Recombinant Antibody Engineering Enables Reversible Binding for Continuous Protein Biosensing

ACS Sensors ◽  
2021 ◽  
Author(s):  
Christian Fercher ◽  
Martina L. Jones ◽  
Stephen M. Mahler ◽  
Simon R. Corrie
Keyword(s):  
Recombinant Antibody ◽  
Antibody Engineering ◽  
Reversible Binding

scholarly journals Affinity Maturation of Tacrolimus Antibody for Improved Immunoassay Performance

2008 ◽  
Vol 54 (6) ◽  
pp. 1008-1017 ◽  
Author(s):  
Robert W Siegel ◽  
Wade Baugher ◽  
Tanya Rahn ◽  
Susan Drengler ◽  
Joan Tyner
Keyword(s):  
Limit Of Detection ◽  
Recombinant Antibody ◽  
Surface Display ◽  
Dissociation Rate ◽  
Affinity Maturation ◽  
Antibody Engineering ◽  
Yeast Surface Display ◽  
Variable Heavy ◽  
Variable Light

Abstract Background: Organic solvents used for extraction of tacrolimus from whole blood samples lower the apparent affinity of the antibody used in a diagnostic immunoassay, thereby affecting the detection limit. Methods: We used in vitro recombinant antibody engineering to screen and isolate clones from diverse libraries with mutagenic complementarity regions (CDRs) from tacrolimus 1-60-46 hybridoma cell line, with improved binding to tacrolimus in the presence of 10% methanol organic solvent solution. Results: We isolated a number of clones with mutations in variable heavy (VH) CDR 2, variable light (VL) CDR 1, and VL CDR 3 with improved binding. Various combinatorial pairings constructed from these individual mutations contained >10-fold improvements in both the dissociation rate and overall equilibrium affinity constants. Selected clones produced as IgG have increased functional sensitivity, with a 3- to 6-fold reduction in the limit of detection relative to the parental tacrolimus 1-60-46 monoclonal antibody in the Architect® Tacrolimus immunodiagnostic assay. Conclusions: The recent advent of recombinant in vitro antibody display technologies in general, and yeast surface display in particular, allows the flexibility to engineer new or augment specific analytical characteristics, such as affinity, specificity, or stability, into previously isolated and otherwise desirable antibodies to enhance assay performance. These in vitro selections can also be performed under conditions meant to mimic the assay in which the reagent will ultimately be used, to increase the likelihood of successful assay development.

scholarly journals A general strategy to control antibody specificity against targets showing molecular and biological similarity: Salmonella case study

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Riccardo Marega ◽  
N. Desroche ◽  
A.-C. Huet ◽  
M. Paulus ◽  
C. Suarez Pantaleon ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Recombinant Antibody ◽  
Cross Reactivity ◽  
Antibody Engineering ◽  
General Strategy ◽  
Large Set ◽  
Antibody Specificity ◽  
Molecular Features ◽  
Analyte Detection ◽  
And Control

Abstract The control of antibody specificity plays pivotal roles in key technological fields such as diagnostics and therapeutics. During the development of immunoassays (IAs) for the biosensing of pathogens in food matrices, we have found a way to rationalize and control the specificity of polyclonal antibodies (sera) for a complex analytical target (the Salmonella genus), in terms of number of analytes (Salmonella species) and potential cross-reactivity with similar analytes (other bacteria strains). Indeed, the biosensing of Salmonella required the development of sera and serum mixtures displaying homogeneous specificity for a large set of strains showing broad biochemical variety (54 Salmonella serovars tested in this study), which partially overlaps with the molecular features of other class of bacteria (like specific serogroups of E. coli). To achieve a trade-off between specificity harmonisation and maximization, we have developed a strategy based on the conversion of the specificity profiles of individual sera in to numerical descriptors, which allow predicting the capacity of serum mixtures to detect multiple bacteria strains. This approach does not imply laborious purification steps and results advantageous for process scaling-up, and may help in the customization of the specificity profiles of antibodies needed for diagnostic and therapeutic applications such as multi-analyte detection and recombinant antibody engineering, respectively.

scholarly journals CEACAM1 is a Privileged Cell Surface Antigen to Design Novel ScFv Mediated-Immunotherapies of Melanoma, Lung Cancer and Other Types of Tumors

2012 ◽  
Vol 6 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Maurizio Cianfriglia ◽  
Valentina Fiori ◽  
Sabrina Dominici ◽  
Silvia Zamboni ◽  
Michela Flego ◽  
...  
Keyword(s):  
Cell Surface ◽  
Recombinant Antibody ◽  
Metastatic Potential ◽  
Antibody Fragments ◽  
Antibody Engineering ◽  
Surface Glycoprotein ◽  
Immunoglobulin Superfamily ◽  
Aberrant Expression ◽  
Cell Surface Glycoprotein ◽  
Drug Conjugates

Carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) is a cell surface glycoprotein involved in intercellular binding, belonging to the immunoglobulin superfamily. It is involved in cell-cell recognition and modulates cellular processes that range from vascular angiogenesis to the regulation of insulin homeostasis and T-cell proliferation. Aberrant expression of CEACAM1 is often associated with progression and metastatic potential in melanoma, lung carcinoma and other types of tumor. Tumor-specific antigens such as CEACAM1 are ideal targets for cancer immunotherapy because they are over-expressed by the cancer cell and not on non-malignant tissues, minimizing the risk of autoimmune destruction. Many of the limitations of therapeutic use of rodent monoclonal antibodies (mAbs) can now be overcome by exploiting the use of recombinant antibody fragments and the advances in antibody engineering methods to improve tumor retention, reduce immunogenicity and modulate pharmacokinetics. In addition, a novel effective model of immunotherapeutic treatments of tumors includes antibody drug conjugates (ADCs) that combine specific mAbs and antibody fragments with cytotoxic drugs, proteins, enzymes, radionuclides and nanoparticles. This review aims to describe how these antibody engineering approaches can meet the challenges for generating new and effective antibody constructs for diagnosis and therapy of CEACAM1 expressing malignancies.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

scholarly journals Trastuzumab Modulates the Protein Cargo of Extracellular Vesicles Released by ERBB2+ Breast Cancer Cells

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 199
Author(s):  
Silvia Marconi ◽  
Sara Santamaria ◽  
Martina Bartolucci ◽  
Sara Stigliani ◽  
Cinzia Aiello ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Breast Cancer Cells ◽  
Culture Supernatant ◽  
High Resolution Mass Spectrometry ◽  
Recombinant Antibody ◽  
Target Cells ◽  
Cellular Processes ◽  
Erbb2 Signaling

Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is an ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles (EVs) released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here, we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Trastuzumab (Tz) induces the expression of a specific glycosylated 50 kDa isoform of the CD63 tetraspanin and modulates the expression of 51 EVs proteins, including TOP1. Because these proteins are functionally associated with organelle organization, cytokinesis, and response to lipids, we suggest that Tz may influence these cellular processes in target cells at distant sites via modified EVs.

Antibody engineering-driven controllable chemiluminescence resonance energy transfer for immunoassay with tunable dynamic range

2021 ◽  
Vol 1152 ◽  
pp. 338231
Author(s):  
Leina Dou ◽  
Yantong Pan ◽  
Mingfang Ma ◽  
Suxia Zhang ◽  
Jianzhong Shen ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Antibody Engineering

scholarly journals Improvement of Biophysical Properties and Affinity of a Human Anti-L1CAM Therapeutic Antibody through Antibody Engineering Based on Computational Methods

2021 ◽  
Vol 22 (13) ◽  
pp. 6696
Author(s):  
Heesu Chae ◽  
Seulki Cho ◽  
Munsik Jeong ◽  
Kiyoung Kwon ◽  
Dongwook Choi ◽  
...  
Keyword(s):  
Computational Methods ◽  
Human Monoclonal Antibody ◽  
Antibody Engineering ◽  
Antigen Binding ◽  
Post Translational Modification ◽  
Biophysical Properties ◽  
Preclinical Development ◽  
Cell Bank ◽  
Aggregation Propensity

The biophysical properties of therapeutic antibodies influence their manufacturability, efficacy, and safety. To develop an anti-cancer antibody, we previously generated a human monoclonal antibody (Ab417) that specifically binds to L1 cell adhesion molecule with a high affinity, and we validated its anti-tumor activity and mechanism of action in human cholangiocarcinoma xenograft models. In the present study, we aimed to improve the biophysical properties of Ab417. We designed 20 variants of Ab417 with reduced aggregation propensity, less potential post-translational modification (PTM) motifs, and the lowest predicted immunogenicity using computational methods. Next, we constructed these variants to analyze their expression levels and antigen-binding activities. One variant (Ab612)—which contains six substitutions for reduced surface hydrophobicity, removal of PTM, and change to the germline residue—exhibited an increased expression level and antigen-binding activity compared to Ab417. In further studies, compared to Ab417, Ab612 showed improved biophysical properties, including reduced aggregation propensity, increased stability, higher purification yield, lower pI, higher affinity, and greater in vivo anti-tumor efficacy. Additionally, we generated a highly productive and stable research cell bank (RCB) and scaled up the production process to 50 L, yielding 6.6 g/L of Ab612. The RCB will be used for preclinical development of Ab612.

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