Parental Myeloma Research Articles

The Potential of O’nyong-nyong Virus Strain SG650 Murine Monoclonal Antibodies for Detection of O’nyong-nyong and Chikungunya Viruses

O’nyong-nyong virus (ONNV) and Chikungunya virus (CHIKV) are antigenically related alphaviruses responsible for febrile illnesses common to the tropics and associated with relatively high morbidity and mortality. Murine monoclonal antibodies (mAbs) targeting alphaviruses like Chikungunya have been developed and used to make commercially available kits. However, few studies have been conducted to develop antibodies specific to ONNV and no commercial kits are available for use in endemic regions where outbreak potential is high. We demonstrate the potential of in-house generated monoclonal antibodies against ONNV to detect both ONNV and CHIKV. The objective of this study was to generate mAbs using hybridoma technology, characterize the developed mAbs, determine their specificity against selected alphaviruses and check their diagnostic potential using an indirect IgG enzyme-linked immunosorbent assay (ELISA) and focus neutralization assay (FRNT50). BALB/c mice were immunized with ONNV purified proteins from ONNV infectious culture fluid. After four rounds of booster injections, the mice were sacrificed, spleen cells harvested and fused with parental myeloma cells then cultured in selective media and the successful hybrid clones with antibody-producing ability purified to yield the desired mAbs. Five monoclonal antibodies targeting the ONNV E1 protein of isotypes IgG2a/kappa, IgG2b/kappa and IgM/kappa (P1B12, P1E9, P1G11, P1B4 and P1G6) demonstrated a potential to detect both ONNV and CHIKV isolates by indirect IgG ELISA but no potential for neutralization of the viruses by FRNT50. This study demonstrates the potential efficacy of in-house serological tools as an alternative in the absence of commercial assays in screening and diagnosis of ONN and CHIK viruses which are often co-circulating. It is our recommendation that this work may be pursued further to design and optimize ELISA assays, using the developed mAbs, for the detection of both ONN and CHIK viruses in the research laboratory set-up

A CK1δ/CK1ε-to-β-Catenin Circuit Is a Therapeutic Vulnerability for Primary and Drug Resistant Multiple Myeloma

Multiple Myeloma (MM) remains an incurable malignancy, despite the advent of several new therapeutic agents, including immunomodulatory drugs (IMiDs, e.g., Lenalidomide (Len)) and proteasome inhibitors (PIs, e.g ., Bortezomib (Btz)). Accordingly, there is an urgent need to identify new targetable vulnerabilities for MM patients. We developed an ex vivo 384-well assay that allows one to define drug sensitivities of primary patient CD138+ MM cells in the context of a reconstructed tumor microenvironment (TME), including allogeneic BMSC, extracellular matrix and MM patient serum. Using this platform and activity-based proteomic profiling (ABPP), we identified shared signaling pathways induced by the interactions of MM with the TME and integrated these data with screens performed using a bank of protein kinase inhibitors (PKI) and current anti-MM therapeutics. These analyses revealed that the serine/threonine kinases casein kinase-1δ (CK1δ) and CK1ε are high priority targets for MM. In support of this data, a highly selective and potent dual inhibitor of CK1δ/CK1ε coined SR-3029 is the most potent PKI versus MM. Further, our studies revealed SR-3029 has potent activity in 75/86 primary patient MM specimens tested thus far, including quad and penta-refractory MM.Previous studies have shown that SR-3029 targets CK1δ in triple negative breast cancer (TNBC) and that the therapeutic response relied on disabling the Wnt/β-catenin signaling. Aberrant activation of the Wnt/β-catenin pathway has well established roles in the development and progression of cancer. Indeed, gain-of-function somatic mutations in β-catenin and upstream receptors such as LRP5, and loss-of-function mutations in components of the β-catenin destruction complex, including the scaffold proteins APC and Axin, are hallmarks of several tumor types.Our analyses of whole exome sequencing (WES) of MM patients revealed that ~40% of tumors have functional protein-altering mutations in the Wnt/β-catenin pathway, and that this is manifest in both treatment-naïve and relapsed MM. Additionally, RNA-seq and gene expression profiling analysis of over 1,300 MCC MM patients specimens have show that components of the Wnt/β-catenin signaling pathway are elevated in some subsets of MM. Further, we have shown that SR-3029 treatment or inducible knockdown of CK1δ or CK1ε rapidly compromises the growth and survival of MM cell lines. Importantly, the anti-MM activity of SR-3029 is augmented in MM cell lines with selected resistance to Btz and Len relative to parental myeloma cells. Further, treatment of MM cells with this kinase inhibitor leads to marked reductions in total β-catenin protein, decreases in β-catenin target genes (e.g., MYC, CCND1 and WNT3), and to marked changes in the expression of components of the Wnt/β-catenin signaling pathway. Finally, using the established 5TGM1/Kal-Ridge (C57B6/KaLwRijHsd) syngeneic mouse model of multiple myeloma, we show that tumors derived from 5TGM1 MM cells, which rapidly die following exposure to SR-3029 ex vivo, are also sensitive to CK1δ/CK1ε inhibition in vivo, where the SR-3029 treated cohort of animals demonstrated decreased tumor burden as assessed by IgG2b levels and imaging, and by significantly improved survival relative tovehicle treated recipients. Collectively, these findings demonstrate that SR-3029 has potent activity in both naïve and therapy resistant multiple myeloma and establish CK1δ and/or CK1ε as attractive targets for anti-myeloma therapy. DisclosuresSiqueira Silva:AbbVie: Research Funding. Teer:Interpares Biomedicine: Consultancy. Shain:Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Consultancy; Takeda Pharmaceuticals: Consultancy.

