Anti-glycan monoclonal antibodies: Basic research and clinical applications

Anti-glycan monoclonal antibodies have important applications in human health and basic research. Therapeutic antibodies that recognize cancer- or pathogen-associated glycans have been investigated in numerous clinical trials, resulting in two FDA-approved biopharmaceuticals. Anti-glycan antibodies are also utilized to diagnose, prognosticate, and monitor disease progression, as well as to study the biological roles and expression of glycans. High-quality anti-glycan mAbs are still in limited supply, highlighting the need for new technologies for anti-glycan antibody discovery. This review discusses anti-glycan monoclonal antibodies with applications to basic research, diagnostics, and therapeutics, focusing on recent advances in mAbs targeting cancer- and infectious disease-associated glycans.     

In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant gram-positive cocci

BACKGROUND: Monoclonal (mAbKT) and recombinant single-chain (scFvKT) anti-idiotypic antibodies were produced to represent the internal image of a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity, including gram-positive cocci. Pathogenic eukaryotic and prokaryotic microorganisms, such as Candida albicans, Pneumocystis carinii, and a multidrug-resistant strain of Mycobacterium tuberculosis, presenting specific, although yet undefined, KT-cell wall receptors (KTR), have proven to be killed in vitro by mAbKT and scFvKT. mAbKT and scFvKT exert a therapeutic effect in vivo in experimental models of candidiasis and pneumocystosis by mimicking the functional activity of protective antibodies naturally produced in humans against KTR of infecting microorganisms. The swelling tide of concern over increasing bacterial resistance to antibiotic drugs gives the impetus to develop new therapeutic compounds against microbial threat. Thus, the in vitro bactericidal activity of mAbKT and scFvKT against gram-positive, drug-resistant cocci of major epidemiological interest was investigated. MATERIALS AND METHODS: mAbKT and scFvKT generated by hybridoma and DNA recombinant technology from the spleen lymphocytes of mice immunized with a KT-neutralizing monoclonal antibody (mAb KT4) were used in a conventional colony forming unit (CFU) assay to determine, from a qualitative point of view, their bactericidal activity against Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae strains. These bacterial strains are characterized by different patterns of resistance to antibiotics, including methicillin, vancomycin, and penicillin. RESULTS: According to the experimental conditions adopted, no bacterial isolate proved to be resistant to the activity of mAbKT and scFvKT. CONCLUSIONS: scFvKT exerted a microbicidal activity against multidrug resistant bacteria, which may represent the basis for the drug modeling of new antibiotics with broad antibacterial spectra to tackle the emergence of microbial resistance.     

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.     

In vitro and in vivo studies on protective and inhibitory antibodies against Plasmodium falciparum in the Saimiri monkey

Immunoglobulins (Ig) were purified from serum or ascitic fluids of Saimiri monkeys infected with Plasmodium falciparum and with differing levels of functional immunity. Their protective activity was assessed in studies of passive transfer of immunity in vivo, and their neutralizing activity was measured in studies of parasite inhibition in vitro in cultures of human or Saimiri RBC. Although protective and inhibitory antibodies were detected in different Ig preparations, the levels of these activities were not directly correlated. Some Ig preparations, showing high protective activity, provide little or no inhibition of the parasite in vitro. Conversely, inhibitory activity was present in Ig preparations unable to confer protection in vivo. These results indicate that protective antibodies are active through a more complex immune mechanism than simple neutralization probably involving cellular immunity.     

