Ultrasound-enhanced monoclonal antibody production.

With the rapidly growing demand for monoclonal antibody (mAb)-based products, new technologies are urgently needed to increase mAb production while reducing manufacturing costs. To solve this problem, we report our research findings of using low-intensity pulsed ultrasound (LIPUS) to enhance mAb production. LIPUS with frequency of 1.5 MHz and pulse repetition frequency of 1 kHz, as well as duty cycle of 20%, was used to stimulate hybridoma cells to enhance the production of mAb, anti-CD4 (hybridoma GK1.5). The enzyme-linked immunosorbent assay results show a 60.42 ± 7.63% increase of mAb expression in hybridoma cells. The evidence of structural changes of the cellular outer membrane in both transmission electron microscopy and scanning electron microscopy images and the more than 20% lactate dehydrogenase release indicates that the increased mAb production is related to the increased cell permeability induced by LIPUS. This value-added ultrasound technology provides a potential cost-effective solution for pharmaceutical companies to manufacture mAb-based drugs. The technology, in turn, can reduce the drug manufacturing costs and decrease health care spending.

Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment.

BACKGROUND: The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF) irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water) produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production. RESULTS: In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts proliferate slower in NPD water. CONCLUSION: Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion. Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.

Neoplastic transformation of a human hybrid cell line by alpha particles in relation to mammography X rays.

The hybrid cell line CGL1 is the only cell line which allows quantitation of neoplastic transformation in human cells. Hybrid cells were exposed to 3.4 MeV alpha particles or to mammography X rays (29 kV(p)) and both survival and neoplastic cell transformation were assayed. For comparison, previously published data obtained with 200 kV(p) X rays are also included. Alpha particles were significantly more efficient than 29 kV(p) X rays in cell killing. Surprisingly, the efficiency of mammography X rays for the induction of neoplastic cell transformation was close to that observed for alpha particles.

Retention and viability characteristics of mammalian cells in an acoustically driven polymer mesh.

A processing approach for the collection and retention of mammalian cells within a high porosity polyester mesh having millimeter-sized pores has been studied. Cell retention occurs via energizing the mesh with a low intensity, resonant acoustic field. The resulting acoustic field induces the interaction of cells with elements of the mesh or with each other and effectively prevents the entrainment of cells in the effluent stream. Experiments involving aqueous suspensions of polystyrene particles were used to provide benchmark data on the performance of the acoustic retention cell. Experiments using mouse hybridoma cells showed that retention densities of over 1.5 x 10(8) cell/mL could be obtained. In addition, the acoustic field was shown to produce a negligible effect on cell viability for short-term exposure.

[Influence of low doses of ionizing radiation on tenascin expression in hybridoma cell systems]. / Vliianie ioniziruiushchei radiatsii v malykh dozakh na ékspressiiu tenastsina v gibridomnykh kletochnykh liniiakh.

One of the achievements of the modern radiation ecology is the preparation and application of stable eukariotic cell lines to solve various problems occurring under exposure to ionizing radiation, especially to low doses. The detection of onco-fetal protein--tenascin in different embryonic and tumor cells of humans and animals supposes the probability of appropriate gene expression in lymphoid cells, including hybridomal cells. Using the immunochemical method, the study of tenascin expression in two mouse hybridomal lines was carried out. Tenascin was revealed in hybridomal lines MLC-1 and K-48. Further hybridomal cell lines were exposed to X-ray radiation (120 KV) with doses 2.10,15 cGy. The obtained results demonstrated the sensitivity of tenascin expression to low doses of ionizing radiation, that may be used as a convenient model of studying of genotoxic effects of various damaging ionizing agents on a cell level.

Heat inactivation of mammalian cell cultures for biowaste kill system design.

A biowaste kill system was implemented to treat biological waste generated from a clinical manufacturing and R&D antibody facility. To confirm that design parameters of this continuous decontamination system are sufficient to inactivate mammalian cell culture waste, bench-scale experiments were conducted. The biowaste kill system heat inactivates mammalian cell cultures before they are piped to a neutralization tank and subsequently released to the sewage system. Heat inactivation of cells is accomplished by exposing cells to 80 degrees C for 1 min. Small-scale heat inactivation studies were performed on CHO, 293-HEK, and hybridoma cells. Cells at 1 x 10(6) cells/mL or 1 x 10(7) cells/mL were exposed to 37, 60, 70, or 80 degrees C for 0, 30, 60, and 120 s. Viability based on trypan blue exclusion method and ability to proliferate was assessed after exposure to heat. Data suggest that exposure of cells to 80 degrees C for 60 s is sufficient to inactivate these cultures before they are released to the sewage system.

