Therapeutic TVs for Crossing Barriers in the Brain

Abstract

Brain disorders are at the leading edge of global disease burden worldwide. Effective therapies are lagging behind because most drugs cannot reach their targets in the brain because of the blood-brain barrier (BBB). The new development of a BBB transport vehicle may bring us a step closer to solve this problem.

If curing brain diseases is equivalent to the Mars mission in biomedicine, then delivering drugs across the blood-brain barrier (BBB) is unquestionably the Moonshot. Since the discovery of the BBB by German physician Paul Ehrlich in the late 1800s, generations of scientists and engineers tried to understand how to breach the BBB for successful delivery of drugs to the central nervous system (CNS), but none have fully succeeded. Recently, biotechnology and pharmaceutical companies have joined the race proposing cutting-edge antibody-based technologies. Two new reports from Denali Therapeutics definitely showcased their success in creating a promising platform (Kariolis et al., 2020; Ullman et al., 2020), which perhaps could be our Falcon 9 for the next step.

The BBB is the gate keeper of the CNS, uniquely separating brain internal milieu from the circulating blood. The BBB limits toxic substances, pathogens, and >99% of drugs from entering the brain, yet at the same time selectively controls transport of ions, nutrients, and essential signaling molecules through its highly specialized transport systems (Sweeney et al., 2019). The receptor-mediated transcytosis (RMT) is one of the secret passages within the BBB. RMT is a series of tightly regulated cellular events in the endothelium, starting with clathrin-mediated receptor endocytosis and followed by intracellular trafficking of the endosomes containing the “cargo” and sorting of the vesicles toward directed exocytosis, rather than lysosomal-mediated degradation, a process guided by small Rab GTPases (Zhao et al., 2015). Many growth factors and signaling peptides, such as leptin, insulin, insulin-like growth factor, lipoproteins, and transferrin, are delivered to the brain across the BBB via RMT. Therefore, targeting the RMT systems offers a tremendous opportunity for CNS drug delivery (Sweeney et al., 2019).

Transferrin receptor (TfR) was the first extensively utilized as an RMT system for transport of antibodies and antibody-drug conjugates across the BBB (Friden et al., 1991) (see Figure 1). A monoclonal antibody OX-26 was the first experimental vessel, which transported methotrexate conjugates across the murine BBB to reach the brain parenchyma side. Following this report, other BBB associated receptors, such as insulin receptors, have been actively tested for their potential for CNS drug delivery (Zuchero et al., 2016). Because RMT efficiency is highly dependent on the abundance and spatial distribution of the receptors, TfR, as one of the top transmembrane proteins enriched at the BBB (Zuchero et al., 2016), has become the principal target for antibody designing and optimization.

Figure 1. A Brief Historical Review of Targeting the TfR Receptor-Mediated Transport System for Trans-Blood-Brain Barrier Drug Delivery.

Proof-of-concept experiments in the 1990s showed that TfR Mab can be delivered into the brain via a TfR RMT system. Antibody engineering greatly accelerated the development, and three generations of antibodies targeting TfR for trans-BBB delivery are now available. The new engineered TV with key residue E³⁸⁷ can deliver therapeutics more efficiently than ever before. TfR, transferrin receptor; RMT, receptor-mediated transport; BBB, blood-brain barrier; Mab, monoclonal antibodies; scFV, single-chain variable fragment; Aβ, Alzheimer’s amyloid-β peptide; BACE1, amyloid processing enzyme β-secretase; sFab, single antigen binding fragment; TV, transport vehicle; ATV, antibody transport vehicle, which can target amyloidosis (brown splotches in the brain); PTV, protein transport vehicle, which brings in the iduronate-2-sulfatase (IDS) for catabolism of glycosaminoglycans (GAGs); MPS II, mucopolysaccharidosis type II lysosomal storage disease.

