Molecular cloning and primary characterization of a single-chain antibody against human sarcoma-associated antigen p200.

BACKGROUND: Monoclonal Antibody (MAb) 29-13 reacts with the human sarcoma-associated antigen (SAA) p200. We report here engineering and primary characterization of a single chain antibody (scFV2913). MATERIALS AND METHODS: The scFV2913 recombinant gene, consisting of VH-linker-VK, was constructed with RT-PCR. This gene was cloned and expressed in E. coli. The renatured scFV2913 was used in the immunostaining study. RESULTS: Consistent with its parent MAb 29-13, purified and renatured scFV2913 showed affinity and specificity to the SAA p200 according to the immuno-histochemical staining study of 99 specimens of human sarcomas and other tissues. CONCLUSIONS: Due to its retained specificity and affinity, scFV2913 may be useful in immunodiagnosis and immunotherapy for sarcoma patients.

Expression of sarcoma-associated antigens p102 and p200 in human sarcoma cell lines.

BACKGROUND: Understanding tumor antigen expression and its correlation with the cell cycle may help in designing immunotherapy by monoclonal antibodies. Therefore, we studied the in vitro expression of sarcoma-associated antigens p102 and p200 in the G, S, and G2/M phases of sarcoma cell lines. METHODS: The expression of human cell surface sarcoma-associated antigens p102 and p200 was studied in 13 human sarcoma cell lines, using flow cytometry. RESULTS: p102 was detected by monoclonal antibody 19-24-6 in all 13 sarcoma cell lines, and p200 was detected by monoclonal antibody 29-13-17 in five of 13. p102 antigen expression was 1.4- to 3.4-fold higher (p < 0.001) than p200 expression. Although sarcoma cell lines showed a wide range of p102/p200 antigen expression, over 99% of the entire in vitro and in vivo cell population was found to be p102- and/or p200-positive. In three cell lines, p102 expression was cell cycle-dependent, with relative fluorescence intensity ranging from 13.8% to 23.9% higher at the G1 phase than at the G2/M phase. In three cell lines, the expression of p200 at the GI phase was 22.4% to 40.9% percent higher than at the G2/M phase. CONCLUSIONS: The heterogeneity and cell cycle dependence of p102/p200 antigen expression in sarcoma cells suggest that monoclonal antibodies 19-24-6 and 29-13-17 might be applied to the immunotherapy of sarcoma.

Isolation and analysis of lectin-reactive sarcoma-associated membrane glycoproteins.

Sarcoma and normal tissue plasma membrane lectin-reactive glycoproteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two peanut agglutinin-reactive N-acetylgalactosamine-containing glycoproteins of 1.05 x 10(6) and 1.25 x 10(5) Da and one lentil agglutinin-reactive mannose/N-acetylglucosamine(-fucose)/sialic acid-containing glycoprotein of 1.7 x 10(5) Da (Gp170) were detected in osteosarcoma and malignant fibrous histiocytoma (MFH), respectively. However, these glycoproteins were not detected in normal tissue plasma membranes. Concanavalin A, wheat germ and Ulex europaeus Type I agglutinins did not reveal any unique sarcoma-associated membrane glycoproteins. Preliminary studies on monoclonal antibodies (mAbs) generated against Gp170 (mAb 64-35-84) and against lentil-reactive glycoproteins from MFH (mAbs 67-34 and 67-117) revealed high specific binding to a number of membranes isolated from MFH and osteosarcoma tissues, with no crossreactivity to normal human tissues tested (liver, spleen and skin). Detailed analysis of mAb 67-102, which was generated against lentil-reactive glycoproteins isolated from MFH plasma membranes, exhibited significant binding to membranes isolated from osteosarcoma, liposarcoma and MFH; moderate binding to synovial sarcoma, aggressive fibromatosis and fibrosarcoma; and minimal to no binding to other soft tissue sarcoma plasma membranes. No binding was observed to twenty normal tissue specimens, with the exception of low positive binding to two of five fat and two of three colon specimens.

The use of daunomycin-antibody immunoconjugates in managing soft tissue sarcomas: nude mouse xenograft model.