Cell replication rates and processes concerning antibody production in vitro are not influenced by 2.45-GHz microwaves at physiologically normal temperatures.

Several contradictory papers concerning the effects of microwaves on living organisms and on in vitro cell suspensions have been published through the years. These papers are difficult to interpret, because temperature measurement data are often lacking. Reliable temperature measurements are important, because they enable one to determine whether the observed microwave effects are thermal or nonthermal. Therefore, a method was developed to investigate microwave effects on cellular processes, in which the temperature was precisely monitored during microwave treatment using a fiberoptic thermometer. This method involved the processes required for in vitro production of monoclonal antibodies. Monoclonal antibodies are vital ingredients in (microwave-stimulated) immunostaining techniques and ELISAs, which have become important techniques in neuroscience. The effects of 2.45-GHz microwaves on mouse myeloma and (neural) hybridoma cell replication rates and on antibody production were investigated. In addition, the effects on the cell fusion abilities of spleen lymphocytes and myeloma cells and on in vitro immunization were studied. The results of this study show no effects of microwaves on either of the processes mentioned using exposure times up to 5 h a day at a physiologically normal temperature of 37 degrees C. It was concluded that the effects of 2.45-GHz microwaves detected at higher temperatures are thermal effects and that no indications for nonthermal 2.45-GHz microwave effects exist under the exposure conditions used in the present study.

UV irradiation of a B-cell hybridoma increases expression of alkaline phosphatase: involvement in apoptosis.

Expression of alkaline phosphatase (APase) by 7TD1 B-cell hybridoma was amplified by ultraviolet irradiation; cell growth was inhibited and cell death by apoptosis was increased. Irradiation induced high levels of APase activity in cycling as well as in apoptotic cells. In contrast, APase activity faded with time in nonirradiated cells and was no longer expressed in spontaneous apoptotic cells appearing after several days in culture. This was demonstrated by cell morphology, DNA fragmentation, and flow cytometry after simultaneous staining of DNA with Hoechst 33342 and APase with naphthol AS-TR phosphate--fast red RC fluorescent reagent. Levamisole, a specific inhibitor of APase activity, almost totally abrogated apoptosis induced by ultraviolet irradiation at doses that failed to affect 7TD1 cell survival. These data suggest that APase could play a role in the signalling cascade that mediates apoptosis in irradiated cells.

Chromophore-assisted laser inactivation of subunits of the T-cell receptor in living cells is spatially restricted.

Chromophore-assisted laser inactivation (CALI) is a molecular photoablation technique that has been used to elucidate the in vivo roles of specific proteins in neural development. The interpretation of its effects on proteins in living cells relies on knowing how spatially restricted the CALI-induced damage is in vivo. To determine the spatial specificity of CALI in living cells, we have applied CALI to individual subunits of the T-cell receptor (TCR) complex on the surface of 2B4 hybridoma cells in culture and have examined the consequent structural and functional integrity of the TCR-alpha, TCR-beta and CD3-epsilon. The CALI of TCR-beta resulted in the disruption of the beta subunit and also resulted in a small effect on antibody binding alone to the neighboring TCR-alpha but caused no effect on another subunit, CD3-epsilon. Reciprocal experiments directing CALI to TCR-alpha and CD3-epsilon gave consistent results. No effects other than a simple loss of function were observed for any of these CALI experiments. These data demonstrate the extent of CALI-induced damage within a multisubunit complex in living cells and provide greater confidence for the future application of this technique to understanding in vivo function of proteins during complex cellular processes.

Alpha-particle radiotherapy with 211At-labeled monodisperse polymer particles, 211At-labeled IgG proteins, and free 211At in a murine intraperitoneal tumor model.

