Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases

Abstract

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

Human antibodies targeting ENPP1 as candidate therapeutics for cancers

“…The purity and structure of the antibodies were analyzed by Superdex 200 Increase 10/300 GL chromatography (GE Healthcare, Chicago, IL, USA) using methods previously established by our laboratory ( 31 ). The standard proteins Ferritin, Aldolase, Conalbumin, Ovalbumin, Carbonic anhydrase and Ribonuclease were used for calibration.…”

“…The binding and specificity of Fab, scFv and IgG1 to ENPP1 were analyzed by ELISA as previously described ( 31 ). Additional analysis by ELISA was performed in a similar manner with IgG1 to confirm non-specificity with other ENPP members.…”

“…The affinity and avidity of the anti-hENPP1 antibody were detected by biolayer interferometry BLItz (ForteBio, Menlo Park, CA, USA) as described previously ( 31 ). Briefly, DPBS was used to establish a baseline for 30s, and streptavidin biosensors (ForteBio) were coated with 16.7 μg/mL recombinant ENPP1-Biotin for 2 min.…”

Human Antibody VH Domains Targeting GPNMB and VCAM-1 as Candidate Therapeutics for Cancers

“…To perform panning antibody candidates against GPNMB and VCAM-1, a large phage-displayed human V H library was used against human IgG1 Fc fused recombinant GPNMB and VCAM-1 separately. The panning was performed as previously described . The V H phage library was first incubated with 50 μL Protein G magnetic beads (Thermo Fisher Scientific) to remove nonspecific binders.…”

“…For the construction of DbTE, humanized OKT3 was inserted at the C terminal of V H followed by the IgG1 Fc with LALAPG mutation. The expression and purification were performed as previously described . Briefly, the V H -Fc and DbTE were transiently transfected and expressed by the Expi293 expression system and purified by protein A resin (Thermo Fisher Scientific).…”