Antibody Affinity and Stability Maturation by Error-Prone PCR

Unkauf, Tobias; Hust, Michael; Frenzel, André

doi:10.1007/978-1-4939-7447-4_22

Cited by 8 publications

(5 citation statements)

References 24 publications

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“…The in vitro a nity maturation was performed as described previously in detail with some modi cations [55]. Figure 5 illustrates the process.…”

Section: In Vitro a Nity Maturationmentioning

confidence: 99%

Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses

Langreder

Schäckermann²,

Meier

et al. 2022

Preprint

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Insect bite hypersensitivity (IBH) is the most common allergic skin disease of horses. It is caused by insect bites of the Culicoides spp. which mediate a type I/IVb allergy with strong involvement of eosinophil cells. No specific treatment option is available so far. One concept could be the use of a therapeutic antibody targeting equine interleukin 5, the main activator and regulator of eosinophils. Therefore, antibodies were selected by phage display using the naïve human antibody gene libraries HAL9/10, tested in a cellular in vitro inhibition assay and subjected to an in vitro affinity maturation. In total, 28 antibodies were selected by phage display out of which eleven have been found to be inhibiting in the final format as chimeric immunoglobulin G with equine constant domains. The two most promising candidates were further improved by in vitro affinity maturation up to factor 2.5 regarding their binding activity and up to factor 2.0 regarding their inhibition effect. The final antibody named NOL2262D10 showed a strong inhibition of the interleukin 5 binding to its receptor (IC50 = 4 nM). Furthermore, a nanomolar binding activity (EC50 = 8.8 nM), stable behavior and satisfactory producibility were demonstrated. This antibody is an excellent candidate for in vivo studies for the treatment of equine IBH.

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“…The in vitro a nity maturation was performed as described previously in detail with some modi cations [55]. Figure 5 illustrates the process.…”

Section: In Vitro a Nity Maturationmentioning

confidence: 99%

Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses

Langreder

Schäckermann²,

Meier

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Successful in vitro affinity maturation is often accomplished through sequential rounds of error‐prone polymerase chain reaction (Unkauf, Hust, & Frenzel, ) or by using degenerate codons to introduce variations within the CDRs (Tiller et al, ). In either approach, semi‐random changes are made in the amino acid sequence and then screened to select for antibodies with increased affinity.…”

Section: Engineering Immunoglobulingmentioning

confidence: 99%

Antibody and antibody derivatives as cancer therapeutics

Arlotta

Owen

2019

WIREs Nanomed Nanobiotechnol

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Antibodies are an important class of therapeutic for treating a wide range of diseases. These versatile macromolecules can be engineered to target many different antigens and to utilize several mechanisms of action to produce a pharmacological effect. The most common antibody platform used for therapeutics is immunoglobulin G (IgG). Advances in protein-display and genetic engineering have enabled the construction and manipulation of IgG to enhance desired activity such as increasing antigen affinity, modulating pharmacokinetics, and enhancing effector functions.IgGs can also be altered to suppress undesired effects, such as immunogenicity. The main approaches to control IgG behavior include engineering the protein sequence and glycosylation of intact IgG; constructing IgG-based derivatives, including bispecific and multivalent binders; and fusing small-drug molecules or proteins to IgG-derived scaffolds. Often, a single modification applied to a given IgG can alter more than one property. The desired effects of an antibody therapeutic should be carefully tailored to the physiology and characteristics of each disease condition.

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“…A plausible alternative that allows for faster production of mAbs is through in vitrodirected protein evolution procedures, which involve (1) the generation of native and immune gene libraries [12] or synthetic [13] that encode single-chain variable fragment (scFv) mAbs displayed through phage (phage display) [14]; (2) the process of screening and selection of isoforms related to an immobilized antigen (target proteins and/or previously identified biomarkers) [15]; and (3) the in vitro maturation of the affinity or specificity of the antibody, through the insertion of random mutations and selection of the isoform with improved affinity [16].…”

Section: Introductionmentioning

confidence: 99%

Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display

Effer,

Ulloa,

Dappolonnio

et al. 2024

Antibodies

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Gallbladder cancer (GBC) is a very aggressive malignant neoplasm of the biliary tract with a poor prognosis. There are no specific therapies for the treatment of GBC or early diagnosis tools; for this reason, the development of strategies and technologies that facilitate or allow an early diagnosis of GBC continues to be decisive. Phage display is a robust technique used for the production of monoclonal antibodies (mAbs) involving (1) the generation of gene libraries, (2) the screening and selection of isoforms related to an immobilized antigen, and (3) the in vitro maturation of the affinity of the antibody for the antigen. This research aimed to construct a human immune library from PBMCs of GBC patients and the isolation of scFv-phage clones with specificity against the larger extracellular loop belonging to claudin 18.2, which is an important biomarker overexpressed in GBC as well as gastric cancer. The immune-library-denominated GALLBLA1 was constructed from seven GBC patients and has a diversity of 6.12 × 1010 pfu mL−1. After three rounds of panning, we were able to identify clones with specificity against claudin 18.2. GALLBLA1 can contribute to the selection, isolation, and recombinant production of new human mAbs candidates for the treatment of gastrointestinal cancers.

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Antibody Affinity and Stability Maturation by Error-Prone PCR

Cited by 8 publications

References 24 publications

Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses

Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses

Antibody and antibody derivatives as cancer therapeutics

Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display

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