Analysis of human fibrosarcoma cells exposed to radiolabeled monoclonal antibody 19-24, which recognizes sarcoma-associated antigen p102, revealed that over 54% of the cell surface-bound radioactivity was internalized. No modulation of cell surface p102 antigen by monoclonal antibody 19-24 was observed in human fibrosarcoma cells. Monoclonal antibody 19-24 coupled to daunomycin via a dextran bridge was found to be most effective. In different preparations, the daunomycin:total protein molar ratio ranged from 1.9 to 6.1. In vitro cytotoxicity studies using human fibrosarcoma cells showed that, at 10 micrograms/ml concentration, this immunoconjugate was 79.4% as efficient as free daunomycin and, at 1 microgram/ml concentration, 36.8% as efficient. Control nonspecific murine monoclonal antibody P3 immunoconjugates were relatively ineffective. The distribution of 14C-Adriamycin, 125I-labeled monoclonal antibody 19-24, and 125I-labeled 19-24 immunoconjugate was also evaluated over a 24-h period in tumor and normal tissues of athymic mice bearing a human fibrosarcoma xenograft. Poor uptake of radiolabeled Adriamycin by the tumor tissue was observed. The level of 14C radioactivity in the tumor tissue never exceeded 1% of the total injected dose and was 24.8-fold lower than the radioactivity found in the spleen tissue. Tumor tissue uptake of radiolabeled monoclonal antibody 19-24 was characterized by the high tumor tissue:blood ratio of 1.62 +/- 0.28 (SD). However, for monoclonal antibody 19-24 immunoconjugates, this ratio decreased to 0.66 +/- 0.05, which was still higher than normal (liver, 0.48 +/- 0.02; lung, 0.48 +/- 0.07; spleen, 0.28 +/- 0.01) or nonspecific monoclonal antibody P3 immunoconjugates (0.22 +/- 0.03). Thus, it appears that, compared to free daunomycin, monoclonal antibody 19-24 immunoconjugates may be more efficient and less cytotoxic to normal tissues.

Quantitative alteration of some aortic intima proteins in fatty streaks and fibro-fatty lesions.

Proteins from grossly and histologically normal human aortic intimas and human aortic intima with fatty streaks or fibro-fatty lesions were extracted with 9 M urea mixture. Protein extracts were mixed with an internal absorbance calibrator (carbonic anhydrase) and subsequently separated by two-dimensional gel electrophoresis, silver stained, and quantitated by a laser beam densitometer. The vascular-origin proteins actin, tropomyosin-like proteins, tubulin, glycoprotein G35, and two myosin light chains were present in the highest amounts in normal aortic intima (27-year-old male). Quantitation of vascular-origin proteins in aortic intima with a fibro-fatty lesion from the same subject showed a slight decrease in relative amount of these proteins as compared to the normal intima. Several polypeptides (P15, P18, P60, P110b) and plasma-derived proteins not observed in the normal intima were found in fibro-fatty lesion (albumin, haptoglobin beta-chain, fibrinogen beta-chain, alpha 1-HS-glycoprotein). Other proteins which were present in very low amounts in the normal intima (transferrin, alpha 1-antitrypsin, apolipoprotein A-1, P56, P190) were found to be major proteins of intima with fibro-fatty lesion. Differences in relative amount of plasma-derived and vascular-origin proteins between normal intima and intima with fatty streaks, studied in a large number of specimens from 38 thoracic intimas and 18 paired abdominal intimas (16-34 years old) were less prominent. Statistically significant increases of the albumin/actin ratio were found in fatty streaks as compared to paired normal intimas as well as in the mean value of albumin/actin ratio in the group of fibro-fatty lesions (mean = 6.1) as compared to the group of fatty streaks (mean = 1.7) or normal intima (mean = 0.7). Several lesion unique proteins were observed; however, the frequency of the occurrence of these proteins in 41 specimens with lesion was low. No significant differences were observed in intima protein pattern and quantities of selected intima proteins between paired thoracic and abdominal aortas.

Production and characterization of a monoclonal antibody to human saposin C.

A murine IgG1 monoclonal antibody, termed 68-12, was produced against purified human saposin C. Immunoprecipitation and binding analysis indicated that the antibody reacted only with saposin C. Dot blotting and Western analysis demonstrated that antibody 68-12 also reacted with prosaposin and a higher molecular weight protein(s) in murine spleen and cerebral grey matter. Solid phase competitive radioimmunoassay against 125I labeled saposin C (0.25 micrograms/ml) showed no cross reactivity for saposin A, B and D up to 15 micrograms/ml. At a concentration of 50 micrograms/ml saposin A, B and D cross reacted 21, 1.5, and 49% respectively. Monoclonal antibody 68-12 appears to have potential utility in the purification, detection and quantitation of human saposin C and its precursor.

In vitro cytotoxicity of the conjugate adriamycin with anti-sarcoma monoclonal antibody 19-24.

Monoclonal antibody (MoAb) 19-24, which recognizes a cell surface sarcoma-associated antigen p102, was linked via a biotin-avidin-biotin bridge to adriamycin (ADR). The molar ratios of ADR: total protein ranged from 2 to 7.5. Significant differences were not observed between the binding ability of the ADR-MoAb conjugate and of the unconjugated MoAb 19-24 to fresh sarcoma tissue membranes. The ADR-MoAb conjugate also retained the ability to compete with MoAb 19-24 for binding to sarcoma-associated antigen p102. In vitro cytotoxicity studies using human fibrosarcoma cells showed that the ADR-MoAb conjugate maintained 40% of the efficacy of free ADR. However, the conjugate was not cytotoxic to p102 antigen negative rat fibrosarcoma SP-24 cells. These data support the suggestion that MoAb-drug conjugates might be helpful in developing highly specific antisarcoma therapeutic agents.

