*2.3. Pharmacokinetics and CNS Distribution of Antibodies*

The PK properties of therapeutic antibodies are an essential factor that determine their in vivo efficacy by impacting their biodistribution and have been extensively studied in recent years [20]. The processes that govern the biodistribution of therapeutic antibodies depends on the species they are administered to and on the properties of the antibody itself. While physiological conditions are frequently constant, various properties of a therapeutic antibody such as its charge or size can be modified during development in order to optimize its PK behavior. Structural modifications such as glycosylation can also impact the biodistribution of an antibody. Of particular importance, however, is the role of FcRn on PK properties of an antibody, which must be considered in designing therapeutic antibodies for neurological disorders. FcRn is a receptor that is highly expressed in various tissues and prolongs an IgG antibody's half-life by protecting it from lysosomal degradation. It has been reported that the receptor contributes to the efflux of IgG therapeutic antibodies at the BBB and can reduce brain uptake following administration despite prolonging its half-life. The crucial role of FcRn on the CNS distribution behavior of antibodies is further discussed in Section 4.

#### *2.4. Mechanisms of Antibody Passage Across the BBB*

In the past few decades, various transport mechanisms have been identified as major pathways for macromolecules to cross the BBB. Generally, approximately 0.1% of circulating antibodies enter the brain. Mechanisms in play include: i) Adsorptive-mediated endocytosis; (ii) Carrier-mediated transport; and (iii) Receptor-mediated transcytosis.

(i) Adsorptive-mediated endocytosis (AMT) is a mechanism of BBB transport that relies on an electrostatic interaction between a cationic molecule in the circulation and the negatively charged cell membrane at the BBB, which will in turn trigger internalization of the positively charged molecule [3]. Cationic modification of proteins such as albumin and IgGs have been used to enhance their uptake into the brain. Studies have demonstrated that cationization of antibodies by covalently linking primary amine groups to their surface enhances their uptake into the brain by AMT. The capacity of AMT is high, but this mechanism is low in affinity and therefore has poor specificity. This is because cationized molecules can interact with negatively charged cell membranes of peripheral organs so that uptake in the brain does not increase proportionally [4,21]. The non-specificity of AMT mechanism should be considered in designing therapeutic antibodies that are targeted to the brain [6].

(ii) Carrier-mediated transport (CMT) is a mechanism by which small molecules such as glucose, amino acids, vitamins, hormones, and other nutrients rapidly cross the BBB [4]. This is a saturable mechanism due to the engagement of carriers and maintains homeostasis in the CNS by transporting these molecules bidirectionally [3]. Carrier-mediated transporters include CLUT1, which mediates transport of glucose and mannose and LTA1, which mediates transport of large neutral amino acids [21]. In principle, molecules can enter the brain using the CMT if they are conjugated to either endogenous ligands of the carriers or their analogues. However, this process has proved to be challenging for transport of antibodies because these carriers transport small molecules and are highly stereoselective.

(iii) Receptor-mediated transcytosis (RMT) is one of the most promising approaches for delivering antibodies to the brain [4]. There are three categories of receptors that mediate RMT: iron transporters such as transferrin receptors (TfR); insulin transporters such as insulin receptor (IR); and lipid transporters such as low-density lipoprotein receptor- related protein 1 (LRP1). This process entails binding of the ligand to the receptor, internalization of the ligand–receptor complex, and exocytosis on the abluminal side of the cell [3]. It is important to keep in mind, however, that high-affinity antibodies toward receptors that mediate RMT will follow the lysosomal pathway when internalized, which results in their degradation [22]. While this phenomenon creates a challenge in using the RMT mechanism, optimizing the affinity of the ligand that is targeting these receptors has proved to be an effective strategy [22].

#### **3. Current In Vitro and In Vivo Methodologies for Measuring Brain Access of Antibodies: Advantages and Limitations**

Implementation of in vitro models of the BBB that correlate with in vivo studies would provide desirable preclinical tools for the mechanistic understanding of drug transport via brain endothelial cells and uptake into the CNS monitored by the BBB. Use of these as a screening tool are of critical importance for the determination of drug permeability, PK, and distribution to brain tissues and cells.

#### *3.1. In Vitro Methods*

To aid in our understanding of the role of the BBB in protecting the brain microenvironment, different types of in vitro models of the BBB have been developed, which are classified into either static or dynamic BBB models [19,23]. Static BBB models are commonly used, but they do not imitate the shear stress, which is usually generated in vivo due to the blood flow. Static BBB models are further divided into monolayer and co-culture models, based on type of cells involved in the BBB design. While the brain microvessel endothelial cell culture model presents many differences compared with the in vivo system, monolayer cultures in a trans-well system allow a simple method for drug screening and permeability studies. The co-culture BBB model, however, is used to mimic the anatomic structure of BBB in vivo, in which BMECs are co-cultured with other CNS cells that directly contribute to the barrier properties of the BBB. As none of these in vitro models can entirely imitate the in vivo conditions, there is no perfect in vitro model of the BBB. Therefore, it is important to choose the in vitro model according to the requirement of the study. More details about the advantages and disadvantages of the different in vitro BBB models are currently covered in a thorough review article by Bagchi et al. (2019) [19].

