**1. Introduction**

Drug uptake into the brain is quite challenging, although not impossible [1]. Since the brain is located in a non-expandable vault (cranium) and is very sensitive to pressure and the environment, cerebrospinal fluid (CSF) flow in and out of the brain is highly regulated and controls the selective uptake of key nutrients and fluid to maintain normal brain function. This regulation also includes the passage of large molecules such as immunoglobulin thereby accounting for the observed difficulties of targeting the central nervous system (CNS) with therapeutic proteins and reagents. Many potentially useful drugs, which, because of their low entrance into the CNS, are not being used to treat brain disease. This lack of access to the brain has been described as a major hurdle in the development of large biomolecules and a reason given for their comparatively long development times and high failure rate [2]. As a consequence, several approaches are currently being investigated to enhance the CNS delivery of various types of large biomolecules, such as antibodies, recombinant proteins, gene vectors, liposomes, and nanoparticles (Table 1). To evaluate CNS delivery, quantitative measurements are used to understand better and potentially even improve upon methods for the targeted delivery of antibody-based therapeutics across the BBB. In particular, scientific and technological advancements that focus on evaluating methods for altering antibody penetration and distribution in the brain have not yet been developed adequately to treat neurological

**Citation:** Kouhi, A.; Pachipulusu, V.; Kapenstein, T.; Hu, P.; Epstein, A.L.; Khawli, L.A. Brain Disposition of Antibody-Based Therapeutics: Dogma, Approaches and Perspectives. *Int. J. Mol. Sci.* **2021**, *22*, 6442. https://doi.org/10.3390/ijms 22126442

Academic Editor: Yong-Seok Heo

Received: 18 May 2021 Accepted: 11 June 2021 Published: 16 June 2021

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diseases. Moreover, even if candidate antibodies for the therapy of CNS diseases may be already available, they cannot currently be utilized because of their poor blood-to-brain penetration due to the presence of the tight-junctioned BBB preventing the passage of antibodies [3]. Thus, increased attention is being placed on novel antibodies capable of successfully enhancing brain tissue concentration as well as targeting specific disease regions within the CNS [4,5]. If proven safe and effective, these new technologies could represent the future of antibody therapy in the treatment of neurologic diseases.

**Table 1.** Overview of large biomolecules in current preclinical development for enhanced delivery across the BBB. Part of this table is reproduced from Tucker (2011) with permission of the copyright owner [1].


Here, we review some of the most important principles and multiple strategies for enhancing antibody delivery to the brain and discuss how they can be applied to the pre-clinical development of CNS therapeutics. The guiding principles and knowledge gained from preclinical evaluation of these different strategies for CNS-targeting antibodies that are currently under development are also discussed, with a particular emphasis on pharmacokinetic (PK) and disposition properties. In addition, this review includes a brief description of the physicochemical and biochemical interactions between antibodies and biological matrices. As such, focus is given to defining the general properties of antibodies, their similarities and differences with regard to charge, neonatal Fc receptor (FcRn) binding, and target affinity. These types of studies provide scientists with the knowledge necessary to select the appropriate antibody characteristics to maximize brain exposure, which in turn, could provide better efficacy of their product. Finally, an improved understanding of the effects of these critical characteristics may allow for the better design of novel antibody therapeutics with unique and useful properties that conceptually are able to efficiently cross the BBB [5]. Hence, the objective of this review was to describe the progress of antibody-based drugs and highlight the principles and existing approaches for enhancing their entrance into the brain to achieve a desirable concentration range for the therapy of CNS disease.

### **2. Delivery of Antibodies into the Brain: Mechanism of Delivery**

Diseases of the CNS are in need of effective biotherapeutics. However, the CNS has been considered off-limits to antibody therapeutics because of the presence of the BBB, which separates the circulating blood from the brain and extracellular fluid in the CNS to prevent brain uptake of most large molecules [11,15]. Recent advances in preclinical and clinical drug development suggest that antibodies can cross the BBB in limited quantities and act centrally to mediate their effects [4]. In particular, immunotherapy studies of AD have shown that targeting beta amyloid with antibodies can reduce disease pathology in both mouse models and patients, with strong evidence supporting a central mechanism of action.

#### *2.1. Physiology and Barriers of the CNS*

The arrangement of cells at the interface between the blood and the CNS restricts both the paracellular and transcellular diffusion of hydrophilic and hydrophobic substances into the CNS [16]. The blood–brain barrier (BBB) is used to describe the barrier between the blood and the brain and spinal cord parenchyma proper. At this interface, cerebral microvessels, lined with endothelial cells, limit the passage of small molecules from the blood into the brain or spinal cord [11]. Microvascular endothelial cells make up a large portion of the brain's surface area, which helps account for its ability to restrict the flow of substances into the brain [17]. A second barrier, referred to as the blood–CSF barrier, exists between the blood and the ventricular CSF. Formed by CSF producing tight-junctioned epithelium of the choroid plexuses, this epithelial cell barrier accounts for a significant surface area of exchange [16]. Additionally, the blood flow rate within the choroid plexuses is higher than any other brain structures, and therefore, the blood flow through these areas significantly contributes to exchanges between the blood and the CNS. A third barrier to the CNS is the arachoid membrane, which completely encircles the CNS and separates the subarachnoid CSF from the bones and *dura mater* extracellular fluids [16,18]. These three barriers to the CNS work to manage the traffic of small and large molecules from the blood into the brain.

#### *2.2. BBB Structure*

As described above, the BBB consists of the network of cells that communicate and associate together to form a barrier between the interstitial fluid of the brain and circulating blood. A thin monolayer of brain microvascular endothelial cells (BMECs) joined together by tight junction forms the physical BBB. The BMECs are supported by the capillary basement membrane, pericytes, astrocytes, and microglial cells. It is the interaction of the BMECs with these other cell types that creates the specific brain microvascular network. The tight junctions are responsible for the selective permeability of the BBB, as they seal the apical region of the endothelial cells together and restrict the entrance of hydrophilic drugs into the brain. Additionally, actin filaments, such as cadherins and catenins, arranged below the tight junctions, link together to form a band of adherence junctions. These adherence junctions contribute to the brain barrier, and also, among other roles, they promote BMECs adhesions, cell polarity, and control paracellular permeability regulations. It is the dynamic interaction between the tight junctions and the adhesion junctions and the other cellular components of the BBB via signaling pathways that regulate the BBB's permeability. The arrangement of cells that form the BBB allow it to have uniform thickness, a negative surface charge, little pinocytotic activity, and no fenestrae [19].