Monoclonal antibody identification and characterization of two human sarcoma-associated antigens.

Two murine monoclonal antibodies, 29-13 (IgG1) and 29-2 (IgG2a), generated against malignant fibrous histiocytoma plasma membranes immunoprecipitated a Mr 200,000 protein (p200), with an isoelectric point between 6.3 and 7.5. Two additional antibodies, 35-16 (IgG1) and 30-40 (IgG2a), generated against Ewing's sarcoma membranes, immunoprecipitated an acidic protein of Mr 160,000 (p160), with an isoelectric point between 5.8 and 6.7. Monoclonal antibodies 29-13 and 29-2 recognize a similar determinant(s) on p200 while 35-16 and 30-40 recognize different determinants on p160. Monoclonal antibody 29-13 exhibited significant binding to membranes isolated from fibrosarcoma and aggressive fibromatosis; moderate binding to osteosarcoma, hemangiopericytoma, and malignant fibrous histiocytoma; and minimal to no binding to other soft tissue sarcoma plasma membranes. The p200 protein was not expressed in 16 other malignant tumors and in only 3 of 35 normal human tissue specimens. High levels of p200 were selectively expressed by leiomyosarcoma, Ewing's sarcoma, and fibrosarcoma cells as well as neonatal fibroblasts in vitro, but not by other carcinoma cell lines or B-lymphoblasts. The p160 protein appeared to be selectively expressed by Ewing's sarcoma with little or no expression on other sarcomas, carcinomas, or normal tissues. However, the p160 antigen was expressed in Ewing's sarcoma, leiomyosarcoma, melanoma, 4 of 9 carcinomas, and neonatal fibroblasts in vitro. The affinity of MoAbs 29-13, 29-2, 35-16, and 30-40 ranged from 5.3 x 10(8) to 4.7 x 10(9) M-1 for sarcoma membranes with approximately 5 x 10(4) binding sites/sarcoma cell.

Monoclonal antibody characterization of sarcoma-associated antigen p102.

A membrane protein of Mr 102,000 Da (p102) was detected in 51 out of 53 human sarcomas, using a murine IgG1 monoclonal antibody 23-26. Antigen expression in sarcoma histologic subtypes appeared dependent on the amount of fibrous tissue matrix present in the original specimen. High levels of p102 antigen were also found in all sarcoma, carcinoma cell lines and neonatal skin fibroblasts tested. Low levels of p102 were also expressed in membranes from some specimens of melanoma, lung and colorectal carcinoma, first trimester fetus, fat, lung and liver while skin specimens expressed slightly higher levels of antigen. Lectin affinity absorption indicated p102 was mannose containing glycoprotein, isoelectric point (pI) 4.7 and affinity constant of 2.3 x 10(9) M-1, with 5.9 x 10(5) binding sites per cultured human HT-1080 fibrosarcoma cell. The overexpression of p102 in sarcomas and other neoplastic tissues suggests that this protein may be associated with the neoplastic state.

Effect of menstrual cycle on protein expression in human uterine leiomyomas.

Tissue proteins in paired samples of uterine leiomyomas and normal myometria from 26 patients were compared after extraction with 9 M urea, separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and visualization by silver staining. A creatine kinase carbamylation train and a rat heart extract were used as internal standards for the relative charge and the relative molecular weight (Mr) scales, respectively. Two groups of four proteins each, a first group of Mr = 34,500 (34K) and relative charge of creatine kinase of -14 to -21(P34a-d) and a second group of Mr = 56,000 (56K) and relative charge of creatine kinase less than -23(P56a-d), were more frequently expressed in uterine leiomyoma tissue than in normal myometrial tissue: P34, 14 of 26 (53.8%) in tumors and zero of 26 (0%) in normal myometria; P56a&d, 17 of 26 (65.3%); P56b&c, 20 of 26 (76.9%) in tumors and P56a&d, zero of 26 (0%); and P56b&c, 12 of 26 (46.1%) in normal myometria. Both P34 and P56 were expressed more frequently in the uterine leiomyoma during the proliferative phase than during the nonproliferative phase (P34 in proliferative phase: 12 of 13, 92.3%; and in nonproliferative phase: two of 13, 15.3%; P56a-d in proliferative phase: 13 of 13, 100%; and in nonproliferative phase: P56a&d, four of 13, 30.7%, and P56b&c, seven of 13, 53.8%). The cyclic expression of P34 and P56 suggests that their synthesis is related to the intrinsic hormonal environment of the tumors.