Protein Engineering Design & Selection最新文献_第9页

Antibody humanization-the Influence of the antibody framework on the CDR-H3 loop ensemble in solution. 抗体人源化——抗体框架对溶液中CDR-H3环系的影响。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa004

Monica L Fernández-Quintero, Martin C Heiss, Klaus R Liedl

{"title":"Antibody humanization-the Influence of the antibody framework on the CDR-H3 loop ensemble in solution.","authors":"Monica L Fernández-Quintero, Martin C Heiss, Klaus R Liedl","doi":"10.1093/protein/gzaa004","DOIUrl":"10.1093/protein/gzaa004","url":null,"abstract":"Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the complementarity determining region (CDR)-H3 loop dynamics. We also studied a Fv without structural information of the anti-idiotypic antibody Ab2/3H6, because it completely lost its binding affinity upon superhumanization, as an example of a failed humanization. Enhanced sampling techniques in combination with molecular dynamics simulations allow to access micro- to milli-second timescales of the CDR-H3 loop dynamics and reveal kinetic and thermodynamic changes involved in the process of humanization. In most cases, we observe a reduced conformational diversity of the CDR-H3 loop when grafted on a human framework and find a conformational shift of the dominant CDR-H3 loop conformation in solution. A shallow side minimum of the conformational CDR-H3 loop ensemble attached to the murine framework becomes the dominant conformation in solution influenced by the human framework. Additionally, we observe in the case of the failed humanization that the potentially binding competent murine CDR-H3 loop ensemble in solution shows nearly no kinetical or structural overlap with the superhumanized variant, thus explaining the loss of binding.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 9","pages":"411-422"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098879/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37703280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Computer-guided library generation applied to the optimization of single-domain antibodies. 计算机引导文库生成应用于单结构域抗体的优化。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa006

Hiroki Akiba, Hiroko Tamura, Jose M M Caaveiro, Kouhei Tsumoto

引用次数: 6

Protein A superantigen: structure, engineering and molecular basis of antibody recognition. 蛋白A超抗原:结构、工程和抗体识别的分子基础。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz026

Ohan Mazigi, Peter Schofield, D. Langley, D. Christ

引用次数: 10

Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents. 针对人类治疗性Fc支架的高选择性IgNAR可变单结构域的分离及其作为定制生物处理试剂的应用。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa002

Magdalena J Buschhaus, Stefan Becker, Andrew J Porter, Caroline J Barelle

{"title":"Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents.","authors":"Magdalena J Buschhaus, Stefan Becker, Andrew J Porter, Caroline J Barelle","doi":"10.1093/protein/gzaa002","DOIUrl":"https://doi.org/10.1093/protein/gzaa002","url":null,"abstract":"The adaptive immune system of cartilaginous fish (Elasmobranchii), comprising of classical hetero-tetrameric antibodies, is enhanced through the presence of a naturally occurring homodimeric antibody-like immunoglobulin-the new antigen receptor (IgNAR). The binding site of the IgNAR variable single-domain (VNAR) offers advantages of reduced size (<1/10th of classical immunoglobulin) and extended binding topographies, making it an ideal candidate for accessing cryptic epitopes otherwise intractable to conventional antibodies. These attributes, coupled with high physicochemical stability and amenability to phage display, facilitate the selection of VNAR binders to challenging targets. Here, we explored the unique attributes of these single domains for potential application as bioprocessing reagents in the development of the SEED-Fc platform, designed to generate therapeutic bispecific antibodies. A panel of unique VNARs specific to the SEED homodimeric (monospecific) 'by-products' were isolated from a shark semi-synthetic VNAR library via phage display. The lead VNAR candidate exhibited low nanomolar affinity and superior selectivity to SEED homodimer, with functionality being retained upon exposure to extreme physicochemical conditions that mimic their applicability as purification agents. Ultimately, this work exemplifies the robustness of the semi-synthetic VNAR platform, the predisposition of the VNAR paratope to recognise novel epitopes and the potential for routine generation of tailor-made VNAR-based bioprocessing reagents.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 9","pages":"385-399"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37695454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR. 利用分子伴侣TAPBPR在哺乳动物细胞中产生可溶性pmhc - 1分子。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa015

