A stomach oncofetal antigen recognized by monoclonal antibody GC302

A monoclonal antibody, GC302, was established by fusing murine myeloma NS/1 cells with the splenocytes of a BALB/c mouse immunized with a human gastric cancer cell line, NU-GC-3. The serological specificity of GC302 was analyzed by an anti-mouse Ig mixed-hemadsorption (MHA) test on a panel of human cell lines, and an immunoperoxidase method using the frozen sections of tumors and normal tissues of adult and fetus. GC302 reacted with cancers of the stomach and colorectum but did not react with hepatocellular carcinomas, melanomas, or astrocytomas in the MHA tests. By the immunoperoxidase method, GC302 was found not to react with normal adult gastric mucosa, but to react with the mucosa in the fetal stomach, intestinal metaplasia, and almost all of the cancer of the stomach. GC302 also reacted with the normal mucosa of the intestine, colon, and rectum as well as with cancers of these origins. In normal liver sections, the antibody reacted with the bile ducts, but not with the hepatic cells. These results indicate that the antigen detected by GC302 is characterized as an oncofetal antigen in the stomach, and also as a differentiation antigen whose localization discriminates between the gastrointestinal tracts of the forgut origin and those of the midgut and hindgut origin. The molecular weight of the GC302 antigen was estimated to beca. 40,000 by the Western blot analysis. Periodic acid treatment on the antigen suggested that the antigenic determinant is a carbohydrate.     

Measurement of prostate-specific membrane antigen in the serum with a new antibody

Work to date has identified prostate-specific membrane antigen (PSMA) as a membrane-bound glycoprotein with high specificity for prostatic epithelial cells. PSMA reacts with the monoclonal antibody 7E11.C5, which is present in serum, seminal fluid, and prostatic epithelial cells, and is increased in its expression in the presence of a hormone refractory state associated with prostatic cancer. This report confirms these results and further documents the presence of the monoclonal antibody 3F5.4G6, which reacts with the extracellular domain of PSMA. This region of PSMA is also an element present in a truncated version of the protein, so-called PSM′. Immune precipitation with either 7E11.C5 or 3F5.4G6 yields an isolated protein species that are reactive with the reciprocal antibody in Western blot analysis. Thus, 3F5.4G6 recognizes the same PSMA protein as does 7E11.C5, but at different epitopes on essentially opposite ends of the molecule. These two antibodies are well suited for use in a sandwich immunoassay, either one as a capture or detection antibody. Current work on this is underway. This report also confirms that 7E11.C5 Western blots for PSMA are negative with normal human brain tissue. The monoclonal antibody 9H10 does not react with 3F5.4G6 or with 7E11.C5 in studies conducted herein. Moreover, 3F5.4G6 reacts with PSMA found in the LNCaP cell line, but not DU-145 or PC3, which lack PSMA. © 1996 Wiley-Liss, Inc.     

Development of new prostate specific monoclonal antibodies

BACKGROUND: Despite the need for new prostate-specific diagnostic and therapeutic targets, very few unique prostate (cancer) specific antigens have been characterized. Monoclonal antibody (mAb) technology is a powerful tool to identify specific antigenic markers, which could be potential targets for cancer diagnostics or therapy. METHODS: Splenocytes from mice immunized with prostate cancer (PCa) homogenates of different origin were fused using standard techniques. Employing a differential high-throughput screening method followed by immediate screening in immunohistochemistry (IHC) a large number of hybridomas were screened for prostate (cancer) specificity. RESULTS: From 25 successful fusions approximately 300 clones were identified excreting PCa-reactive antibodies. Subsequent immunohistochemical fine-specificity analysis reduced this number to 26. Eventually, after extensive fine-specificity analysis, the number of mAbs appearing to define prostate-specific antigenic structures that might serve as new diagnostic or therapeutic targets was reduced to three. CONCLUSIONS: Using mAb technology combined with a high throughput screening method we have developed three mAbs (1.8, 2.26, and 3.10) directed against prostate associated antigens that might identify potential new therapeutic targets.     

