共查询到20条相似文献,搜索用时 15 毫秒
1.
Laura M. Haynes Zachary M. Huttinger Andrew Yee Colin A. Kretz David R. Siemieniak Daniel A. Lawrence David Ginsburg 《The Journal of biological chemistry》2022,298(12)
Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants. 相似文献
2.
《MABS-AUSTIN》2013,5(2):440-450
Panitumumab, as a commercially available antibody, is an effective anticancer therapeutic against epidermal growth factor receptor (EGFR), although it exerts weak antibody-dependent cell-mediated cytotoxicity (ADCC) activity owing to its IgG2 nature. Here, we firstly engineered panitumumab by grafting its variable region into an IgG1 backbone. The engineered panitumumab (denoted as Pan) retained binding activity identical to the parental antibody while exhibiting stronger ADCC activity in vitro and more potent antitumor effect in vivo. To further enhance the target selectivity of Pan, we generated Pan-P by tethering an epitope-blocking peptide to Pan via a tumor-specific protease selective linker. Pan-P showed almost 40-fold weaker affinity compared with Pan, but functional activity was restored to a similar extent as Pan when Pan-P was selectively activated by urokinase-type plasminogen activator (uPA). More importantly, targeted localization of Pan-P was observed in tumor samples from colorectal cancer (CRC) patients and tumor-bearing nude mice, strongly indicating that specific activation also existed ex vivo and in vivo. Furthermore, Pan-P also exhibited effective in vivo antitumor potency similar to Pan. Taken together, our data evidence the enhanced antitumor potency and excellent target selectivity of Pan-P, suggesting its potential use for minimizing on-target toxicity in anti-EGFR therapy. 相似文献
3.
Valencio Salema Carmen Mañas Lidia Cerdán Carlos Piñero-Lambea Elvira Marín Rob C. Roovers 《MABS-AUSTIN》2016,8(7):1286-1301
Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. 相似文献
4.
Yun Yang Qingcheng Guo Mao Xia Yantao Li Xiaoyun Peng Tao Liu Xin Tong Jin Xu Huaizu Guo Weizhu Qian Sheng Hou Jianxin Dai Hao Wang Rong Liu Yajun Guo 《MABS-AUSTIN》2015,7(2):440-450
Panitumumab, as a commercially available antibody, is an effective anticancer therapeutic against epidermal growth factor receptor (EGFR), although it exerts weak antibody-dependent cell-mediated cytotoxicity (ADCC) activity owing to its IgG2 nature. Here, we firstly engineered panitumumab by grafting its variable region into an IgG1 backbone. The engineered panitumumab (denoted as Pan) retained binding activity identical to the parental antibody while exhibiting stronger ADCC activity in vitro and more potent antitumor effect in vivo. To further enhance the target selectivity of Pan, we generated Pan-P by tethering an epitope-blocking peptide to Pan via a tumor-specific protease selective linker. Pan-P showed almost 40-fold weaker affinity compared with Pan, but functional activity was restored to a similar extent as Pan when Pan-P was selectively activated by urokinase-type plasminogen activator (uPA). More importantly, targeted localization of Pan-P was observed in tumor samples from colorectal cancer (CRC) patients and tumor-bearing nude mice, strongly indicating that specific activation also existed ex vivo and in vivo. Furthermore, Pan-P also exhibited effective in vivo antitumor potency similar to Pan. Taken together, our data evidence the enhanced antitumor potency and excellent target selectivity of Pan-P, suggesting its potential use for minimizing on-target toxicity in anti-EGFR therapy. 相似文献
5.
Christian Schr?ter Ralf Günther Laura Rhiel Stefan Becker Lars Toleikis Achim Doerner Janine Becker Andreas Sch?nemann Daichi Nasu Berend Neuteboom Harald Kolmar Bj?rn Hock 《MABS-AUSTIN》2015,7(1):138-151
There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics. 相似文献
6.
《MABS-AUSTIN》2013,5(1):138-151
There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics. 相似文献
7.
