Rat Hybridoma Research Articles

Melatonin attenuates acute and chronic itch in mice: the antioxidant and anti-inflammatory effects of melatonin receptors.

BackgroundItch is a common symptom of skin diseases and significantly reduces patients’ quality of life. Melatonin has anti-inflammatory and antioxidant effects. Our study examined the potential anti-itch effects of melatonin (N-acetyl-5-methoxytryptamine) in mice.MethodsWe detected the effects of melatonin and its receptors on acute and chronic itch by conducting itching behavioral experiments in male C57 mice. Reactive oxygen species (ROS) levels and calcium ion (Ca2+) mobilization during acute itching production were explored using flow cytometry and calcium imaging techniques. Melatonin expression in the serum of the chronic itch model mice was determined by enzyme-linked immunoassays. Hematoxylin and eosin staining show the effects of melatonin on skin thickness in a chronic itch model. Cytokine and chemokine levels were determined by quantitative polymerase chain reaction.ResultsWe discovered that compound 48/80 (C48/80)- and chloroquine (CQ)-induced scratching were significantly decreased by intraperitoneal (i.p), intradermal, and intrathecal administration of melatonin in a dose-dependent manner in mice, and the co-administration of melatonin receptor antagonists abolished the anti-itch effects of i.d melatonin. The incubation of melatonin significantly decreased the intracellular ROS levels induced by C48/80 and CQ in cultured ND7/23 cells from a mouse x rat hybridoma nerve as neuron. Melatonin inhibited intracellular Ca2+ increases induced by CQ (but not C48/80) in cultured dorsal root ganglia (DRG) neurons. Melatonin (50 mg/kg i.p) attenuated imiquimod (IMQ)- or acetone and diethyl ether followed by acetone-ether-water (AEW)-induced chronic itch and epidermal hyperplasia in mice. Finally, melatonin treatment reduced the IMQ-induced expression of ST2 and interleukin-33 (IL-33) or the AEW-induced expression of interleukin 31 (IL-31) and interleukin 31 receptor A (IL-31 RA) in the mice.ConclusionsCollectively, our results indicate that melatonin attenuates acute and chronic itch, possibly via melatonin receptors, and its antioxidant, and anti-inflammatory effects in mice.

Novel ROR1 Antibody Is Able to Trigger Specific and Superior Complement-Dependent Cytotoxicity (CDC) on Primary CLL Cells

Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia of the elderly, remains largely incurable despite chemotherapy and small molecule inhibitors. Treatment targeting CD19 and CD20 has achieved important clinical benefits; however, these antigens are also present on normal B-cells rendering the patients immunodeficient and at a higher risk of infections. In view of this, our immunotherapy strategy is to produce therapeutic antibodies targeting the receptor-tyrosine-kinase-like orphan receptor 1 (ROR1), a protein present on the surface of malignant B-cells and cancer stem cell-like cells (CSC), but absent on most normal tissue.In order to produce new ROR1 monoclonal antibodies (MAbs), we generated a rat hybridoma library and screened >150 anti-ROR1+clones. We then cloned 16 of our own novel antibodies as well as previously published clones: 4a5 and R12 to human IgG1, kappa constant regions. A total of 13 chimeric antibodies recognised human ROR1 on different cell types, out of which, clone SA1 showed superior complement-dependent cytotoxicity (CDC) than other ROR1 MAbs, including clones 4a5 and R12.To determine the binding domain of our ROR1 MAbs, we generated stable cell lines expressing either full length-ROR1 or various forms of truncated ROR1 extracellular domains. Flow cytometry evaluation using these cells, and Epitope Mapping ELISA using a ROR1 peptide library, showed that our antibodies preferentially bind the Ig-like and Frizzled domains. Additionally, we fused clone SA1 to a mouse IgG2a, kappa constant regions in order to perform competition assays amongst our ROR1 MAbs and previously reported clones. We found that there was no epitope overlap amongst any of the tested clones. Moreover, by substituting single amino acids in a relevant region within the Ig-like domain, which we previously determined to be critical for SA1 binding, we found that this epitope is unique to SA1 and, notably is not shared by other clones previously reported in the literature; including clone D10, the prototype of Cirmtuzumab. Importantly, further characterisation of clone SA1 revealed that it can get internalised by both SKW 6.4 cells and CLL cells.As a whole, we have generated and identified a novel anti-human ROR1 monoclonal antibody able to trigger specific CDC of ROR1+ cells. More than that, clone SA1 has a unique epitope and it is able to get internalised by both cell lines and CLL cells that endogenously express ROR1. Additional characterisation and functional analyses are ongoing in order to also develop this antibody as a drug-antibody conjugate (ADC). Importantly, effective targeting of ROR1 expressed on malignant cells and CSC cells but not healthy tissue would provide a safer and more effective treatment of CLL and, in turn, other ROR1+ malignancies. DisclosuresParedes-Moscosso:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Gohil:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies.

