scholarly journals Expeditious Generation of Biparatopic Common Light Chain Antibodies via Chicken Immunization and Yeast Display Screening

2020 ◽  
Vol 11 ◽  
Author(s):  
Jan P. Bogen ◽  
Stefania C. Carrara ◽  
David Fiebig ◽  
Julius Grzeschik ◽  
Björn Hock ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Light Chain ◽  
Surface Display ◽  
Growth Factor Receptor ◽  
Yeast Surface Display ◽  
Single Digit ◽  
Dependent Cell ◽  
Epidermal Growth ◽  
Domain Iii

Bispecific (BsAb) and biparatopic (BpAb) antibodies emerged as promising formats for therapeutic biologics exhibiting tailor-made functional properties. Over recent years, chicken-derived antibodies have gained traction for diagnostic and therapeutic applications due to their broad epitope coverage and convenience of library generation. Here we report the first generation of a biparatopic common light chain (cLC) chicken-derived antibody by an epitope binning-based screening approach using yeast surface display. The resulting monospecific antibodies target conformational epitopes on domain II or III of the epidermal growth factor receptor (EGFR) with lower double- or single-digit nanomolar affinities, respectively. Furthermore, the domain III targeting variant was shown to interfere with epidermal growth factor (EGF) binding. Utilizing the Knob-into-Hole technology (KiH), a biparatopic antibody with subnanomolar affinity was generated that facilitates clustering of soluble and cell-bound EGFR and displayed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parental antibodies. This strategy for generating cLC-based biparatopic antibodies from immunized chickens may pave the way for their further development in therapeutic settings.

Tumor cell lysis and synergistically enhanced antibody-dependent cell-mediated cytotoxicity by NKG2D engagement with a bispecific immunoligand targeting the HER2 antigen

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Christian Kellner ◽  
Sebastian Lutz ◽  
Hans-Heinrich Oberg ◽  
Daniela Wesch ◽  
Anna Otte ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Nk Cell ◽  
Growth Factor Receptor ◽  
Cell Cytotoxicity ◽  
Dependent Cell ◽  
Epidermal Growth ◽  
Nk Cell Cytotoxicity ◽  
Tumor Cell Lysis

Abstract Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC.

scholarly journals EHD1 and RUSC2 Control Basal Epidermal Growth Factor Receptor Cell Surface Expression and Recycling

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Eric C. Tom ◽  
Insha Mushtaq ◽  
Bhopal C. Mohapatra ◽  
Haitao Luan ◽  
Aaqib M. Bhat ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Surface ◽  
Surface Display ◽  
Growth Factor Receptor ◽  
Cell Surface Display ◽  
Cell Surface Expression ◽  
Golgi Compartment ◽  
Epidermal Growth

ABSTRACT Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.

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