A hidden antigenic determinant on membrane-bound human placental alkaline phosphatase

Two sets of monoclonal antibodies (mAbs) specific for human placental alkaline phosphatase (PLAP) were compared. One set of four mAbs was generated against solubilized and purified PLAP; the other set of seven mAbs was generated against the malignant cell line Hela TCRC-1 in which PLAP is an ectopically synthesized membrane-bound enzyme. Double immunodiffusion and competitive enzyme-linked immunosorbent assays were used to examine the relative spatial arrangement of the antigenic determinants to which each of the eleven mAbs binds. Significant differences in immunoreactivity of the antibodies were demonstrated. The mAbs to the solubilized and purified enzyme bound in either of two regions of the molecule. By contrast, all of the mAbs to PLAP as presented on the tumor cell surface bound in only one of these two regions. One of the major antigenic determinants on the solubilized enzyme is apparently unavailable for recognition by immunoreactive cells during immunization with whole cells. Furthermore, when mAbs are generated to this region using purified PLAP as the immunogen, they do not recognize membrane-bound PLAP. The 'hidden' determinant can be exposed in vitro after partial solubilization using butanol to extract the enzyme from HeLa TCRC-1 cells and subsequent treatment with 0.5% Nonidet P-40 detergent. The results of this study have implications for the potential use of mAbs in studies of other cell surface antigens and in tumor immunolocalization and drug targeting.     

Characterization of the placental alkaline phosphatase-like (Nagao) isozyme on the surface of A431 human epidermoid carcinoma cells

A431 human epidermoid carcinoma cells monophenotypically express the placental alkaline phosphatase (PLAP)-like enzyme shown by its catalytic and antigenic characteristics, properties which are shared by the Nagao isozyme. More specifically, it is L-leucine sensitive just as is the rare placental D-variant of PLAP and the testicular heat-stable enzyme. Collectively, these are all referred to as PLAP-like enzymes. The enzyme was localized to the surface of the plasma membrane since it was released in an active form by bromelain treatment of cells. The number of molecules per A431 cell was estimated by radioimmunoassay at 7.5 X 10(5), a value significantly higher than that observed for HeLa TCRC-1 cells (5 X 10(4) which express the S-variant of PLAP, also referred to as the Regan isozyme. The quantity of the enzyme was increased significantly (10-fold) by treating the cells with modulating agents including sodium butyrate, prednisolone, and hyperosmolar sodium chloride. The identification of a cell line such as A431 with enhanced expression in the amount of the PLAP-like enzyme and which can be further enhanced by modulating agents will facilitate studies of the differences and the similarities between this protein and other variants of PLAP. The A431 cell line now takes its place with other cell lines which are phenotypically restricted in their expression of alkaline phosphatase. Finally, the A431 cell line is also shown here to be a suitable model system for in vivo tumor studies such as immunolocalization.     

Tumour and cellular localization by use of monoclonal and polyclonal antibodies to placental alkaline phosphatase

Monoclonal and polyclonal antibodies against placental alkaline phosphatase (PLAP) were evaluated for tumour immunolocalization of human PLAP-producing Hep 2 tumours in nude mice. The antibodies were labelled with 125I and injected i.p. in mice with developing Hep 2 tumours. The distribution of 125I-anti PLAP in various tissues showed that the labelled antibody was enriched in the tumour, the mean concentration ratio being 7.1 and 6.8 for polyclonal and monoclonal antibodies, respectively. A PLAP negative tumour (RD) showed a mean ratio of 1.2. There was a positive correlation between PLAP content and uptake of labelled antibody in the tumours. Hep 2 tumour cells in tissue culture showed 100% positivity for PLAP, while imprints of the tumour after passage in nude mice showed 40-50% positivity. PLAP offers potential as a useful marker for localizing tumours in humans.     