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8±5.2ng/ml in proximal small intestine to 54.3±10.3ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.

Abstract 1832: Anti-nicastrin monoclonal antibodies as therapeutics for invasive breast cancer: Characterization and function

Abstract Background: Nicastrin (NCT) is a crucial structural and functional constituent of the gamma-secretase (GS) enzyme which activates a range of substrates (Notch1-4, CD-44, Her-4, E-cadherin, EpCAM etc.), with a causative role in development and progression of malignant disease. NCT is the only GS component with a single-pass transmembrane- and a large extracellular domain (ECD). This makes NCT a potential target for monoclonal antibody (Ab) therapy. NCT has a GS-substrate recognition role, while other relevant functional regions of NCT ECD are still unknown. We have previously determined that NCT is overexpressed in breast cancers (BC) where its abundance confers worse overall survival in the estrogen receptor negative patients and that its genetic silencing inhibit BC cell invasion (Filipovic et al, Br Can Res Tr 2011). Methods: Using genetic immunization with NCT cDNA, we raised and cloned rat hybridomas expressing anti-NCT monoclonal Abs (NMA). Four clones (NMA1-4) were selected and purified, based on their ability to recognise cell surface NCT on BC cells and potency to inhibit BC cell invasion/proliferation in vitro. Non-cell based binding of whole Ab IgGs was measured by ELISA. Cell-based binding was assessed by FACS. BIACore 3000 was used for initial epitope mapping. Antigen for ELISA and BIACore was the NCT-ECD protein.Results: From the four clones, three induce functional cellular effects (NMA2-4). NMA1 clone recognises cellular, cell surface NCT but has no functional effects on BC cells and was used as negative control in all experiments. In MDA-MB-231 cells, Abs NMA2 and NMA4 were equally potent at inhibiting invasion (>60%). NMA4 and NMA2 both arrested MDA-MB-231 cell proliferation up to 50%. In 3D Matrigel, NMA2 and NMA4 conserved growth inhibitory effect and induced re-polarization of BC acini. By ELISA, the highest Kd was determined for NMA4. Cell-based quantitative FACS on non-permeabilized MDA-MB231s allowed determination of Ab binding affinity to the endogenous antigen. Binding affinity could be ranked in the following order: NMA1 = NMA2 = NMA4 > NMA3. BIACore competitive binding determined that NMA2 and NMA4 Abs bind spatially distinct regions on NCT. Cell fractionation and western blotting showed that NMA4 recognizes cell-membrane NCT. NMA2 and NMA4 reduced Notch activation and vimentin levels in MDA-MB231 cells by >50%. Conclusion: Our results suggest that anti-NCT Abs may be potential therapeutics for invasive breast cancer. Data will be presented which compare the relationship between BIAcore binding affinity, cross-competition for NCT binding site and functional inhibition of cell proliferation and invasion, indicating relevance of distinct NCT domains for distinct cellular processes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1832. doi:1538-7445.AM2012-1832

Effects of BCL-2 over-expression on B cells in transgenic rats and rat hybridomas

The rat is an important biomedical experimental model that benefited from the recent development of new transgenic and knockout techniques. With the goal to optimize rat mAb production and to analyze the impact of Bcl-2 on B-cell development, we generated bcl-2 transgenic rats. Transgenic rats showed Bcl-2 over-expression in B cells, increased B cell numbers in lymphoid organs, elevated production of immunoglobulins (Igs) and prolonged B-cell survival in vitro. Transgenic rats remained healthy, reproduced normally and did not develop autoimmunity. Fusions with bcl-2 transgenic splenocytes did not result in increased hybridoma generation. A comparison of on- and off-rates of 39 mAbs generated with bcl-2 transgenic and wild-type animals revealed no significant differences. Over-expression of Bcl-2 in hybridomas did not change cell proliferation but resulted in increased Ig production. Bcl-2 transgenic rats will be a useful tool for the generation of rat mAbs, the analysis of B cells in different pathophysiological models, such as autoimmunity, cancer or organ transplantation, and the study of rat B-cell biology.

