Protein engineering approaches for antibody fragments: directed evolution and rational design approaches

Arslan, Merve; Karadag, Dilara

doi:10.3906/biy-1809-28

Cited by 21 publications

(15 citation statements)

References 75 publications

(119 reference statements)

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“…YSD with the eukaryotic quality control system in the protein secretion pathway is a powerful tool to simultaneously optimize stability and/or expression of a targeted Ab in addition to the affinity during the affinity maturation, which is difficult to be achieved by a rational design ( 43 , 44 ). The affinity-matured 5R65 and 5R65.7 Abs using YSD technology showed higher colloidal stability against 3 freeze-thaw cycles than that of the initial Ab 2B7 ( Supplementary Figure 5 ) and comparable expression levels by transient expression in mammalian cells with purification yields of approximately 30–70 mg/L of transiently transfected HEK293F cells to those of commercialized therapeutic Abs ( 16 ).…”

Section: Discussionmentioning

confidence: 99%

Engineering of Humanized Antibodies Against Human Interleukin 5 Receptor Alpha Subunit That Cause Potent Antibody-Dependent Cell-Mediated Cytotoxicity

et al. 2021

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Patients with severe eosinophilic asthma (SEA; characterized by persistent eosinophilia in blood and airway tissues) experience frequent asthma exacerbations with poor clinical outcomes. Interleukin 5 (IL-5) and IL-5 receptor alpha subunit (IL-5α) play key roles in eosinophilia maintenance, and relevant therapeutic strategies include the development of antibodies (Abs) against IL-5 or IL-5α to control eosinophilia. Benralizumab, an anti–IL-5α Ab that depletes eosinophils mainly via Ab-dependent cell-mediated cytotoxicity and through blockage of IL-5 function on eosinophils, has been clinically approved for patients with SEA. Here, we report engineering of a new humanized anti–IL-5Rα Ab with potent biological activity. We first raised murine Abs against human IL-5Rα, humanized a leading murine Ab, and then further engineered the humanized Abs to enhance their affinity for IL-5Rα using the yeast surface display technology. The finally engineered version of the Ab, 5R65.7, with affinity (KD ≈ 4.64 nM) stronger than that of a clinically relevant benralizumab analogue (KD ≈ 26.8 nM) showed improved neutralizing activity toward IL-5–dependent cell proliferation in a reporter cell system. Domain level Ab epitope mapping revealed that 5R65.7 recognizes membrane-proximal domain 3 of IL-5Rα, distinct from domain I epitope of the benralizumab analogue. In ex vivo assays with peripheral eosinophils from patients with SEA and healthy donors, 5R65.7 manifested more potent biological activities than the benralizumab analogue did, including inhibition of IL-5–dependent proliferation of eosinophils and induction of eosinophil apoptosis through autologous natural-killer-cell–mediated Ab-dependent cell-mediated cytotoxicity. Our study provides a potent anti–IL-5Rα Ab, 5R65.7, which is worthy of further testing in preclinical and clinical trials against SEA as a potential alternative to the current therapeutic arsenal.

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Section: Discussionmentioning

confidence: 99%

Engineering of Humanized Antibodies Against Human Interleukin 5 Receptor Alpha Subunit That Cause Potent Antibody-Dependent Cell-Mediated Cytotoxicity

et al. 2021

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“…Recently, the use of HER2 targeting antibody fragments gained attention. The small size allows better tumor penetration, lower immunogenicity, and cheaper and larger scale production compared to full length antibodies [ 76 ]. In a Phase I study, [ 68 Ga]Ga-HER2-Nanobody was evaluated for PET imaging of HER2 expressing tumors in 20 women with primary or metastatic HER2 + BCa (IHC proven).…”

Section: Direct Targeting Of Tumor Cellsmentioning

confidence: 99%

Development of Radiotracers for Breast Cancer—The Tumor Microenvironment as an Emerging Target

Heesch

Maurer

Stickeler

et al. 2020

Cells

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Molecular imaging plays an increasingly important role in the diagnosis and treatment of different malignancies. Radiolabeled probes enable the visualization of the primary tumor as well as the metastases and have been also employed in targeted therapy and theranostic approaches. With breast cancer being the most common malignancy in women worldwide it is of special interest to develop novel targeted treatments. However, tumor microenvironment and escape mechanisms often limit their therapeutic potential. Addressing tumor stroma associated targets provides a promising option to inhibit tumor growth and angiogenesis and to disrupt tumor tissue architecture. This review describes recent developments on radiolabeled probes used in diagnosis and treatment of breast cancer especially in triple negative type with the focus on potential targets offered by the tumor microenvironment, like tumor associated macrophages, cancer associated fibroblasts, and endothelial cells.

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“…The F(ab) fragment is an antibody structure that still binds to antigens but is monovalent and lacks the Fc portion. An antibody digested by the enzyme papain yields two F(ab) fragments of about 50 kDa each and an Fc fragment [15].…”

Section: Antibody Fragments For Cancer Diagnosticsmentioning

confidence: 99%

Ion Channel Targeting with Antibodies and Antibody Fragments for Cancer Diagnosis

Duranti

Arcangeli

2019

Antibodies

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The antibody era has greatly impacted cancer management in recent decades. Indeed, antibodies are currently applied for both cancer diagnosis and therapy. For example, monoclonal antibodies are the main constituents of several in vitro diagnostics, which are applied at many levels of cancer diagnosis. Moreover, the great improvement provided by in vivo imaging, especially for early-stage cancer diagnosis, has traced the path for the development of a complete new class of antibodies, i.e., engineered antibody fragments. The latter embody the optimal characteristics (e.g., low renal retention, rapid clearance, and small size) which make them ideal for in vivo applications. Furthermore, the present review focuses on reviewing the main applications of antibodies and antibody fragments for solid cancer diagnosis, both in vitro and in vivo. Furthermore, we review the scientific evidence showing that ion channels represent an almost unexplored class of ideal targets for both in vitro and in vivo diagnostic purposes. In particular, we review the applications, in solid cancers, of monoclonal antibodies and engineered antibody fragments targeting the voltage-dependent ion channel Kv 11.1, also known as hERG1.

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Protein engineering approaches for antibody fragments: directed evolution and rational design approaches

Cited by 21 publications

References 75 publications

Engineering of Humanized Antibodies Against Human Interleukin 5 Receptor Alpha Subunit That Cause Potent Antibody-Dependent Cell-Mediated Cytotoxicity

Engineering of Humanized Antibodies Against Human Interleukin 5 Receptor Alpha Subunit That Cause Potent Antibody-Dependent Cell-Mediated Cytotoxicity

Development of Radiotracers for Breast Cancer—The Tumor Microenvironment as an Emerging Target

Ion Channel Targeting with Antibodies and Antibody Fragments for Cancer Diagnosis

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