A Putative Mechanism for the Non-Specific Uptake of Intact Radiolabelled Monoclonal Antibodies in the Testes and Prostate

Non-specific testicular accumulation of radiolabeled intact anti-CEA monoclonal antibody (MAb), (A431/26, Behringwerke AG) was observed in 11 out of 12 patients with the testes and prostate included in the examination field at radioimmunoscintigraphy (RIS). Previous studies have shown that placental alkaline phosphatase (PLAP) serves as an Fc-receptor, mediating IgG transport through the placenta. A closely related protein, the germ cell alkaline phosphatase (GCAP), is expressed in the testes. The testicular uptake of IgG is observed only when intact but not fragmented MAbs are used, indicating involvement of Fc-receptors. MDCK cells (dog kidney cell line) transfected with the plasmid pSVT7 containing the GCAP gene were shown to acquire the capacity to both express membrane bound GCAP and to bind IgG on the cell surface. This might indicate that GCAP is responsible for the non-specific accumulation of intact MAb in the testes and prostate often observed when intact murine MAbs are used for radioimmunolocalization (RIL)     

Private idiotypes located on light and heavy chains of human myeloma proteins characterized by monoclonal antibodies

Idiotypic determinant, an epitope located on the variable region of the heavy or light chain of an immunoglobulin molecule, could be classified into private and public forms. The private idiotype is a marker unique to a single clone of B cell and hence a fingerprint of an individual clone. It could therefore be exploited to monitor expansion of normal or malignant B cells and to target clonally expanded tumorous B cells specifically. In the present study, five murine monoclonal anti-idiotypic antibodies were generated against two human immunoglobulin G (IgG) myeloma proteins. These monoclonal antibodies (MAbs) are produced by hybridoma clones obtained by the fusion of myeloma cells with splenocytes from BALB/c mice immunized with either human IgG1 (three clones) or IgG2 (two clones) myeloma proteins. All MAbs reacted only with the immunizing antigens and had no reactivity with a panel of purified myeloma proteins of four IgG subclasses with different light chains, including IgG1 (n = 9), IgG2 (n = 4), IgG3 (n = 4) and IgG4 (n = 5). They reacted with the Fab, but not the Fc fraction of the immunizing antigen and displayed no reactivity with normal human serum or polyclonal IgG. Immunoblotting analysis demonstrated that two of the MAbs react with linear idiotypes on light chain, whereas the remaining three MAbs recognize heavy chain associated idiotopes, either conformational (n = 2) or linear (n = 1). Such MAbs with specificity for private idiotypes could have potential implications for monitoring and specific immunotherapy of B cell malignancies. They also are useful tools to study structural correlates of idiotypes.     

Modulation of placental alkaline phosphatase activity and cytokeratins in human HN-1 cells by butyrate, retinoic acid, catecholamines and histamine

The effects of butyrate and retinoic acid in combination with catecholamines or histamine on the HN-1 human head and neck squamous carcinoma cell line were investigated analysing cell proliferation, placental alkaline phosphatase (PLAP) activity, and relative cytokeratin content. Butyrate inhibited cell proliferation in agar, whereas retinoic acid induced a small inhibitory effect. Butyrate enhanced PLAP activity in a time related manner in contrast to retinoic acid, which had no significant effect. However, retinoic acid inhibited the efficacy of butyrate to induce PLAP activity. A synergistic enhancement of PLAP activity was demonstrated after treatment of butyrate pretreated cells with catecholamines or histamine. The beta-adrenergic antagonist propranolol partly inhibited the aforementioned enhancement of PLAP activity, whereas the alpha-adrenergic antagonist phentolamine further enhanced PLAP activity. Indirect labeling of keratins with a polyclonal antibody showed that cytokeratin content was enhanced by butyrate but not by retinoic acid. Further analysis of cytokeratin content using four monoclonal antibodies showed that labeling of cytokeratins (5 + 8) was increased by butyrate. PLAP activity could be modulated by a concerted action of either butyrate plus retinoic acid or butyrate plus catecholamines or histamine, indicating a possible role for PLAP in tumour cell proliferation.     

