Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture.These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium.An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production andvice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.     

Improvement in the proliferative activity of human-human hybridomas at low cell density by transfection with bFGF gene

Highly purified recombiaant basic fibroblast growth factor (rbFGF) and acidic FGF (aFGF) stimulated the proliferation of human-human (h-h) hybridomas to the extent of over four-fold from a low cell density such as 1×10³ cells per ml in a serum-free medium in 24-well plates. The stimulatory effect of rbFGF was also observed in various lymphoid cell lines. Expecting that FGF could be an autocrine growth factor, we introduced bFGF gene into a h-h hybridoma using an expression plasmid induced by dexamethasone. The transformed cells thus obtained, HPO-75.11bbFGF-7, were able to grow well from a low inoculum density in a serum-free medium and antibody production was also increased when bFGF gene expression was induced. The transformed cells could grow at clonal density in a serum-free medium in 96-well plates, though the original cells could not. We also obtained a more practical transfectant, HPO-75.29-H74, using a high-shear stress adapted clone as the recipient and an expression plasmid having bFGF gene under the control of metallothioneine-I promoter. The HOP-75.29-H74 cells were capable of growing and producing human monoclonal antibody against hepatitis B virus surface antigen from an inoculum density of 1×10³ cells per ml in an agitation vessel without addition of an inducer.     

HO-323, a human B-lymphoblastoid cell line useful for making human-human hybridomas

A 6-thioguanine-resistant human B-lymphoblastoid cell line, HO-323, was isolated for making human-human hybridomas with high efficiency. Fusions with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients and lymphocytes isolated from lymph nodes of lung and breast cancer patients yield constantly more than one hybridoma clones per 10(5) HO-323 cells plated. HO323 cells also fused with lymphocytes from normal peripheral blood to give hybridomas in the same efficiency. The HO-323 cells were diploid with 46 chromosomes and non-secretors of immunoglobulins. This parent cells doubled every 15 hr and could proliferate in serum-containing medium, even if they were plated at low cell density of less than 10(3) cells/ml. The cells could grow in serum-free medium as well as in serum-containing medium, and the resultant human-human hybridomas could also grow in the same media.     

Screening of Immunoglobulin Production Stimulating Factor (IPSF) in Foodstuffs Using Human-human Hybridoma HB4C5 Cells

We screened the immunoglobulin production stimulating factor (IPSF) in foodstuffs, using human-human hybridoma HB4C5 cells cultured in serum-free 1TES-ERDF medium, and found that egg yolk lipoprotein (YLP), lactoferrin, Block Ace, and casein had IPSF activity. The maximum IPSF activity was obtained at concentrations over 100^g/ml in YLP, 10μg/ml in lactoferrin, and 25 μg/ml in Block Ace and casein. These IPSFs stimulated the IgM production of human-human and mouse-mouse hybridomas, but their effect on IgG producers was very small. This suggests that IgG production of hybridomas is regulated differently from their IgM production.     

The cultivation of mouse and human lymphoid cells on serum-free media]

The optimum composition of several serum-free media has been established for a long-term cultivation of hybridomas, lymphoid and erythroleukemic cells. The medium DME/F12 appeared to be the medium of choice. It is necessary to supplement the basic medium with lipid and iron transport proteins (bovine serum albumin, transferrin) and peptide hormone (insulin) for obtaining stable results. However, there are differences in successful growth of examined cell lines under serum-free conditions: some of them acquire saturation density comparable with that of the control medium (hybridomas derived from myeloma Sp2/0-Ag14, cell lines K-562, Raji) but other lines do not (hybridoma derived from myeloma NS0/1, cell lines Namalwa, RPMI 1788, Molt-4). Thus, these serum-free media are not universal, therefore each new hybridoma and cell line should be tested to determine the suitability for them of some proposed media. The high effectiveness of cultivation under serum-free conditions can be presumably achieved by optimization of both qualitative and quantitative composition of the serum replacement and of the basic medium.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Transferrin receptor and ferritin expressions of a human-human hybridoma in protein-free media

Summary The expressions of transferrin receptor and ferritin in a human-human hybridoma in protein-free media were examined. The regulation of the expressions of the two proteins appeared to be decoupled in protein-free cultures. Such cultures also exhibited much higher ferritin expression and endogenous iron pool than a culture in serum-free medium containing transferrin.     

Development of a Protein-free Medium with Iron Salts Replacing Transferrin for a Human-Human Hybridoma

Many protein-free media have been deveoped, because protein-free media are usually more economical than serum-free or serumcontaining media and facilitate the purification ofbioactive materials. We evaluated various iron salts and chelating agents replacing transferrin to develop a protein-free medium for a human-human hybridoma, HB4C5, and found out that ferric citrate was favorable for the production and the productivity of monoclonal antibodies.     

