Detection of high CD44 expression in oral cancers using the novel monoclonal antibody,C44Mab-5

CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C₄₄Mab-5 (IgG₁, kappa), recognized both CD44s and CD44v3-10. C₄₄Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C₄₄Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C₄₄Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.     

The anatomy of the nervous system of the hydrozoan jellyfish,Polyorchis penicillatus,as revealed by a monoclonal antibody

Dissociated cells from the margin and tentacles of the hydromedusa Polyorchis penicillatus were centrifuged in a Percoll gradient to remove cnidocytes. The resulting formaldehyde-fixed cells were used to inoculate mice to produce monoclonal antibodies. One of the hybridomas, which secreted antibodies against all neurons, was cloned and designated as mAb 5C6. Immunohistochemical labelling with mAb 5C6 of whole-mount preparations and paraffin sections provided a far more complete picture of the organisation of the hydromedusan nervous system than was previously available when using neuronal labelling techniques that restrict labelling to certain neuronal types. Besides confirming anatomical features described in earlier studies these techniques allowed us to discover a number of new structures and to determine connections that were only suspected. Such findings included: 1.The discovery of an arch-like connection between the swimming motor neuron network at the apices of the subumbrellar muscle sheets 2.An orthogonal network connecting each pair of radial nerves in each radius 3.Continuity of a central branch of the radial nerve with the radial innervation of the manubrium 4.Details of the sensory neuronal contribution to the microanatomy of the ocelli and cnidocyte batteries 5.Presence of specialised receptor cells in the margin at the bases of tentacles 6.Neurons apparently innervating the radial muscles of the velum 7.Isolated neurons in the peduncle and gonads Electronic Publication     

Identification of bovine CD52-like molecule by monoclonal antibody IVA-543: distribution of CD52-like molecule in the bull genital tract

The bovine maturation-associated sperm membrane antigen CD52-like molecule has been analysed using a mouse anti-sperm monoclonal antibody developed against bull spermatozoa. The antigen recognised by monoclonal antibody IVA-543 was detected on blood mononuclear cells (including lymphocytes and monocytes) and on a minor population of polymorphonuclear leukocytes. The bovine CD52-like molecule is secreted by the epididymal epithelium and then it is inserted into the sperm membrane during the epididymal transport in the distal part of epididymis. The CD52-like molecule was absent from spermatozoa derived from testes, and the highest proportion of IVA-543-reactive sperm was observed in the cauda epididymis (91.6%).This study has shown that the new molecule identified on bovine cells has properties analogous to those previously described for CD52 molecules in man, mouse, rat, monkey, and dog.     

Anti-(glioma surface antigen) monoclonal antibody G-22 recognizes overexpressed CD44 in glioma cells

We raised an anti-glioma monoclonal antibody, named G-22, that specifically recognizes a human gliomaassociated surface antigen. Proven to be useful for target imaging of malignant gliomas after radioisotope labeling and cerebrospinal fluid diagnosis by enzyme-linked immunospecific assay, G-22 was found to immunoprecipitate an 83-kDa glycoprotein of the human glioma U-251MG cell. We purified this antigen by G-22-coupled cyanogenbromide-activated Sepharose affinity chromatography, and sequence analysis demonstrated that the 54 amino acid residues were identical to positions 55–108 of human CD44. The results show that the smallest spliced form (85 kDa) of CD44 is strongly expressed in glioma cells.     

Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.     

Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4

Programmed cell death-ligand 1 (PD-L1), which is a ligand of programmed cell death-1 (PD-1), is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1) expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb), L₁Mab-4 (IgG_2b, kappa), using cell-based immunization and screening (CBIS) method and investigated hPD-L1 expression in oral cancers. L₁Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4) in flow cytometry and stained oral cancers in a membrane-staining pattern. L₁Mab-4 stained 106/150 (70.7%) of oral squamous cell carcinomas, indicating the very high sensitivity of L₁Mab-4. These results indicate that L₁Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers.     

Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.     

De novo protein sequencing,humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody

QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs), suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications, QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart, including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs.     