Expression of placental alkaline phosphatase in gastric and colorectal cancers. An immunohistochemical study using the prepared monoclonal antibody

The authors developed monoclonal antibodies (MoAb) against human placental alkaline phosphatase (PLAP). Four specific MoAb reacting only with PLAP and two nonspecific MoAb reacting equally with isozymes of alkaline phosphatase (hepatic, intestinal, and placental) were obtained. Immunohistochemical staining with the specific MoAb showed that the cell membrane and cytoplasm of cancer cells were stained in gastric and colorectal carcinoma. The incidence of PLAP positivity was 23% (25 of 107) of all gastric carcinomas. Among gastric carcinomas, the 42% (13 of 31) positivity of highly differentiated carcinoma (papillary adenocarcinoma and well-differentiated tubular adenocarcinoma) was a significantly higher rate than that found in poorly differentiated carcinoma (poorly differentiated adenocarcinoma and signet-ring cell carcinoma, five of 41, 12%). The incidence of PLAP positivity was 11% (four of 35) in colorectal carcinoma. In contrast, gastric adenoma, intestinal metaplasia, and noncancerous tissue adjacent to cancer did not show staining. These results indicated that expression of PLAP was apt to occur in more highly differentiated gastric carcinoma and was highly specific for carcinoma in the gastrointestinal tract, although its incidence was not high.     

Levels of alkaline phosphatase isozymes in human seminoma tissue

The three human isozymes of alkaline phosphatases were quantitatively determined in normal testis and seminoma tissues. The highly selective assays were based on isozyme specific monoclonal antibodies. In the normal testis approximately 90% of the catalytic activity originates from the tissue unspecific alkaline phosphatase, and the remaining activity was due to trace expression of both intestinal (approximately 5%) and placental alkaline phosphatase (PLAP) or PLAP-like isozyme (approximately 5%). In homogenates of seminoma tissues, highly increased levels of all three isozymes were identified. Both the tissue unspecific alkaline phosphatase and PLAP-like enzymes displayed relative increases of 10- to 100-fold and intestinal alkaline phosphatase 2- to 10-fold compared with normal testis. This finding indicates that the entire genome coding for alkaline phosphatases may be activated in seminomas. The PLAP-like enzyme from seminoma cells comprises a heterogenous population of molecules demonstrating partial heat sensitivity and microheterogeneity upon starch gel electrophoresis in contrast to the pregnancy related PLAP. These findings have implications for the different PLAP assays used in the clinical monitoring of seminoma patients.     

Construction, characterisation and kinetics of a single chain antibody recognising the tumour associated antigen placental alkaline phosphatase.

The murine monoclonal antibody H17E2 recognises placental alkaline phosphatase (PLAP), an antigen present in the human term placenta and also expressed by many tumours. The antibody is of value in both immunoscintigraphy and radioimmunotherapy in testicular and ovarian cancer. The small size of genetically engineered single chain antibodies (SCAs) should give diagnostic and therapeutic advantages of improved tumour penetration and increased blood clearance compared to IgG. Employing recombinant DNA techniques a SCA based on H17E2 has been expressed in Escherichia coli and has been shown to bind placental alkaline phosphatase specifically. When administered to nude mice bearing human tumour xenografts, the H17E2 SCA effectively localised to tumour whilst a co-administered non-specific SCA did not. H17E2 SCA achieves tumour: blood ratios that are superior to those achieved with whole IgG, probably owing to its rapid blood clearance. We conclude that the H17E2 SCA is suitable for further investigation as an agent for clinical imaging and therapy. Additionally, the SCA can also be used for the construction of antibody based fusion proteins to target other effector functions to tumour cells.     

Establishment and characterization of calcyclin binding protein (CacyBP) monoclonal antibody

Anti-calcyclin binding protein (CacyBP) monoclonal antibodies (MAb) were produced using an in vitro immunization method. BALB/c mouse splenocytes were immunized with purified 6 x His-CacyBP fusion protein and fused with myeloma cells using polyethylene glycol (PEG) 4000. By selection using enzyme-linked immunosorbent assay (ELISA), three anti-CacyBP MAbs were obtained. The MAb BD1, whose isotype was IgG1, interacted with the fusion protein. Western blot and immunofluorescence microscopy showed that the MAb BD1 against CacyBP could recognize CacyBP protein derived from human gastric cancer cell lines in both native and denatured forms. This MAb would act as a useful tool for the detection of CacyBP protein in future studies.     