Characterization and applications of lymphocytic clonal growth factor in human plasma

Human plasma was found to contain a macromolecular protein which can grow even a single cell of human lymphocytic cell lines (B-lymphoblastoid cell line HO-323-3 and T-lymphoblastoid cell line CCRF-CEM) and human-human hybridoma clones (SH-9, SU-1 and HB4C5) in a dish, but it has no effect on the growth of epithelial cell lines (lung cancer cell lines PC-8, QG-56 and QG-90). The proliferating activity for lymphocytic cell lines was gradually decreased at 4 or -20°C and dramatically decreased by heating at more than 60°C for 15 min. From human plasma, active fractions were purified by a successive application of Ca²⁺ treatment, ammonium sulfate fractionation, DEAE-5PW column chromatography (FPLC) at pH 7.6. These active fractions were divided into at least three proteins by DEAE-5PW chromatography at pH 8.5 and chromatofocusing. These purified factors, named lymphocytic clonal growth factors (LCGFs), had similar molecular weights of about 600 K and each factor consisted of a 180 K and two 210 K subunits associated with hydrogen bondings. By the addition of 5 g/ml of each factor into culture media, incidences of human-human hybridomas and cloning efficiencies of the hybridomas increased several-fold.     

Human-human hybridomas secreting antibodies specific to human lung carcinoma

Summary Human Namalwa cells were screened in serum-free medium and in 6-thioguanine, then fused with human lymphocytes from lymph nodes of lung adenocarcinoma cancer patients. Extensive testing using 14 lung cancer cell lines, 11 other cancer cell lines and 4 normal fibroblast lines identified monoclonal antibodies produced by 4 hybridoma clones that reacted specifically with lung adenocarcinoma cells. These monoclonal antibodies also reacted with lung adenocarcinoma tissues and not normal tissues or erythrocytes of any blood type. These hybridoma clones grew and stably secreted the antibodies in serum-free medium as well as in serum-containing medium. Editor's Statement Identification of monoclonal antibodies that recognize human lung adenocarcinoma cells with reasonable specificity represents a potentially important development that may prove useful in diagnosis and therapy of neoplastic disease. The selection procedures and methods for propagation of the human-human hybridomas described in this paper also represent some novel approaches that may be of general application. David W. Barnes     

An alternative method for the isolation of NS-1 hybridomas using cholesterol auxotrophy of NS-1 mouse myeloma cells

Summary We have used the cholesterol auxotrophy of NS-1 mouse myeloma cells as the basis for selecting NS-1 hybridomas. The outgrowth of nascent NS-1 hybridomas in cholesterol-free serum-free medium was 3- to 9-fold more efficient than that in HAT medium and resulted in 3- to 13-times as many antigen-reactive hybridoma wells. This method of hybridoma selection can be applied with any sterol-dependent parent cell line. Hybridomas established under serum-free culture conditions were growth inhibited by fetal calf serum. This work was supported by the Japanese Ministry of Education, Science and Culture and in part by grants from the National Institutes of Health. Editor's Statement This article reports a creative technical application of the author's previous work on lipid metabolism in lymphoid cells allowing an efficient, alternative selection procedure for isolation of hybridomas.     

Monoclonal cytotoxic T lymphocyte hybridomas capable of specific killing activity, antigenic responsiveness, and inducible interleukin secretion

We have recently described the production of cytotoxic T lymphocyte (CTL) hybridomas that grow continuously in culture, exhibiting constitutive, allospecific (anti-H-2b) killing activity. We now report on the response of these monoclonal CTL hybridomas to specific antigen (H-2Db) and to mitogenic lectins. Both specific antigen and T cell mitogens enhance hybridoma-mediated specific target cell killing. In addition, stimulated, but not unstimulated hybridoma cells secrete considerable amounts of IL 2 into the culture medium. Repeated cloning of the hybridomas provides strong evidence that both killing activity and IL 2 secretion can be attributed to one cell. Unfractionated Con A supernatants, containing IL 2 and other factors known to influence T cell responsiveness, or IL 2-containing media of stimulated hybridomas affect neither the growth nor the lytic activity of the hybridomas. Anti-LFA-1 monoclonal antibody, a potent inhibitor of CTL and CTL hybridoma-mediated target cell lysis, abolishes antigen- or mitogen-induced IL 2 secretion by the CTL hybridomas. Involvement of a single hybridoma receptor in antigen recognition (afferent and efferent) and in initiating IL 2 secretion is proposed. The CTL hybridomas displaying retarded killing activity before the antigenic or mitogenic stimulation appear to represent an intermediate stage in CTL differentiation, reminiscent of "memory" CTL.     

