A Fully-Human Antibody Specifically Targeting a Membrane-Bound Fragment of CADM1 Potentiates the T Cell-Mediated Death of Human Small-Cell Lung Cancer Cells

Abstract

Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer and the leading cause of global cancer-related mortality. Despite the earlier identification of membrane-proximal cleavage of cell adhesion molecule 1 (CADM1) in cancers, the role of the membrane-bound fragment of CAMD1 (MF-CADM1) is yet to be clearly identified. In this study, we first isolated MF-CADM1-specific fully human single-chain variable fragments (scFvs) from the human synthetic scFv antibody library using the phage display technology. Following the selected scFv conversion to human immunoglobulin G1 (IgG1) scFv-Fc antibodies (K103.1–4), multiple characterization studies, including antibody cross-species reactivity, purity, production yield, and binding affinity, were verified. Finally, via intensive in vitro efficacy and toxicity evaluation studies, we identified K103.3 as a lead antibody that potently promotes the death of human SCLC cell lines, including NCI-H69, NCI-H146, and NCI-H187, by activated Jurkat T cells without severe endothelial toxicity. Taken together, these findings suggest that antibody-based targeting of MF-CADM1 may be an effective strategy to potentiate T cell-mediated SCLC death, and MF-CADM1 may be a novel potential therapeutic target in SCLC for antibody therapy.

Empowering SARS‐CoV‐2 variant neutralization with a bifunctional antibody engineered with tandem heptad repeat 2 peptides

“…After centrifugation for 40 min at 12 000 g at 4°C, the phages were resuspended in 3% (w/v) bovine serum albumin (BSA) in PBS. Biopanning was performed using the rescued scFv‐displayed phage pool to select SARS‐CoV‐2 BA.2 RBD‐specific scFvs, as previously described 24 . Briefly, six rounds of biopanning were performed with M‐270 epoxy Dynabeads™ (Invitrogen), which were covalently immobilized with 4‐μg recombinant His‐tagged SARS‐CoV‐2 BA.2 RBD (BA.2 RBD‐His) (Sino Biological).…”

“…Biopanning was performed using the rescued scFvdisplayed phage pool to select SARS-CoV-2 BA.2 RBD-specific scFvs, as previously described. 24 Briefly, six rounds of biopanning were performed with M-270 epoxy Dynabeads™ (Invitrogen), which were covalently immobilized with 4-μg recombinant His-tagged SARS-CoV-2 BA.2 RBD (BA.2 RBD-His) (Sino Biological). Subsequently, 96 phage clones were randomly selected from the output colonies, and their reactivity to BA.2 RBD-His was assessed using a phage enzyme-linked immunosorbent assay (ELISA).…”

Development and Characterization of Phage Display-Derived Monoclonal Antibodies to the S2 Domain of Spike Proteins of Wild-Type SARS-CoV-2 and Multiple Variants

“…After centrifugation for 40 min at 12,000× g at 4 °C, the phages were resuspended in 3% ( w / v ) bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Using the rescued scFv-displayed phage pool, biopanning was performed to select SARS-CoV-2 extracellular domain of S protein (SARS-CoV-2 S-ECD)-specific scFvs from a synthetic scFv antibody library, as previously described [ 27 ]. Briefly, four rounds of biopanning were conducted to select SARS-CoV-2 S-ECD-specific scFv clones using magnetic beads (Dynabeads M-270 Epoxy; Invitrogen, Waltham, MA, USA) coated with 4 μg recombinant SARS-CoV-2 S-ECD (Sino#40589-V08B1; Sino Biological Inc., Beijing, China).…”

Development and Characterization of Phage-Display-Derived Novel Human Monoclonal Antibodies against the Receptor Binding Domain of SARS-CoV-2

“…Biopanning was performed to isolate human antibodies specific to the SARS-CoV-2 RBD from a human synthetic scFv antibody library, as previously described [ 33 ]. Briefly, four rounds of biopanning were conducted to select SARS-CoV-2 RBD-specific scFv clones using Dynabeads™ M-270 epoxy magnetic beads (Invitrogen, Carlsbad, CA, USA) coated with 4 μg recombinant SARS-CoV-2 RBD (Cat#40592-V08B; Sino Biological, Beijing, China).…”