Immunodetection of partially glycosylated isoforms of alpha-dystroglycan by a new monoclonal antibody against its beta-dystroglycan-binding epitope

The alpha/beta dystroglycan (DG) complex links the extracellular matrix to the actin cytoskeleton. The extensive glycosylation of alpha-DG is believed to be crucial for the interaction with its extracellular matrix-binding partners. We characterized a monoclonal antibody, directed against the beta-DG-binding epitope ( approximately positions 550-565), which recognizes preferentially hypoglycosylated alpha-DG. In Western blot, the antibody was able to detect a number of partially glycosylated alpha-DG isoforms from rat brain and chicken skeletal muscle tissue samples. In addition, we demonstrated its inhibitory effect on the interaction between alpha- and beta-DG in vitro and preliminary immunostaining experiments suggest that such hypoglycosylated alpha-DG isoforms could play a role within cells.     

An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.     

Atorvastatin reduces CD68, FABP4, and HBP expression in oxLDL-treated human macrophages

With the aim of identifying new target genes that could contribute to limit foam cell formation, we analyzed changes in the pattern of gene expression in human THP-1 macrophages treated with atorvastatin and oxidized-LDL (oxLDL). To this end, we used a human cDNA array containing 588 cardiovascular-related cDNAs. Exposure to oxLDL resulted in differential expression of 26 genes, while coincubation with atorvastatin modified the expression of 29 genes, compared to treatment with oxLDL alone. Changes in the expression of candidate genes, potentially connected to the atherosclerotic process, were confirmed by quantitative RT-PCR and Western blot. We show that atorvastatin prevents the increase in the expression of scavenger receptor CD68 and that of fatty acid binding protein 4 caused by oxLDL. In addition, atorvastatin reduces the expression of HDL-binding protein, apolipoprotein E, and matrix metalloproteinase 9. These findings are relevant to understand the direct antiatherogenic effects of statins on macrophages.     

Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody,LL2

LL2 is a murine monoclonal antibody IgG2a reactive with B cells and non-Hodgkin's B-cell lymphoma, which, in a radioiodinated form, induces responses in lymphoma patients [Goldenberg et al. (1991) J Clin Oncol 9:548–564]. In this report we identify LL2 as a member of the CD22 cluster. The molecular size of the antigen, its expression profile, and competitive blocking studies were used to establish this identification. By Western blot analysis and immunoprecipitation studies using the Raji Burkitt's lymphoma cell line metabolically labelled with [³H]leucine, the LL2 antigen was determined to correspond to a molecular mass of 140 kDa. The molecular mass of the LL2 antigen, and the B-cell-restricted reactivity of the LL2 antibody, were consistent with both the CD21 and CD22 clusters. To assess additional similarities and differences between LL2 and anti-CD22 and anti-CD21, the binding of these mAb to cultured cell lines. Nalm-6 and Molt-4, was compared by flow cytometry. The binding profile of LL2 on these cell lines was consistent with anti-CD22, but not anti-CD21. Sequential immunoprecipitation and cross-blocking studies with anti-CD22 monoclonal antibodies recognizing established CD22 epitopes were performed to confirm that LL2 reacts with CD22 and to determine which epitope LL2 recognizes. Binding of¹³¹I-LL2 to Raji cells is inhibited over 90% by prior incubation of the target cells with unlabelled RFB4, indicating that LL2 belongs to the same epitope group as RFB4, i.e., epitope B.This work was supported in part by USPHS grant CA39841 from the NIH     

A new monoclonal antibody, 4-1a,that binds to the amino terminus of human lipoprotein lipase

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5–25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL–GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.     

Protection of mice against Clostridium chauvoei infection by anti-idiotype antibody to a monoclonal antibody to flagella

Abstract Polyclonal rabbit anti-idiotypic antibody (anti-Id) against the protective monoclonal antibody specific to the flagella of Clostridium chauvoei was produced, purified, and characterized. Anti-Id inhibited the binding of its related monoclonal antibody to the flagellar antigen, suggesting that the anti-Id bore an internal image of the flagellar antigen. When mice were immunized with anti-Id intraperitoneally, the survival rate increased significantly, compared with mice immunized with normal rabbit IgG ( P < 0.01), and specific anti-flagellar antibodies were induced.     

