A human hybrid hybridoma

Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. We have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. We fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1 kappa antibody directed against tetanus toxoid and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1 lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture medium containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassays. Our results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies.     

The effect of the catalytic topoisomerase II inhibitor dexrazoxane (ICRF-187) on CC9C10 hybridoma viability and productivity

The effect of dexrazoxane on monoclonal antibody (Mab) production by CC9C10 hybridoma cells was investigated. Dexrazoxane is a catalytic inhibitor of DNA topoisomerase II. DNA topoisomerase II has a critical role in DNA metabolism and its inhibition by dexrazoxane can prevent completion of cytokinesis. Incubation of hybridomas with dexrazoxane was found to increase specific monoclonal antibody production by up to four-fold. However, due to the growth inhibitory effects of dexrazoxane the total Mab yield decreased by 40%. Under high density culture conditions(defined here as 10⁶ cells ml^-1) specific monoclonal antibody production increased by up to 37%, which was, however, accompanied by up to a 48% decrease in Mab yield. Hybridomasthat were incubated with dexrazoxane significantly increased in size due to the inhibition of cytokinesis. Dexrazoxane was also observed to induce a delayed apoptosis in the hybridomas. The caspase inhibitor Z-VAD-fmk slightly decreased the apoptotic effects of dexrazoxane. Preincubation with the caspase inhibitorZ-Asp-CH2-DCB had no effect on dexrazoxane-treated hybridomas, but it did have antiapoptotic effects on the untreated hybridomas which normally undergo a significant basal level of apoptosis. In conclusion, dexrazoxane-induced growth inhibition (which results in higher specific antibody production) and apoptosis inhibition (which results in prolonged viability) has the potential to significantly enhance the productivity of hybridoma cell cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.     

Transfection of NS0 myeloma fusion partner cells with HSP70 gene results in higher hybridoma yield by improving cellular resistance to apoptosis

Mouse myeloma NS0 cells widely used in hybridoma technology lack the expression of a major stress protein Hsp70 which is the principal component of the basic cellular defense mechanism. These cells rapidly undergo apoptosis at the late-stationary phase of batch culture following nutrient exhaustion. Since Hsp70 was recently demonstrated to protect cells against numerous apoptotic stimuli, the aim of the present study was to examine the protective potential of the protein expression in engineered myeloma NS0 cells and in resulting hybridomas. Myeloma cells were transfected with the hsp70 gene under beta-actin gene promoter. To imitate harmful conditions that hybridoma or myeloma cells often experience when cultivated in large scale for an antibody production, NS0(wt) and NS0(hsp70) cell cultures were maintained without changing the medium for a few days, and the expression of apoptotic markers has been studied. It was found that long-term cultivation induced apoptosis in original cells manifested by typical nuclei fragmentation, DNA ladders and activation of caspase-3. In contrast, in transfected cells under the same conditions the outcome of apoptosis was postponed for 24 hours. Most relevant was that the fusion of transfected myeloma cells with immune splenocytes resulted in twofold hybridomas output compared with wild-type fusion partner. Almost half of the hybridomas continued to be hsp70-positive and maintained higher robustness in culture. The level of monoclonal antibodies production by hybridoma cells obtained with the use of NS0(wt) and NS0(hsp70) was similar, however, the secreted product was better preserved in culture supernatants of Hsp70-positive cells. It is concluded that transfection of mouse myeloma cells with the hsp70 gene can be a novel means to increase hybridoma yield and reduce the sensitivity of myeloma and hybridoma cells to culture conditions insults accompanying monoclonal antibody production.     