Purification of an acrosomal antigen recognized by a monoclonal antibody and antifertility effects of isoimmune serum

A highly conserved acrosomal antigen reactive to a monoclonal antibody (HS-63), generated against human sperm, was purified to homogeneity with a combination of conventional procedures and immunoaffinity chromatography using a soluble extract of mouse and rabbit testes. The molecular weight of the purified antigen was 42-50 kD when analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis. The high specificity of the purified antigen to monoclonal antibody HS-63 was shown by indirect immunofluorescent inhibition assay, enzyme-linked immunosorbent assay, Western blot analysis and radioimmunosorbent assay. The purified antigen was used for isoimmunization of mice and rabbits. Following successive immunizations, antisera of high titres were raised and reacted specifically with antigen on the sperm acrosome and in testes of several mammalian species, but not with somatic tissues. These isoimmune sera exhibited strong inhibition on mouse in-vitro fertilization and human sperm penetration of zona-free hamster eggs. The results of this study suggest that the sperm-specific acrosomal antigen reacting with HS-63 could be a good candidate for the development of immunocontraceptive vaccines in humans and in other animals.     

A germ cell-specific nuclear antigen recognized by a monoclonal antibody raised against mouse testicular germ cells

A monoclonal antibody (mAb TRA104) raised against mouse testicular germ cells was able to recognize the nuclei of testicular germ cells at all the stages of differentiation from embryonic gonocytes to spermatids and did not react with any somatic cells. The antigen recognized by mAb TRA 104 was exclusively present in testicular extracts. The molecular weights and isoelectric point (pI) of the antigens determined by Western blotting analysis were 60–110 kDa and 7.2, respectively. This antigen(s) is referred to as a germ cell-specific nuclear antigen(s) (GENA) since GENA was first detected specifically in the genital ridge at around 12 days of gestation by Western blotting analysis. In the testis, the expression increased gradually until adulthood whereas it was lost in the ovary by postpartum day 5. Thus, GENA is a molecule(s) exclusively present in the nuclei of germ cells and may be a useful marker with which to study the mechanism of germ cell development and differentiation at the molecular level.     

Monoclonal antibody that defines the prostate specific antigen

A monoclonal antibody to a protein with a single band in the 30-kD region was obtained from fusion of Balb/C mouse spleen cells immunized with epithelial fractions of BPH, with the myeloma cell line P3 × 63 Ag8 6.5.3 by standard procedures. This antigen was characterized in immunobinding studies with various cellular and target antigens and in immunoperoxidase staining.     

Tissue Resonance Interaction Method (TRIMprob) has the potential to be used alongside the recognized tests in the screening protocols for prostate cancer

Abstract:   The objective of this study was to evaluate the accuracy of the magnetic induction technique with a nonlinear tunable oscillator (the Tissue Resonance Interaction Method [TRIMprob]) in the diagnosis of prostate cancer (CaP). Overall, 148 men were split into two groups (patients at risk of CaP [Group 1] and controls [Group 2]) and evaluated with the TRIMprob. Group 1 consisted of 100 patients (mean age: 63.8 ± 7.2 years) with elevated prostate-specific antigen (>4 ng/mL) levels and/or abnormal digital rectal examination. Eleven patients (Group 2a, mean age: 59.5 ± 7.3) with previously biopsy-proven CaP served as positive controls. In addition, 37 voluntary men (Group 2b, mean age: 39.8 ± 10.4) with normal prostate-specific antigen and digital rectal examination without lower urinary tract symptoms served as negative controls. Non-linear resonance was analyzed at 465 MHz and a cut-off value of 40 units was detected as the resonance value for the best threshold to distinguish benign conditions from CaP after transrectal ultrasonography-guided biopsy with a standard 10–12 core technique in Group 1. Mean resonance values (±standard deviation) with the TRIMprob examination for patients in Groups 1 and 2b were 36.72 ± 22.35 and 73.64 ± 10.06, respectively, whereas for patients in Group 2a, it was 13.73 ± 12.12 ( P  < 0.01). Sensitivity, specificity, positive and negative predictive values of the TRIMprob using the study cohort of Group 1 were found as 76%, 61.3%, 39.6% and 88.5%, respectively. Despite some technical limitations, the non-invasive TRIMprob examination may have a role in screening protocols for CaP.     

