Der p 1-based immunotoxin as potential tool for the treatment of dust mite respiratory allergy

Abstract

Immunotoxins appear as promising therapeutic molecules, alternative to allergen-specificimmunotherapy. In this work, we achieved the development of a protein chimera able to promote specific cell death on effector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purified from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were effectively conserved in proDerp1αS. Immunotoxin impact was assayed by using effector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purified basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic effect on these cells, apparently due to its lack of internalization after their surface IgEbinding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specific, second-generation of immunotoxins following proDerp1αS, is further discussed. Allergy or type I hypersensitivity reactions are widely described as a loss of tolerance to certain harmless exogenous antigens, such as airborne allergens, foods or insect venoms. Allergies prevalence currently reaches the 30% among the population in the industrialized countries, experiencing an alarming increment over the last few decades 1. Allergies cause a substantial social and economic impact, being the development of new treatments a crucial goal for biomedical research. Common symptoms of allergies, such as asthma and rhinitis, share a strong T-helper type 2 (Th2) immune polarisation, in combination with a significant decreased activity of the IFNγsecreting Th1 cells. Because of this shift, several active mediators are released (e.g. histamine, leukotrienes and prostaglandins, among others) by basophils and mast cells, ultimately resulting in the main immune-pathological features of the allergic response 2,3. Despite the well accepted fact that our understanding of the mechanisms underlying allergies has dramatically improved lately, we are still far from developing an effective therapy because of the multifactorial origin of this pathology. Accordingly, oral tolerance induction has shown to be highly effective in patients at early ages, but it dramatically fails once most immune responses have already been established. Within this same idea, allergen-specific-immunotherapy (AIT) arises as a unique alternative 4,5 , but carries powerful limitations that completely offset its clinical benefits. These limitations include: the variable q...

A recombinant Der p 1-specific allergen-toxin demonstrates superior killing of allergen-reactive IgG+ hybridomas in comparison to its recombinant allergen-drug conjugate

“…Notably, these IC 50 values are relatively comparable to those obtained in cancer immunotherapy: the anti-EGFR(scFv) immunotoxins; scFv1711-ETAʹ and scFv2112-ETAʹ developed by Niesen et al , for the treatment of solid tumours demonstrated IC 50 values ranging from 4 pM to 0.46 nM [ 45 ]. In a recent study, Rodrigo et al also reported on the development and evaluation of a Der p 1 allergen-toxin based on the ribotoxin: α-sarcin [ 46 ]. This construct named proDerp1αS was expressed in yeast P. pastoris and proof of principle evaluated on human basophil-like cells (humRBL-2H3) sensitized with sera from Der p 1 allergic patients.…”

Nanobody-Based EGFR-Targeting Immunotoxins for Colorectal Cancer Treatment

“…Ribotoxins recognize a conserved motif in the large rRNA subunit, namely the sarcin/ricin loop (SRL), and produce a single cleavage, leading to protein biosynthesis inhibition and cell death by apoptosis [38,39]. Among ribotoxins, the most characterized member is α-sarcin, a small basic ribotoxin of 150 amino acids that has been employed as the toxic domain in the design of several ITXs against allergies [40] and cancers, showing potent antitumoral activity both in vitro and in vivo [41][42][43][44].…”