Fully human antibodies against the Protease-Activated Receptor-2 (PAR-2) with anti-inflammatory activity

Abstract

PAR-2 belongs to a family of G-protein coupled Protease-Activated Receptors (PAR) which are activated by specific proteolytic cleavage in the extracellular N-terminal region. PAR-2 is activated by proteases such as trypsin, tryptase, proteinase 3, factor VIIa, factor Xa and is thought to be a mediator of inflammation and tissue injury, where elevated levels of proteases are found. Utilizing the HuCAL GOLD® phage display library we generated fully human antibodies specifically blocking the protease cleavage site in the N-terminal domain. In vitro affinity optimization resulted in antibodies with up to 1000-fold improved affinities relative to the original parental antibodies with dissociation constants as low as 100 pM. Corresponding increases in potency were observed in a mechanistic protease cleavage assay. The antibodies effectively inhibited PAR-2 mediated intracellular calcium release and cytokine secretion in various cell types stimulated with trypsin. In addition, the antibodies demonstrated potent inhibition of trypsin induced relaxation of isolated rat aortic rings ex vivo. In a short term mouse model of inflammation, the trans vivo DTH model, anti-PAR-2 antibodies showed inhibition of the inflammatory swelling response. In summary, potent inhibitors of PAR-2 were generated which allow further assessment of the role of this receptor in inflammation and evaluation of their potential as therapeutic agents.

Update on protease‐activated receptor 2 in cutaneous barrier, differentiation, tumorigenesis and pigmentation, and its role in related dermatologic diseases

“…Potential for PAR2 as a therapeutic target has initiated a series of investigations into PAR2 modulators . Anti‐PAR2 human antibodies have also begun development . Development of safe tanning agents may be possible as the PAR2 peptide ligand LIGR was found to increase pigmentation without inducing inflammation .…”

The development of proteinase-activated receptor-2 modulators and the challenges involved

“…The antibody SAM11 was directed against the tethered ligand sequence of PAR2 and has been widely used as a tool to observe the expression of PAR2 on cell surfaces, block trypsin mediated cleavage in cellular studies and reduce PAR2 joint inflammation in murine models of arthritis [33]. In 2011 a series of humanised antibodies were developed which were shown to have a good binding affinity for PAR2 and to inhibit intracellular Ca 2+ release and prevent cytokine secretion mediated by PAR2, as well as inflammatory oedema in vivo [77]. Intra-articular injection (5-10 ng) resulted in inhibition of joint inflammation and arthritic index in murine models.…”

Toward Drugs for Protease-Activated Receptor 2 (PAR2)

“…These anti-PAR2 antibodies were also effective in inhibiting inflammatory edema in vivo. 205 Table 9 summarizes some PAR2binding antibodies reported in patents with potential therapeutic applications. However, not all antigenic epitopes were disclosed.…”

“…They inhibited trypsin-induced iCa 2+ release, cytokine secretion, and relaxation of isolated rat aortic rings. These anti-PAR2 antibodies were also effective in inhibiting inflammatory edema in vivo . Table summarizes some PAR2-binding antibodies reported in patents with potential therapeutic applications.…”