Characterization of New Alternaria alternata–Specific Rat Monoclonal Antibodies

In this study, three different rat hybridoma cell lines secreting monoclonal antibodies (mAbs) recognizing the spores from Alternaria alternata, a plant pathogenic fungus, contaminant of food products and important cause of both allergic rhinitis and asthma, have been characterized. These three mAbs are all of IgM isotype. Two antibodies, A1 and F10, were cross-reactive antibodies recognizing spores from Alternaria, Cladosporium, Penicillium, Aspergillus and Stachybotrys genera, but not the yeasts Saccharomyces cerevisiae or Candida albicans. Competitive and sandwich assays demonstrated that these two mAbs were directed against the same or very close repetitive(s) epitope(s). A1-based sandwich ELISA efficiently detected this epitope in various mould (but not yeast)-soluble extracts prepared from strains grown in the laboratory. Moreover, this A1-based sandwich ELISA detected its cognate epitope in air and dust samples obtained from dwellings. The third antibody, E5, recognized only the spores of Alternaria and the phylogenetically very close Ulocladium botrytis. This E5 antibody is directed against a repetitive epitope found in Alternaria and Ulocladium laboratory extracts and can be used in a sandwich assay for the quantification of these moulds. Therefore, E5 antibody is a promising tool for the development of Alternaria-Ulocladium-specific immunoassays, while A1 and F10 could be interesting tools for the quantification of the total mould biomass.

Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity

Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r (2) values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions.

Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

BackgroundThe rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.ResultsThe cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.ConclusionAltogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.

Galectin-3 Gene Inactivation Reduces Atherosclerotic Lesions and Adventitial Inflammation in ApoE-Deficient Mice

This study has examined the role of galectin-3 (GaL3), a multicompartmented N-acetyllactosamine-binding chimeric lectin, on atherogenesis in the ApoE-deficient mouse model of atherosclerosis. Pathological changes consisting of atheromatous plaques, atherosclerotic microaneurysms extending into periaortic vascular channels, and adventitial and periaortic inflammatory infiltrates were assessed in an equal number (n = 36) of apolipoprotein (Apo)E-deficient mice and ApoE-GaL3 double-knockout mice. These mice were divided into three age groups, 21 to 23 weeks, 25 to 31 weeks, and 36 to 44 weeks of age. Results of this morphological analysis have shown an age-related increase in the incidence of aorta atheromatous plaques and periaortic vascular channels in ApoE-deficient mice. By contrast ApoE/GaL3 double-knockout mice did not show an increase in pathological changes with age. The 36- to 44-week group of ApoE(-/-)/GaL3(-/-) mice had a significantly lower number of atherosclerotic lesions (P < 0.004) and fewer atheromatous plaques (P < 0.008) when compared with ApoE(-/-)/GaL3+/+ mice of the same age. ApoE(-/-)/GaL3(-/-) mice had a lower number of perivascular inflammatory infiltrates and mast cells than those found in ApoE(-/-)/GaL3+/+ mice. The reduced number of perivascular mast cells may have resulted in a low level of interleukin-4 that contributed to the reduction in the morphological parameters of atherogenesis correlated with the lack of GaL3 expression. The effect of GaL3 deficiency on atherogenesis decrease could be related to its function as a multifunctional protein implicated in macrophage chemotaxis, angiogenesis, lipid loading, and inflammation.

Comparison of cell lines for stable production of fucose‐negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.

Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv–expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion

Tumor-destructive immune responses can be generated by engaging CD137 (4-1BB) via infusing a monoclonal antibody specific for CD137 or vaccinating with a single-chain Fv (scFv) CD137-expressing whole-cell tumor vaccine. We assessed whether such a vaccine can induce tumor rejection in the neu-transgenic (neu-Tg) mouse breast cancer model and compared the antitumor efficacy of vaccination with the infusion of a CD137-specific antibody. Mammary carcinoma cells (MMC) from a neu-Tg mouse were transfected to stably express surface scFv derived from the anti-CD137 rat hybridoma 1D8 or 3H3. The anti-CD137 scFv-expressing cells were rejected when transplanted into neu-Tg mice by a mechanism that involved both CD4(+) and CD8(+) T cells, and vaccination with such cells delayed the outgrowth of MMC cells transplanted 3 days previously. T cells from neu-Tg mice that had been vaccinated proliferated and produced IFN-gamma when stimulated by MMC but not by antigen-negative variant breast cancer cells that did not express the neu tumor antigen. In addition, antibodies binding to the MMC but not to antigen-negative variant cells were detected in sera from some but not all of the immunized mice. Complete regression of s.c. transplanted MMC tumors was observed in mice repeatedly immunized against MMC-1D8 starting on the day the MMC cells were transplanted. In contrast, repeated administration of either of two different anti-CD137 monoclonal antibodies did not induce complete tumor regression, although tumor growth was delayed.