Four different chemical forms of the alpha-particle emitting radionuclide 211At were injected intraperitoneally in mice inoculated intraperitoneally 30 hr in advance with 10(6) cells of the K13 murine hybridoma cell line. The different 211At forms were (a) free 211At, (b) 211At-labeled TP-3 nonspecific monoclonal antibody (211At-TP-3), (c) 211At-labeled human IgG kappa (211At-hIgG kappa), and (d) 211At-labeled monodisperse polymer particles (211At-MDPP). A significantly prolonged survival (P < 0.05) was observed with injected doses down to 7 kBq for the 211At-MDPP, and down to 25 kBq for 211At-hIgG kappa. There were no significant differences in survival between 211At-MDPP, 211At-hIgG kappa, and 211At-TP-3 at the dose level of 200 kBq. The group receiving 250 kBq free 211At per animal had a shorter survival than the three other forms at 200 kBq. The groups treated with 500, 200, and 65 kBq 211At-MDPP had a similar survival. The group given the highest dose of 211At-hIgG kappa (275 kBq) had the highest fraction (50%) of long-term survivors of all groups. Biodistribution measurements and total body scintigrams in mice without tumor revealed that the free 211At was distributed all over the body within 10 min after injection while at 2 hr a high fraction of the 211At-TP-3 and 211At-hIgG kappa was still present intraperitoneally. In conclusion this study indicates that 211At-labeled MDPP and 211At-labeled IgG's may be efficient tools for treatment of intraperitoneal superficial tumor cells and malignant ascites.

Subset-specific co-stimulatory signals are required for IL-2 production but not growth inhibition responses by T cell hybrids specific for myelin basic protein.

Two distinct types of T cell hybridomas (designated THYB-1 and T-HYB-2) were derived by fusing BW5147 thymoma cells with encephalitogenic T helper cells from Lewis rats. Both subsets required MHC-restricted presentation of determinants within the 72-86 peptide sequence of myelin basic protein (MBP) as a requisite signal for IL-2 production. Unlike THYB-1 hybrids, however, THYB-2 hybrids required additional accessory cell activities that were mediated by radiosensitive nonadherent (RS-NAdh) splenocytes (SPL). In this study, we describe two observations indicating that RS-NAdh SPL enable MBP-specific responses of THYB-2 hybrids by providing subset-specific co-stimulatory signals that act independently of antigen recognition pathways. First, RS-NAdh SPL were required by THYB-2 hybrids for MBP-stimulated IL-2 production but were not needed when MBP-specific inhibition of hybrid growth was used as an alternative measure of cellular activation. Second, PMA and ionomycin induced optimal IL-2 production by both THYB-1 hybrids and BW5147 thymoma cells but only stimulated low or marginal levels of IL-2 production by THYB-2 hybrids. Together, these observations indicate that RS-NAdh SPL were required for the specific response of IL-2 production regardless of whether the response was stimulated by antigen or by mitogens that bypass initial antigen recognition events. This study thereby provides additional evidence that distinct stimulus-response relationships define two T-helper cell lineages in experimental autoimmune encephalomyelitis.

Radiation-induced apoptosis in a murine T-cell hybridoma.

Induction of an apoptotic cell death was studied in a mouse T-cell hybridoma. Apoptosis was induced in these cells following exposure to dexamethasone, X-radiation, 43 degrees C heat shock, A254 light, and hydrogen peroxide. In 5-Gy-exposed cells, a radiation-induced G2 phase cell cycle progression block was maximum by 8 h. The cells began to escape this progression block by 10 h. Nuclear DNA fragmentation and uptake of the vital dye trypan blue began at 12 and 14 h, respectively, and were complete by 28 h. X-radiation-induced cell death was diminished when cells were irradiated in the presence of dimethyl sulfoxide, indicating that cell death was induced by oxidative cell damage. Substitution of nuclear DNA with bromodeoxyuridine enhanced death in cells exposed to either X-radiation or A310 light, indicating that apoptosis could be induced by DNA damage. The results are consistent with radiation-induced apoptosis being stimulated by oxidative DNA damage. DNA damage stimulates a long-lived signal which controls the expression of apoptosis. Apoptosis is expressed in the G1 phase of the cell cycle subsequent to the cell irradiation.

Spontaneous and radiation-induced genetic instability of heteromyeloma hybridoma cells.

