Murine/bovine hybridomas producing monoclonal alloantibodies to bovine red cell antigens

In attempts to produce stable lines secreting bovine monoclonal antibodies, murine/bovine hybridomas (1 degree xenohybridomas) were selectively cultured in 8-azaguanine to derive HAT-sensitive lines that were then used as myeloma partners for further fusions with bovine lymphocytes. The resulting 2 degrees xenohybridomas were further selected to produce 3 degrees xenohybridomas. Four stable lines secreting bovine monoclonal antibodies recognizing blood group determinants X1 (an IgG1), E'2 (an IgM) and SU" (an IgGI) and another (an IgGI) as yet unidentified were produced from fusions of 2 degrees xenohybridomas with lymphocytes from calves that had been immunized with bovine red cells.     

Murine hybridomas secreting natural monoclonal antibodies reacting with self antigens

Spleen cells from nonimmunized BALB/c mice were fused with two nonsecreting myeloma lines. The hybrids were selected in HAT medium and screened for Ig production and for antibody activity against actin, tubulin, myosin, thyroglobulin, myoglobin, spectrin, dsDNA, fetuin, and transferrin. Among 161 hybrids secreting Ig, three were found to react with DNA, one with thyroglobulin, and one mainly with myosin. Two of these hybrids could be propagated and further characterized. On the basis of inhibition experiments, one was found to be directed against dsDNA; the other was directed mainly against myosin but at the same time reacted significantly with actin, tubulin, spectrin, and dsDNA. Reactivity with myosin seemed to be concentrated in the light meromyosin subfragment, known to be rich in alpha-helical structure. These results indicate: 1) There are reactive B cell clones directed against self antigens. 2) The antibody specificities found for these antibodies are very similar to those found for natural antibodies in normal human serum and for human monoclonal Ig. 3) The widespread reactivity found for the clone mainly reacting with myosin raises the possibility that the determinant recognized by this antibody is a conformational structure that possibly is associated with alpha-helical structures.     

Utilization of leucine methyl ester for the generation of hybridomas producing monoclonal antibodies specific to tumor-associated antigens

In an attempt to obtain monoclonal antibodies specific to tumor-associated antigens. C3H/He mice were immunized with syngeneic MM2 tumor cells, and the primed spleen cells were fused with P3-X63-Ag8.653 myeloma cells. The outgrowth of hybridomas, however, was extremely low and monoclonal antibodies were not obtained. The reason for the low hybridoma growth was studied. It was found that MM2 cells used as the immunogen, the fusion partner myeloma cells and the resulting hybridomas shared at least one tumor-associated antigen, namely Q5 antigen. Because of this common antigen, cytotoxic cells, presumably cytotoxic T lymphocytes, which were lytic to the hybridomas, were induced during the culture for generation of the hybridomas. Removal of lysosome-rich cells, including cytotoxic T lymphocytes, from the primed spleen cells before the fusion by treatment with leucine methyl ester, a lysosomotropic agent, drastically improved the outgrowth of hybridomas. By this method, seven stable hybridoma clones producing monoclonal antibodies specific to tumor-associated antigens were obtained. Two of the seven clones were found to secrete monoclonal IgM species, which reacted with the extra-cellular region of the Q5 antigen. This procedure will be an option when production of monoclonal antibodies specific to cell-surface antigens is intended and outgrowth of hybridomas is unexpectedly low.     

Storage and reconstruction of hybridomas producing monoclonal antibodies against viral antigens

The conditions were optimized for freezing storage, restoring and further cultivation of hybridoma cells producing antibodies to viral antigens. The effect of density of cellular suspension frozen,concentration of calf embryo serum in cryoprotected medium and mild conditions of the restoring of hybridoma were studied. To restore deeply frozen hybridoma 24 hole plastic panels with a layer of feeding cells of the HEPES and insulin containing medium were used. The fulfilling of these requirements makes possible restoration of intact antibody-producing hybridoma from 10(2)-10(3) frozen cells.     