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening

Hybridoma methods for monoclonal antibody (mAb) cloning are a mainstay of biomedical research, but they are hindered by the need to maintain hybridomas in oligoclonal pools during antibody screening. Here, we describe a system in which hybridomas specifically capture and display the mAbs they secrete: On-Cell mAb Screening (OCMS?). In OCMS?, mAbs displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS? demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG “cap”, as a universal assay for anti-viral mAbs. We produced and characterized OCMS?-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. Lastly, we used OCMS? to assess expression and antigen binding of a recombinant mAb produced in 293T cells. As a novel method to physically associate mAbs with the hybridomas that secrete them, OCMS? overcomes a central challenge to hybridoma mAb screening and offers new paradigms for mAb discovery and production.     

Genetic analysis of human B cell hybridomas expressing a cross-reactive idiotype

We have examined the genetic basis for the expression of a human cross-reactive idiotype (CRI) commonly found on monoclonal IgM rheumatoid factors. The CRI was identified with a monoclonal antibody (17.109) and has been localized previously to the kappa-variable region. By using the human lymphoblastoid cell line WI-L2-729-HF2, and mononuclear cells from several sources, a panel of hybridomas was generated that produced 17.109 CRI-positive Ig. A recently cloned human germ-line V kappa III gene, Humkv305, served as a probe to identify genes which were rearranged and expressed in 17.109 CRI-positive and -negative hybridomas. This probe, when hybridized to human genomic DNA under stringent conditions, identified only two to five germ-line bands. In 10 separate 17.109 CRI-positive hybridoma clones, an additional rearranged V kappa band was identified. The probe did not anneal to rearranged V kappa bands in hybridoma clones that produced kappa-chains lacking the CRI. RNA dot-blot studies provided evidence for expression of genes hybridizing to the Humkv305 probe. The results indicate that the 17.109 CRI is a serologic marker for a single V kappa gene, or a small family of closely related V kappa genes, which is identified by the Humkv305 probe.     

Cell-cycle-dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: a population analysis

Single-cell rates of accumulation of cellular protein have been determined as a function of total protein content using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G(1), S(1) and G(2) + M cell cycle phases. A novel flow cytometric technique for the identification of hybridoma cells in mitosis was developed and implemented. The data were obtained from a producer cell line which synthesizes and secretes high levels of monoclonal antibodies, and from a nonproducer clone which does not synthesize and secrete substantial amounts of antibody. The results indicate that the kinetics of single-cell protein accumulation in these two cell lines are considerably different. In particular, low protein content G(1) phase producer cells were characterized by a rate of protein accumulation which was approximately five times higher than the mean rate observed for higher protein content producer cells cycle phase. In contrast, the rate of accumulation of protein increased continuously with totalprotein content for the G(1) phase nonproducer cells. S phase hybridoma cells were characterized by a considerably lower rate of protein accumulation which did not vary much with protein content for either cell line. Finally, G(2) + M phase producer cells demonstrated a negative rate of protein accumulation which indicates that the rates of protein synthesis. It was hypothesized that these differences in total protein accumulation are caused by differences in monoclonal antibody accumulation. The distribution of rates suggests the need for a segregated approach to the modeling of the kinetics of antibody production in hybridomas.     

Characterization of higher avidity monoclonal antibodies produced by murine B-cell hybridoma variants selected for increased antigen binding of membrane Ig

Somatic mutation in the Ig genes plays a major role in the increase of antibody affinity observed in secondary immunologic responses. It has been shown that the mechanism responsible for the high rate of somatic mutation in the Ig genes was active not only in normal B lymphocytes but also in B-cell hybridomas secreting mAb. Also, it has been reported that B-cell hybridomas were positive for membrane Ig of the same specificity as the secreted mAb. The presence of membrane Ig suggested that somatic variants secreting mAb of higher affinity could be selected by the increased capacity of these hybridoma cells to bind immobilized Ag. This hypothesis was tested with hybridoma cells secreting an IgM mAb reacting with the A Ag of the ABO blood group system. In two selection experiments, we have isolated several variant cell lines secreting mAb of increased avidity for the A Ag under similar IgM concentrations. Biochemical characterization of one of the variant mAb indicated that the mutation responsible for the increased avidity has occurred in the heavy chain gene. The method developed may have profound implications for the diagnostic and therapeutic use of mAb and will permit the study, in an in vitro system, of the role of somatic mutations in antibody diversity.     

