Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously we have shown that the V(H) and V(L) fragments of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 are weakly associated but can be driven together by antigen. By joining these antibody variable domains to the cytoplasmic portion of the murine erythropoietin receptor, we created a chimeric growth factor receptor that could be activated by HEL. After co-transfection with two plasmids encoding the respective chimeric receptors in IL-3 dependent murine pro-B Ba/F3 cells, a portion of the cells survived under antigen dependent stimulation without IL-3. These surviving cells all showed coexpression of the two chimeric receptor chains and demonstrated HEL dose-dependent growth stimulation without IL-3. When another IL-3 dependent cell line 32D was transfected with a variant of such chimeric receptor with a linker peptide (Gly-Ser-Gly) inserted between V(H)/V(L) and EpoR domains, an improved growth response was attained. These observations suggest the utility of heterodimeric Fv chimeric receptors in creating cells that respond to monomeric antigen.
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PMID:Cell-growth control by monomeric antigen: the cell surface expression of lysozyme-specific Ig V-domains fused to truncated Epo receptor. 1091 58

Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for industrial applications such as protein production is often not practical. Based on our previous design of a binary hen egg lysozyme (HEL)-specific receptor composed of portions of the anti-HEL antibody and the erythropoietin receptor, a new pair of chimeric receptors having the intracellular domain of gp130 were made and transfected to an interleukin-6 (IL-6)-dependent hybridoma, 7TD1. The clone expressing the two new receptors showed clear HEL dose-dependent cell growth and monoclonal antibody production in both serum-based and serum-free media without IL-6. These results establish the feasibility of applying receptor design to tailor cells for the inexpensive induction of cell growth for the purpose of producing therapeutic products.
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PMID:Replacing factor-dependency with that for lysozyme: affordable culture of IL-6-dependent hybridoma by transfecting artificial cell surface receptor. 1142 43

We report a strategy for generating efficient signal transduction with unnatural heterologous receptor combinations. As previously described [Ueda, H., Kawahara, M. et al. (2000) J. Immunol. Methods 241, 159-170], chimeric receptors composed of the V(H)/V(L) domains of anti-hen egg lysozyme antibody HyHEL-10 and N-terminally truncated erythropoietin receptor (EpoR) can be activated by lysozyme. When the cytoplasmic domains of these receptors were substituted with one derived from gp130, IL-3 dependent Ba/F3 cells expressing both V(H)-gp130 and V(L)-gp130 grew dose-dependently when given lysozyme without IL-3. However, cells expressing the heterologous pair of V(H)-gp130 and V(L)-EpoR also showed more efficient and stricter lysozyme-dependent proliferation in the absence of IL-3, indicating this combination is as an efficient and strict signal transducer as wild-type EpoR. The immunoprecipitation data indicated the existence of a preformed V(H)-gp130 and V(L)-EpoR heterodimer in the absence of lysozyme, suggesting the crucial role of a receptor conformational change in signal triggering as well as wild-type EpoR and gp130. Phosphorylation of JAK2, STAT3, and STAT5 was observed upon the addition of lysozyme, suggesting the activation of both EpoR- and gp130-derived signals.
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PMID:A growth signal with an artificially induced erythropoietin receptor-gp130 cytoplasmic domain heterodimer. 1148 Oct 50

While antibiotic selection has been routinely used for the selection of genetically modified cells, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. In this study, we propose an Antigen-MEdiated Genetically modified cell Amplification (AMEGA) system employing antibody/receptor chimeras without antibiotic selection. Based on a rational design where the extracellular domains of dimeric erythropoietin receptor (EpoR) or gp130 were substituted with heterodimeric V(H)/V(L) regions of anti-hen egg lysozyme (HEL) antibody and EpoR D2 domains, the genes encoding the chimeras as well as a model transgene, enhanced green fluorescent protein (EGFP), were retrovirally infected into IL-3-dependent Ba/F3 cells followed by direct HEL selection in the absence of IL-3. After a single round of selection, EGFP-positive cells were selectively amplified, resulting in a population of almost 100% positive cells. The AMEGA without antibiotic selection will not harm normal cells, which will be especially useful for increasing the efficacy for stem cell-based gene therapy.
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PMID:Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch. 1265 20

Selection of genetically modified cells is a critical step to engineer the cells with desired properties. While antibiotic selection has been commonly used, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. To overcome this problem, a positive screening method for genetically modified cells is proposed using a pair of chimeric receptors that trigger a growth signal in response to a specific antigen. Either V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 was fused to extracellular D2 domain of erythropoietin receptor (EpoR) and transmembrane/cytoplasmic domains of either EpoR or gp130. A model transgene, enhanced green fluorescent protein (EGFP) and the chimeric receptor genes that reconstituted functional Fv were retrovirally co-infected to interleukin (IL)-3-dependent Ba/F3 cells, followed by direct HEL selection in the absence of IL-3. Consequently, a single round of selection led to a single population of EGFP-positive cells. The detailed protocol of the method termed antigen-mediated genetically modified cell amplification (AMEGA) is described.
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PMID:AMEGA: antigen-mediated genetically modified cell amplification. 1473 29