Murine interleukin-2 receptor subunits differentially detected with anti-interleukin-2 monoclonal antibodies

Cross-linking of radioiodinated interleukin-2 to murine CTLL-2 cells enabled detection of 70 kDa, 85 kDa and 105 kDa complexes of IL-2 and its binding proteins under the high-affinity binding condition. A series of anti-interleukin-2 monoclonal antibodies (L15, L20, L23, L34, and L61) were tested for their activity to immunoprecipitate these cross-linked complexes. L61, which had strong neutralizing activity, precipitated only the 70 kDa complex. L15, L20, and L34, which also had neutralizing activity, precipitated not only the 70 kDa complex but also the 85 kDa complex. L23, which had practically no neutralizing activity, precipitated the 105 kDa complex as well as the 85 kDa complex. These results suggest that there are at least three distinct receptor binding sites for each receptor subunit on the interleukin-2 molecule, which are discernible by these monoclonal antibodies and that the 105 kDa complex may play a significant role in the formation of the high-affinity receptor complex and the signal transduction.     

Generation of hybridomas producing human monoclonal antibodies against human cytomegalovirus

In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens.     

A new human fusion partner,HK-128, for making human-human hybridomas producing monoclonal IgG antibodies

A hypoxanthine-aminopterin-thymidine (HAT) sensitive human fusion partner cell line, HK-128 was established from a human plasmacytoma line, LICR-LON-HMy2 (HMy2). The HK-128 cells showed a 100% cloning efficiency. Fusion efficiency of HK-128 was so high that one hybridoma cell was produced by fusion of 10⁵ cells of HK-128 with lymphocytes, obtained from lymph nodes of breast cancer patients. About 90% of the resulted hybridomas were IgG producers. The remainder revealed IgM producing activity, which was lost by long term culture. This result indicates that the HK-128 cell line has an advantage for making hybridoma cells producing IgG. Among ca. 7,000 hybridomas obtained by fusion of HK-128 with lymphocytes of a breast cancer patient, we could establish a hybridoma cell line which produced IgG specifically reacting to a human breast cancer cell line, MCF-7.     

In vivo interaction of antibodies with cell surface proteins used as antigens

Antibody interactions with cell membrane glycoproteins in vivo exhibit features of aggregation and capping with resultant shedding similar to those events described in several in vitro isolated cell systems. Requirements for divalent ligand binding and participation of cytoskeletal elements are demonstrated in vivo as well. Persistence of antigen in immune complexes with complement interaction in situ appear to be necessary to induce an inflammatory response in vivo. Abrogation of this response occurs when circumstances permit antigenic modulation with disappearance of the immune complex.     

Karyological analysis of hybridoma lines producing monoclonal antibodies to viral antigens

The number of Hybridomes obtained from various sources rapidly increases at present. Clones producing monoclonal antibodies to influenza virus A/USSR/090/77 and to VEE-230 are generated in the laboratory of the Institute of Virology (Academy of Sciences of the USSR). The present work is devoted to the study of Hybridome karyotype by means of C-method for chromosome staining with the aim to reveal specific characteristics of these lines. Results of the investigation have shown that the count of chromosomes together with an examination of their C-staining picture permit proving a hybrid nature of the clones and identifying various Hybridomes by chromosome markers.     

Generation of hybridomas producing human monoclonal antibodies against herpes simplex virus after in vitro stimulation

Hybridomas producing human monoclonal antibodies against herpes simplex virus were generated by in vitro antigen stimulation before cell fusion. The cell fusion with tonsillar lymphocytes which were stimulated with antigen and/or pokeweed mitogen generated many hybridomas producing human IgG against the virus. A combination of antigen and pokeweed mitogen synergistically enhanced the generation of virus-specific hybridomas. Furthermore, the higher the antibody response of the tonsil, the more virus-specific hybridomas were generated by the cell fusion. These results suggest that cell fusion with in vitro stimulated lymphocytes can be applied to a variety of clinically relevant viruses.     