Antiprostate carcinoma monoclonal antibody (D83.21) cross reacts with a membrane antigen expressed on cytomegalovirus-transformed human fibroblasts

Virological and epidemiological studies have implicated human cytomegalovirus (HCMV) as a possible etiological agent of prostate cancer. Because of the suspected associations, this laboratory tested the reactivity of a prostate-associated monoclonal antibody with HCMV-transformed cells. This mouse monoclonal antibody, D83.21, reacts with a membrane antigen on prostate and bladder tumor cells and does not bind to a variety of other malignant or normal cells. The results of this study indicated that the prostate-associated antibody bound to a membrane antigen on HCMV-transformed cells as detected by radioimmunoassay, immunofluorescence, and complemented-dependent cytotoxicity. This cross-reactivity appeared to be specific for HCMV-transformed cells and did not react with HCMV-infected cells or those transformed by other viruses. Antibody affinity chromatography, used to isolate the D83.21-reactive protein, revealed two peptides of 60 and 28 kd on both prostate tumor and HCMV-transformed cells. The results suggest that D83.21 reacts with a common cell surface protein expressed on HCMV-transformed cells and urogenital tumors.     

Prostate cancer imaging with a new monoclonal antibody: A preliminary report

Background: Optimal treatment of prostate cancer depends on accurate staging. Computed tomography (CT) and magnetic resonance imaging have severe limitations, and standard bone scanning can show only destructive osseous metastases. A radiolabeled antibody specific to prostatic adenocarcinoma could theoretically find evidence of soft-tissue metastases and lymph node involvement. Methods: An immunoconjugate (CYT-356) consisting of a murine monoclonal antibody against human prostatic adenocarcinoma bound to a linkerchelator and radiolabeled with indium 111 was administered intravenously to seven patients with documented Stage D adenocarcinoma of the prostate. Planar imaging was done on days 1, 2, and 3 after injection. The CYT-356 scans were compared with standard technetium Tc 99m sulfur colloid bone scans and CT scans. Results: Optimal imaging results were obtained on the 72-h scans. All patients had lesions on both the^99mTc-sulfur colloid bone scan and the CYT-356 scan. The location of the lesions correlated to a great extent. Two patients had positive lesions biopsied, and both biopsies showed the presence of metastatic prostatic carcinoma. There were no side effects from administration of the antibody. Conclusion: In this preliminary study, CYT-356 scanning appears to be a promising agent to accomplish specific staging of prostatic carcinoma.     

前列腺特异性膜抗原在前列腺癌诊治中的研究进展

近年来,前列腺癌特异性的分子标志物——前列腺特异性膜抗原(PSMA)已成为前列腺癌临床研究中的热点之一。PSMA在前列腺癌的早期诊断、基因治疗、预后评估中所起的作用变得越来越重要。本文就PSMA蛋白的结构、功能、表达特点、基因表达以及基于PSMA的前列腺癌放射免疫显像、DNA疫苗、自杀基因治疗的相关研究进展及其在前列腺癌诊治中的作用进行了综述。     

Identification of differentiation antigens in mouse testicular germ cells recognized by monoclonal antibody TRA 55

We have isolated a monoclonal antibody (mAb) TRA 55, which recognizes mouse testicular germ cells from mid-pachytene spermatocytes to the early stages of haploid spermatids during differentiation. Immunohistochemical analysis produced strong positive staining of the nuclei and faint staining in the cytoplasm of germ cells. At meiotic division, when the nuclear membrane disappeared, a specific positive signal could be observed on metaphase chromosomes. When germ cells produced haploid spermatids, antigenicity became suddenly weak and soon disappeared. TRA 55 did not react with testicular somatic cells, such as Sertoli cells or Leydig cells. Western blot analysis of the whole testis showed four positive bands with molecular weights of 43, 46, 49 and 55 kDa. Three bands of 43, 49 and 55 kDa, and a single band of 46 kDa were recovered in cytoplasmic and nuclear fractions of testicular germ cells, respectively. Chronological changes in the Western blot pattern indicated that these antigens became detectable in the testis at the age of 10 days. Furthermore, all antigens were resistant to periodate treatment, suggesting that the epitope was in an amino acid rather than a sugar moiety. These antigen molecules may play important roles in the differentiation of germ cells at the later stages of meiotic prophase and meiotic division in the mouse testis.     

