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Monoclonal antibodies have been applied in a wide range of biological and medical studies since the advent of cell fusion technology. Although cell fusion techniques have been improved by using myelomas and reagents, researchers still find it difficult to produce monoclonal antibodies because of the long protocols, high costs, and low efficiency of obtaining hybridomas. To solve these problems, we first developed an iliac lymph node method in 1995 using rats. In this method, an antigen emulsion is injected intramuscularly into the tail base, and then B lymphocytes are isolated from the enlarged iliac lymph nodes. This method is approximately 10 times more productive than the conventional spleen method. Here, we present further improvements to the iliac lymph node method to render it easily applicable in both mice and rats. We found that the frequency of hybridomas secreting specific antibodies was over five times higher using the electro cell fusion method than using the polyethylene glycol (PEG) fusion method. This frequency using the iliac lymph node method with electro cell fusion is at least 50 times higher than that using the traditional spleen method, thereby leading to the reduction in the number of mice or rats to be sacrificed. In addition, only a single injection for immunization is necessary for the iliac lymph node method, opposed to three for the spleen method. Therefore, this method is rapid, inexpensive, and ethical for producing monoclonal antibodies. [ABSTRACT FROM AUTHOR]

Background: Calcitonin gene‐related peptide (CGRP) therapeutics introduce new excitement and possibly yet to be determined distressing discords in the field of Headache Medicine. Growth in knowledge of CGRP in the pathophysiology of migraine introduced CGRP antagonism to headache treatment. Potential adverse effects on the other circulatory and neurovascular diseases have been foremost concerns. Failures in development of gepants and growth in knowledge of monoclonal antibody therapeutics combined to deliver the anti‐CGRP monoclonal antibodies (mAbs). Current Situation as of July 2018: Erenumab, eptinezumab, fremanezumab, and galcanezumab are approved, submitted to, or preparing for submission at both the European Medicines Agency and the US Food and Drug Administration (FDA). Methods: This Headache Currents update emanates from a symposium on CGRP and immunology in Headache Medicine, and reviews both. Results and Conclusion: Understanding CGRP in Headache Medicine requires information on aspects of the CGRP ligand, cell surface G protein receptor, CGRP receptor specifics, and antagonism by CGRP small and large molecules. Recent reports of CGRP's high affinity for amylin receptors dictate some attention to this family‐related peptide. To better understand potential immunogenic risks and off‐target toxicities of the anti‐CGRP monoclonal antibodies, this review discusses immunology and CGRP and reviews IgG structure and function, monoclonal antibody production, ligand–antigen–antibody relationships, and clinical CGRP mAb specifics. Upon completion, the reader should better summarize CGRP antagonist fundamentals, recall antibody structure and function, restate therapeutic mAbs attributes, and appraise immunogenic risks. [ABSTRACT FROM AUTHOR]

Growth in knowledge about calcitonin gene‐related peptide (CGRP) in the pathophysiology of migraine brought CGRP antagonism to headache medicine. Failures in development of small molecule CGRP receptor antagonists and increasing knowledge and use of monoclonal antibodies (mAbs) in medicine led to the breakthrough development of large molecule anti‐CGRP mAbs: eptinezumab, erenumab, fremanezumab, and galcanezumab. This specifics about CGRP immunology aims to outline: (1) knowledge needed for CGRP antagonism and (2) developmental issues of specific CGRP antagonists for provider use. This clinically oriented review documents IgG structure and function; state of the art of monoclonal IgG production and ligand‐antigen‐antibodies in migraine therapeutics contributing to immunogenic risks and off‐target toxicities. Specifics to CGRP ligand, receptor, antagonism, and molecules, small and large, complete this review. Completion will facilitate assessment of the similarities, differences, and application of the forthcoming anti‐CGRP receptor and ligand antagonists for patients. [ABSTRACT FROM AUTHOR]