Remission Induction in Patients with Lymphoid Malignancies Using Unconjugated CAMPATH-1 Monoclonal Antibodies

CAMPATH-1 (CDw52) antigen is a heavily glycosylated, non-modulating glycoprotein expressed abundantly on the cell surface of nearly all normal and malignant lymphocytes but not on hemopoietic stem cells. A series of rat monoclonal antibodies (MAbs) with CDw52 specificity but of varying immunoglobulin isotype has been produced, and assessed for ability to deplete lymphoid cells in patients with lymphoproliferative disorders. Although all IgM, IgG2a and IgG2b rat MAbs were able to elicit lysis with human complement in vitro, only the IgG2b MAb (CAMPATH-1G) could elicit substantial lymphoid depletion in vivo. The efficacy of CAMPATH-1 G probably results from its ability to bind human Fc receptors and activate cell-mediated lysis of antibody-coated cells. Twenty-nine patients with lymphoid malignancies have received between 250 mg and 680 mg of CAMPATH-1G; nine of these attained complete remission

A human MAb with CDw52 specificity has been produced by “grafting” the complementarity-determining regions (CDRs) of the rat MAb into human IgG1 heavy- and kappa light-chain genes. These constructs were transfected into a rat myeloma cell line. Sufficient human MAb (CAMPATH-1H) has been purified to treat two patients with non-Hodgkin's lymphoma in leukemic phase. Although each patient only received approximately 100 mg of MAb, remission was induced in both patients with recovery of normal hemopoiesis during the course of therapy

CDw52 MAbs are the first MAbs to demonstrate consistent anti-tumor effects in vivo and may have roles in the therapy of a wide range of lymphoid malignancies and as powerful immunosuppressive agents     

Placental alkaline phosphatase, alphafetoprotein and carcinoembryonic antigen in testicular tumors. Tissue typing by means of cytologic smears.

Indirect immunofluorescence and radioimmunoassay with specific rabbit antisera demonstrated the occurrence of alphafetoprotein (AFP), carcinoembryonic antigen (CEA) and placental alkaline phosphatase (PLAP) in primary testicular tumor cells. Embryonal carcinomas had AFP- and CEA-containing cells, sometimes PLAP. PLAP and sometimes CEA were found in seminoma cells. Sera from patients with advanced non-seminomatous tumors could contain any of these antigens or any combination of them. Sera from patients with seminomas had raised PLAP or CEA. PLAP appears to be a new marker for seminoma.     

Alkaline phosphatase isozymes in human testicular germ cell tumors, their precancerous stage, and three related cell lines

The four known isozymes of the human alkaline phosphatase (ALP) were detected by isoelectric focusing in extracts of various types of germ cell tumors, three related cell lines, and their precancerous elements (atypical germ cells). In seminoma, placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (PLAP-like) could be separated by isoelectric focusing following isolation by immunoaffinity. The occurrence of both isozymes in seminoma could explain partial heat sensitivity and variation in electrophoretic patterns of the seminoma isozyme frequently observed upon starch gels, in comparison to the normal placental phenotype. The four ALP isozymes are produced not only in germ cell tumors, but already in precancerous tissues. Quantitative analysis showed that the amount of the four isozymes varies in parallel in the tumors tested. Maximal expression was found in seminoma. The relation between ALP gene overexpression and gene amplification by polyploidy of chromosomes 1 and 2 in these lesions is discussed. On the other hand, the ectopic expression of intestinal alkaline phosphatase and PLAP associated with overexpression of PLAP-like in tumor cells as well as in their precancerous stage indicates gene activation by some unknown mechanisms, probably a regulatory process affecting the three tissue-specific ALP genes simultaneously.     

Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase

Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.     