A self determinant recognizing T cell hybridoma

A T cell hybridoma (53(113)) obtained by fusion of BALB/c spleen cells and the BW 5147 lymphoma T cell line is described. This hybridoma recognizes mouse RBC (MRBC) and rat RBC, but not human, rabbit, guinea pig, or SRBC. The culture supernatant possesses hemagglutinating activity for the same indicator RBC. In addition to this, 53(113) cells are able to form protein A plaques in the presence of guinea pig complement and normal mouse serum (NMS) or purified mouse immunoglobulins (Ig). Because mouse Ig as well as sonicates from MRBC are able to inhibit the rosettes between the hybridoma cells and the MRBC, and because the sonicates inhibit protein A plaque formation, it seems likely that the same product can recognize a similar determinant expressed on MRBC and mouse Ig. The hypothesis that a 53(113) structure recognizes identical or cross-reactive carbohydrate determinants shared by murine Ig and C is considered.     

Human monocyte-mediated cytotoxicity: the use of Ig-bearing hybridomas as target cells to detect trigger molecules on the monocyte cell surface

We recently reported the preparation and characterization of a monoclonal antibody, 32.2, specific for the high-affinity Fc receptor (FcR) for IgG on human monocytes. We have utilized the hybridoma cell line producing this antibody as a target for monocyte-mediated cytotoxicity. The hybridoma was selected for stable sublines that expressed high quantities of surface 32.2 immunoglobulin (Ig) through flow cytometry. Monocyte-mediated cytotoxicity, with these sublines used as targets, was evaluated with the use of a 51Cr-release assay. It was found that monocytes could efficiently lyse the hybridoma cells (HC 32.2) bearing surface Ig directed to the high-affinity FcR. Consistent with the specificity of the 32.2 antibody for an epitope on the high-affinity receptor outside of the ligand binding site, human IgG did not block monocyte killing of HC 32.2. In contrast, monocytes could not mediate lysis of hybridoma cells bearing high levels of antibody directed to other monocyte cell surface molecules, in particular, class I MHC molecules, the C3bi receptor, and the My 23 antigen. The effect of IFN-gamma on the ability of monocytes to mediate lysis of the 32.2 Ig-bearing hybridomas was also assessed. Monocytes cultured in the absence of IFN-gamma could lyse the hybridoma line expressing high levels of 32.2 Ig as efficiently as monocytes cultured in the presence of IFN-gamma. However, untreated monocytes were less able than IFN-gamma-treated monocytes to kill HC 32.2 expressing lower levels of Ig. Thus, IFN-gamma may enhance the efficiency of monocyte-mediated antibody-dependent killing under conditions where limited antibody is available on the target. These studies demonstrate that the high-affinity FcR on monocytes can act as a cytotoxic trigger molecule for killing of tumor cell targets and that this trigger does not require specific binding to the Fc binding epitope. These results further encourage possible clinical application of the 32.2 monoclonal antibody in tumor therapy.     

Delivery of immunostimulatory monoclonal antibodies by encapsulated hybridoma cells