Abstract: Photodynamic therapy (PDT) is a noninvasive, highly selective approach to the treatment of tumors. However, its therapeutic effect is limited by long‐lasting skin phototoxicity. Therefore, to compromise this shortcoming, it is preferable to deliver photosensitizers selectively to tumor cells with the aid of antibodies specific against tumor‐associated antigens. Cancer/testis antigens 83 (CT83), also called KK‐LC‐1 or CXorf61, recognized by cytotoxic T lymphocytes (CTL), has become a promising target for immunotherapy. Herein, we developed and characterized a novel mouse CT83 mAb 7G4 with a high affinity with Gallium (III) 5, 10, 15‐tris (ethoxycarbonyl) corrole (1‐Ga), a new and promising photosensitizer in PDT. The enzyme‐linked immunosorbent assay (ELISA), flow cytometry and cytotoxicity activity assays revealed that 7G4‐1‐Ga was able to recognize human CT83 with high specificity. Furthermore, 7G4‐1‐Ga showed greater cytotoxicity to CT83‐expressing human cancer cells in vitro than 1‐Ga. These results suggest that the antibody‐conjugated photosensitizer between anti‐CT83 mAb and 1‐Ga may have a good application in PDT, where the destruction of CT83‐expressing tumor is required. [ABSTRACT FROM AUTHOR]

Amino acids and glucose consumption, cell growth and monoclonal antibody (mAb) production in mammalian cell culture are key considerations during upstream process and particularly media optimization. Understanding the interrelations and the relevant cellular physiology will provide insight for setting strategy of robust and effective mAb production. The aim of this study was to further our understanding of nutrient consumption metabolism, since this could have significant impact on enhancing mAb titer, cell proliferation, designing feeding strategies, and development of feed media. The nutrient consumption pattern, mAb concentration, and cell growth were analyzed in three sets of cell cultures with media supplementation of glucose, methionine, threonine, tryptophan, and tyrosine. The amino acids metabolism and its impact on cell growth and mAb production during the batch and fed‐batch culture were closely analyzed. It was shown that the phenylalanine, tyrosine and tryptophan biosynthesis pathways were significantly altered under different culture conditions with different media. These changes were more apparent in the fed‐batch process in which higher mAb titer was observed due to the metabolic changes than mAb titer in the batch process. The pathway analysis approach was well utilized for evaluating the impact on the relevant pathways involved under different cell culture conditions to improve cell growth and mAb titer. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:793–805, 2018 [ABSTRACT FROM AUTHOR]

Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016 [ABSTRACT FROM AUTHOR]