Understanding the receptor activation mechanism is essential for the rational design of pharmacologically active ligand molecules. However, the activation mechanism of most cytokine receptors remains still unclear, and while agonism and antagonism have been described for ligand-mimetic peptides, there has been no report of inverse agonism that has been characterized for G protein-coupled receptors (GPCRs). To explore the activation mechanism of cytokine receptors, here we tried to investigate how agonism and antagonism could be altered by randomizing antibody variable region of an antibody/cytokine receptor chimera recognizing hen egg lysozyme (HEL) as an agonist. Based on our previous finding that the co-expression of V(H)-gp130 and V(L)-erythropoietin receptor (EpoR) chimeras transduced strict and efficient HEL-dependent cell growth signal, a V(H)-gp130 library encoding four randomized CDR2 residues was retrovirally infected to IL-3-dependent Ba/F3 cells already transfected with V(L)-EpoR. The selection without IL-3 resulted in a clonal expansion of the transduced cells, and interestingly some of which showed HEL dose-dependent growth suppression. Our results clearly indicate that agonism and antagonism of the antibody/cytokine receptor chimera can be readily switched by a subtle modification of the ligand binding domain as well as that of GPCRs, also implying the existence of inverse agonism in cytokine receptor superfamily.
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PMID:Reversal of antigen-dependent signaling by two mutations in antibody/receptor chimera: implication of inverse agonism in cytokine receptor superfamily. 1524 20

In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V(H) portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (V(H)-EpoR), and (ii) a V(L)-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a model recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.
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PMID:Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera. 1623 21

We have previously designed antibody-cytokine receptor chimeras that could respond to a cognate antigen. While these chimeric receptors were functional, it has not been investigated exactly how they mimic signal transduction through corresponding wild-type receptors. In this study, we compared the growth properties and the phosphorylation status of intracellular signal transducers between the erythropoietin receptor (EpoR)- or gp130-based chimeric receptors and wild-type EpoR or EpoR-gp130 chimera, respectively. Expression plasmids, encoding V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2 domain of EpoR and transmembrane/cytoplasmic domains of either EpoR or gp130, were constructed, and pairs of chimeric receptor combinations (V(H)-EpoR and V(L)-EpoR, V(H)-gp130 and V(L)-gp130, V(H)-EpoR and V(L)-gp130, V(H)-gp130 and V(L)-EpoR) were expressed in an IL-3-dependent myeloid cell line, 32D. Growth assay revealed that the transfectants all grew in a HEL-dependent manner. As for phosphorylation of Stat3, Stat5, ERK and Akt, the chimeric receptors showed similar activation pattern of signalling molecules with wild-type receptors, although the chimeric receptors showed ligand-independency and a little lower maximal phosphorylation than the corresponding wild-type receptors. The results demonstrate that antibody-receptor chimeras could substantially mimic wild-type receptors.
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PMID:Mimicry of erythropoietin and interleukin-6 signalling by an antibody/cytokine receptor chimera in murine myeloid 32D cells. 1744 4

Although adoptive transfer of tumor-specific T cells is a plausible approach for cancer immunotherapy, the therapeutic application was hampered due to severe side effects caused by administration of high-dose interleukin (IL)-2, which was used for long-lasting maintenance of tumor-specific T cells in vivo. To solve this problem, here we propose to use an antibody/IL-2 receptor chimera, which can transduce a growth signal in response to a cognate antigen. As a model system, V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 was tethered to extracellular D2 domain of erythropoietin receptor and transmembrane/cytoplasmic domains of IL-2 receptor beta or gamma chain. When the pairs of chimeric receptors (V(H)-IL-2Rbeta and V(L)-IL-2Rgamma, or V(H)-IL-2Rgamma and V(L)-IL-2Rbeta) were expressed in IL-3-dependent pro-B cell line Ba/F3 and IL-2-dependent T cell line CTLL-2, the cognate antigen HEL induced selective expansion of gene-modified cells in the absence of IL-3 and IL-2, respectively. Growth assay revealed that the combination of V(H)-IL-2Rbeta and V(L)-IL-2Rgamma transduced a more stringent HEL-dependent growth signal, indicating some conformational effects of the chimeras. Furthermore, STAT3, STAT5 and ERK1/2, which are hallmarks for IL-2R signaling, were all activated by the antibody/IL-2R chimeras. These results clearly demonstrate that the antibody/IL-2R chimeras could substantially mimic the wild-type IL-2R signaling, suggesting the potential application in expansion of gene-modified T cells.
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PMID:Selective expansion of genetically modified T cells using an antibody/interleukin-2 receptor chimera. 1858 35

IL-6 has been known to modulate the growth of many hybridoma cells and also promote resultant antibody productivity. However, IL-6 is so expensive that the use of IL-6-dependent hybridomas for industrial antibody production is not practical. In this study, we aimed at designing antibody/gp130 and antibody/EpoR chimeras which could tightly control cell growth in response to more affordable cognate antigen. Retroviral vectors encoding V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2/transmembrane domains of erythropoietin receptor (EpoR) and cytoplasmic domains of either EpoR or gp130, were constructed, and a homodimeric or a heterodimeric pair of chimeric receptor combinations (V(H)-gp130 and V(L)-gp130 or V(H)-gp130 and V(L)-EpoR) were expressed in an IL-6-dependent hybridoma 7TD1. The chimeric receptor-derived growth signal was observed in both combinations, while some residual growth signal was observed in the absence of HEL. To reduce interchain interaction between the two receptor chains, we introduced mutations to the transmembrane domain of both chimera combinations. Consequently, the heterodimeric combination of V(H)-gp130 and V(L)-EpoR showed clear HEL-dependent cell growth, while the homodimeric combination of V(H)-gp130 and V(L)-gp130 showed reduced cell growth in the absence of HEL. This is the first report that an EpoR-gp130 cytoplasmic domain heterodimer could transduce a growth signal in hybridoma cells, indicating tight and economical growth control of hybridoma cells via our chimeric receptors.
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PMID:Growth control of hybridoma cells with an artificially induced EpoR-gp130 heterodimer. 1900 75


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