Immunostimulatory monoclonal antibodies are immunoglobulins directed toward surface proteins of immune system cells that augment the immune response against cancer in a novel therapeutic fashion. Exogenous administration of the recombinant humanized immunoglobulins is being tested in clinical trials with agents of this kind directed at a variety of immune-controlling molecular targets. In this study, the encapsulation of antibody-producing hybridoma cells was tested in comparison with the systemic administration of monoclonal antibodies. Hybridomas producing anti-CD137 and anti-OX40 mAb were encapsulated in alginate to generate microcapsules containing viable cells that secrete antibody. Immobilized cells in vitro were able to release the rat immunoglobulin produced by the hybridomas into the supernatant. Microcapsules were implanted by injection into the subcutaneous tissue of mice and thereby provided a platform for viable secreting cells, which lasted for more than 1 week. The pharmacokinetic profile of the rat monoclonal antibodies following microcapsule implantation was similar to that attained following an intraperitoneal administration of the purified antibodies. The rat–mouse hybridoma cells did not engraft as tumors in immunocompetent mice, while they lethally xenografted in immunodeficient mice, if not microencapsulated. The antitumor therapeutic activity of the strategy was studied on established CT26 colon carcinomas resulting in complete tumor eradication in an elevated fraction of cases and strong tumor-specific CTL responses with either anti-CD137 or anti-OX40 producing hybridomas, thus offering proof of the concept. This form of administration permitted combinations of more than one immunostimulatory monoclonal antibody to exploit the synergistic effects such as those known to be displayed by anti-CD137 and anti-OX40 mAb.     

A modified hybridoma technique for production of monoclonal antibodies having desired isotypes

In the present study, we describe a modified hybridoma technique for production of monoclonal antibodies (mAbs) having a desired isotype. Mice were immunized with the antigen of interest. After having reached a high antibody titer, cells expressing IgM or IgG molecules were isolated from spleen cells of the immunized mice using a Magnetic Cell Sorting System. The isolated cells were fused with myeloma cells using the conventional fusion protocol. With the isolated IgM⁺ spleen cells, more than 75% (85 ± 7%; means ± SD) were IgM producing cells and a large number of IgM mAbs specific to the protein of interest were obtained. With the isolated IgG⁺ spleen cells, 41 ± 40% of the generated hybridomas produced IgG antibody and no IgM producing hybridoma was generated. A large number of IgG mAbs specific to the protein of interest could be produced. The results indicate that the generated hybridomas produce corresponding antibody isotypes as expressed on the surface of their starting cells. The technique that we have developed will be very useful for production of desired mAbs having a specific isotype.     

Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.     

Phosphorylcholine is favorable for antibody production from hybridoma cells

Growth of antibody-secreting hybridomas requires special conditions such as serum-free defined media containing growth factors and vitamins. However, the surface on which these cells can proliferate has been shown to play an important role. Phosphorylcholine (PC)-based polymers are zwitterionic compounds with nonbiofouling properties. These polymers are characterized by having reduced protein absorption properties. Our aim was to determine whether well-established hybridoma cell lines were able to proliferate and produce measurable amounts of monoclonal antibodies when grown on PC-polymer-coated surfaces. Comparative experiments using four well-known hybridoma cell lines (PAb421, PAb246, PAb1801 which recognize p53, and PAb280 which recognizes SV40 small t antigen) grown on PC-polymer-coated, uncoated, and two commercially available tissue culture plates showed that PC-polymer-coated plates were more efficient than uncoated plates in sustaining cell growth and monoclonal antibody production/secretion as defined by growth assays and ELISA. Also, results demonstrated that PC-polymer-coated plates were able to perform better than commercially available plates. These observations suggest that PC polymers could be used as an alternative, efficient surface coating to grow hybridoma cell lines and allow detectable antibody secretion.     

An IL 2-secreting T cell hybridoma that responds to a self class I histocompatibility antigen in the H-2D region