Patterns of seminoma tissue markers and deletions

Seminomas and control tissues were analyzed for several tumor markers. Very high levels of placental alkaline phosphatase (PLAP)-like enzyme levels were found in all 18 seminomas studied. The majority of the seminomas were of phenotype I, thus differing from palcental PLAP. The mean amount of enzyme protein as measured by monoclonal antibodies, was 100 times higher than in non-malignant tissues and 10 times lower than in placental tissue. The specific enzymatic activity in seminomas was about half of that observed in placenta. Similarly, the specific activity of PLAP-like enzymes in sera of patients with seminoma was only about half of that found in pregnancy sera. HCG was strongly elevated in 3 seminomas, but not obviously related to PLAP. Thirteen of the 17 pure seminomas had HCG over 100 IU/g, which was not seen in normal testes. Liver alkaline phosphatase (LAP) and intestinal alkaline phosphatase (IAP) were high in seminomatous tissues, the mean increases being 60-fold and 20-fold, respectively. The highest IAP levels were found in 2 yolk-sac tumors. Ferritin was moderately elevated in seminomas, but high in several control tissues. Carcinoembryonic antigen (CEA) was not elevated and alpha-fetoprotein (AFP) was not detected at all in pure seminomas. A decrease in carbohydrate antigen 50 (CA-50) content was noted in seminomas as compared to normal testes, yolk-sac tumors and choriocarcinomas. Defects in tumor-related enzymes may account for increase of PLAP and decrease of CA-50.     

Preparation of novel anti-ski monoclonal antibodies

Ski is an avian sarcoma virus oncogene homolog best known for inhibiting TGF beta signaling through its association with the SMAD proteins. Anti-Ski antibodies (MAbs) of high titer were prepared by immunizing BALB/c mice with multifocal intradermal injections and fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Three MAbs were selected for further characterization as classes and subclasses. Antibodies were produced by these three clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin IgG1 and IgG2b subclass with kappa light chain. They could recognize Ski as determined by Western blot analysis. The produced MAbs will be a useful tool for further investigation of Ski functions in organisms.     

Flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin, its subunits, and fragments on human cancer cells.

A quantitative flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin (hCG), its subunits, and fragments on human cancer cells was developed using a double-antibody reaction; a flow cytometry with a 2-W argon laser, standard settings, and filters for fluorescein isothiocyanate use; commercially available software; and the ectopic hCG producer CCL 2 HeLa cells from the American Type Culture Collection (ATCC) as a cell control to standardize the reagents and for overall quality control. Twenty-two monoclonal antibodies (MoAb) and immunoglobulin G fractions from three rabbit polyclonal antisera were tested for effects of antibody concentration (titration), reproducibility at different levels of epitope expression, and variability of epitope expression to select appropriate primary antibodies. Based on the results of the various tests, three polyclonal immunoglobulin G antibodies and a panel of nine MoAb directed to epitopes located in five different regions on the hCG molecule were selected as first antibodies. Their specificity was determined by using two unrelated MoAb of the same isotype at the same concentration to replace the primary MoAb and by a competition experiment. The unrelated MoAb also were used for the selection of the appropriate control fluorescence profile needed for the software. The unique characteristics of this method were: the use of living cells, standardized reagents, internal and external quality control, and the highest sensitivity, which could detect as few as 10(3) molecules of fluorochrome per cell. Serial analyses of the ATCC CCL 2 HeLa cells and two of its variants and of the eutopic hCG producer JEG-3 choriocarcinoma cells revealed the expression of membrane-associated epitopes of intact hCG, its subunits, and fragments by a high percentage of the cells, indicating that the expression of these sialoglycoproteins by these two different types of cancer cells is a common phenotypic characteristic.     

Placenta-like alkaline phosphatases from human osteosarcoma cells.

Hormone-induced alkaline phosphatases in human osteosarcoma cells (LM) were extracted and purified. Characterization of the purified enzyme showed two distinct isoenzymes. One isoenzyme was heat labile, was homoarginine inhibited, and had the electrophoretic migration of alkaline phosphatase of human osseous origin. Immunodiffusion showed that this isoenzyme reacted positively only against anti-bone alkaline phosphatase antibodies. The second isoenzyme was heat stable, was inhibited by phenylalanie, and had the same electrophoretic migration as did alkaline phosphatase extracted from mature normal human placenta. This second isoenzyme had the same antigenicity as did the normal placental enzyme. Like the D-variant placental phenotype, this second isoenzyme was inhibited by L-leucine and ethylenediaminetetraacetic acid.