Nathan Robertson Nancy Lopez-Anton Shalom A. Gurjar Hena Khalique Zainab Khalaf Siobhan Clerkin Vaughan R. Leydon Richard Parker-Manuel Alexander Raeside Tom Payne Tim D. Jones Len Seymour Ryan Cawood 《The Journal of biological chemistry》2020,295(52):18436
Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies. 相似文献
8.
Rita Pellegrini Filippo Centis Stefania Martignone Antonio Mastroianni Elda Tagliabue Emanuela Tosi Sylvie Ménard Maria I. Colnaghi 《Cancer immunology, immunotherapy : CII》1991,34(1):37-42
Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 104 receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression. 相似文献
9.
10.
Receptor modulating properties of an antibody directed against the epidermal growth factor receptor 总被引:2,自引:0,他引:2
A murine antiserum with specificity for the human epidermal growth factor (EGF) receptor was used to investigate EGF receptor function. The IgG fraction of this antiserum displayed no EGF-like mitogenic activity, even when cross-linking was ensured by sequential treatment with rabbit anti-(mouse IgG). The interaction of antibody with solubilized purified EGF receptor was characterized in detail. The binding of 125I-antibody to the receptor was not blocked by EGF, but the binding of 125I-EGF to the receptor was blocked by the immune IgG. Scatchard analysis of this reaction revealed a reduction in maximal EGF binding but an enhanced EGF binding affinity. In addition, at low concentrations, the immune IgG was found to enhance receptor kinase activity in the absence of EGF. The enhancement of kinase activity, as measured by receptor phosphorylation, was due to a decreased Km for ATP, and an increased V. These results suggest that the antibody is capable of altering conformations at receptor active sites by binding to non-active species-specific epitopes. 相似文献
11.
M Pogorzelski S Ting T C Gauler F Breitenbuecher I Vossebein S Hoffarth J Markowetz S Lang C Bergmann S Brandau J A Jawad K W Schmid M Schuler S Kasper 《Cell death & disease》2014,5(2):e1091
Infection with human papillomaviruses (HPVs) characterizes a distinct subset of head and neck squamous cell cancers (HNSCCs). HPV-positive HNSCC preferentially affect the oropharynx and tonsils. Localized HPV-positive HNSCCs have a favorable prognosis and treatment outcome. However, the impact of HPV in advanced or metastatic HNSCC remains to be defined. In particular, it is unclear whether HPV modulates the response to cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), which is a mainstay of treatment of advanced HNSCC. To this end, we have examined the sensitivity of HPV-positive and -negative HNSCC models to cetuximab and cytotoxic drugs in vitro and in vivo. In addition, we have stably expressed the HPV oncogenes E6 and E7 in cetuximab-sensitive cancer cell lines to specifically investigate their role in the antibody response. The endogenous HPV status or the expression of HPV oncogenes had no significant impact on cetuximab-mediated suppression of EGFR signaling and proliferation in vitro. Cetuximab effectively inhibited the growth of E6- and E7-expressing tumors grafted in NOD/SCID mice. In support, formalin-fixed, paraffin-embedded tumor samples from cetuximab-treated patients with recurrent or metastatic HNSCC were probed for p16INK4a expression, an established biomarker of HPV infection. Response rates (45.5% versus 45.5%) and median progression-free survival (97 versus 92 days) following cetuximab-based therapy were similar in patients with p16INK4A-positive and p16INK4A-negative tumors. In conclusion, HPV oncogenes do not modulate the anti-EGFR antibody response in HSNCC. Cetuximab treatment should be administered independently of HPV status. 相似文献
12.