We have examined the genetic stability of heteromyeloma cells both spontaneously and following ionizing radiation. Clones of E10 cells (SHM-D33 heteromyeloma X human lymphoblastoid) were examined for the stability of human immunoglobulin (Ig) production (mu, lambda), relative human and mouse DNA, and total DNA content. The stability of recloned E10 cells was improved more than fourfold relative to the stability of the nascent E10 cells. The spontaneous loss of human Ig production in the established E10 cells was approximately 1.5 x 10(-3) events/cell per generation, which is comparable to mouse hybridomas. In contrast to the relative stability of antibody production, the relative human DNA content of antibody producing clones of E10.26 cells showed considerable variation (median, 15%; range, 4 to 23% for 30 clones) although the total DNA content of the clones was relatively constant (1.2(+/- 0.1) x 10(-12)g/cell). The frequency of Ig(mu-) antibody loss variants was increased in three subclones of E10 cells following irradiation (P less than 0.05, 20 to 90 Ig(mu-) variants/10(5) cells per Gray. In addition, the human DNA content per cell was significantly reduced (P less than 0.001) in a sample of irradiated E10 clones, while the total DNA content per cell was constant. We conclude that, although the antibody production is relatively stable in heteromyeloma cells, the relative human DNA content is constantly drifting by small amounts while maintaining a constant DNA content.

Production of hapten-specific T cell hybridomas and their use to study the effect of ultraviolet B irradiation on the development of contact hypersensitivity.

We produced a series of T cell hybridomas that produce IL-2 when cultured with syngeneic APC coupled to FITC or TNP. These hybridomas are hapten specific and Ia restricted. The hybridomas were used to detect hapten-bearing APC in draining lymph nodes of mice sensitized with trinitrochlorobenzene or FITC in vivo. Hapten-bearing APC capable of stimulating the hybridomas were detectable in draining lymph nodes of hapten-painted mice within 3 h after sensitization. The ability of lymph node APC to stimulate the hybridomas peaked at 24 h and declined by 48 h. The dendritic cell subpopulation was the subpopulation of cells that were found in the regional lymph nodes of hapten-painted animals that were capable of stimulating the hybridomas to produce IL-2. Prior treatment of the skin with low dose UVB irradiation before epicutaneous application of contact sensitizers significantly reduced the capacity of hapten-bearing APC to stimulate the hybridomas. This observation was corroborated by results obtained from flow microfluorometry analysis of lymph node cells from FITC-sensitized mice. Lymph node dendritic cells obtained from FITC-painted mice contain a brightly staining group of cells by flow microfluorometry analysis. Lymph node dendritic cells from FITC-painted, UVB-irradiated mice did not contain this brightly staining population. These results indicate that low dose, local UVB irradiation may affect APC migration and/or function. We believe that these hybridomas will prove to be useful tools in the study of the development and regulation of contact hypersensitivity.

[The possibility of using outbred mice for the production of monoclonal antibodies]. / Vozmozhnost' ispol'zovaniia besporodnykh myshei dlia polucheniia monoklonal'nykh antitel.

Outbred laboratory mice, pretreated with an ascites-forming stimulator and an irradiation dose of 6 Gy, can be used for the production of monoclonal antibodies. However, the passage of hybridomas in irradiated outbred mice rapidly leads to the death of the cells. The properties of antibodies obtained from ascitic fluid of outbred mice are no different from those of antibodies obtained from BALB/c mice.

VH repertoire in progeny of long term lymphoid-cultured cells used to reconstitute immunodeficient mice.

VH gene utilization in the progeny of long term lymphoid-cultured cells used for reconstitution of severe combined immunodeficient mice under varying conditions was determined. Hybridomas made from the spleens of these animals were evaluated for clonality and donor origin and a panel of 146 independent hybridomas were subsequently examined for VH expression. Hybridomas derived from the spleens of SCID mice reconstituted with fresh cells, used as a control, utilized VH families in proportion to their numerical representation in the genome. However, hybridomas from the spleens of mice reconstituted with long term cultured cells utilized a predominance of the two VH gene families most proximal to JH, characteristic of cells early in B lymphocyte development. Coinjection of thymocytes with cultured fetal liver cells, to provide good levels of T lymphocytes, did not alter this pattern of VH utilization. Irradiation (3 Gy) of the mice before cultured cell injection, which leads to more complete reconstitution of the B cell compartment, was effective in removing this bias in the VH repertoire. Hybridomas derived from these mice expressed their VH genes more in proportion to family size, characteristic of cells later in B lymphocyte development. In this manner, long term lymphoid-cultured cells can be used to study the transitions that occur in VH repertoire expression which appear to be mediated by either B lymphocyte developmental microenvironment or population size.

Isotype restriction of murine antibodies towards the loop region of hen's egg white lysozyme.

