Specific localization of In-111-labeled monoclonal antibody versus 67-Ga-labeled immunoglobulin in mice bearing human breast carcinoma xenografts

Summary A murine monoclonal antibody reacting with more than 95% of all breast cancers was radiolabeled with In-111 and injected IP into nude mice bearing human breast carcinoma xenografts, together with Ga-67-labeled normal mouse immunoglobulin. Images were produced with a gamma camera in dual isotope mode. Tumors could be localized clearly with In-111-labeled specific monoclonal antibody, but improved visualization was obtained after computer-assisted subtraction of the image with Ga-67-labeled nonspecific immunoglobulin. The tumor-to-tissue contrast was improved from 2.3 to 5.9 after subtraction. Imaging with In-111-radiolabeled monoclonal antibody was superior to imaging with iodinated antibody. For the first time it was shown that images of two chemically related isotopes, Ga-67 and In-111, coupled to nonspecific and specific antibody, respectively, and simultaneously injected, can be subtracted to show the preferential uptake of the specific antibody in the tumor. As these isotopes are routinely used in clinical practice this technique may prove to be more practical for immunodetection of tumors in patients than existing imaging techniques.     

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.     

The immunotherapeutic effects in tumor-bearing mice of Ly-6 monoclonal antibodies

In vivo administration of Ly-6 mAb which recognize lymphoid differentiation Ag encoded for by the Ly-6 gene complex were found to have significant beneficial immunotherapeutic effects in tumor-bearing mice. The effectiveness of the mAb treatment in mice bearing sarcomas, leukemias, or melanomas was dependent on the host and not the tumor Ly-6 phenotype. The treatment was effective in nu/nu mice, although a more pronounced inhibition of tumor growth occurred in immunocompetent mice. The effectiveness of the therapy in immunocompetent mice was dependent on the dose of mAb and was influenced by the immunogenicity of the tumor. It ranged from significant growth inhibition of weakly immunogenic tumors to complete rejection of strongly immunogenic tumors. The results of cell-mediated cytotoxic assays of splenocytes from mAb-treated mice indicated that Ly-6 mAb treatment induced and/or augmented tumor-specific CTL as well as NK cell activity in these mice. Ly-6 mAb treatment represents a novel method for tumor immunotherapy using mAb recognizing lymphoid differentiation Ag with functional activities.     

Modulation of the M2 muscarinic acetylcholine receptor activity with monoclonal anti-M2 receptor antibody fragments

Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.     

Characterization of monoclonal antibody fragments produced by plant cells

Production of a murine IgG1 was investigated using hairy roots, shooty teratomas, and suspended cells of transgenic tobacco. In all cases, in addition to complete assembled antibody, two to four major antibody fragments accumulated in the biomass. A range of protease inhibitors, protein-stabilizing agents, inhibitors of N-glycosylation and protein secretion, glycan-reactive agents, and affinity probes was used to characterize these fragments and investigate their sites and mechanisms of formation. The fragments were not experimental artifacts caused by antibody degradation during tissue homogenization and sample preparation, nor did they represent glycosylation variants. All of the molecules were actively secreted into the culture media and some showed evidence of Golgi-associated glycan processing, indicating they were not assembly intermediates. Antibody fragments of 50 and 80 kDa were identified mainly as the products of extracellular degradation in the root and shoot apoplast; the 80-kDa fragment was also present in cell suspension medium, and in suspended cell biomass toward the end of the growth phase. Larger 120- and 135-kDa fragments were most likely produced by proteolytic degradation along the secretory pathway outside of the endoplasmic reticulum (ER) and Golgi apparatus; the carbohydrate residues of the 135-kDa antibody suggest formation between these organelles. Inhibition of protein secretion and retention of antibody in the ER and/or Golgi reduced fragmentation and increased antibody accumulation levels, probably by reducing exposure to the principal sites of protease activity. This work highlights the importance of foreign protein degradation in plant tissues as a mechanism for posttranslational product loss. Identifying the nature of these degradative processes is a first step toward alleviating their effects, improving protein yields, and enhancing the feasibility of plants as a commercial means for large-scale protein production.     