Sara M O'Rourke, Giora I Morozov, Jacob T Roberts, Adam W Barb, Nikolaos G Sgourakis

{"title":"Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.","authors":"Sara M O'Rourke, Giora I Morozov, Jacob T Roberts, Adam W Barb, Nikolaos G Sgourakis","doi":"10.1093/protein/gzaa015","DOIUrl":"10.1093/protein/gzaa015","url":null,"abstract":"Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 12","pages":"525-532"},"PeriodicalIF":2.6,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451022/pdf/gzaa015.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38213003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Designing an improved T-cell mobilising CXCL10 mutant through enhanced GAG binding affinity. 通过增强GAG结合亲和力设计改进的t细胞动员CXCL10突变体。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz043

Tanja Gerlza, Michael Nagele, Martha Gschwandtner, Sophie Winkler, Andreas Kungl

{"title":"Designing an improved T-cell mobilising CXCL10 mutant through enhanced GAG binding affinity.","authors":"Tanja Gerlza, Michael Nagele, Martha Gschwandtner, Sophie Winkler, Andreas Kungl","doi":"10.1093/protein/gzz043","DOIUrl":"https://doi.org/10.1093/protein/gzz043","url":null,"abstract":"The chemokine CXCL10 is released by a plethora of cells, including immune and metastatic cancer cells, following stimulation with interferon-gamma. It acts via its GPC receptor on T-cells attracting them to various target tissues. Glycosaminoglycans (GAGs) are regarded as co-receptors of chemokines, which enable the establishment of a chemotactic gradient for target cell migration. We have engineered human CXCL10 towards improved T-cell mobilisation by implementing a single site-directed mutation N20K into the protein, which leads to a higher GAG binding affinity compared to the wild type. Interestingly, this mutation not only increased T-cell migration in a transendothelial migration assay, the mutant intensified T-cell chemotaxis also in a Boyden chamber set-up thereby indicating a strong role of T-cell-localised GAGs on leukocyte migration. A CXCL10 mutant with increased GAG-binding affinity could therefore potentially serve as a T-cell mobiliser in pathological conditions where the immune surveillance of the target tissue is impaired, as is the case for most solid tumors.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 8","pages":"367-373"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz043","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37572249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability. 静电相互作用调节IgG1和IgG4P抗体的不同聚集倾向，并通知带电残基取代以提高可发育性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz046

James T Heads, Richard Lamb, Sebastian Kelm, Ralph Adams, Peter Elliott, Kerry Tyson, Sarfaraj Topia, Shauna West, Ruodan Nan, Alison Turner, Alastair D G Lawson

{"title":"Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability.","authors":"James T Heads, Richard Lamb, Sebastian Kelm, Ralph Adams, Peter Elliott, Kerry Tyson, Sarfaraj Topia, Shauna West, Ruodan Nan, Alison Turner, Alastair D G Lawson","doi":"10.1093/protein/gzz046","DOIUrl":"https://doi.org/10.1093/protein/gzz046","url":null,"abstract":"Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 6","pages":"277-288"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz046","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37484111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 20

Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design. 通过共识设计获得的具有改进的蛋白质热稳定性和酶活性的分选酶突变体。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa018

Magdalena Wójcik, Susana Vázquez Torres, Wim J Quax, Ykelien L Boersma

引用次数: 9

FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection. FluoroCalins:具有新型结合功能与荧光蛋白融合的工程脂钙蛋白，用于生物分子成像和检测。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz047

Evelyn Eggenstein, Antonia Richter, Arne Skerra

{"title":"FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection.","authors":"Evelyn Eggenstein, Antonia Richter, Arne Skerra","doi":"10.1093/protein/gzz047","DOIUrl":"https://doi.org/10.1093/protein/gzz047","url":null,"abstract":"FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 6","pages":"289-296"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37533945","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Engineered variants of a lipase from Yarrowia lipolytica with improved trypsin resistance for enzyme replacement therapy. 酶替代疗法中改良胰蛋白酶抵抗的脂肪酶工程变体。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa001

Huitu Zhang, Huan Liu, Ying Zhang, Tongwei Sun, Guoguo Wu, Cuixia Zhou, Xiaonong Wu, Jing Zhang, Rong Yue, Haikuan Wang, Yujie Dai, Fufeng Liu, Fuping Lu

引用次数: 1