Monoclonal antibodies specific for the loop determinant (residues 64-80) of hen's egg white lysozyme demonstrated an immunoglobulin class restriction. Only IgM response against the loop could be evoked in mice, irrespective of whether the immunogen was the intact native lysozyme as such, or the loop peptide covalently conjugated to a synthetic carrier, poly-DL-alanyl-poly-L-lysine (A-L). Despite the fact that in polyclonal antisera from mice immunized with lysozyme, the ELISA-titre of anti-loop reactivity was very low, 26% of the total anti-lysozyme response could be accounted for by the loop when expressed as the percentage of anti-lysozyme hybridoma colonies producing monoclonal antibodies reactive with the loop. The results can be interpreted either as determinant controlled isotype restriction, or alternatively, as an affinity restriction leading to the phenomenon that antibodies of isotypes other than IgM are formed, but are of too low avidity to be detected by the ELISA method.

Mapping of SJL/J reticulum cell sarcoma tumor-associated Ia antigens by T cell hybridomas: characterization of tumor-specific and shared epitopes detected on IE+ allogeneic cells.

Previous studies have suggested that reticulum cell sarcoma (RCS) tumor cells of SJL/J (IA + IE-) mice express neospecificities that are related to antigenic specificities characteristic of IE+ allogeneic cells. These neospecificities have also been suggested to play a role in the strong syngeneic antitumor proliferative response as well as in regulating RCS growth in vivo. The present studies characterize four RCS tumor-specific T cell hybridoma clones prepared from the fusion of BW5147 thymoma with T cells derived from lymph nodes of tumor-bearing mice. Upon stimulation, these hybridomas secrete IL 2 in the supernatant. Two hybridomas responded to RCS to IE+k and to IE+d allogeneic cells, respectively, and the other two hybridomas were tumor specific. The specificity of these hybridomas was assessed by response to both spontaneous and transplantable RCS lines and failure to stimulate a response by either normal or LPS-induced B cell blasts from the host SJL/J cells. The epitopes recognized by the T cell hybridomas were examined by the ability of several monoclonal antibodies to inhibit the IL 2-induced response by the T cell hybridomas. Antibodies directed against the IABs polypeptide of the IA hybrid molecule blocked the antitumor response by all four hybridomas. However, the response to allogeneic IE+ cells was not blocked by anti-IAs antibody but was blocked by antibodies directed against either the IAk,d or IEk,d hybrid molecules or the corresponding alpha- or beta-chains. The response to both RCS and allogeneic cells was blocked by monoclonal antibodies directed against L3T4 antigens on the T cells. Based on the exquisite specificity of the T cell receptors, the results here demonstrate that RCS tumor cells express on their surface both tumor-specific I-A-associated epitopes and Ia-associated antigenic specificities that are shared with IE+ allogeneic cells. The present studies of adapting T cell hybridomas and blocking antibodies proved useful to characterize and map distinct tumor-associated epitopes on the surface of tumor cells. These findings, when combined with structural studies, should help unravel the molecular complexity of tumor-associated antigens.

Expression of T cell receptor genes in an antigen-specific hybridoma and radiation-induced variants.

We have analyzed a series of mutants derived from a KLH-specific, I-E-restricted T hybridoma (FN1-18) which have lost antigen-reactivity while retaining both T cell receptor idiotypic determinants and the ability to respond to Con A. The variants have not gained any detectable alloreactivity, nor is there an obvious lesion in the mutants' beta chain DNA containing the utilized beta chain genes. This loss of antigen reactivity is due to a failure of stable production of the specific V beta-containing mRNA. Our results indicate that in FN1-18, the T cell receptor antigenic determinants are most likely carried by the alpha chain alone or by a complementation product of the V alpha FN1-18 with the V beta of BW5147. V beta FN1-18 represents a previously undescribed T cell receptor V region.

Gamma ray-induced mutants as a tool for the production and characterisation of monoclonal antibodies against HLA-alloantigens.

To simplify the screening procedure for murine monoclonal antibodies specific for polymorphic HLA determinants, spleen cells from a mouse immunized with the human cell line BJAB-B95.8.6 were fused with NS1 mouse myeloma cells, and hybridoma supernatants were screened for their reactivity on BJAB-B95.8.6 and two gamma ray-induced HLA-loss mutants of this line. The use of these HLA-loss mutants allowed the rapid identification of two new allospecific MOABs designated TU160 and TU161. Serological as well as biochemical studies revealed TU160 to be specific for HLA-A2, and TU161 for HLA-B13 molecules, respectively. Both MOABs were determined to be antibodies of the IgG class and were able to precipitate their antigens from lysates of radioactively labeled cells.