Is cancer progression caused by gradual or simultaneous acquisitions of new chromosomes?

BackgroundFoulds defined, “Tumor progression (as a) permanent, irreversible qualitative change in one or more of its characters” (Cancer Res. 1954). Accordingly progressions, such as metastases and acquired drug-resistance, were since found to be subspecies of cancers with conserved and numerous new chromosomes. Here we ask whether cancers acquire numerous new chromosomes gradually or simultaneously in progressions. The currently prevailing theory of Nowell (Science, 1976) holds that unexplained “genetic instability” generates “variant sublines (with) changes in chromosome number” and that “clonal” progressions arise by “stepwise selection of more aggressive sublines”. The literature, however, contains many examples of “immediate” selections of progressions with numerous new chromosomes - notably experimentally initiated fusions between cancers and heterologous cells. Furthermore, the stepwise progression theory predicts intermediate sublines of cancers with multiple non-clonal additions of new chromosomes. However, the literature does not describe such intermediates.ResultsIn view of these inconsistencies with stepwise progression we test here a saltational theory, in which the inherent variability of cancer-specific aneuploidy generates “immediate” progressions with individual clonal karyotypes, transcriptomes and phenotypes in single steps. Using cell fusion as an established controllable model of “immediate” progression, we generated seven immortal murine hybridomas by fusing immortal murine myeloma cells and normal antibody-producing B-cells with polyethylene glycol within a few minutes. These immortal hybridomas contained individual sets of 71 to 105 clonal chromosomes, compared to the 52 chromosomes of the parental myeloma. Thus the myeloma had gained 19 to 53 new clonal chromosomes in seven individual hybridomas in a single step. Furthermore, no stable intermediates were found, as would be predicted by a saltational process.ConclusionsWe conclude that random fusions between myelomas and normal B-cells generate clonal hybridomas with multiple, individual chromosomes in single steps. Similar single-step mechanisms may also generate the “late” clonal progressions of cancers with gains of numerous new chromosomes and thus explain the absence of intermediates. Latency would reflect the low probability of rare stochastic progressions. In conclusion, the karyotypic clonality of hybridomas and spontaneous progressions suggests karyotypic alterations as proximate causes of neoplastic progressions. Since cancer-specific aneuploidy catalyzes karyotypic variation, the degree of aneuploidy predicts the clinical risk of neoplastic progression, confirming classical predictions based on DNA content.

Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas

Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igα and Igβ of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igα expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).

Preclinical development of hybrid cell vaccines for multiple myeloma

Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We propose that hybrid cells, formed by fusing professional antigen-presenting cells with malignant plasma cells, would induce immune responses capable of mediating tumour regression. The human B-lymphoblastoid cell line, HMy2, was fused in vitro with CD138+ bead-separated myeloma plasma cells from five patients with MM. The hybrid cell lines generated in these studies grew stably in tissue culture, and maintained their phenotypic and functional characteristics, providing self-renewing cell lines with potential for therapeutic vaccination. The hybrid cells stimulated allogeneic and autologous T-cell proliferative responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, and directly activated both CD4+ and CD8+ T-cell responses. The enhanced T-cell stimulation correlated with expression of CD80 on the hybrid cells, and was inhibited by CTLA4-Ig fusion protein. The hybrid cell lines expressed several tumour-associated antigens known to be expressed in myeloma. These data show that self-replicating cell lines with enhanced immunostimulatory properties and potential for therapeutic vaccination can be generated by in vitro fusion of ex vivo myeloma cells and B-lymphoblastoid cell lines.

Immunoprecipitation: Purifying the Immune Complexes

INTRODUCTIONIn this protocol, immune complexes are formed by the addition of antibodies to cell lysates. The antibodies bind to their cognate antigen from a lysate. Protein A or protein G beads are then added to the solution containing antibody-antigen complexes, and the proteins that do not bind to the beads are removed by washing. The correct controls for immunoprecipitation should include nonimmune antibodies that are as close as possible to the specific antibody; for example, polyclonal serum should be compared to polyclonal serum from the same species (ideally a prebleed from the same animal used for immunization). For monoclonal antibodies, the control must be from the same source as the specific antibody. Do not use tissue culture supernatants from parental myeloma cells as they do not contain antibodies. Suitable control hybridoma cell lines can be obtained from the American Type Culture Collection and the European Collection of animal cell cultures.

Rapid specific amplification of rat antibody cDNA from nine hybridomas in the presence of myeloma light chains

Most monoclonal antibodies to mouse antigens have been derived from rat spleen-mouse myeloma fusions. Many resultant hybridomas express one of several myeloma kappa chain transcripts, even though the parent myeloma may have been ascribed as not expressing light chain protein. Previous reports have only differentiated against one of these mouse light chains. We have found at least three different myeloma kappa transcripts in the panel of nine hybridomas that were derived from four different myeloma parents. We have designed an amplification strategy that differentiates the rearranged rat kappa chain from all mouse light chains. Moreover, this method is expedient as it requires minimal downstream manipulation.

Development of Cell Based Therapeutic Vaccines for the Treatment of Multiple Myeloma.

Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We proposed that semi-autologous myeloma hybrid cells, formed by fusing malignant plasma cells with the human lymphoblastoid cell line, HMy2, would generate immortalised cell lines with enhanced immunogenicity for T cell responses, which could be used as potential therapeutic vaccines. The specific goals of the project were: to generate stable hybrid cell lines in vitro, by fusion of HMy2 with ex vivo tumour cells from patients with MM; to characterise the hybrid cells with respect to growth in culture, stability, and surface marker phenotype; and to investigate the ability of the hybrid cells to stimulate T cell immune responses in vitro as compared with parental tumour cells. The parent HMy2 cell line was fused with ex vivo myeloma plasma cells (purified from the marrow using CD138+ magnetic beads) from 5 patients, and chemically selected in HAT medium (to which HMy2 is sensitive), to form stable cell lines that grew continuously in culture. Efforts of growing myeloma cell lines from all of the patients were unsuccessful. The hybrid status of 4 of the 5 fused cell lines was confirmed by microsatellite testing using polymorphic loci on chromosomes X, 16q, 7q, 13q, 5q, 2p, 11p, 12p. Furthermore, the microsatellite analysis was checked after 3 months of continuous culture (approximately 40 passages), and all of the hybrids remained genetically stable over this period. Hybrid cells were analysed for expression of immunologically relevant accessory molecules using flow cytometry, in comparison with parent tumour cells and HMy2. The hybrids retained CD138 expression, although expression of CD56 was reduced in hybrids from two of three CD56+ myeloma cases. CD54 (ICAM-1) and CD50 (ICAM-3) were uniformly expressed in all hybrids. CD58 (LFA-3) was expressed more strongly in the hybrids than in original tumour cells, but CD11a (LFA-1) was weakly expressed or absent, in line with expression on HMy2 cells. All of the hybrids expressed CD40, CD80 (B7-1) and CD86 (B7-2), although CD80 was weakly or not expressed by myeloma plasma cells. Both MHC class I and II were expressed in all the hybrids, suggesting the potential for antigen presentation to both CD8+ and CD4+ T cells, in contrast to the majority of myeloma plasma cells, which failed to express MHC class II. The hybrid cells therefore expressed important markers to facilitate effective antitumour responses, which were absent on the parent tumour cells. The hybrid cells stimulated allogeneic T cell proliferative (3H thymidine uptake) responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, which failed to induce T cell proliferation above background levels. In addition, T cells from patients from whom hybrid cell lines had been derived responded to stimulation with both autologous and allogeneic hybrid cell lines, whilst failing to proliferate in response to autologous or allogeneic myeloma plasma cells. These data indicate that myeloma patients are immunocompetent, suggesting that active immunisation with semi-autologous hybrid cell lines represents a valid approach to immunotherapy in these patients. We have shown the generation of myeloma hybrid cell lines that grow continuously in tissue culture and have increased immunogenicity for T cell responses in vitro. This approach represents a novel strategy for the development of therapeutic vaccines for the treatment of multiple myeloma.

Immunomodulatory gene therapy for haematological malignancies.

Immunomodulatory gene therapy for haematological malignancies.

Early phase karyotype analysis of chromosome segregation after formation of mouse-mouse hybridomas with chromosome painting probes.

FISH analysis with chromosome painting probes allows, better than karyotyping after Giemsa banding, the study of chromosome segregation after hybridoma formation. FISH is particularly useful for intraspecies hybrids and allows visualization of small chromosome fragments. Cell hybrids were constructed between P3 x 63Ag8.653 mouse myeloma cells and lymphocytes from BALB/c mice by PEG fusion and by selection in hypoxanthine azaserine medium. Three hybridomas (A4, D8, F10) were selected and, after cloning, the cells were cultivated in vitro over a period of 28 days. During this time in culture, air-dried metaphase spreads were prepared by standard methods. For FISH chromosome painting, digoxigenin- and biotin-labeled mouse chromosome painting probes and rhodamine-antidigoxigenin antibodies and fluorescein-avidin were used for dual color detection. Total chromosome numbers and the numbers of mouse chromosomes 1, X, 6 and 12 were estimated as function of days in culture. Mean chromosome numbers of 78 (D8), 82 (F10) and 150 (A4) were observed. The major rearrangements of chromosome numbers occured in the first 28 days in culture and did not change significantly between day 28 and day 56. Mouse chromosome #12, which had the largest chromosome fragments in the parent myeloma, remained stable while the number of X chromosomes, which were significantly fragmented already in the parent myeloma, decreased by approximately 50%.

Prolongation of murine hybridoma cell survival in stationary batch culture by Bcl-xL expression.

While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures.

Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19K adenoviral gene.

