Development of target antigen-displaying virus-like particles (VLPs) for the generation of antibodies using hybridoma technology

Abstract

Formaldehyde-fixed, paraffin-embedded (FFPE) cell and tissue samples are of great importance for immunohistochemical studies of histological specimens. However, antibodies for FFPE samples pose a challenge to antibody discovery as current immunization strategies rely predominantly on soluble proteins that cannot adequately reflect the changes in target antigens during the FFPE process. Enveloped virus-like particles (VLPs) allow for the presentation of membrane-anchored target antigens on the VLP surface and elicit a strong target antigen-specific antibody response after immunization. This proof-of-concept study presents a novel FFPE-like fixation methodology for VLP preparation aiming at the generation of FFPE-compatible monoclonal antibodies (mAbs). Human 293-F-derived VLP-producing suspension cell pools were established to produce human immunodeficiency virus (HIV)-like particles decorated with the truncated human low affinity nerve growth factor receptor (trNGFR) as model antigen. The trNGFR antigen was efficiently incorporated into VLPs with an average of 284 } 24 trNGFR molecules per VLP. To develop a fixation protocol applicable to VLPs, trNGFR-expressing cells were subjected to a variety of fixation treatments. Changes in epitopes introduced by fixation were monitored using two mAbs recognizing either an epitope present in native NGFR or an epitope present in native and FFPE NGFR. The novel simplified fixation procedure consisted of only formaldehyde and 90 °C heat fixation (FF90). Transmission electron microscopic and dynamic light scattering analysis of FF90 VLPs revealed that the fixed VLPs withstood the FF90 treatment and showed no morphological changes, allowing for the FF90 trNGFR-VLPs to be used to immunize mice for hybridoma cell generation. Hybridoma clones were screened for mAbs specifically recognizing native, FF90 and FFPE trNGFR-expressing cells in a flow cytometric assay. The isolated hybridoma mAbs did not recognize native epitopes but were reactive with FF90 and FFPE epitopes. The use of FF90-trNGFR VLPs for immunization led to the discovery of nine FFPE-NGFR-specific mAbs. This proofof-concept study demonstrated that FF90-treated VLPs decorated with a membrane-anchored target antigen are suitable antigens to preferentially generate FFPE-compatible mAbs. The FF90-VLP platform should be useful for the future discovery of specific mAbs directed against a variety of FFPE cell surface antigens.