For several cancer cell types, the lack of an adequate supply of the amino acid l-glutamine (Gln) triggers apoptosis, a phenomenon termed Gln addiction. In this report, we examined the role of the anti-apoptotic proteins of the B-cell lymphoma 2 (BCL-2) protein family in the survival of Sp2/0-Ag14 (Sp2/0) mouse hybridoma cells, a cell line that undergoes apoptosis within minutes of Gln deprivation. Western blot analysis revealed that myeloid cell leukaemia 1 (MCL-1) was expressed at much higher levels than BCL-2, B-cell lymphoma extra-large and BCL-2-like protein 2 making it the prominent pro-survival BCL-2 family member in this hybridoma. Gln deprivation triggered a progressive decrease in MCL-1 protein levels, which coincided with the decrease in Sp2/0 cell survival. Moreover, Sp2/0 cells were much more sensitive to the broad Bcl-2 homology domain-3 (BH3) mimetic obatoclax (which targets MCL-1) than to the more selective drug ABT-737 (which does not target MCL-1). Finally, we show that obatoclax sensitizes Sp2/0 cells to apoptosis following Gln starvation. All together, the data presented here reveal that modulation of the pro-survival protein MCL-1 is an important step in the sequence of events leading to the initiation of apoptosis in Gln-starved Sp2/0 cells. Cancer cells require an adequate supply of l-glutamine for their survival. Using a mouse hybridoma cell line that is exquisitely sensitive to glutamine starvation, we show that the levels of the pro-survival BCL-2 family protein MCL-1 decrease upon glutamine starvation in a manner that correlates with the loss of cell viability. Moreover, inhibiting MCL-1 with the drug obatoclax sensitizes hybridoma cells to glutamine starvation. Thus, in some cancer cells, glutamine starvation triggers the inactivation of pro-survival proteins. Our data suggest that the combined inhibition of glutamine biosynthesis pathways and BCL-2 proteins may prove effective against some cancers. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Passively administered antibodies to conserved epitopes on the attachment (G) glycoprotein of human respiratory syncytial virus (hRSV) have potential in the immunoprophylaxis of human infections. This study set out to generate monoclonal antibodies (MAbs) recognizing all prevalent lineages of HRSV and capable of immunoprophylaxis in mice. Two murine MAbs of broad specificity for prevalent virus strains were generated by immunization of mice with hRSV of sub-group A followed by selection of hybridomas on recombinant G glycoprotein from a sub-group B virus. The anti-G hybridomas generated secreted antibody of high affinity but negligible neutralizing capacity one of which was tested in mice and found to be protective against live virus challenge. Western blotting and partial epitope mapping on transiently expressed G-glycoprotein fragments indicate that these antibodies recognize a complex epitope on the protein backbone of the molecule involving residues both C′- and N-terminal to the central conserved motif. J. Med. Virol. 86:1267-1277, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Three heavy-chain and three kappa (κ)-chain transcripts were amplified from hybridoma cells secreting a monoclonal antibody (m Ab) against transferrin receptor. Sequence analysis via IMGT/ V- QUEST yielded the functional/aberrant prediction. Two functional κ-chain transcripts, Vκ2 and Vκ3, and one functional V H1 were revealed. Comprehensive bioinformatics analyses including sequence alignment, phylogenetic tree, somatic hypermutation prediction, and three-dimensional-molecular structure modeling were used to predict the origin of the two κ-chain transcripts. The results of bioinformatics analysis suggest that Vκ3 is derived from the myeloma partner of the hybridoma; Vκ2 is derived from B-cell. Functional transcripts V H1 and Vκ2 and Vκ3 were then used to construct two chimeric antibodies chi- C2 ( Vκ2- V H1) and chi- C3 ( Vκ3-V H1), respectively. Antigen-binding experiments showed that only chi- C2 remained the same affinity as its parental mAb. Possible explanations for the coexistence of two functional κ-chain transcripts and the different affinity of the two chimeric antibodies are discussed. [ABSTRACT FROM AUTHOR]

Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools. Published 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:745-753, 2013 [ABSTRACT FROM AUTHOR]

The rapidly expanding market for monoclonal antibody and Fc-fusion-protein therapeutics has increased interest in improving the productivity of mammalian cell lines, both to alleviate capacity limitations and control the cost of goods. In this study, we evaluated the responses of an industrial CHO cell line producing an Fc-fusion-protein to hyperosmotic stress, a well-known productivity enhancer, and compared them with our previous studies of murine hybridomas (Shen and Sharfstein, Biotechnol Bioeng. 2006;93:132–145). In batch culture studies, cells showed substantially increased specific productivity in response to increased osmolarity as well as significant metabolic changes. However, the final titer showed no substantial increase due to the decrease in viable cell density. In fed batch cultures, hyperosmolarity slightly repressed the cellular growth rate, but no significant change in productivity or final titer was detected. To understand the transcriptional responses to increased osmolarity and relate changes in gene expression to increased productivity and repressed growth, proprietary CHO microarrays were used to monitor the transcription profile changes in response to osmotic stress. A set of osmotically regulated genes was generated and classified by extracting their annotations and functionalities from online databases. The gene list was compared with results previously obtained from similar studies of murine-hybridoma cells. The overall transcriptomic responses of the two cell lines were rather different, although many functional groups were commonly perturbed between them. Building on this study, we anticipate that further analysis will establish connections between productivity and the expression of specific gene(s), thus allowing rational engineering of mammalian cells for higher recombinant-protein productivity. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [ABSTRACT FROM AUTHOR]

