Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics

Abstract

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation. Preclinical proof-of-concept studies suggest that human plasma-derived Hp is a promising therapeutic candidate for several Hb-driven diseases. Optimizing the efficacy and safety of Hb-targeting biotherapeutics may require structural and functional modifications for specific indications. Improved Hp variants could be designed to achieve the desired tissue distribution, metabolism, and elimination to target hemolytic disease states effectively. However, it is critical to ensure that these modifications maintain the function of Hp. Using transient mammalian gene expression of Hp combined with co-transfection of the pro-haptoglobin processing protease C1r-LP, we established a platform for generating recombinant Hp-variants. We designed an Hpβ-scaffold, which was expressed in this system at high levels as a monomeric unit (mini-Hp) while maintaining the key protective functions of Hp. We then used this Hpβ-scaffold as the basis to develop an initial proof-of-concept Hp fusion protein using human serum albumin as the fusion partner. Next, a hemopexin-Hp fusion protein with bispecific heme and Hb detoxification capacity was generated. Further, we developed a Hb scavenger devoid of CD163 scavenger receptor binding. The functions of these proteins were then characterized for Hb and heme-binding, binding of the Hp–Hb complexes with the clearance receptor CD163, antioxidant properties, and vascular nitric oxide sparing capacity. Our platform is designed to support the generation of innovative Hb scavenger biotherapeutics with novel modes of action and potentially improved formulation characteristics, function, and pharmacokinetics.

Hemolysis, free hemoglobin toxicity, and scavenger protein therapeutics

“…The entire body of work, beginning from the studies conducted by Jayle et al 99 , Smithies O et al 100,101 , and Bunn HF et al 102,103 to the first protein-ligand complex crystal structures of haptoglobin-Hb 57 and hemopexin-heme 104 recombinant platforms are being exploited to conceptualize next-generation scavengers. These proteins will be structurally homogenous and contain specific functional subunits to prolong plasma half-life, provide dual Hb and heme-binding specificities, and enhance bioavailability in specific compartments 60,105,106 . These approaches will improve drug formulation, delivery, and tissue distribution.…”

“…It exists in humans as a polymorphic protein with genetically determined dimeric or multimeric assemblies of Hb-binding subunits (haptoglobin beta-chains) 58 . Even the smallest possible configuration of an Hbhaptoglobin complex has a molecular weight of more than 100 kDa, which far exceeds size restrictions of translocation pathways that permit extravasation of cell-free Hb in the kidney and vasculature 30,59,60 . This simple 'size-exclusion mechanism' of haptoglobin is highly effective at protecting the kidney and vascular NO homeostasis against disruption by cell-free Hb 9 .…”

Zonulin as a Potential Therapeutic Target in Microbiota-Gut-Brain Axis Disorders: Encouraging Results and Emerging Questions

“…In this process, the cleavage enzyme protease complement C1r subcomponent-like protein (C1r-LP) plays a crucial role [ 36 , 37 , 38 ], and thus is also hypothesized by Fasano to modulate the amount of zonulin in the circulation [ 39 ]. Serum level of pre-haptoglobin 2 or zonulin is approximately one thousandth of mature haptoglobins [ 7 ] and do not form complexes with hemoglobin [ 36 , 40 ].…”

Engineering Therapeutics to Detoxify Hemoglobin, Heme, and Iron

“…The final recombinant Hp protein was shown to be fully functional Hp through Hb-binding kinetics, preservation of vascular NO signaling in vitro and in vivo, and prevention of heme release and lipid peroxidation (41). In a later study, selective amino acid mutations enabled the generation of an Hp1-1 protein that was devoid of CD163 binding, which could be useful for certain therapeutic applications such as intracerebral hemorrhage, where capture of Hb-Hp in the brain could lead to toxic iron buildup (116).…”

“…Fusion proteins are a promising engineering strategy to modulate the properties and function of biomolecules. With an established system to generate recombinant Hp, its β chain was used as a scaffold to associate with the fusion protein partners, HSA and Hpx (116). While most of the α chain could be omitted from the protein sequence, expression of Hpβ (termed mini-Hp) required a portion from the α chain containing the C1r-LP cleavage site for proper expression.…”