Production and characterization of mouse ureteric bud cell-specific rat hybridoma antibodies utilizing subtractive immunization and high-throughput screening

The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used. A subtractive immunization strategy was performed in rats to generate monoclonal antibodies that preferentially reacted with antigens on UB, but not IMCD cells. In addition, the technique of antibody printing, a novel high-throughput antibody screening method for determining the specificities of a large number of monoclonal antibodies, is described. The methodologies outlined in this manuscript have broad applicability as they demonstrate that subtractive immunization can be performed in rats with cells derived from mice. Additionally, the high-throughput screening methods should facilitate the use of subtractive immunization for identifying antibodies that can distinguish differences in proteins expressed in closely related cell types.

The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity

An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes alpha1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody.

Expression of a Functional IL-13Rα1 by Rat B Cells

IL-13 mediates its effects through a complex receptor system including IL-4Rα and a functional IL-13Rα1. IL-13 has been reported to have no effects on mouse B cells due to a lack of receptor expression. However, on human B cells a functional IL-13Rα1 has been described. Here, we identified the rat IL-13Rα1 in order to analyze its expression and function in rat B cells. The expression of IL-13Rα1 has been shown by the presence of mRNA and the corresponding protein in purified rat B cells and in rat hybridoma B cell line. Rat B cells are able to bind IL-13 and to proliferate when cultured with CD40 ligand and IL-13. In vivo experiments showed that administration of IL-13 did enhance IgE production. These results suggest a direct interaction of rat B cells with IL-13 through a functional receptor with an increase of IgE production and provide a relevant model to further study the activity of IL-13 and to better understand its role in human diseases.

A monoclonal antibody, MIN/3/60, that recognizes the sulpho-Lewis(x) and sulpho-Lewis(a) sequences detects a sub-population of epithelial glycans in the crypts of human colonic epithelium.

Monoclonal antibodies (MAbs) directed to Lewis(x) (Le(x)) and related carbohydrate sequences have been invaluable in anticipating biological roles for these oligosaccharides by detecting the remarkable changes that occur in their expression from the earliest stages of embryogenesis, through development and sequential stages of cell differentiation and maturation. A notable impact has been in the molecular dissection of ligand-receptor interactions in key cell adhesion events at the initial stages of leukocyte recruitment in inflammation, and almost certainly in the metastasis of epithelial tumours. Antibodies that recognise Le(x) and the 3'-sialyl forms were observed to identify leukocyte subsets; these were subsequently found to match those recognized by the leukocyte-endothelium adhesion molecules, the E- and P-selectins. We now describe a MAb (rat hybridoma MIN/3/60) raised to 3'-sulpho-Le(x), a carbohydrate sequence which, in vitro, is bound not only by the E-, L-, and P-selectins, but also by the cysteine-rich domain of the macrophage endocytosis receptor. We observe that MIN/3/60 is bispecific, however; it binds 3'-sulpho-Le(a) as well as 3'-sulpho-Le(x). Nevertheless, our exploratory studies reveal that it may be a useful histochemical reagent when used in conjunction with a monospecific antibody to 3'-sulpho-Le(a). The MIN/3/60 antibody reveals a sub-population of epithelial glycans in the crypts of Lieberkühn in normal human colon.

Preparation and functional properties of polyclonal and monoclonal antibodies to murine MD-1

Rabbits, rats and hamsters were immunized with KLH-coupled synthetic peptide sequences of the murine MD-1 molecule. Serum from immunized animals bound in Western gels to a 25 KDa protein extracted from LPS stimulated mouse spleen cells, as did a rat hybridoma (SH1.2.47) prepared from peptide-immunized rats. CHO cells transfected with a plasmid cDNA construct encoding murine MD-1, the target antigen for the antibodies in question, were also stained (in FACS) by the same antibodies. Patching and capping of the antigen(s) detected by any one of these sera abolished binding of all antibodies in subsequent FACS analysis, consistent with the hypothesis that they all detected the same antigen. In a final study to assess the possible involvement of MD-1 in regulation of cell activation for cytokine production following allostimulation, we found that all of the antibodies inhibited IL-2 and IFNγ production, while enhancing IL-4 and IL-10 production, in mixed leukocyte reactions (MLR) in vitro.