It is now well documented that apoptosis represents the prevalent mode of death in lymphoid cultures and occurs spontaneously in late-exponential phase of batch cultures following nutrient exhaustion. In an attempt to enhance the cell survival of these cell lines, we have initially engineered nonproducing NS/0 myeloma cells with a vector expressing the adenoviral E1B-19K protein. NS/0 cells transfected with E1B-19K were found to be more resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. In this study, we have characterised a number of NS/0 subclones constitutively expressing different levels of E1B-19K, as well as several subclones in which the expression of E1B-19K was regulated by a tetracycline-controllable gene switch. We have found that a threshold E1B-19K level was required in order to achieve protection against apoptosis. The extent of resistance against cell death induced by nutrient deprivation in glutamine-free medium and in the late phase of batch cultures correlated with the level of E1B-19K expression up to an optimal level where further increases in E1B-19K levels did not result in significant additional protection. To assess the effects of E1B-19K on antibody productivity, an apoptosis-resistant NS/0 clone was then transfected with a chimeric antibody construct. Despite their improved viability, the antibody productivity of E1B-19K clones in batch culture was not significantly improved. Moreover, while the use of E1B-19K considerably delayed cell death, cells eventually died by apoptosis. Surprisingly, E1B-19K had no beneficial effect on the efficiency of fusion of NS/0 myelomas and splenocytes for the generation of hybridoma cells. Furthermore, the resulting hybridomas, although expressing E1B-19K at levels comparable to the myeloma parent, were no longer resistant to apoptosis. This indicates that the ability of E1B-19K to prevent apoptosis is not only dose-dependent but also seems to be cell-type dependent.

Cholinergic function in the hippocampus of juvenile rats chronically deprived of NGF

Intracellular and extracellular recordings were used to assess the cholinergic function in hippocampal slices from juvenile rats chronically deprived of NGF. NGF was neutralised by implanting into the lateral ventricle of postnatal (P) day 2 rats, αD11 hybridoma cells (secreting monoclonal antibodies specific for NGF). Parental myeloma cells (P3U) were used as controls. At P15–P18, slow cholinergic EPSPs could be elicited in cells from both αD11- and P3U-treated rats. However, slices from αD11-implanted rats exhibited a 50% reduction in acetylcholine release following stimulation of cholinergic fibres. This effect was associated to a significant increase in the sensitivity of pyramidal cells to carbachol, as suggested by the shift to the left of the dose/response curve. This may reflect a compensatory mechanism for the reduced efficacy of cholinergic innervation in NGF-deprived rats. In both αD11- and P3U-treated rats, carbachol was able to induce a similar concentration-dependent depression of the field EPSPs, evoked by Schaffer collateral stimulation, suggesting that presynaptic muscarinic receptors were not altered. In rats implanted with αD11 cells at P15 and sacrificed at P21–P24, no changes in the sensitivity to carbachol were found. At this developmental stage, no differences in acetylcholine release were observed between P3U- and αD11-treated animals. These results provide physiological evidence for a regulatory role of NGF in the cholinergic function of the hippocampus during postnatal development.

Transfection of murine myeloma cells to produce a chimeric antibody to the interleukin-2 receptor.

Murine myeloma X63Ag8.653 cells were transfected with heavy and light-chain expression vectors for a chimeric antibody (Ab) to the human interleukin-2 receptor. A cell line producing low quantities of the chimeric Ab was obtained and was transfected with either the cytomegalovirus (CMV) immediate-early gene ie1 or Epstein-Barr virus (EBV) BMLF1 DNA, together with the hygromycin B resistance (HyR) encoding gene for selection to improve productivity. Two cell lines with a four to eightfold increase in productivity were obtained. They showed higher levels of heavy- and light-chain mRNA expression. CMV ie1 or EBV BMLF1 DNA was not detected and no integration pattern changes for the heavy- and light-chain DNA were seen. The long-term productivity of one of the cell lines showed hygromycin B (Hy) requirement. Transfection with the HyR DNA alone also resulted in cells with increased productivity. The expression vectors contained the immunoglobulin light-chain enhancer kappa B DNA sequences (kappa B site). Nuclear extracts from parent myeloma cells showed one kappa B-binding protein band on a polyacrylamide gel, but nuclear extracts from transfected cells showed two additional slower-migrating bands. Increased Ab production correlated with an increased ratio of the medium-mobility kappa B-binding protein band to the high-mobility band. The possibility that Hy used for selection activated kappa B-binding proteins and increased Ab expression is discussed.

