Inhibition of attachment and growth of tumor cells on collagen by a monoclonal antibody

Summary A murine monoclonal antibody, VM-1, which binds to basal cells of normal human epidermis, reduces the ability of human squamous cell carcinoma cells (SCL-1) derived from the skin to attach and spread on collagen by about 50% and causes cell rounding. Similar effects have been previously shown using normal human keratinocytes. The attachment of cell lines derived from human lung squamous cell carcinomas (SW1271 and SW900), melanoma A375, glioblastoma 126, and fibrosarcoma HT1080 is also inhibited by this antibody. VM-1 antibody does not bind to normal human fibroblasts, benign nevus cells, or the human B-cell-derived line 8866. VM-1 antibody inhibits the growth of SCL-1 cells in vitro as measured by cell numbers and [³H]thymidine ([³H]TdR) incorporation. It is not cytolytic in the presence of complement as measured by⁵¹Cr release. Repeated treatment of SCL-1 cells with VM-1 antibody significantly reduces the proportion of SCL-1 cells that attach to collagen. In addition, after treatment of SCL-1 cells with VM-1 antibody, several proteins can no longer be demonstrated by gel electrophoresis of the cell-free supernatant. The VM-1 antibody effect on attachment and spreading is partially reversed by pretreatment of the collagen surface with laminin and fibronectin, but not with the carbohydrates chondroitin-6-sulfate or hyaluronic acid or with the protein lysozyme. By fluorescence staining, the antigen recognized by VM-1 antibody is membrane-bound and Triton X-100 extractable. The VM-1 antigen is excluded from Bio-Sil TSK-400 and sediments at about 10.5 S. It has a covalent molecular weight on the order of 10⁶. Proteinase K digestion produces VM-1 antibody reactive fragments, assumed to be polysaccharides, with a polydisperse molecular weight distribution in the range 5000 to 30 000. The VM-1 antigen is partially lost from solution on boiling and is no longer detectable in the aqueous or organic phase after chloroform-methanol extraction. The properties of the VM-1 antigen are consistent with those of a proteoglycan involved in attachment and spreading of kerationcytes and certain tumor cells on collagen. This research was supported by a grant from the Elsa U. Pardee Foundation, a Training Grant from the National Institutes of Health, Bethesda, MD, and the Psoriasis Research Institute. Part of this work has appeared as an abstract in Fed. Proc. 43:1929, Abst. #2994, 1984.     

Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin

A hybrid hybridoma secreting a bispecific hybrid mAb (bsmAb), which recognizes both the epidermal growth factor receptor (EGF-R) and the drug doxorubicin, was produced by somatic hybridization of two hybridomas. The bsmAb obtained was able to retarget doxorubicin cytotoxicity in vitro specifically on EGF-R-positive cells exerting at the same time an antidotal effect on cells that did not overexpress the EGF-R. Distribution studies in mice indicate that the bsmAb selectively delivers the drug to tumour cells and modifies doxorubicin biodistribution with a statistically significant decrease of drug concentration in the intestine, which is the main target of early anthracycline toxicity. In keeping with this finding is the remarkable antidotal activity exerted by bsmAb in mice treated with doxorubiein, which is proved by retardation in loss of body weight and mortality. The effectiveness on tumour growth of the mAb followed by the administration of doxorubicin appears to be equal to that of the drug alone; however, the bsmAb exerts a remarkable antidotal activity.     

