recipients against subsequent homologous but not heterologous tumor cell challenge. The CBE-extracted SV40-induced TSTA (tumor-specific) was observed to be free of detectable, cross-protective embryonic antigens (EA) by tumor transplantation assays. The induction of cytotoxic lymphocyte-mediated immunity with the CBE-released TSTA was dependent on the administration of a single sensitizing injection of 12-20 micrograms antigen protein. Higher concentrations (50-1,000 micrograms) of the CBE tumor cell extract, given in a single injection, enhanced tumor growth as did two injections of 12.5 micrograms CBE-extracted SV40-induced TSTA at 1-week intervals. A cross-protective antigen(s), not detected in the CBE tumor extracts, was retained in the intact, 1-butanol-extracted SV40 and Adv-7-induced tumor cell lines after completion of the CBE extraction procedure and in similarly extracted 10-day hamster fetal cells. Some alterations in the normal immunogenicity of EA extracted with CBE followed by KCI from SV40-induced sarcoma cells could be detected in the transplantation assays and lymphocyte transformation assays, whereas EA extracted from CBE-KCI-treated Adv-7 cells or 10-day hamster fetal cells retained normal immunogenicity in vivo and in vitro. These procedures provide a means for successful separation of immunogenic SV40- and Adv-7-induced TSTA from detectable, biologically active, cross-protective EA from the surfaces of these sarcoma cells.     

Three novel mouse monoclonal antibodies, OM-A, OM-B, and OM-C, reactive with mucinous type ovarian tumors

Three mouse IgG1 monoclonal antibodies (MoAbs) reactive predominantly with cytoplasmic antigens of mucinous type ovarian tumors were produced. OM-A MoAb was reactive with 11 of 14 mucinous type and two of three endometrioid type ovarian tumors, although only a minor population of the tumor cells was positive in the latter type. This MoAb was not reactive with serous type, clear cell type, or other types of ovarian tumors, nor with various types of uterine carcinoma. Normal adult and fetal tissues of female genital organs were not positive with this MoAb. Among nongynecological carcinomas, three of six metastatic tumors to the ovary from the gastrointestinal tract, one of five gastric carcinomas, and one of eight lung adenocarcinomas were positive. As for normal adult and fetal tissues of nongynecological origin, epithelium of the normal stomach, small bowel, and bronchus as well as epithelium of fetal small and large bowel and secretory products were weakly positive. Thus, this MoAb showed a selected specificity against mucinous and endometrioid types of ovarian tumors. OM-B MoAb showed a broader specificity than OM-A, reacting with all mucinous type, two of three endometrioid type, and three of 16 serous type ovarian tumors, but not with clear cell type tumors. Adenoma type, but not squamous type, cervical carcinomas and one-half of endometrial carcinomas were positive. This antigen is present in cervical mucosa, but not in ovary or endometrium. OM-C MoAb showed a specificity similar to, but broader than that of, OM-B; i.e., 11 of 14 mucinous type, two of three endometrioid type, nine of 16 serous type, and one of nine clear cell type ovarian tumors were positive. It is reactive with adenoma type uterine carcinoma and normal mucosa of the uterine cervix and with normal surface epithelium of the oviduct. Among nongynecological tumors, OM-B antigen was present in metastatic tumors to the ovary as well as in gastric and pancreatic carcinomas, while OM-C was in metastatic tumors to the ovary and gastric and colonic carcinomas. Thus, the serological analysis showed that these three MoAbs showed selective specificities to mucinous and endometrioid types of ovarian tumors. Preliminary characterization of these three OM antigens suggested that these are distinct from carcinoembryonic antigen or ABH blood group-related antigens.     

Mouse monoclonal antibodies detecting disialogangliosides on mouse and human T lymphomas

Six monoclonal antibodies (MAbs) (4 IgG3 and 2 IgM) were produced by hybridomas obtained from A/J mice immunized with EL4(C57BL/6 derived-T lymphoma). They were found to react with antigens expressed on both mouse and human T-lymphomas but not on B lymphomas or normal cells. All of these antibodies reacted with the disialoganglioside GD2, GalNAc beta I----4(NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta I----4Glc-Cer, by 3 different assay systems including the immune adherence inhibition test, enzyme-linked immunosorbent assay, and enzyme immunostaining on thin-layer chromatography. The binding specificities of these MAbs to disialogangliosides differed. Four MAbs (AI-201, AI-287, AI-410, and AI-425) showed restricted specificities, detecting only GD2, whereas the other 2 (AI-245 and AI-267) had a broader specificity, recognizing GD2, GD3, and GDlb. No evidence was obtained for the presence of the antigenic epitope in glycoproteins of mouse and human tumor cells. The ganglioside content of EL4 was low in comparison with that of M14 (a human melanoma cell line).     