A self-reactive T cell hybridoma that secretes IL-2 in response to H-2d haplotype cells resulted from a fusion of BALB/cBy lymph node cells with the AKR thymoma BW5147. The lymph node cells used had been enriched for cells reactive to (TG)-A--L, but neither this antigen nor fetal calf serum were required for stimulation of the hybridoma designated 3DT52.5. The gene product responsible for stimulation mapped to the H-2D region. Allogeneic cells of the b, f, k, q, and s haplotypes failed to stimulate. Not all H-2d haplotype cells were effective stimulators of 3DT52.5. Peritoneal cells and splenic B cells were much more stimulatory than splenic T cells. Most tumor cell lines of H-2d derivation and of B cell or macrophage/monocyte lineage were stimulatory, whereas H-2d T cell lines were not. The capacity to stimulate 3DT52.5 did not correlate with the ability to stimulate I region-restricted hybridomas, or with the ability to be induced to stimulate such hybridomas. Stimulatory cell lines did not apparently produce a soluble factor required for stimulation, and negative cell lines were not inhibitory. The monoclonal antibody 27-11-13, which reacts with H-2D of the b, d, and q haplotypes, inhibited stimulation of 3DT52.5 but did not inhibit stimulation of the sibling hybridoma 3DT18.11, which responds to (TG)-A--L plus I-Ad. Conversely, the monoclonal anti-I-Ad antibody MK-D6 inhibited stimulation of 3DT18.11 but not 3DT52.5. Although it is clear that 3DT52.5 recognizes a class I antigen coded for in the H-2D region, the precise molecular nature of the antigen is unknown. The structure of the antigen receptor on this hybridoma may prove to be of interest when it can be compared with receptors found on T cell hybridomas restricted by class II histocompatibility antigens.     

Accumulation of unusual sterile transcripts of TCRβ in mouse hybridoma, murine tumour and non-human primate marmoset tumour

Accumulation of immunoglobulin Ig RNA (from several loci viz., C_H, C_α, J_k-C_k and S_μ during Igμ isotype switching) in B cells and T cell receptor (TCR) RNAs (α, β andγ) in T cells of unusual sizes emanating from germline and rearranged genes were reported to accumulate in human and mouse (and murine too). The precise mechanism and function of these sterile RNA species are yet to be delineated. Similar accumulation of RNA species of unusual sizes were identified with DNA-RNA hybridization and isolation of cDNA employing with DNA and antibody probes in mouse hybridoma, murine tumour, non-human primate marmoset tumour and human leukemic cells.     

Increased antigen binding strengths of hybrid antibodies produced by human hybrid hybridomas

Two cell lines of human hybridomas were fused to generate hybrid antibodies. One human hybridoma cell line was HT2 producing IgM monoclonal antibody (MAb) reactive to carboxy peptidase A (Cpase) and double stranded DNA (ds DNA) and another was SU-1-D2 secreting IgM MAb reactive to ds DNA but not to Cpase. Most hybrid hybridomas obtained by fusion of the two hybridomas secreted hybrid antibodies exhibiting increased antigen binding strengths. All of the hybrid antibodies with increased binding strengths against Cpase and ds DNA contained only the light chains derived from SU-1-D2. These results suggested that increase in the binding strength of the hybrid antibodies resulted from heterogeneous association of H and L chains derived from HT2 and SU-1-D2 cells.     

Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative “cloning-free” approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.     

Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative “cloning-free” approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.     

Demonstration of a novel tumor-killing factor secreted from human macrophage-monocyte hybridomas

We constructed human macrophage-monocyte hybridomas between a thymidine kinase-deficient human macrophage-like cell line, designated as TAM-2, and human peripheral blood monocytes in order for the study of cytokines from human monocytes. The hybrid and macrophage-monocyte nature of the growing cells was confirmed by the following facts: 1) All of the hybridomas established possess TK activity, whereas the TAM-2 cells are TK negative. 2) Most but not all of the hybridomas express the MaG-1 Ag which was shown to be a human macrophage-granulocyte specific Ag, but not T- and B-specific Ag. In the assay for cytokine, a few of the hybridomas produced a novel tumor-killing factor (TKF) after stimulation with PMA, polypeptone, and retinoic acid. Chemical nature of the TKF from the 3-63 hybrid clone was characterized and compared to those of well-known TNF and lymphotoxin. The TKF from a hybridoma was basic protein and had binding capacity to Con A-Sepharose, whereas TNF had an opposite nature. Moreover, TKF activity was not neutralized by both a murine monoclonal antibody against human TNF and rabbit antisera against human lymphotoxin. Thus, these results strongly indicate that the TKF is a novel TKF produced by human monocytic cells.