Production of a monoclonal antibody directed against the nerve growth factor receptor from sympathetic membranes

Spleen cells from BALB/c mice immunized with a plasma membrane-enriched fraction from rabbit sympathetic ganglia were fused with the mouse myeloma NS1. A hybrid clone was obtained that produced monoclonal antibody directed against the receptor for nerve growth factor (NGF). The antibody, identified as IgG, was able to immunoprecipitate solubilized NGF receptor in the presence or absence of bound NGF. The antibody bound specifically to sympathetic membranes with high affinity but did not affect the binding of 125I-NGF to its receptor in sympathetic or sensory neurons or PC12 cells.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

The development and purification of a bispecific antibody for lymphokine-activated killer cell targeting against the rat colon carcinoma CC531

In vivo targeting of lymphokine-activated killer (LAK) cells to tumour deposits by bispecific monoclonal antibodies (bimAb) may be a way to improve adoptive immunotherapy. We developed a bimAb against adherent LAK (ALAK) cells and colon tumour CC531 in Wag rats. The bimAb was produced by somatic hybridization of two mouse hybridomas, one producing monoclonal antibodies (mAb) against CD8 (IgG2b, OX8), and the other producing mAb against a CC531-associated antigen (IgG1, CC52). A bimAb-producing clone was selected by an enzyme-linked immunosorbent assay with CC531 tumour cells. BimAb were purified from ascitic fluid by protein A affinity chromatography. Each of five pooled peak fractions was analysed by flow cytometry for the presence of bimAb. Most bimAb were found in a fraction that was eluted at pH 4.5 from protein A. FPLC analysis of this fraction revealed that no parental antibodies were present. The OX8 × CC52 bimAb greatly increased conjugate formation in vitro between ALAK cells and CC531. Results of⁵¹Cr-release assays with CC531 as target cells and ALAK cells as effector cells were not significantly different in the presence or in the absence of the bimAb. The methods we used here, a cell enzyme-linked immunosorbent assay and flow cytometry, are simple methods for development and purification of a bimAb when a functional selection method is not a priori available. The OX8 × CC52 bimAb we developed this way may increase in vivo tumour targeting of ALAK cells and thus augment antitumour effect in vivo.     

Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma

The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc.     

Pancreatic fate of a (125)I-labelled mouse monoclonal antibody directed against pancreatic B-cell surface ganglioside(s) in control and diabetic rats

The possible use of a mouse monoclonal antibody directed against rat pancreatic B-cell surface ganglioside(s) and labelled with radioactive iodine for selective imaging of the endocrine pancreas by a non-invasive procedure was investigated by following its pancreatic fate in experiments conducted either in vitro by incubation of rat isolated pancreatic islets, acinar tissue and pancreatic pieces or in vivo after intravenous injection of the (125)I-labelled antibodies ([(125)I]gamma-G). Although the binding of [(125)I]gamma-G per microg protein was about one order of magnitude higher in isolated islets than in acinar tissue, no significant difference was detected when comparing pancreatic pieces or isolated islets from control animals and rats rendered diabetic by one or two prior administrations of streptozotocin (STZ rats). Likewise, except in one set of experiments, no significant difference was found between control animals and STZ rats, when measuring the radioactive content of the pancreatic gland, relative to that of plasma, 1-4 days after the intravenous injection of [(125)I]gamma-G. These findings indicate that under the present experimental conditions, the mouse monoclonal antibody labelled with radioactive iodine does not appear to be a promising tool for selective imaging of the endocrine pancreas, e.g. by single photon emission computerized tomography.     

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Pharmacokinetics and brain uptake in the rhesus monkey of a fusion protein of arylsulfatase a and a monoclonal antibody against the human insulin receptor

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder of the brain caused by mutations in the gene encoding the lysosomal sulfatase, arylsulfatase A (ASA). It is not possible to treat the brain in MLD with recombinant ASA, because the enzyme does not cross the blood‐brain barrier (BBB). In the present investigation, a BBB‐penetrating IgG‐ASA fusion protein is engineered and expressed, where the ASA monomer is fused to the carboxyl terminus of each heavy chain of an engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor‐mediated transport on the endogenous BBB insulin receptor, and acts as a molecular Trojan horse to ferry the ASA into brain from blood. The HIRMAb‐ASA is expressed in stably transfected Chinese hamster ovary cells grown in serum free medium, and purified by protein A affinity chromatography. The fusion protein retains high affinity binding to the HIR, EC50 = 0.34 ± 0.11 nM, and retains high ASA enzyme activity, 20 ± 1 units/mg. The HIRMAb‐ASA fusion protein is endocytosed and triaged to the lysosomal compartment in MLD fibroblasts. The fusion protein was radio‐labeled with the Bolton–Hunter reagent, and the [¹²⁵I]‐HIRMAb‐ASA rapidly penetrates the brain in the Rhesus monkey following intravenous administration. Film and emulsion autoradiography of primate brain shows global distribution of the fusion protein throughout the monkey brain. These studies describe a new biological entity that is designed to treat the brain of humans with MLD following non‐invasive, intravenous infusion of an IgG‐ASA fusion protein. Biotechnol. Bioeng. 2013; 110: 1456–1465. © 2012 Wiley Periodicals, Inc.     