前列腺特异性膜抗原在前列腺癌诊治中的研究进展

近年来，前列腺癌特异性的分子标志物—前列腺特异性膜抗原（PSMA）已成为前列腺癌临床研究中的热点之一。PSMA在前列腺癌的早期诊断、基因治疗、预后评估中所起的作用变得越来越重要。本文就PSMA蛋白的结构、功能、表达特点、基因表达以及基于PSMA的前列腺癌放射免疫显像、DNA疫苗、自杀基因治疗的相关研究进展及其在前列腺癌诊治中的作用进行了综述。     

A tumor-associated antigen in the scirrhous gastric carcinoma cell line MK-01 defined by monoclonal antibody S202

A monoclonal antibody (MAb) S202, with an IgG₁ isotype, that reacted strongly with the scirrhous gastric carcinoma cell line MK-01 was established. MAb S202 reacted with the colonic cancer cell line, SW116, and the pancreatic cancer cell line, PK-1, when tested by indirect immunofluorescence. The S202 reactive antigen was expressed in the majority of acetone-fixed fresh frozen cancer tissues. Eighty to 100 per cent of the paraffin-embedded sections of stomach, colon and pancreatic adenocarcinoma were positive for the S202 antigen, with diffuse cytoplasmic staining, whereas esophageal and breast cancers demonstrated markedly less immunostaining. Supplemented serum-free medium collected from 7 day old tumor cell cultures were assayed for the presence of antibody-defined antigens. Antigens detected by MAb S202 were released by the cell lines SW1116 and PK-1. The binding of MAb S202 to the colonic adenocarcinoma sections was reduced after treatment with sodium periodate which suggests that respective antigenic determinants are of carbohydrate nature without sialic acid residues.     

Improved immunohistochemical detection of prostatic acid phosphatase by a monoclonal antibody

A monoclonal antibody with high affinity to acid phosphatase isoenzyme 2 (Ab-AcP2) was selected to examine its binding to different normal and tumor tissues using the indirect immunohistochemical method. Both mature prostatic epithelial cells in the prostate and the highly dedifferentiated prostatic cancer cells in the bone marrow showed strong binding to the antibody. Among nonprostatic tissues, only bone marrow, breast, and kidney showed trace staining in some specimens. The specificity of Ab-AcP2 was much better than that of the polyclonal antibody to acid phospatase previously reported. When the antibody to the prostate-specific antigen (Ab-PSA) was used, weak background staining was often encountered, and weak to moderate stains were seen in the prostatic stroma, bone marrow, lung, skin, and melanoma.     

8点及12点前列腺穿刺活检诊断前列腺癌的价值比较研究

目的比较8点及12点前列腺穿刺活检诊断前列腺癌的价值,分析前列腺特异性抗原(PSA)、前列腺特异性抗原密度(PSAD)及前列腺体积(PV)对前列腺癌检出率(PCDR)的影响。方法回顾性分析260例因PSA异常增高而接受首次直肠超声引导下前列腺穿刺活检的患者相关资料,其中132例患者接受8点穿刺,128例患者接受12点穿刺。结果依据PSA、PV、PSA与PV及PSAD,患者被进一步分组。8点及12点的总的PCDR没有显著的差异,在PV≥45mL、PSA≥10ng/mL且PV≥45mL及0.15ng/(mL·cm3)≤PSAD≤0.25ng/(mL·cm3)组中,12点的PCDR明显高于8点。结论 8点及12点前列腺穿刺总的PCDR没有显著区别(P0.05),但在PV较大同时PSA较高或者PSAD处于中等大小时(0.15~0.25)ng/(mL·cm3),12点的PCDR明显高于8点(P均0.05)。     