Pharmacokinetics and biodistribution of a new anti-episialin monoclonal antibody 139H2 in ovarian-cancer-bearing nude mice

Summary The new murine anti-episialin monoclonal antibody (mAb) 139H2 has been selected for its strong reactivity with a series of human ovarian cancer xenografts. In the present report we describe the characteristics of mAb 139H2 investigated in vitro as well as in vivo. Scatchard plot analysis using the human ovarian cancer cell line NIH:OVCAR-3 showed an affinity constant of 1 × 10⁸ M^–1 and the expression of 7 × 10⁶ antigenic sites/cell. Reactivity with OVCAR-3 xenograft tissue was intense, localized at the cell membrane, heterogeneously distributed, and mainly detectable at the apical site of the cell. Administration of radiolabelled mAb 139H2 to nude mice bearing s.c. OVCAR-3 xenografts showed specific uptake in the tumour up to 9% of the injected dose/g. The maximum uptake in the tumour was retained for 3.5 days and mAb 139H2 cleared from the tumour with a half-life of 5.5 days. The half-life in blood was 50 h and no antibody-antigen complex formation could be detected. Poor uptake and no retention in episialin-negative WiDr colon cancer xenografts demonstrated specificity. Administration of an excess of an unlabelled irrelevant mAb did not influence the uptake in the OVCAR-3 xenografts or in other tissues. In contrast, tumour uptake decreased after addition of 300 µg or more unlabelled mAb 139H2 to a tracer dose of radiolabelled mAb 139H2. The uptake of mAb 139H2 in OVCAR-3 xenografts appeared inversely related to the tumour size.Supported by the Dutch Cancer Society     

Production of monoclonal antibody against a hemolysin (Vp-TRH) produced by Vibrio parahaemolyticus

The antigenicity of a hemolysin (Vp-TRH: Vp-TDH related hemolysin) produced by Kanagawa phenomenon-negative clinical isolates of Vibrio parahaemolyticus was studied using monoclonal antibodies (MAbs). A total of 12 hybridoma clones which produced MAbs against Vp-TRH were established. All MAbs contained the Kappa light chain and were IgG type. These MAbs were divided into a minimum of 5 different specificity groups, including antibodies specific to Vp-TRH and common to both Vp-TRH and Vp-TDH, a possible pathogenic toxin of Kanagawa phenomenon-positive V. parahaemolyticus. These results clearly show the immunological similarity and dissimilarity (specificity) of Vp-TRH and Vp-TDH.     

Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary (CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA, a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding to its epitope. Received: 5 October 1998 / Accepted: 19 November 1998     

Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas

 Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (K _d = 1.7×10^–10 M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans. Received: 9 September 1996 / Accepted: 25 September 1996     

Effector functions of CD8-positive guinea pig T lymphocytes

The response of guinea pig T lymphocytes to different stimuli was analysed with focus on the functions of CD8-positive T cells, which so far had been poorly defined in this animal model. For identification and purification of guinea pig cytotoxic T lymphocytes, three monoclonal antibodies, directed against the CD8 differentiation antigen were characterized and compared with respect to expression pattern and biochemical characteristics of the corresponding cell surface antigen. The antibodies were used for the identification of the cytotoxic T lymphocyte subpopulation within alloreactive T cell lines, and for the depletion of CD8-positive cells in in vitro assays. Purified CD4- and CD8-positive cells were tested for their ability to proliferate in response to antigen, mitogen or anti-guinea pig Thy-1 monoclonal antibodies. Both, CD4- and CD8-positive cells showed IL-2 release and subsequent proliferation after polyclonal stimulation. Cytotoxic activity in CD8-positive alloreactive T cells was expressed in vitro only after repeated stimulation.     

Interaction of a monoclonal antibody with the urease of Ureaplasma urealyticum

The monoclonal antibody (mAb) U.u. 5B2 against the urease of U. urealyticum (U.u) serotype 8, was affinity purified and was found to be an IgG 1 type, with an apparent dissociation constant of 2.9 × 10⁻¹⁰ M. Immunoblot analysis of the cytoplasmic proteins of U.u electrophoresed under non-denaturing conditions showed a reaction with a major and a minor band corresponding to the urease activity. The mAb, U.u.5B2 inhibited the urease activity up to 93% and precipitated a protein from the cytoplasm with a molecular weight of 75 kDa, corresponding to the purified urease subunit. This mAb also reacted with six other U.u serotypes but not with Jack bean urease, other urease containing bacteria or genital mycoplasma.