Development of a bispecific monoclonal antibody against a gallium-67 chelate and the human melanoma-associated antigen p97 for potential use in pretargeted immunoscintigraphy

A bispecific monoclonal antibody (bsmAb) has been developed against the human melanoma-associated antigen p97 and an octahedral gallium chelate (Ga-HBED) using the hybrid hybridoma technology. As tetradomas were expected to produce a maximum of ten different molecular species of immunoglobulins, the bispecific antibody was purified from this mixture by consecutive protein A affinity and cation-exchange chromatographic techniques. Although it was established by sodium dodecyl sulphate/polyacrylamide gel electrophoresis that the heavy (H) and light (L) chains of the two parental immunoglobulins were mismatched in the bispecific antibody, results from cell enzyme-linked immunosorbent assay indicated significant dual specific binding to both the melanoma cells and⁶⁷Ga-HBED. Other in vitro techniques further confirmed that the bsmAb Bi 5-56-II-17 still retained about 30%–40% simultaneous binding capacity to both the antigens, as would have been expected in a bsmAb that has ideally matched H and L chains. Preliminary in vivo experiments using nude mice bearing the human melanoma xenografts showed that the bsmAb Bi 5-56-II-17 was able to target the radioactive gallium chelate to the tumours twice as efficiently compared to the monospecific, bivalent gallium chelate antibody.     

Development of a bispecific F(ab′)2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity

 A bispecific F(ab′)₂ antibody conjugate (BAC) was constructed against the complement receptor CR3 of macrophages and a variant CD44 (CD44v6) antigen of rat pancreatic adenocarcinoma cells to redirect macrophage-mediated tumor cytotoxicity. The Fab′ fragments of monoclonal antibodies (mAb) 1.1ASML and OX42, recognizing the CD44v6 and the CR3 antigens respectively, were chemically coupled at the hinge region using 5,5′-dithiobis(2-nitrobenzoate). The BAC was characterized in vitro for its specific, dual binding capacity to CD44v6 and CR3 antigens. Although the monovalence of the BAC resulted in lower avidities to both the antigens as expected, it was still able to form stable cross-linkages between tumor cells and macrophages in culture leading to the formation of “clump-like” cell aggregates. The in vitro and in vivo tumor-targeting capacity of the BAC was compared with that of the parental antitumor mAb 1.1ASML, which mediates tumor killing by antibody-dependent cell cytotoxicity. These results showed that, even though the bivalent mAb 1.1ASML did not mediate stable cross-linking of target and effector cells, its Fc-receptor-mediated killing of tumor cells was more effective when compared to the BAC. Thus, this study strongly supports the hypothesis that firm persistent binding between effector and target cells per se is not as important as the choice of trigger molecule used for macrophage activation to redirect their tumor cytotoxic potential effectively. Received: 2 May 1996 / Accepted: 21 May 1996     

Anticancer activity of TTAC-0001, a fully human anti-vascular endothelial growth factor receptor 2 (VEGFR-2/KDR) monoclonal antibody,is associated with inhibition of tumor angiogenesis

Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.     

Anticancer activity of TTAC-0001, a fully human anti-vascular endothelial growth factor receptor 2 (VEGFR-2/KDR) monoclonal antibody,is associated with inhibition of tumor angiogenesis

Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.     

Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer showed that scFv M97 transfection significantly prolonged the survival time of nude mice. The results indicate that intracellular antibody technology represents a novel and efficient way to abrogate selectively the activity of type IV collagenase.     

Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer     

Effect of feed rate on growth rate and antibody production in the fed-batch culture of murine hybridoma cells

Batch and fed-batch cultures of a murine hybridomacell line (AFP-27) were performed in a stirred tankreactor to estimate the effect of feed rate on growthrate, macromolecular metabolism and antibodyproduction. Macromolecular composition was foundto change dynamically during batch culture ofhybridoma cells possibly due to active production ofDNA, RNA and protein during the exponential phase.Antibody synthesis is expected to compete with theproduction of cellular proteins from the amino acidpool. Therefore, it is necessary to examine therelationship between cell growth in terms of cellularmacromolecules and antibody production. In this study,we searched for an optimum feeding strategy bychanging the target specific growth rate in fed-batchculture to give higher antibody productivity whileexamining the macromolecular composition. Concentratedglucose (60 mM) and glutamine (20 mM) in DR medium(1:1 mixture of DMEM and RPMI) with additional aminoacids were fed continuously to the culture and thefeed rate was updated after every sampling to ensureexponential feeding (or approximately constantspecific growth rate). Specific antibody productionrate was found to be significantly increased in thefed-batch cultures at the near-zero specific growthrate in which the productions of cellular DNA, RNA,protein and polysaccharide were strictly limited byslow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.     

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.     

Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of (125)I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized (125)I-225 mAb is recycled to the surface much more efficiently than internalized (125)I-EGF. Also, we found that internalization of (125)I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidenced by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.     

Protection and suppression of the humoral immune response in mice mediated by a monoclonal antibody against the M epitope of Brucella

Abstract The capacity of the BmE10-5 monoclonal antibody (mAb), with specificity for the Brucella spp. M epitope, to confer protection against infection with B. abortus 2308 (A-dominant strain) has been evaluated. Injected before infection, the BmE10-5 mAb diminished the bacterial counts in spleen from week 1 to week 8 postinfection and in liver from week 4 to week 7. Thus, protection mediated by the BmE10-5 mAb, as measured by a reduction in the bacterial counts in both spleen and liver, was demonstrated from week 2 to week 8 postinfection. The humoral immune response of IgG, IgM, IgG1 and IgG3 antibodies, specific against the B. abortus 2308 smooth lipopolysaccharide, was clearly suppressed in all the mice protected with the BmE10-5 mAb, thus demonstrating the importance, in protecting against infection, of the existence in serum of M-epitope-specific antibodies at the same time the infection is acquired. The development of subcellular vaccines including the Brucella M epitope could constitute an interesting alternative to attenuated living vaccines.     

Effect of weak combined static and extremely low‐frequency alternating magnetic fields on tumor growth in mice inoculated with the Ehrlich ascites carcinoma

It has been shown that the ultralow‐frequency extremely weak alternating component of combined magnetic fields (MFs) exhibits a marked antitumor activity. The parameters of this component have been found (frequency 1, 4.4, 16.5 Hz or the sum of these frequencies; intensity 300, 100, 150–300 nT, respectively) at which this MF in combination with a collinear static MF of 42 µT inhibits or suppresses the growth of Ehrlich ascites carcinoma (EAC) in mice. It was shown that the exposure of mice with EAC to combined MFs causes structural changes in some organs (liver, adrenal glands), which are probably due to the total degradation of the tumor tissue. In mice with transplanted EAC, the tumor tissue after exposure to weak MFs was practically absent, as distinct from control animals in which the invasion of the tumor into the adipose tissue surrounding the kidneys, mesenteric lymph nodes, and spermatic appendages was observed. In animals without tumors, no pathological deviations from the norm in the structure of organs and tissues occurred after exposure to weak MF, indicating that this factor per se is not toxic to the organism. Bioelectromagnetics 30:343–351, 2009. © 2009 Wiley‐Liss, Inc.