In vitro and in vivo B lymphocyte-activating properties of monoclonal anti-delta antibodies. I. Determinants of B lymphocyte-activating properties

To appreciate better the mechanisms by which B lymphocytes are activated by anti-Ig antibodies, we characterized seven monoclonal mouse allo-antibodies to IgD of the a allotype for their isotypes, fine specificities, IgD-cross-linking abilities, avidities, and abilities to activate B cells in vitro and in vivo. Three of the monoclonal antibodies tested bound to the Fc fragment of IgD with relatively high avidity and were effective at cross-linking IgD, since they precipitated soluble IgD and rapidly capped B cell membrane IgD. These were the only antibodies tested that induced B cell DNA synthesis in vitro and were the most effective antibodies at inducing in vivo increases in B cell size and DNA synthesis and in vitro and in vivo increases in B cell surface Ia expression. Two antibodies bound to the Fd fragment of IgD with relatively high avidity but could not rapidly cap cell membrane IgD or precipitate soluble IgD even in the presence of 2% polyethylene glycol. These high-avidity, poorly cross-linking antibodies were unable to stimulate B cell DNA synthesis in vitro and were much less effective than the first group of anti-delta antibodies at stimulating in vivo increases in B cell DNA synthesis, size, or surface Ia expression or in vitro increases in surface Ia expression. One antibody, which bound to the Fc fragment of IgD with an intermediate avidity, was unable to rapidly cap B cell membrane IgD or precipitate soluble IgD in saline, but could precipitate soluble IgD in the presence of 2% polyethylene glycol. This antibody failed to induce B cell DNA synthesis in vitro and was as effective as the higher-avidity, poorly cross-linking antibodies at stimulating increases in B cell size, surface Ia expression, and DNA synthesis in vivo, and surface Ia expression in vitro. One antibody, which bound to the Fd fragment of IgD with low avidity and was unable to precipitate soluble IgD or to cap cell membrane IgD, had little ability to activate B cells by any of the parameters studied. Each of the monoclonal anti-delta antibodies, regardless of isotype or fine specificity, when bound to agarose to increase its ability to cross-link IgD, was mitogenic for B cells in vitro. None of the monoclonal antibodies to IgD of the a allotype stimulated B cells from b allotype mice to increase their size, surface Ia expression, or synthesis of DNA in vitro or in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

Properties and applications of new monoclonal antibodies raised against calf DNA polymerase alpha

A panel of 12 hybridoma cell lines secreting monoclonal antibodies against alpha-polymerase were prepared by fusion of mouse myeloma cells and spleen cells of a rat immunized with homogeneous calf thymus alpha-polymerase. Hybridomas were selected and cloned on the basis of immunobinding to pure alpha-polymerase in solid phase radioimmunoassay. Antibodies secreted by these cells eventually were purified in milligram quantities from ascites fluids. These antibodies, all of the rat immunoglobulin M class, cross-reacted with alpha-polymerases from calf and monkey cells as revealed by immunobinding in radioimmunoassay and by immunoprecipitation of DNA polymerase activity. The antibodies were not capable of neutralizing the enzyme activity. With the methods described these antibodies may be used to immunoprecipitate alpha-polymerase from crude extracts of mammalian cells and to measure levels of the enzyme protein.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

In vitro and in vivo antagonists against parathyroid hormone-related protein

Four analogues of parathyroid hormone-related protein (PTHrP), PTHrP(7-34)NH2, (10-34)NH2, (15-34)NH2 and (20-34)NH2, were synthesized and their antagonistic activity against PTHrP(1-34) was examined in vitro and in vivo. In vitro studies revealed that all four analogues antagonized PTHrP-stimulated cyclic AMP production in rat osteosarcoma cells (ROS 17/2.8), and that PTHrP(7-34)NH2 and PTHrP(10-34)NH2 had potent antagonistic activity. In vivo experiments in nude mice also revealed that PTHrP(7-34)NH2 completely inhibited hypercalcemia induced by PTHrP(1-34), indicating that these analogues antagonize the effects of PTHrP(1-34) in vitro and in vivo.     

Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo.

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line. Supernatants from growing hybridomas were screened by indirect immunofluorescence on frozen sections of a portion of 21-day fetal rat heads that included the calvaria bone, periosteum, muscle, fibrous connective tissue, and skin. Six MAb were selected with bone-associated staining and limited ability to label other tissues. Either cell surface or cytoplasmic molecules were recognized by five of the MAb; one recognized a molecule detectable both in the cytoplasm, on the cell surface, and in the extracellular matrix. Of the antibodies selected, one identified both preosteoblasts and osteoblasts and has been found to be against alkaline phosphatase. The others recognized the mature osteoblasts, osteocytes, and chondrocytic cells. The pattern and distribution of the labeling in vivo extended to primary cells and cell lines in vivo. These results support earlier observations on molecules differentially expressed by cells at different stages of the osteoblast lineage and extend the available cell surface and cytoplasmic epitopes identifiable as marker molecules.     

