Affinity maturation of a novel antagonistic human monoclonal antibody with a long V_HCDR3 targeting the Class A GPCR formyl-peptide receptor 1

Abstract

(2015) Affinity maturation of a novel antagonistic human monoclonal antibody with a long V H CDR3 targeting the Class A GPCR formyl-peptide receptor 1, mAbs, 7:1, 152-166, DOI: 10.4161/19420862.2014.985158 To link to this article: https://doi.org/10. 4161/19420862.2014 Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding V H CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long V H CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long V H CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.

Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody–Drug Conjugate Targeting 5T4

“…29) with recombinant human 5T4 using a 48-day rest/boost immunization protocol (30) and was identified from a biochemical assay of hybridoma supernatants for binding to 5T4. This antibody underwent affinity optimization by CDR-directed mutagenesis (31) and phage display selection (32,33).…”

A review of antibody-based therapeutics targeting G protein-coupled receptors: an update

“…These can be broadly divided into those obtained by XFEL, such as 5-HT 2B [75] and GCG [76], and X-ray crystallography, such as TSHR [77], GIP [78] and EP4 [79]. Other studies have conducted homology modeling in combination with intensive epitope mapping to identify putative important contact residues of functional antibodies, e.g., CXCR4 [80], or by obtaining a crystal structure of the Fab fragment and modeled in combination with a GPCR homology model, e.g., FPR1 [81], but the greatest accuracy is obtained from high-resolution crystallography approaches. The knowledge gained from such studies can inform decisionmaking in the drug discovery process.…”

Formation of ultralong DH regions through genomic rearrangement

“…The third complementary determining region of the heavy chain (CDR H3) is particularly important in antibody molecules as it contains the greatest diversity and also usually makes the most extensive contact with antigen. Long CDR H3 regions are often found in broadly neutralizing antibodies targeting human immunodeficiency (HIV), influenza, and polio viruses[4–8], and are also thought to be important in binding challenging antigens like G-protein coupled receptors and protease active sites[9, 10]. Thus, genetic mechanisms to form long CDR H3s may be very important in immune responses against key antigens.…”