Determination of cell surface membrane antigens common to both human neuroblastoma and leukemia-lymphoma cell lines by a panel of 38 monoclonal antibodies

The surface membrane antigens of 7 neuroblastoma and 91 leukemia-lymphoma cell lines were studied with the use of a total of 36 murine monoclonal antibodies (MoAb) primarily developed against hematopoietic cells and 2 MoAb developed against human fetal brain. Five of the MoAb against hematopoietic cells (BA-1, BA-2, DU-ALL-1, J-5, and BA-3) consistently bound to common acute lymphoblastic leukemia cell lines, and 2 others (MCS-2 and OKM-1) reacted uniformly with acute myeloblastic-acute monoblastic leukemia cell lines. However, these 7 MoAb also reacted with 1-7 neuroblastoma cell lines. All the human neuroblastoma cell lines bound MoAb BA-2 and DU-ALL-1. Six of the 7 lines reacted with BA-1. Only 1 neuroblastoma cell line (SJ-N-CG) gave positive staining with J-5 and BA-3, and another line (SK-N-AS) bound MoAb MCS-2 and OKM-1. Anti-fetal brain MoAb (UJ-13A and UJ-127-11) were highly positive for all the neuroblastoma cell lines. By contrast, 4 of 43 leukemia-lymphoma cell lines tested bound these anti-fetal brain antibodies. Both B3/25 and OKT-9, anti-transferrin receptor antibodies, reacted with all of the hematopoietic and neuroblastoma cell lines. These results demonstrate that neuroblastoma and hematopoietic cell lines possess common antigenic determinants despite their different embryologic origins. The neuroblastoma cell lines may be classified into subgroups on the basis of phenotype profiles determined by the MoAb. MoAb may be useful in characterization and classification of neuroblastoma cells, as has already proved to be the case for cells of the hematopoietic lineages.     

Growth and antigenic properties of a biopsy-derived Burkitt's lymphoma in thymus-less (nude) mice

A Burkitt's lymphoma was transferred to the congenitally athymic mouse mutant nude with biopsy material from a 7-year-old Kenyan girl. The tumor grows locally at the site of inoculation with no distant metastases. The established tumor has been maintained for six passages so far with preservation of histological and cytological appearance. The mouse-passaged tumor has a normal human diploid female chromosome complement. Isozyme studies have shown tumor to be of the same glucose-6-phosphatedehydrogenase (G-6-PD) and phosphoglucomutase₁ phenotype (B and 2-1 respectively) as tumor biopsy from the patient. The mouse-passaged line maintained surface IgM, similarly to the original tumor and the derived tissue culture line, but lost the IgG coating characteristic for the original tumor but absent from two subsequent biopsies and from the derived tissue culture line. This is in line with previous observations indicating that surface-associated IgG on Burkitt biopsies is due to coating from the outside, whereas surface-associated IgM is a cell marker. Whereas the biopsy cells of the patient were positive for EBV-associated membrane antigen (MA), but not for early antigen (EA) and viral capsid antigen (VCA), the mouse-passaged line was positive for all three. This suggests that the restrictive influence of the human host on the production of EA and VCA in the Burkitt tumor is raised in the mouse host. The serum of the tumor-bearing nude mice contained anti-human antibodies, but no detectable EBV (anti-MA, EA and VCA)-antibodies.     

Differential inducibility of Epstein-Barr virus in cloned, non-producer Raji cells.

Cells of the human lymphoblastoid non-producer line Raji were cloned in soft agar. Individual colonies were isolated and analyzed for their inducibility of the Epstein-Barr virus-associated early antigen (EA). The induction of EA by the tumor promoter TPA varied among the different cell clones. Clones with very high and very low inducibility of the resident Epstein-Barr virus genome were further analyzed. Constant differences in the inducibility of EA were observed after activation by tumor promoters, 5-iododeoxyuridine or antibodies to human IgM. Induction of EA synthesis by superinfection with Epstein-Barr virus from the P3HR-1 line, however, did not vary among the clones tested. No differences in expression of the Epstein-Barr virus-associated nuclear antigen (EBNA) were noted in cells of clones with high or low susceptibility to EA induction. DNA reassociation kinetics demonstrated that Raji cells with high susceptibility to EA induction contained a significantly higher number of Epstein-Barr virus genome equivalents per cell than cells with low susceptibility. Treatment of Raji cells with the tumor promoter TPA did not change the ratio of Epstein-Barr virus-specific DNA to cellular DNA.     