ERBB oncogene proteins as targets for monoclonal antibodies

General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.     

Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF

Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.     

Obtaining hybridomas producing monoclonal antibodies with different specificities against Mycobacterium tuberculosis of the human and bovine types

A procedure for isolation of hybridomes producing monoclonal antibodies (McAB) to tubercle bacilli is described. Specificity of the McABs was studied with the solid phase radioimmune and immunoenzyme tests. Supernatant of tubercle bacilli destroyed with ultrasound was used as antigens. The McABs did not practically react with antigens of the tubercle bacilli atypical forms. Five ascitic monoclonal hybridomes were isolated. Four of them produced antibodies with selective specificity to antigens of bovine tubercle bacilli (M. bovis-8 and BCG) and one produced antibodies to antigens of human tubercle bacilli (H37Rv).     

Comparison of culture methods for human-human hybridomas secreting anti-HBsAg human monoclonal antibodies

Human-human hybridomas which secrete a human monoclonal antibody (h-MoAb) against hepatitis B virus surface antigen showed growth associated production kinetics. The rate of h-MoAb production rapidly decreased after cell growth was arrested in a perfusion culture, even if the perfusion rate was increased. A continuous suspended-perfusion culture, in which both culture broth and culture supernatant are continuously harvested and the same volume of fresh medium is continuously fed into the reactor, was developed to maintain continuous growing conditions during cultivation. In this culture system, the production of h-MoAb continued for more than 50 days with an average productivity of 5.0 mg/l of working volume/day. A semicontinuous immobilized-perfusion culture in which parts of the cells are repeatedly removed from the immobilized reactor was another useful technique for the long term cultivation of these h-h hybridomas. As an average h-MoAb production rate, 62 mg/l of immobilized-bed volume/day was achieved for 65 days of cultivation using a ceramic matrix reactor, and 327 mg/l/day was achieved over 47 days of cultivation using a hollow fiber reactor equipped with Cultureflo M^TM Thus, the antibody productivity per reactor volume per day by the semicontinuous immobilized-perfusion culture was much higher than that of the continuous perfusion culture in an agitation reactor.     

Screening of monoclonal antibodies using antigens labeled with acetylcholinesterase: application to the peripheral proteins of photosystem 1

An original immunoenzymatic screening method, based on the use of antigens labeled with the stable enzyme acetylcholinesterase (AChE, EC 3.1.1.7), is described. The high turnover of this enzyme results in a very sensitive detection of antibodies. In this method, monoclonal antibodies from the supernatants of hybridoma cultures are immobilized on a solid phase coated with anti-mouse immunoglobulins and react simultaneously with the appropriate antigen labeled with biotin molecules. In a second step, biotinylated acetylcholinesterase is in turn associated to the system via avidin interactions and subsequently detected by a colorimetric assay. The method appears more sensitive and easier to use than either the corresponding radioimmunological test using a 125I-iodinated antigen or the same type of enzymatic immunoassay performed with biotinylated horseradish peroxidase instead of biotinylated AChE. The combined use of microtiter plates, solid-phase separation, and colorimetric detection allows a high level of automation of the method which makes it very efficient to process a large number of samples. This technique has been successfully applied to the screening of monoclonal antibodies directed against peripheral proteins of the photosystem 1 (PS1) membrane complex in photosynthesis. A complete set of antibodies recognizing these PS1 components was selected. The same technique was also tested in competition immunoassays and appears to be a very precise and useful tool for quantifying PS1 polypeptides in different biological extracts, including sodium dodecyl sulfate-denatured membranes. This can be of special interest for studying the biogenesis of membrane complexes.     