Sherry L La Porte Charles Eigenbrot Mark Ultsch Wei-Hsien Ho Davide Foletti Alison Forgie Kevin C Lindquist David L Shelton Jaume Pons 《MABS-AUSTIN》2014,6(4):1059-1068
Nerve growth factor (NGF) is indispensable during normal embryonic development and critical for the amplification of pain signals in adults. Intervention in NGF signaling holds promise for the alleviation of pain resulting from human diseases such as osteoarthritis, cancer and chronic lower back disorders. We developed a fast, high-fidelity method to convert a hybridoma-derived NGF-targeted mouse antibody into a clinical candidate. This method, termed Library Scanning Mutagenesis (LSM), resulted in the ultra-high affinity antibody tanezumab, a first-in-class anti-hyperalgesic specific for an NGF epitope. Functional and structural comparisons between tanezumab and the mouse 911 precursor antibody using neurotrophin-specific cell survival assays and X-ray crystal structures of both Fab-antigen complexes illustrated high fidelity retention of the NGF epitope. These results suggest the potential for wide applicability of the LSM method for optimization of well-characterized antibodies during humanization. 相似文献
13.
《MABS-AUSTIN》2013,5(4):1059-1068
Nerve growth factor (NGF) is indispensable during normal embryonic development and critical for the amplification of pain signals in adults. Intervention in NGF signaling holds promise for the alleviation of pain resulting from human diseases such as osteoarthritis, cancer and chronic lower back disorders. We developed a fast, high-fidelity method to convert a hybridoma-derived NGF-targeted mouse antibody into a clinical candidate. This method, termed Library Scanning Mutagenesis (LSM), resulted in the ultra-high affinity antibody tanezumab, a first-in-class anti-hyperalgesic specific for an NGF epitope. Functional and structural comparisons between tanezumab and the mouse 911 precursor antibody using neurotrophin-specific cell survival assays and X-ray crystal structures of both Fab-antigen complexes illustrated high fidelity retention of the NGF epitope. These results suggest the potential for wide applicability of the LSM method for optimization of well-characterized antibodies during humanization. 相似文献
14.
F. Nadiya N. Anjali J. Thomas A. Gangaprasad K. K. Sabu 《Plant biology (Stuttgart, Germany)》2019,21(1):3-14
- Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses.
- In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer.
- We identified 161 potential miRNAs representing 42 families, including monocot/tissue‐specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars.
- Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT‐PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA–mRNA target pairing using RNA ligase‐mediated 5′ Rapid Amplification of cDNA Ends (5′RLM‐RACE) PCR.
15.
Photonic modulation of epidermal growth factor receptor halts receptor activation and cancer cell migration
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Cláudia M. Botelho Odete Gonçalves Rogério Marques Viruthachalam Thiagarajan Henrik Vorum Andreia C. Gomes Maria Teresa Neves‐Petersen 《Journal of biophotonics》2018,11(9)
Epidermal growth factor receptor (EGFR) plays a key role in regulating cell survival, proliferation and migration, and its overexpression and activation has been correlated with cancer progression. Cancer therapies targeting EGFR have been applied in the clinic with some success. We show, by confocal microscopy analysis, that illumination of adenocarcinomic human alveolar basal epithelial cells (Human A549—EGFR biosensor cell line) with 280 nm at irradiance levels up to 20 times weaker than the Ultraviolet B (UVB) solar output for short periods of time (15‐45 minutes) prevents epidermal growth factor‐mediated activation of EGFR located on the cell membrane, preventing or reducing cellular disaggregation, formation of filopodia and cell migration. This effect of Ultraviolet (UV) light illumination was confirmed further in a functional scratch assay, and shown to be more effective than that of a specific EGFR‐signaling inhibitor. This new photonic approach may be applicable to the treatment of various types of cancer, alone or in combination with other therapies. 相似文献
16.
Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor 总被引:3,自引:0,他引:3
Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility. 相似文献
17.
Rodríguez M Ramírez NI Ayala M Freyre F Pérez L Triguero A Mateo C Selman-Housein G Gavilondo JV Pujol M 《Biotechnology and bioengineering》2005,89(2):188-194
When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells. 相似文献
18.
In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors
are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation
we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results
demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and
round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no
staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly,
the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since
their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic
processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators
of spermatogenesis in bovine. 相似文献
19.
20.
Vajdos FF Adams CW Breece TN Presta LG de Vos AM Sidhu SS 《Journal of molecular biology》2002,320(2):415-428
Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions. 相似文献