A novel monoclonal antibody-defined antigen which distinguishes human non-small cell from small cell lung carcinomas

Spleen cells from BALB/c mice hyperimmunized with the human epidermoid lung carcinoma cell line T222 were fused with NS-1 mouse myeloma cells to produce monoclonal antibodies to human lung cancer antigens. Hybridoma culture supernatants were tested by an enzyme-linked immunosorbent assay for reactivity against a panel of human lung tumor cell lines. Supernatant from hybridoma EA1 (immunoglobulin G1) displayed strong reactivity with four of four non-small cell lung carcinomas but did not react with three of three small cell lung carcinoma (SCLC) cell lines. This hybridoma was cloned by limiting dilution and utilized to generate ascites antibody for subsequent immunohistochemical and antigen characterization studies. Evaluation of fresh frozen tumor tissue sections by immunoperoxidase staining methods revealed EA1 reactivity with the vast majority of non-SCLCs tested (21 of 21 epidermoid, 17 of 18 adenocarcinomas, four of four large cell, two of two bronchioloalveolar) and no reactivity with nine of nine small cell lung carcinomas. EA1 also stained bronchial epithelium and other benign and malignant epithelial tissues. The EA1 antigen was determined to have a molecular weight of 75,000 by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human non-SCLC tumor extracts. These data imply that EA1 recognizes a novel antigen expressed by non-SCLCs and other epithelial tissues. The absence of EA1 reactivity with SCLCs suggests that this monoclonal antibody may find future application in distinguishing non-SCLC from SCLC and prove useful in furthering our understanding of the histogenesis of lung carcinomas.     

Histopathological characterization of a novel monoclonal antibody, MLuC1, reacting with lung carcinomas

A monoclonal antibody (MoAb), MLuC1, derived from the fusion of P3-X63-Ag 8-U1 mouse myeloma cells with spleen cells from an HR mouse immunized with the carcinoma cell line SW626, was studied to define its reactivity profile on normal and neoplastic human tissues and its potential clinical applications in lung cancer. Evaluation of paraffin sections using the ABC immunoperoxidase method showed a "pan-epithelial" reactivity; a large majority of epithelial components of organs in the respiratory, digestive and urogenital systems (except liver, rectum and ovary) were immunostained. As regard to neoplastic tissues MLuC1 recognized 84% of lung carcinomas (82% of small cell, 100% of squamous cell, 74% of adenocarcinomas), 86% of breast and 62% of ovarian carcinomas. On the contrary, MLuC1 was non-reactive with the other normal and tumoral non-epithelial tissues. Due to its spectrum of reactivity this MoAb could be useful for different diagnostic purposes such as differential diagnosis and lung cancer cytology.     

High levels of Met-72 antigen expression: correlation with metastatic activity of B16 melanoma tumor cell variants

In the present study, both poorly and highly metastatic clones derived from the C57BL/6 mouse B16 melanoma were used with cyclophosphamide in an attempt to elicit host antibody responses against cell surface markers expressed on highly metastatic tumor variants. The immunizations, performed in both syngeneic and xenogeneic combinations in Lewis rats, resulted in the production of 3 mouse and 2 rat monoclonal antibodies (MoAb) that preferentially react with highly metastatic clones derived from the B16 melanoma. These MoAb all immunoprecipitated a 72,000-dalton, cell surface-expressed glycoprotein, referred to as Met-72. In this study, 2 of the mouse anti-Met-72 MoAb were examined in detail for a) tumor specificity, b) reactivity against normal mouse tissue by in vivo absorption, and c) their ability to discriminate highly metastatic clones derived from the B16 melanoma.     

Monoclonal antibodies against cell surface antigens present on human urinary bladder cancer cells

After hybridomas were prepared by cell fusion between the mouse myeloma cell line P3x63Ag8.653 and the spleen cells of a BALB/c mouse hyperimmune to the human bladder cancer KU-1 cell line, they were cloned. Monoclonal antibodies (HBA4, HBE3, HBE10, HBF2, HBG9, and HBH8) from the hybridoma clones were selected for their serologic reactivity to cell surface antigens of KU-1 cells and for their unresponsiveness to 2 human lymphoma cell lines. They were cross-reactive with a characteristic portion of human epithelial tumor cell lines and with surgically resected bladder cancer tissues. With regard to reactivity to normal human cells and tissues, HBG9 alone was reactive to epidermis and both HBF2 and HBH8 were reactive to erythrocytes, but none of the other 3 monoclonal antibodies was reactive to any normal cells or tissues. The biochemical analysis of the antigens defined at least three antigenic systems. One system was a glycopeptide complex recognized by HBA4, HBE3, and HBG9, and it consisted of molecules having molecular weights of 78,000 (major) and 40,000 and 130,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 125I-radiolabeled extracts of KU-1 cells. Treatment of KU-1 cells with tunicamycin or neuraminidase lowered the reactivity to these antibodies, suggesting that the antigenic determinants reside in sialoglycoproteins. Antigenic determinants against HBG9 and the other two antibodies (HBA4 and HBE3) appeared to be different, as judged by the serologic reactivities. The other two antigenic systems detected by HBE10, HBF2, and HBH8 were defined as neutral glycolipids, according to heat stability, solubility in organic solvents, and characters in lipid fractionation. When examined by thin-layer chromatography in chloroform-methanol-0.25% KCl (30:15:4), a lipid component recognized by HBE10 migrated to a single zone [retardation factor (Rf), 0.33]. The other two antibodies (HBF2 and HBH8) did detect two lipid components (Rf, 0.23 and 0.33). Although the lipid component detected by HBE10 showed the same migration distance as one of the components detected by HBH8 (or HBF2), the antigenic determinants defined by these antibodies appeared to be different, as judged by their serologic reactivities.     