Effects of monoclonal antibodies against lymphocyte surface antigens on interleukin 2 excretion by Epstein-Barr virus-specific human T-cell hybridomas

Monoclonal antibodies were used as probes to study the role of cell surface antigens in the response of Epstein-Barr virus (EBV)-specific human T-T hybridomas to autologous EBV-infected B lymphoblasts. Somatic cell hybrids were generated by fusing EBV-primed peripheral blood T lymphocytes with a mutant clone of the JM human T-lymphoblastoid-cell line. When exposed to autologous EBV-infected B lymphoblasts, the resulting hybrid clones released Interleukin 2 into the culture medium. Incubation of the EBV-infected B cells with two monoclonal antibodies against human Ia-like molecules blocked their ability to trigger the hybridomas. Under the same conditions, monoclonal antibodies against beta 2-microglobulin, and a 45,000 MW surface antigen common to EBV-infected B lymphoblasts, did not alter the capacity of the B cells to stimulate the hybridomas. None of four monoclonal antibodies against surface antigens on the T-cell hybridomas impaired their responsiveness to EBV-infected B lymphoblasts. These results suggest the possibility that naturally occurring or exogenously administered antibodies against Ia molecules might interfere with T-cell regulation of EBV-induced B-cell activation.     

T-cell surface antigens defined by monoclonal antibodies, involved in the induction of human interferon-gamma and interleukin 2

Monoclonal antibodies specific for human T-cell-differentiation antigens were used to investigate the mechanism of induction of interferon-gamma (IFN-gamma) and interleukin 2 (IL-2). High levels of IFN-gamma, accompanied by IL-2 production, were detected in the lymphocyte cultures stimulated by pan T monoclonal antibodies that were mitogenic. These antibodies recognize an antigen complex Tp 19-29 (a complex of T-cell proteins of 19-29 kDa). However, it was possible to induce IL-2 without concomitant production of IFN-gamma using some antibodies specific for other T-cell surface antigens, e.g., Tp 32-45, Tp 41, and Tp 100-120. These antibodies were not mitogenic. The production of lymphokines, therefore, appears to be regulated at the cell surface by receptors or interaction molecules involved in cell triggering. Binding of antibodies to T3 receptor was obligatory for both IFN-gamma induction and mitogenesis but was not required in the induction of IL-2 activity.     

An alternative strategy for high throughput generation and characterization of monoclonal antibodies against human plasma proteins using fractionated native proteins as immunogens

Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two-round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high-abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety-five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, alpha2-macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high-and medium-abundant plasma or tissue proteins.     

Proteomics-based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins

Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.     

Construction and characterization of single-chain antibodies against human insulin-like growth factor-I receptor from hybridomas producing 1H7 or 3B7 monoclonal antibody

Recombinant antibody consisting of the single-chain variable fragment (scFv) of 1H7 monoclonal antibody against insulin-like growth factor-I receptor (IGF-IR) and human IgG(1) Fc domain, scFv-Fc, has been found to exhibit inhibitory effects on breast cancer growth in vitro and in vivo [Li et al. (2000) Cancer Immunol. Immunother. 49, 243; Sachdev et al. (2003) Cancer Res. 63, 627]. Various types of scFvs from hybridomas producing 1H7 or 3B7 mAb were constructed using conventional phage display technology to further characterize the specificity and affinity of anti-IGF-IR mAbs. Binding studies performed using either phage antibodies or soluble scFv proteins to IGF-IR or insulin receptor (IR) and IGF-IR pre-incubated with mAbs suggested that (i) 1H7 and 3B7 bind to IGF-IR but do not bind to its structurally related IR, (ii) either the VL-VH or VH-VL sequence order does not apparently affect specificity for IGF-IR and (iii) 1H7 and 3B7 bind the independent epitopes, located in or near the N-terminal (440-514) and C-terminal (62-184) domains of the alpha subunit, respectively. This study not only revealed new information on binding regions for two anti-IGF-IR mAbs, but also provided the scFv genes as tools for further manipulation of the affinity or development of new IGF-IR-targeted cancer therapeutics.