Manufacture and quality control of CAMPATH-1 antibodies for clinical trials

CAMPATH-1 Abs have been used for T-cell depletion in stem-cell transplantation since the early 1980s. During that time there has been substantial progress in manufacturing techniques and quality control procedures. This article summarizes the methods used to produce the Abs for clinical use and describes results of quality control tests on representative batches. Rat hybridoma and recombinant CHO cells were cultured in hollow-fiber fermentors. Antibodies were purified from the culture supernatant by fractionation with ammonium sulphate, or by column chromatography. Additional steps were added to assure the removal of DNA and viruses. A range of analytical methods was used to characterize the antibodies. Samples were stored frozen at -70 degrees C and re-analyzed many years later to assess the long-term stability. Hollow-fiber fermentors provided a simple and reliable means for antibody production, with yields between 3-10 mg/h and a convenient concentration for further processing (0.6-2.0 mg/mL). All of the CAMPATH-1 Abs (rat IgM, rat IgG2b and human IgG1) could be purified by affinity chromatography on Protein A, but the low pH required for elution caused unacceptable aggregation of the IgM. CAMPATH-1H contained approx. 20% dimeric IgG, which could be removed by size exclusion chromatography. Antibodies were stable for at least 6 years at -70 degrees C, but there was unacceptable aggregation of CAMPATH-1M in one batch stored for 9 years. Pilot-scale production of MAbs for clinical studies is feasible in a small academic center, but regulatory requirements now demand that great attention is paid to all aspects of manufacturing and quality assurance. Although the underlying principles of cell culture and protein chemistry remain the same, the level of documentation, validation and quality control has increased greatly over the last 20 years.

Preparation and functional properties of monoclonal antibodies to human, mouse and rat OX-2

We have prepared mouse and rat hybridomas to a 43-kDa molecule expressed in the thymus, on a subpopulation of dendritic cells, and in the brain, in mammalian tissue derived from mouse, rat and human. Using CHO cells transiently transfected with adenovirus vector(s) expressing a cDNA construct for the relevant OX-2 gene, we show these monoclonal antibodies (Mabs) detect a molecule encoded by this construct (rat OX-2 (rOX-2), mouse OX-2 (mOX-2) and human OX-2 (huOX-2), respectively). Furthermore, at least some of the anti-rat Mabs detect determinants expressed on the murine OX-2 molecule, as we predicted in an earlier publication. Previous studies have implied that this molecule might serve an important role in regulation of cell signaling for cytokine production. Using one-way mixed leukocyte reactions we show that when cells are cultured in the presence of the species-specific Mab, cytokine production becomes polarized ‘away from’ type-2 cytokine production, with preferentially increased expression of type-1 cytokine production.

Intracellular expression and functional properties of an anti-p21 Ras scFv derived from a rat hybridoma containing specific λ and irrelevant κ light chains

A rat single-chain Fv (Y238 scFv) was derived from the Y13–238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13–238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vk chain derived from the rat fusion partner and of a rat Vl chain. Primers designed for rat Vl amplification allowed the cloning of a functional scFv that could bind p21 Ras. The kinetics of interaction of purified Y238 scFv with the p21 Ras protein was evaluated by BIAcore® with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range ( K D=4.58±0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13–259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocqué, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170–1176.]. Finally, BIAcore® analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13–238 epitope.

The cytoplasmic domain of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit is essential for both GM-CSF-mediated growth and differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage progenitor cells. The biologic actions of GM-CSF are mediated by binding to a specific receptor consisting of two chains designated as alpha and beta subunits. We have demonstrated that the murine FDC-P1-derived cell line WT-19 transfected with the human GM-CSF receptor alpha and beta subunits (GM-CSFRalpha and beta) can be induced to differentiate by the addition of human GM-CSF (hGM-CSF). By expressing a series of GM-CSFRalpha mutants in WT19 cells, we have determined the amino acid domains of the GM-CSFRalpha cytoplasmic domain that regulate cell differentiation, proliferation, and survival. We found that the membrane proximal proline-rich domain and adjacent 16 residues are essential for both hGM-CSF-dependent cell proliferation and differentiation. In contrast, the C-terminal region of the GM-CSFRalpha cytoplasmic domain was not necessary for cell differentiation mediated by hGM-CSF, but the removal of this region severely impaired the ability of hGM-CSF to support cell survival. While the activation of JAK2, Shc, Erk, and STAT5 proteins correlated with hGM-CSF-mediated cell growth, cellular differentiation occurred in the absence of activation of these signal transduction pathways.