Abstract: Ten years after the first clinical application of Rituximab, an anti-CD20 recombinant monoclonal antibody, immunotherapy has become common practice in oncology wards. Thanks to the great diversity of the immune system and the powerful methodology of genetic engineering, the pharmacologic potential of antibody-based therapy is far from exhaustion. The recent application of Trastuzumab, an antibody to a receptor tyrosine kinase, in adjuvant breast cancer therapy marks the beginning of a new phase in cancer treatment. Here we discuss molecular mechanisms of antibody-based therapy, the emerging ability to target minimal disease and the therapeutic potential of combining antibodies with other modalities. [Copyright &y& Elsevier]

Sodium butyrate (NaBu) can increase the specific Mab production rate of hybridomas by enhancing histone hyperacetylation and influencing the cell cycle, but it can also inhibit cell growth and induce apoptosis. Thus, the beneficial effect of NaBu on Mab secretion is compromised by its cytotoxic effect. In the present study, expression of the anti-apoptotic protein human Bcl-XLwas made inducible in hybridoma H18 to overcome the cytotoxic effect of NaBu, circumventing the detrimental effects of constitutive high-level expression. We constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drove the expression of bcl-XLin response to metal exposure. The vector was then used to exogenously control the expression of bcl-XLin H18 hybridoma cells. Our data showed that stably transfected H18.D4 cells expressed high levels of Bcl-XL, which was induced within 24 h of addition of ZnSO4. NaBu (0.4 mM) increased antibody production by more than 3-fold in H18.D4. This effect resulted from the suppression of NaBu-induced apoptosis, allowing the H18.D4 cells to grow at higher viability and extending culture longevity by >3 days. [Copyright &y& Elsevier]

Five stable hybridoma cell lines secreting monoclonal antibodies (Mabs) specific for GLRaV-1, one of the agents involved in the aetiology of grapevine leafroll disease, were produced by fusing a nonsecreting myeloma cell line with spleen cells from BALB/c mice immunized with purified GLRaV-1. The Mabs were characterized for their recognition of virus coat protein by DAS-ELISA and Western blotting. Mab (1G10) reacted specifically in ELISA, immuno-electron microscopy and immunoblotting with both GLRaV-1 and GLRaV-3 coat proteins. Mab 1C4 detected 25 of the 33 GLRaV-1 isolates, while Mab 1B7 reacted with 32 isolates including the eight isolates not recognized by Mab 1C4. Two of these hybridoma lines (2F11 and 2F3) are now used routinely for the immunodiagnosis of GLRaV-1. [ABSTRACT FROM AUTHOR]

In 1975 O'Farrell described, in detail, a procedure to separate proteins by polyacrylamide gel electrophoresis in two dimensions. This powerful new technique relied on two characteristics of proteins charge and molecular mass In the first dimension, proteins: were separated on the basis of net charge in a pH gradient by isoelectric focusing, and in the second dimension the proteins were further fractionated exclusively on the basis of their molecular mass by SDS gel electrophoresis. Since two-dimensional gel electrophoresis (2DGE) has a resolving power of at least 20 fold greater than one-dimensional electrophoresis, it has found wide spread application in modern biological research. However, beyond the detection of a given protein, 2DGE provides little additional information about a specific protein other than molecular mass, isoelectric point, and approximate relative abundance in recent years, the development of new technologies have made it possible to directly obtain sequence information, and produce specific antisera for proteins resolved by 2DGE. These new technological developments serve to further increase the power and utility of 2DGE in the analysis of proteins of importance to plant physiology. [ABSTRACT FROM AUTHOR]