The second generation of therapeutic antibodies that recognize TfR for transverse BBB delivery were based on antibody engineering. One approach used protein fusion, where the high-affinity TfR monoclonal antibody (e.g., 8D3) was fused to an anti-amyloid single chain Fv (ScFv) antibody (Zhou et al., 2011) or to a recombinant protein for replacement therapy (Ullman et al., 2020). Genentech applied the bispecific antibody concept and created an antibody with two different Fab domains, one recognizing TfR and the other neutralizing amyloid processing enzyme β-secretase (BACE1) (Yu et al., 2011). Both approaches showed improved but not optimal antibody uptake by the brain, yet despite this, amyloid pathology was significantly reduced in Alzheimer’s disease mouse lines treated with these antibodies (Yu et al., 2011; Zhou et al., 2011). The later versions improved the BBB uptake by either reducing the bispecific antibody’s affinity to TfR (Yu et al., 2014) or only fusing to one Fc domain that can interact with TfR (Niewoehner et al., 2014).

Now Kariolis et al. (2020) reported a new generation of tools. Aiming to create a versatile platform capable of delivering a broad range of cargos, the Denali team engineered one of the human IgG1 Fc domains. They first selected nine amino acids in the CH3 domain of Fc that are distant to other functional binding sites of the receptor for randomization, and luckily identified four unique combinations that endowed Fc’s new ability to bind TfR. Using the YTEWSQ-EDH sequence as a foundation for affinity maturation, Kariolis et al. (2020) finally found a transport vehicle (TV) with the key residue E³⁸⁷ that directly binds to human TfR. With this platform, Kariolis et al. (2020) were able to deliver BACE1 Fabs across the BBB into the brain in both transgenic mice and cynomolgus monkeys, which downregulated BACE1 levels and production of endogenous amyloid-β peptide. Because the TfR binding site is engineered on the Fc domain, it is now possible to have bispecific Fab domains to pursue multiple drug targets at the same time, as for example both BACE1 and Tau for Alzheimer’s disease.

The TfR-TV can also fuse with any therapeutic protein or peptide. Ullman et al. (2020) reported linking the iduronate-2-sulfatase (IDS) to the TV and treated a mouse model of mucopolysaccharidosis type II (MPS II) lysosomal storage disease, also known as Hunter syndrome. Compared to classic enzyme replacement therapy, TfR-TV not only reduced peripheral symptoms but also increased brain IDS levels, successfully restored lysosome function, and prevented neurodegeneration in IDS knockout mice expressing human TfR. Although these are proof-of-concept studies, they clearly demonstrate the improved efficacy of delivering cargo to the brain via TfR-TV and endless capability in adapting diverse therapeutics with potential to target different CNS diseases.

Is the TV the final answer for the BBB challenge? It is probably too early to tell. Using the TfR-TV for antibody delivery will likely require regular dosing, which will be very costly for chronic CNS conditions such as Alzheimer’s disease, but can also cause potential immune responses including first infusion reactions acutely and neutralizing antibodies long-term. Additionally, BBB impairment in different CNS disorders is accompanied by focal and regional microenvironment changes in the affected sites. This includes perivascular accumulation of blood-derived macromolecules and infiltrating immune cells, vascular regression, and blood flow reduction, which could all present a challenge for successful drug delivery and CNS distribution (Sweeney et al., 2018). Additionally, RMT deficiency is expected to occur often, if not always, in the affected brain regions in neurological and/or neurodegenerative disorders associated with vascular dysfunction (Sweeney et al., 2019). This might include deficiency of TfR itself and/or abnormalities in the endocytic and exocytotic molecular machinery, which may potentially contribute to variations in TfR-TV’s efficacy in the population affected by brain diseases.

Although the new RMT-targeting antibody platform is exciting, it might be worthwhile to take a careful look at other strategies developed to circumvent the BBB. Different routes of drug administration have been adapted in clinic already, including intracerebroventricular administration of Cerliponase for Batten’s disease and/or intrathecal administration of anti-sense oligos Spinraza (developed by IONIS and Biogen) for infantile spinal muscular atrophy. Other approaches, although still in preclinical development, have also shown great potential, such as encapsulation into nanoparticles or liposomes. Utilizing the carrier-mediated transport systems can also be very effective for the delivery of certain therapeutics (Zuchero et al., 2016). Additionally, viral vector-mediated gene delivery to the CNS, particularly via the AAV9-PHP variants (Wang et al., 2019), has demonstrated high efficiency in crossing the BBB in rodents. The journey to find a perfect solution may be still out there, but the TfR-TV system deserves a big round of applause!