Purification and characterization of sperm-coating antigen, identified by a monoclonal antibody

Summary. A procedure was designed for purification of a human seminal plasma-specific antigen (HSP-antigen) identified by a monoclonal antibody produced in this laboratory (mAb 4E6). Pooled human seminal plasma was fractionated by consecutively applied methods: affinity chroma-tography on Lentil lectin sepharose, gel chromatography on Ultrogel AcA 34 and immunoaffinity on mAb 4E6 coupled CNBr-Spharose 4B. The antigen-containing fraction obtained after this procedure was proved to be homogeneous when analysed by electrophoresis in polyacrylamide gel. After the consecutive purification procedures the degree of purification was 128 times as compared to the starting material. Electrophoretic analysis of the purified 4E6 antigen under reducing conditions showed that it consisted of 3 polypeptide subunits with molecular weight 70 kDa, 64 kDa and 60 kDa respectively. On the basis of the data obtained from competitive elisa testing of sera from infertile patients it has been suggested that the identified antigen may be involved in pathogenesis of immunologic infertility.     

Anti-tumor effects and lack of side effects in mice of an immunotoxin directed against human and mouse prostate-specific membrane antigen

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a transmembrane protein that is largely restricted to prostatic epithelial cells in humans and is strongly upregulated on prostatic carcinoma cells. It is also expressed on the endothelium of tumor vasculature in humans, but not on the vasculature of normal tissues. Expression of low levels of PSMA has also been found on non-vascular cells in several normal tissues, most prominently on the brain and kidney in humans. PSMA is an excellent candidate for targeting prostate cancer or targeting tumor vasculature of various solid tumors. The high potential clinical benefit of these agents has prompted the search for an animal model in which to assess the efficacy and safety of anti-PSMA monoclonal antibody (mAb)-based therapies. METHODS: A rat monoclonal antibody, E6 that recognizes both mouse and human PSMA was generated using conventional hybridoma techniques. The antibody was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry. An immunotoxin composed of E6, antibody and deglycosylated ricin A-chain (dgA) was prepared chemically. The anti-tumor effects of the immunotoxin were determined in vitro and in mice bearing subcutaneous LnCaP human prostate tumors, which express PSMA on the tumor cell surface. RESULTS: E6 recognizes the extracellular domain of both human and mouse PSMA in ELISA, immunoblot and by immunohistochemistry. E6 strongly stained the vascular endothelium of tumors from humans but not from mice. E6 stained proximal tubules in mouse and human kidneys, and neurons in the mouse and human hippocampus but, unlike the human, did not detectably stain epithelial cells in mouse prostate or small intestine. An E6-dgA immunoconjugate strongly inhibited the growth of LnCaP tumor xenografts without causing apparent toxicity to the mice. Histological observation indicated that the anti-tumor effects were mediated through direct cytotoxic effects on the tumor cells. CONCLUSIONS: We have generated and characterized a rat mAb (E6) that reacts specifically with both human and mouse PSMA and have demonstrated that an immunotoxin constructed from E6 is safe and effective against human prostatic carcinoma cells growing subcutaneously in nude mice.     

Production of monoclonal antibody to human esophageal cancer cell line

The immunization of Balb/C mice with esophageal cell line KYSE-50 established from poorly-differentiated esophageal squamous cell carcinoma, resulted in obtaining the monoclonal antibody KYMN-28-5. This monoclonal antibody is of the IgM class and recognizes a carbohydrate antigen contained in glycoproteins with molecular weights of 53 and 56K, and in neutral glycolipids extracted from teratomas. Tissue staining revealed that this monoclonal antibody reacts strongly with malignant tumors but only weakly, or not at all, with normal tissues, apart from squamous epithelial tissue. KYMN-28-5 is thus a useful tumor marker which will improved the accuracy of serological diagnosing squamous cell carcinoma when combined with the measurement of SCC antigen.