东北虎免疫球蛋白单克隆抗体的制备与鉴定

【目的】提纯东北虎免疫球蛋白并制备其单克隆抗体(McAb),为东北虎传染性疾病诊断试剂盒研制、疫苗免疫效果评估及基础免疫学研究奠定基础。【方法】以饱和硫酸铵和重组的Protein G相结合,提纯东北虎免疫球蛋白作为抗原,免疫C57BL/6小鼠,和骨髓瘤细胞系Sp2/0-Ag14相融合制备杂交瘤细胞,ELISA和蛋白免疫印迹筛选及鉴定阳性克隆,应用杂交瘤产生的McAb鉴定提纯免疫球蛋白及制备的McAb免疫学活性,非竞争性ELISA法测定了其亲和常数。【结果】得到3株稳定分泌抗东北虎免疫球蛋白McAb的杂交瘤细胞,产生的抗体全部识别免疫球蛋白的重链,通过对猫泛白细胞减少症病毒株(FPV-HLJ)灭活疫苗免疫东北虎的抗体消长的检测,证明提纯的免疫球蛋白及其McAb的免疫活性。【结论】Protein G能够用于东北虎免疫球蛋白的提纯,ATD11杂交瘤产生的McAb与抗原有较强的结合能力和免疫学活性,为今后开展东北虎传染病的诊断方法及疫苗研究奠定基础。     

Discrimination between rat thiostatin (T-kininogen) and one of its cystatin-like inhibitory fragments by a monoclonal antibody, and localization of the epitope.

A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats.     

Immunofluorescent localization of Schistosoma mansoni circulating cathodic antigen in tissues of infected mice using monoclonal antibody

In the present study the kinetics of the uptake and deposition of the major circulating cathodic antigen (CCA) of Schistosoma mansoni in liver, spleen, and kidney of S. mansoni infected Swiss mice was investigated in relation to the duration of infection and infection dose (50, 100, 200 cercariae). The presence of antigen was studied with a direct immunofluorescence reaction on frozen sections of the mouse organs, using a fluorescein isothiocyanate (FITC)-labelled mouse IgM monoclonal antibody recognizing a repeating epitope of CCA. CCA was demonstrable from 2 weeks post infection (p.i.) onwards in Kupffer cells in the liver, from 3-4 weeks p.i. onwards in macrophages in the marginal zones in the spleen and from 8 weeks p.i. onwards in kidney glomeruli. The immunofluorescence reactions on CCA in kidney glomeruli, however, remained relatively weak.     

Polyethylene glycol modification of the monoclonal antibody A7 enhances its tumor localization

The F(ab')2 fragment of murine monoclonal antibody A7 was covalently bonded to polyethylene glycol (PEG, molecular weight: 5000) and the conjugate was compared to the parent F(ab')2 fragment by in vitro and in vivo studies. PEG-conjugated antibody fragment retained its antigen-binding activity in a competitive radioimmunoassay. The conjugate had a longer half-life and showed increased accumulation in tumors. Although the tumor: blood ratio for parent F(ab')2 fragment was higher than that for the conjugate, it showed higher value than whole MAb A7. The tissue: blood ratios were kept low with the conjugate, indicating that the conjugate was uptaken to normal organ with lesser extent, as compared with parent F(ab')2 fragment. Our findings indicate that this PEG-conjugated F(ab')2 fragment could be a promising carrier for use in targeting cancer chemotherapy.     

