scholarly journals Soluble Expression of Small Antibody Fragments against PD-L1 Using Escherichia coli with High Yield and Purity

2021 ◽  
Vol 11 (19) ◽  
pp. 9149
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Escherichia Coli ◽  
Tumor Cells ◽  
Surface Protein ◽  
Antibody Fragments ◽  
High Yield ◽  
Soluble Form ◽  
Single Chain ◽  
High Penetration ◽  
Variable Heavy ◽  
Variable Light

Programmed death-ligand 1 (PD-L1) is a surface protein overexpressed in tumor cells. Recently, targeted therapy using PD-L1 antibodies to reconstitute the antitumor activity of T cells has received considerable attention as a cancer treatment. Among the several types of anti-PD-L1 antibodies, small-sized antibody fragments are useful agents to block PD-L1 for experimental and therapeutic purposes owing to their high penetration efficacy toward dense tumor cells. Herein, we expressed and purified recombinant single chain fragment of variable domain, variable heavy chain, and variable light chain, against PD-L1 in a soluble form using Escherichia coli, resulting in their high yield and high purity. We confirmed the antigen-binding efficiency of these antibody fragments, which showed antigen concentration-dependent responses. These results suggest that these small antibody fragments can serve as new agents for blocking or detecting PD-L1.

scholarly journals High-yield expression of periplasmic single-chain variable fragments by solid Escherichia coli cultures

BioTechniques ◽  
2021 ◽  
Author(s):  
Yoshiro Hanyu ◽  
Mieko Kato
Keyword(s):  
Escherichia Coli ◽  
Liquid Culture ◽  
Recombinant Antibody ◽  
Antibody Fragments ◽  
High Yield ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Antibody Fragments ◽  
Single Chain Variable Fragments ◽  
High Yields

High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in Escherichia coli using liquid cultures; however, their yields and quality are often poor. Here the authors expressed a single-chain variable fragment in E. coli cultivated on the wet surface of a solid support. Compared with a liquid culture, the authors obtained 2.5-times more single-chain variable fragments with membrane-cultivated E. coli. This method has two important advantages: it enables high yields of periplasmic single-chain variable fragments compared with liquid culture and offers simple and rapid expression and extraction.

scholarly journals On-target restoration of a split T cell-engaging antibody for precision immunotherapy

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Agnes Banaszek ◽  
Thomas G. P. Bumm ◽  
Boris Nowotny ◽  
Maria Geis ◽  
Kim Jacob ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cell ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Preclinical Models ◽  
Variable Heavy ◽  
Suitable Target ◽  
Single Positive ◽  
Variable Light ◽  
Tumor Types

AbstractT cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.

scholarly journals Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

2022 ◽  
Vol 44 (1) ◽  
pp. 301-308
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Fusion Protein ◽  
Expression System ◽  
Antibody Fragment ◽  
Therapeutic Index ◽  
High Yield ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Fv ◽  
Programmed Death

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

scholarly journals Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1592
Author(s):  
Monika Cserjan-Puschmann ◽  
Nico Lingg ◽  
Petra Engele ◽  
Christina Kröß ◽  
Julian Loibl ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Expression ◽  
Biochemical Properties ◽  
Soluble Form ◽  
Single Chain ◽  
Terminal Amino Acid ◽  
Expression In Escherichia Coli ◽  
Terminal Amino ◽  
Caspase 2

Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at −20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.

Generation of single-chain Fv antibody fragments against Mu-2-related death-inducing gene in Escherichia coli

2019 ◽  
Vol 46 (4) ◽  
pp. 4027-4037
Author(s):  
Dimuthu Dhammika Wickramanayake ◽  
Jun-Ha Choi ◽  
Juhyun Shin ◽  
Jae-Wook Oh
Keyword(s):  
Escherichia Coli ◽  
Antibody Fragments ◽  
Single Chain ◽  
Single Chain Fv ◽  
Fv Antibody ◽  
Single Chain Fv Antibody

Spontaneous assembly of bivalent single chain antibody fragments in Escherichia coli

1994 ◽  
Vol 31 (3) ◽  
pp. 219-226 ◽  
Author(s):  
D.P. Mcgregor ◽  
P.E. Molloy ◽  
C. Cunningham ◽  
W.J. Harris
Keyword(s):  
Escherichia Coli ◽  
Antibody Fragments ◽  
Single Chain ◽  
Single Chain Antibody

scholarly journals Generation and diversification of recombinant monoclonal antibodies

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Keith F DeLuca ◽  
Jeanne E Mick ◽  
Amy Hodges Ide ◽  
Wanessa C Lima ◽  
Lori Sherman ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Low Cost ◽  
Antibody Fragments ◽  
Species Specificity ◽  
High Yield ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ethical Concerns ◽  
Commercial Sources ◽  
Kinetochore Function

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

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