Interspecies-, species- and type-specific T antigenic determinants of human papovaviruses (JC and BK) and of Simian virus 40.

Immunofluorescence tests, absorption studies and quantitative analysis by a very sensitive 51Cr microcomplement fixation (CF) technique were used to define the degree of relatedness between the tumor (T) antigens induced by human papovaviruses, strain JC and BK, with simian virus 40(SV40) and mouse polyoma virus (PyV). Antisera against JCV, BKV, SV40 and PyV T were raised in tumor-bearing hamsters. The data obtained indicate that T antigens of JCV, BKV and SV40 possess various subspecificities which can be distinguished and looked upon as interspecies-, species- and type-specific antigenic determinants. It was found that JCV T and BKV T synthesized in transformed hamster cells share about the same amount (20%) of interspecies cross-reacting antigen with SV40 T from H-50 cell extracts (transformed hamster cells). Although hamster cells transformed by PyV showed definite PyV T reactivity, no cross-reactivity, at least with the sera used, was found with human papovavirus and SV40 T antigens. Furthermore, degree of heterogeneity was observed within the T antigen complex derived from different SV40-transformed cells.     

Anti-idiotypic antibody as the surrogate antigen for cloning scFv and its fusion proteins

Single-chain variable fragment (ScFv) is a versatile building block for novel targeting constructs. However, a reliable screening and binding assay is often the limiting step for antigens that are difficult to clone or purify. Anti-idiotypic antibodies may be useful as surrogate antigens for cloning scFv and their fusion proteins. 8H9 is a murine IgG(1) monoclonal antibody (MAb) specific for a novel antigen expressed on the cell surface of a wide spectrum of human solid tumors, but not in normal tissues. Rat anti-8H9-idiotypic hybridomas (clones 2E9, 1E12, and 1F11) were produced by somatic cell fusion between rat lymphocytes and mouse SP2/0 myeloma. In direct binding assays enzyme-linked immunosorbant assay--(ELISA)--they were specific for the 8H9 idiotope. Using 2E9 as the surrogate antigen, 8H9-scFv was cloned from hybridoma cDNA by phage display. 8H9scFv was then fused to human-gamma1-CH2-CH3 cDNA for transduction into CHO and NSO cells. High expressors of mouse scFv-human Fc chimeric antibody were selected. The secreted homodimer reacted specifically with antigen-positive tumor cells by ELISA and by flow cytometry, inhibitable by the anti-idiotypic antibody. The reduced size resulted in a shorter half-life in vivo, while achieving comparable tumor to nontumor ratio as the native antibody 8H9. However, its in vitro activity in antibody-dependent cell-mediated cytotoxicity was modest.     

Monoclonal antibodies that detect different antigenic determinants of the same human osteosarcoma-associated antigen

Monoclonal antibodies (MoAbs) against human osteosarcoma cells were obtained by the fusion of NS/1 mouse myeloma cells with spleen cells from the human osteosarcoma cell line-immunized BALB/c mice. Two hybrid clones were established and designated as 2H10 and 2D3. Both MoAbs reacted strongly with all osteosarcoma tissues but not with other bone and soft tissue tumors such as chondrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, and rhabdomyosarcoma. In addition, neither MoAb reacted with tumor cell lines and tissues obtained from other cancers. Immunohistochemical analysis demonstrated that 2H10 and 2D3 reacted with endothelial cells in sarcoma tissues, but not with those of other tumors and normal tissues. 2H10 also reacted with cells on the basal layer of epidermis of the skin. 2H10- and 2D3-defined antigen has an approximate molecular weight of 75,000 under nonreducing and reducing conditions, indicating that the antigen has a single chain structure and there is no intramolecular disulfide bond. 2H10- and 2D3-defined antigen has a pI value between 5.5 and 6.2. Sequential immunoprecipitation analysis clearly demonstrated that 2H10 and 2D3 recognized the same antigen molecule. However, further analysis suggested the possibility that 2H10 and 2D3 MoAbs recognized the different antigenic determinants on the same antigen molecule.