Cell-cell adhesion by homophilic interaction of the neuronal recognition molecule axonin-1.

The axonal surface glycoprotein axonin-1, which occurs both as a glycosyl-phosphatidylinositol-anchored membrane-bound form and a secreted form, promotes neurite outgrowth and is thought to be involved in axon-guidance mechanisms in the developing nervous system. Recently, we have demonstrated that the neurite-outgrowth-promoting activity of axonin-1, presented as a substratum for cultured neurons, is mediated by a heterophilic interaction with the axonal glycoprotein neuronglia cell-adhesion molecule (Ng-CAM). Here we present evidence for homophilic (like-like) binding among axonin-1 molecules. Axonin-1 was heterologously expressed in myeloma cells. Clonal cell lines, with exposed membrane-bound axonin-1 at their surface, formed large multicellular aggregates. Incubations of transfected and parental myeloma cells, under a series of different conditions, revealed homophilic axonin-1/axonin-1 interactions across the intermembrane space as the molecular mechanism promoting stable cell-cell contacts. Using structural and functional characterisation, recombinant axonin-1 was very similar to native axonin-1, suggesting that homophilic axonin-1 interactions are also established in neurons. The capability of axonin-1 to interact with both Ng-CAM and other axonin-1 molecules might contribute to the formation of macromolecular networks at contact sites of growth cones and axons, comprising molecules of both membranes, and thus represent a mechanism for regulating neurite outgrowth and pathfinding.

The effects of hybridoma growth factor in conditioned media upon the growth, cloning, and antibody production of heterohybridoma cell lines

Interleukin 6 (IL-6)/hybridoma growth factor (HGF) has been shown to be the requirement for growth of murine hybridomas in vivo or in vitro. In this paper, two kinds of conditioned media (CM) from the culture supernatants of a human fibroblast cell line CRL1506 and a cloned Epstein-Barr virus (EBV) transformed human lymphoblastoid cell line (LCL) N23 were found to have IL-6 activity by strongly promoting the growth, antibody secretion (increase of one- to three-fold), and cloning efficiencies of heterohybridomas secreting human monoclonal anti-hemorrhagic fever with renal syndrome virus antibodies and LCL. Since these CM contained no detectable IL-2, and IL-4 had no effects on the growth of the cell lines, IL-6 was considered to be the main active component of the CM responsible for promoting hybridoma growth. This effect was further confirmed by IL-6-dependent cell line 7TD1 bioassay (IL-6 activity in the CM ranging from 1,000 to 10,000 units/ml). Moreover, we successfully established four EBV-transformed lymphoblastoid cell lines at single-cell level by adding an equal volume of CRL1506-CM to 10% FCS-RPMI1640 in limiting dilution. Finally, it is worth noting that the sensitivity of the heterohybridomas to the two kinds of CM was not the same and was not consistent with that of their parental myeloma cell lines. Thus, it suggests that the CM might contain more than one factor, and the choice of proper conditioned media should be very useful for human monoclonal antibody production.

Herpes simplex virus-specific hybridoma cells cannot be persistently infected with this virus.

Mouse hybridoma clones were examined for their ability to support replication of herpes simplex virus (HSV). Infection of hybridoma clone 1 cells producing an antibody not specific for HSV resulted in a persistent infection with a continuous production of infectious virus, whereas infection of the parental myeloma cells X63-Ag8.653 led to an abundant virus production and extinction of the culture. In contrast, infection of hybridoma cells producing HSV-specific antibodies was restricted to a few weeks. Infectious virus was isolated for a maximum of 10 days and, afterwards, viral antigens were detected by immunofluorescence for a maximum of 18 days. The neutralizing capacity of the antibodies was not essential since the pattern of infection in clone III E8 cells, producing a non-neutralizing antibody, did not differ from that observed in clones 2c and VI A6, which produced highly and weakly neutralizing antibodies, respectively. After loss of viral antigen, HSV DNA was no longer detected by Southern blot hybridization in hybridoma clone 2c cells. Since no difference other than the specificity of the produced antibodies is suspected between the hybridoma clones, the results suggest that the presence of HSV-specific antibodies in the B-lymphoid cell cultures is responsible for virus elimination from the cells.

