Citation

  • Authors: Xu, Z.. et al.
  • Year: 2023
  • Journal: Nat Commun . 14 5949
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi

Method

Recombinant antibodies against the F4 epitope were constructed using single-chain variable fragments (scFv) sequences selected from two phage display libraries. The constructs were then transformed into DH5α chemical competent cells for plasmid amplification, and large-scale production of full-length antibodies was carried out by transfecting Expi293F cell line using FectoPro with corresponding plasmids. Recombinant antibodies in the cell culture supernatants were harvested 5 days after transfection, purified using GammaBind Plus Sepharose antibody purification resin, followed by dialysis against 0.01 M glycine solution containing 0.15 M NaCl to avoid aggregates, and thereafter concentrated, buffered in the same glycine solution, and finally stored at 4 °C before injection. Once converted to full-length IgGs, the recombinant antibodies were denoted R69- clone number.

Abstract

Rheumatoid arthritis (RA) involves several classes of pathogenic autoantibodies, some of which react with type-II collagen (COL2) in articular cartilage. We previously described a subset of COL2 antibodies targeting the F4 epitope (ERGLKGHRGFT) that could be regulatory. Here, using phage display, we developed recombinant antibodies against this epitope and examined the underlying mechanism of action. One of these antibodies, R69-4, protected against cartilage antibody- and collagen-induced arthritis in mice, but not autoimmune disease models independent of arthritogenic autoantibodies. R69-4 was further shown to cross-react with a large range of proteins within the inflamed synovial fluid, such as the complement protein C1q. Complexed R69-4 inhibited neutrophil FCGR3 signaling, thereby impairing downstream IL-1β secretion and neutrophil self-orchestrated recruitment. Likewise, human isotypes of R69-4 protected against arthritis with comparable efficiency. We conclude that R69-4 abrogates autoantibody-mediated arthritis mainly by hindering FCGR3 signaling, highlighting its potential clinical utility in acute RA.

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