Enhancing effects of retinoic acid on monoclonal antibody production of human-human hybridomas

The effects of retinoids on the production of monoclonal antibody of human-human hybridomas were examined. IgG antibody secretion of a hybridoma CLNH11 was enhanced up to about two- to fourfold by retinoic acid (RA) at concentrations ranging from 10(-9) to 10(-5) M, where RA had little effect on the growth rate and saturation density of the cell. Among other retinoids, retinol magnified the antibody production as well as RA. Retinal and retinyl acetate had weak effects. Retinyl palmitate showed no effect. RA also enhanced the production of monoclonal antibodies from other human-human hybridomas: SLNF10, IgG-producing; CoLNE10, IgA-producing; TOS/H8, IgM-producing. RA and human hybridomas provide a defined system to study the effects of retinoids on immune responses at a molecular level.     

Stimulation of monoclonal antibody production by human-human hybridoma cells with an elevated concentration of potassium or sodium phosphate in serum-free medium

Potassium or sodium phosphate was found to stimulate the production of human monoclonal antibody by human-human hybridoma HB4C5. The addition of 15 mM Na-phosphate (pH 7.4) into serum-free culture medium increased the antibody production up to 4-fold, when seeded at cell density of 1×10⁵ cells/ml in dishes. At the higher cell density of 5×10⁵ cells/ml, K-phosphate was more effective than Na-phosphate, at the same concentration. In large-scale continuous culture, the addition of 10 mM Na-phosphate into serum-free culture medium stimulated antibody production by HB4C5 cells 6-fold.     

Growth stimulating activity of heat shock protein 90α to lymphoid cell lines in serum-free medium

A growth stimulating factor was purified from the culture supernatant of human-human hybridoma SH-76 cells in serum-free RPMI 1640 medium by the serial use of DEAE anion and heparin affinity chromatographies. The factor was a 85 kDa protein on SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence (PEETQTQDQPME) of the protein was coincident with that of heat shock protein 90α (HSP90α). The protein reacted with anti-HSP90 monoclonal antibody. These results suggest that the protein was a member of HSP90α family after taking other circumstantial evidence into account. The protein stimulated the growth of some lymphoid cell lines such as human B-lymphoblastoid cell line HO-323-3 hybridomas derived from HO-323, and several other lymphoid cell lines. There were several lymphoid cell lines which did not respond to the protein. Growth stimulating activity of the protein was heat-unstable and significantly decreased at above 60°C. These are the first data that describe growth-stimulating activity of heat shock protein 90α.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

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.     

Serum-free medium for murine and human lymphoid and hybridoma cell lines

We established a serum-free medium of low protein content(125g/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.Abbreviations FBS Fetal Bovine Serum - IMDM Iscove's Modification of Dulbecco's Medium - PBS Phosphate-Buffered Saline - TPA Tissue Plasminogen Activator     

Strategies for the design and use of tumor-reactive human monoclonal antibodies

Human hybridomas secreting monoclonal antibodies (MoAb) reactive with tumor cell antigens were produced in our laboratory by the immortalization of UC 729-6 with B lymphocytes isolated from regional draining lymph nodes of cancer patients. MoAbs were purified from the hybridoma supernates by standard biochemical procedures for in vivo studies and by affinity methods for in vitro experiments. Using a novel method in the preparation of slides containing adherent tumor cells, immunoreactivities of the MoAbs were evaluated by an indirect immunofluorescence assay. Based on this data, human MoAbs can, therefore, be used as research reagents.     

Stimulation of IgM production in human-human hybridoma HB4C5 cells by chitosan.

We screened for immunoglobulin production stimulating factors (IPSFs) in polysaccharides using human-human hybridoma cells, HB4C5, cultured in serum-free medium. Among polysaccharides, citrus pectin, locust bean gum, and chitosan stimulated IgM production of HB4C5 cells. Especially chitosan showed the strongest IPSF activity; 100 ng/ml of chitosan stimulated IgM production approximately 5-fold. Chitosan had several characteristics as IPSF, as follows. 1) For the IPSF activity, 70-90% deacetylation was essential. 2) Chitosan oligomers (n = 5, 6, 7) and chitin oligomers (n = 5, 6, 7) showed no IPSF activities. 3) The IPSF activity of chitosan was inhibited by glucosamine, one of the constitutive sugars of chitosan. 4) Chitosan stimulated IgM production of human lymphocytes in serum-free culture, but not IgG or IgA, nor in serum-supplemented culture.