A novel monoclonal antibody induces the differentiation of monocyte leukemic cells

A cell surface antigen (possibly a receptor) on human myeloid cell lines, that may play a crucial role in the differentiation of promonocytic cells, was detected by a murine monoclonal antibody (MAb 710F). When the myeloid cell lines, HL-60, THP-1 and U937, were cultured with the MAb 710F following pretreatment with phorbol 12-myristate 13-acetate (PMA), they displayed both cellular spreading and a strong adherence to the culture dish substratum. On transforming from their original round shape, the induced cells displayed the well-developed microvilli, spindles, or raffles that are characteristic of macrophages or dendrocytes. Similar morphological changes were not induced by treatment with either PMA or MAb 710F alone. By contrast, the lymphoid cell lines did not respond to these reagents. These results suggested that a signal for cellular adhesion and cytoskeletal reconstitution was triggered by the binding of MAb 710F to an antigen on the PMA-primed cells. Thus, the antigen 710F, which is preferentially expressed on peripheral blood monocytes, may represent a cell surface receptor closely associated with differentiation. The antigen/receptor 710F was shown to be a N-glycosylated protein with Mr. of 35-70k. This MAb may prove useful as a tool for both studies on the differentiation of myeloid lineage cells as well as on monocyte/macrophage adhesion function.     

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)₂, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications.     

A monoclonal antibody against human urokinase: characterization of the epitope and its localization in human kidney

Monoclonal antibodies against human plasminogen activator urokinase have been produced. A G62 hybridoma-producing antibody (IgG) was purified on a DEAE-cellulose column, and it proved useful for the measurement, identification and purification of antigens that had approximate molecular weights of 55- and 33-Kdaltons. For immunochemical measurements and purification, a competitive enzyme-linked immunosorbent assay (ELISA) and affinity chromatography using antibody-immobilized Sepharose 4B were developed. The ELISA has sensitivity to 20 p mole antigen molecules. The binding capacity of the antigen on the affinity column was evaluated on SDS-polyacrylamide slab gels as well as by fibrin autography and ELISA. Results showed that there was quantitative purification with no loss of enzyme activity in the one-step procedure. Western blotting and affinity binding showed antigenic bands with apparent molecular weights of 55- and 33-Kdaltons. Because the 55-Kdalton form contains 33- and 22-Kdalton components connected by a disulfide bond, the epitope domain is present on the 33-Kdalton chain. Using this antibody, we examined human kidney sections by direct immunofluorescence to locate the antigen. It was found in epithelial cells convoluted segments, in glomerulus cells and in capillary endothelial cells, evidence that renal tubular cells synthesize the antigen which then is secreted in urine.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

Use of monoclonal antibody and colloidal gold in E.M. localization of von Willebrand factor in megakaryocytes and platelets

The subcellular localization of Factor VIII/von Willebrand protein (VIII R:Ag) is studied with monoclonal antibody and gold immunocytochemical technique. Monoclonal antibody against purified VIII R:Ag is brightly fluorescent on megakaryocytes and platelets. In E.M., gold immunolabeling is performed on thin cell sections of human megakaryocytes and platelets. Different embedding materials are used to preserve the antigenicity : Epon embedded megakaryocytes show a high concentration of VIII R:Ag in alpha-granules using 4F9 monoclonal antibody. In comparison, lowicryl K4M embedded material does not improve the same specificity, only a few platelets granules were stained. This subcellular localization, in full agreement with biochemical results appears visualized for the first time in E.M.     

Crystallization of the complexes between M315 idiotope and its monoclonal anti-idiotopic antibody Fab fragment

The Fab fragment of a monoclonal anti-idiotopic antibody against M315 has been isolated and its complexes with Fv and Fab' fragment of M315 have been crystallized by using poly(ethylene glycol) 6000 or ammonium sulfate. X-ray diffraction photographs showed that the crystal of the complex with Fv diffracts better than that with Fab'. The Fv-complexed crystal was shown to be tetragonal I4, with cell dimensions a = 152 A and c = 69 A, and to contain one complex molecule of about 75,000 molecular weight in the crystallographic asymmetric unit.