Stability of radioiodinated monoclonal antibodies: In vitro storage and plasma analysis

The in vitro stability and immunointegrity of four radioiodinated monoclonal antibodies was evaluated in various storage conditions and also in plasma samples. The monoclonal antibodies studied included T101, B72.3, Lyml, and 16.88. Stabilities of typical monoclonal antibody therapy solutions, with radioactivities ranging from 2220 to 3700 MBq (60–100 mCi) were assessed using conventional instant thin layer chromatography and size exclusion high performance liquid chromatography. Radioimmunoreactivity was assessed using a live cell attenuated cell, or mucin-linked bead assay. Results of the study demonstrated that therapy solutions were stable to degradation, if properly stored in 5 or 10% human serum albumin at 4 °C for the duration of the study (5 days).Minor losses in immunoreactivity were also measured in stabilized therapy solutions. When incubated in plasma samples, radioiodinated monoclonal antibodies generally remained stable for the duration of the study (3 days). However, significant decreases in immunoreactivity were measured for specific radioiodinated monoclonal antibody preparations.     

Isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.     

In vitro immunization of human lymphocytes. I. Production of human monoclonal antibodies against bombesin and tetanus toxoid

A procedure for in vitro sensitization of human lymphocytes against bombesin conjugated to tetanus toxoid (BTT) is described. Bombesin is a tetradecapeptide associated with small cell lung carcinoma. We found that antibody responses against bombesin as well as tetanus toxoid could be generated in vitro by culturing nylon-separated human splenic lymphocytes for 6 days with lipopolysaccharide, phytohemagglutinin-activated lymphocyte supernatants, human AB serum, and bombesin conjugated to tetanus toxoid. Cells sensitized by this procedure were fused to murine myeloma cells, NS-1. The specificities of resulting hybrids were analyzed by enzyme-linked immunoassays and competitive inhibition experiments. Hybrids secreting anti-bombesin (IgM) or anti-tetanus toxoid (IgM or IgG) were obtained. The ratio of IgG to IgM antibodies against tetanus toxoid could be increased by using antigen coupled to Sepharose beads. The sensitization procedure described here offers a system for the study of antigenic stimulation of human B lymphocytes in vitro and for the production of human monoclonal antibodies with the desired specificities.     

Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells

Monoclonal antibodies against the highly conserved ubiquitous calcium- binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.     

The in vitro and in vivo protective activity of monoclonal antibodies directed against Hantaan virus: potential application for immunotherapy and passive immunization

Hantaan virus (HTNV), a member of the genus Hantavirus, family Bunyaviridae, is an etiologic agent causing a serious human disease, hemorrhagic fever with renal syndrome (HFRS), with a mortality as high as 15% and is also a potential bioterrorism agent. It is urgently needed to develop anti-HTNV-neutralizing monoclonal antibodies (MAbs) for treatment and prevention of HTNV infection. In the present study, 18 murine MAbs directed against HTNV strain Chen were generated and characterized. Among these MAbs, 13 were directed against viral nucleocapsid protein (NP), four recognized the viral envelope glycoprotein G2 and one reacted with both NP and G2. Only those MAbs that recognize the epitopes on G2 were positive in hemagglutination inhibition (HI) test and had in vitro virus-neutralizing activity and in vivo protective activity against HTNV infection of susceptible mice. Since all the mice were protected by administration of the virus-neutralizing MAbs one day before and two days after HTNV challenge, these neutralizing MAbs are potentially useful for pre- and post-exposure prophylaxis and for immunotherapy of HTNV infection. Phase II clinical trials of these neutralizing MAbs for emergent treatment of patients with HTNV infection in early stages of HRFS are carried out in endemic areas in China.