Preclinical development of a long-acting trivalent bispecific nanobody targeting IL-5 for the treatment of eosinophilic asthma

Abstract

Background Eosinophilic asthma is a common subtype of severe asthma with high morbidity and mortality. The cytokine IL-5 has been shown to be a key driver of the development and progression of disease. Although approved monoclonal antibodies (mAbs) targeting IL-5/IL-5R have shown good safety and efficacy, some patients have inadequate responses and frequent dosing results in medication nonadherence. Results We constructed a novel trivalent bispecific nanobody (Nb) consisting of 3 VHHs that bind to 2 different epitopes of IL-5 and 1 epitope of albumin derived from immunized phage display libraries. This trivalent IL-5-HSA Nb exhibited similar IL-5/IL-5R blocking activities to mepolizumab (Nucala), an approved targeting IL-5 mAb. Surprisingly, this trivalent Nb was 58 times more active than mepolizumab in inhibiting TF-1-cell proliferation. In primate studies, the trivalent IL-5-HSA Nb showed excellent pharmacokinetic properties, and peripheral blood eosinophil levels remained significantly suppressed for two months after a single dose. In addition, the trivalent IL-5-HSA Nb could be produced on a large scale in a P. pastoris X-33 yeast system with high purity and good thermal stability. Conclusions These findings suggest that the trivalent bispecific IL-5-HSA Nb has the potential to be a next-generation therapeutic agent targeting IL-5 for the treatment of severe eosinophilic asthma. Graphical Abstract

A humanized trivalent Nectin-4-targeting nanobody drug conjugate displays potent antitumor activity in gastric cancer

“…Due to the non-recognition of murine albumin by our HSA Nb, the majority of Nectin-4 NDCs are cleared through renal elimination within 48 h. In our previous study, trivalent Nbs containing HSA Nb demonstrated a prolonged residence time of up to 12 days in cynomolgus monkeys [ 55 ]. Consequently, our Nectin-4 NDCs may potentially maintain a comparable half-life in cynomolgus monkeys and the human body.…”

Identification and Characterization of a Novel Nanobody Against Human CTGF to Reveal Its Antifibrotic Effect in an in vitro Model of Liver Fibrosis

“…Human CTGF domain II (AA101-167) was optimized using a human codon and synthesized into the pFUSE vector (Invitrogen, Carlsbad, CA, USA), as previously described. 21 , 24 CTGF domain II-Fc was expressed in HEK-293F cells (American Type Culture Collection) and purified by protein A affinity chromatography. The purified antigen was mixed with Freund’s adjuvant and immunized with two young camels.…”

“…Anti-CTGF Nb was screened through phage display bio-panning according to established methods. 21 , 26 , 27 To identify positive colonies, individual colonies from the library were randomly selected for periplasmic extract ELISA verification. After sequencing of the screened colonies, the CTGF-specific Nb gene was amplified and subcloned into the pFUSE-mFc vector.…”

“…20 Nanobodies (Nbs) have been used as imaging agents and therapeutics in preclinical and clinical studies. [21][22][23][24][25] Nbs are novel type of single-domain antibody fragments (VHHs) derived from natural heavy chain-only antibodies in the Camelidae family of mammals. 21,24 It possesses several advantages for therapeutic applications including tumor targeting, owing to low immunogenicity, high physicochemical stability and refolding capacity, and good tissue penetration.…”

NANOBODIES®: A Review of Diagnostic and Therapeutic Applications

“…These treatments include (i) Sonelokimab (M1095) targeting interleukin-17A/F for psoriasis, (ii) Gefurulimab (ALXN1720) targeting autoantibodies against acetylcholine receptors for generalized Myasthenia gravis, (iii) Nb M6495 targeting A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 for osteoarthritis, and (iv) Nb V565 against tumor necrosis factor for Crohn’s Disease [ 138 , 139 , 140 , 141 ]. To further illustrate the potential of Nbs in therapeutics against autoimmune disorders, potential Nb candidates against eosinophilic asthma and multiple sclerosis have also been identified [ 142 , 143 ].…”