Increased frequency of both total and specific monoclonal antibody producing hybridomas using a fusion partner that constitutively expresses recombinant IL-6

The addition of auxiliary feeder cells or conditioned medium has been shown to augment the yield of mouse hybridomas obtained following the cell-cell fusion of myeloma and B lymphocytes. The addition of one of these factors, interleukin-6 (IL-6) has been found to increase the proportion of hybridomas secreting monoclonal antibodies of desired specificity. As an alternative genetic approach, we have examined the efficacy of retroviral infectant of Sp2/0 cells that constitutively expresses recombinant murine IL-6 (Sp2/mIL-6) as fusion partner. The results demonstrated that the yields of both viable Ig-secreting hybridomas, and antigen-specific monoclonal antibodies were increased 3–15-fold and 5–9-fold, respectively, with the Sp2/mIL-6 relative to Sp2/0 or Sp2/ neo cells as fusion partner. Sp2/mIL-6 cells generated hybridomas with comparable growth rates, stability, and Ig production. The results of staining nascent hybridoma colonies immunohistochemically for Ig production suggest that Sp2/mIL-6 cells as a fusion partner increased the viability and/or stability of nascent hybrid cells that are producing Ig. Thus the Sp2/mIL-6 cells are an improved myeloma parent for the generation of large numbers of antibody-producing hybridomas against specific antigens.

Ascites produced in recalcitrant hybridomas by backcrossing to a parental myeloma

Hybridoma lines frequently lose their ability to produce ascites upon extended cultivation in vitro. We have reintroduced genes for tumor formation into three independent hybridoma lines by backcrossing them to the parental myeloma. The parental myeloma cells were eliminated by a brief in vitro selective period in HAT medium, after which the cells were inoculated into pristane-primed mice. In two out of three cases the resultant lines were able to produce ascites without further subcloning or other manipulations. The antibody retained its specificity as judged by immunoblotting. This method is a rapid and efficient approach for the reestablishment of ascites production in hybridoma lines.

Suppression of the translocated myc gene and expression of intracisternal A‐particle genes in tumorigenic and non‐tumorigenic hybrids between murine myeloma and normal fibroblasts

We have studied the tumorigenic potential of a series of independent intraspecies hybrid clones derived from fusion of murine myeloma (BALB/c) and normal fibroblasts (C3H). All of these hybrids grew as adherent cells and thus resembled the fibroblast phenotype. As judged by chromosome enumeration, these hybrids appear to retain the full complement of their parental cells. Three out of 4 hybrids tested were able to form colonies in soft agar and to grow as tumors in either nude or (BALB/c x C3H) F1 mice, albeit at a reduced rate. The 4th hybrid did not grow in agar, was non-tumorigenic and may have had a 2:1 fibroblast to myeloma genomic equivalence ratio. In contrast to the parental myeloma cells, all the hybrids exhibited restricted growth rates in serum-free medium. As in our previous sets of hybrids formed between myeloma and L-cells, expression of the Ig genes was inhibited in the new hybrids and the derived tumors. The constitutive expression of the translocated myc gene in the myeloma parental cells was decreased in the hybrids and in all their derived tumors. In contrast, all of the hybrid cell lines and the tumors express high levels of the intracisternal A particle mRNAs. Our results show that the tumorigenic phenotype of myeloma cells is either fully or partially suppressed in myeloma x fibroblast hybrids and that this may be due to the fact that expression of the translocated c-myc is suppressed. We suggest that, in addition to the translocated myc gene, myeloma cells contain other activated oncogene(s), and that the latter are responsible for the residual tumorigenic potential of the myeloma x fibroblast hybrids.