#### *3.2. In Vivo Methods*

In contrast to in vitro methods, various in vivo methods have been employed to determine the kinetics of drug transport across the BBB. These include intravenous injection, in situ brain perfusion, microdialysis, quantitative whole-body autoradiography (QWBA), and molecular imaging such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), and optical imaging. Brain perfusion is the most widely used technique for obtaining in vivo permeability values for drugs [24,25]. As such, brain perfusion allows injection of a solute into the brain vasculature at higher flow rates and solute concentrations than can be achieved by systemic circulation and hence allows a wider range of solute permeabilities to be measured at a fixed perfusate concentration. Direct injection of the solute into the brain minimizes metabolic loss and plasma protein binding. In this technique, the common carotid artery is cannulated and connected to a perfusion system. Immediately after the animal's heart is stopped, the molecule of interest dissolved in a physiological solution is infused into the brain typically for 5–300 s. Subsequently, the brain is removed, and the ipsilateral hemisphere is dissected, weighed, and the solute concentration determined by chromatography (LC-MS, HPLC, GC) or by radioactive counting methods (gamma or liquid scintillation counting) if the drug is radiolabeled.

In vivo microdialysis is another well-established quantitative technique in neuroscience for measuring small molecule concentrations in brain interstitial fluid (ISF) and CSF with minimal invasion into live animals. This technique essentially began with the push–pull method, which examined the possibility of using a semi-permeable membrane to sample free amino acids and other electrolytes in neuronal extracellular fluid. The technique was further improved by the development of the dialysis bag as a means of collecting the dialysate [26]. Since multiple microdialyis probes can be implanted in the brain, the disposition of drug within different regions of the brain can be simultaneously characterized. The use of this technique to measure macromolecule concentrations in brain, however, has been very limited. This is mainly due to the lack of availability until recently of large molecular weight cutoff (MWCO) probes and the need for a complicated push–pull system to perform microdialysis with large pore probes [27]. Although the push–pull microdialysis procedure for antibodies is challenging and requires extensive training, recent studies have shown that it can provide direct in vivo measurement of free antibody concentration in selected regions of the brain in freely moving animals [28,29]. This technique can avoid the detection of bound antibodies to the brain capillary endothelial cells and the neurons, and

readouts of free antibody concentration in the brain interstitial ISF tend to better represent the required therapeutic concentration at the site-of-action in the brain. The theory and underlying general principles of in vivo microdialysis in general and brain microdialysis in particular are discussed in a review by Darvesh et al. [26].

On the other hand, to evaluate the in vivo PK and tissue distribution of antibodies, intravenous injection of radiolabeled antibody followed by collection of blood and tissue samples from the CNS at different time points ("cut and count") can be used as assays for sensitive uptake analysis [30]. Such an approach, however, is tedious and requires a large number of animals to ensure the reproducibility and reliability of the results. Today, QWBA, which relies on the use of X-ray film and phosphor imaging technology or radioluminography, is another standard method for conducting tissue distribution studies throughout the body of laboratory animals. These studies suggest that QWBA helps study the spatial and regional differences in areas as fine as 50–100 μm and is a good method for studying the targeted delivery of therapeutic proteins across the BBB [31,32]. The main advantage of QWBA is the minimal sample processing at true tissue-level (as opposed to organ-level) concentrations from an in situ preparation.

Furthermore, the continuing development of high-resolution PET and SPECT scanners and the availability of suitable radionuclides (e.g., Cu-64, Zr-89, In-111, I-131, I-124) are providing a non-invasive in vivo alternative that simplifies considerably the visualization and measurement of the whole body and organ PK, as well as brain uptake of antibodies. In this way, real-time dynamics can be obtained on whole body biodistribution of radiolabeled antibodies in the same animal or patient. The major advantages of these radionuclide-based molecular imaging techniques (SPECT and PET) are that they are very sensitive (down to the picomolar level), quantitative, and there is no tissue penetration limit. As a result, new applications of brain molecular imaging in animals are continually being established, which show a correlation between brain uptake of radiolabeled antibodies and brain target levels [33–36]. Another advantage is that molecular imaging methods have good spatial resolution (0.35–1.5 mm), allowing differentiation of tracer uptake on the suborgan level. Accordingly, the importance of spatial resolution in understanding therapeutic protein distribution within the brain has been the subject of several studies in which differences in brain penetration and distribution related to drug format are characterized [30,37,38].

Thus, significant effort has been made to radiolabel protein drugs with radionuclides, which in turn provides a method for tracking the location and quantifying the total radioactivity in tissues. However, the main limitation, which is shared by all these in vivo studies (e.g., "cut and count", QWBA, and molecular imaging) that rely on the usage of a radiolabeled compound, is that these technologies provide data on total radioactivity only and not specifically of the parent compound. In other words, the concentration of radioactivity does not always equate with the identity of the original compound that was radiolabeled, and it may also include radioactivity associated with metabolites and/or degradation products. The reader is referred to a comprehensive review by Tibbitts et al. (2016) of the different radiolabeling methods and the different in vivo technologies and approaches in order to gain a better mechanistic understanding of PK and protein distribution as a way to drive forward the selection of successful drug candidates [31].

In summary, in vitro BBB model selection parameters using human derived cells are critical for predicting drug transport because the disease in question may affect the barrier properties. Although many in vivo experiments have been traditionally performed, drug permeability tests are now carried mostly by in vitro BBB models due to ethical problems, differences between species, and expensive in vivo experiments. Nevertheless, a combinatorial approach of in vitro BBB models and in vivo tests will be the key to the development of CNS therapeutics with improved PK properties and better BBB penetrability [19,39].

#### **4. Approaches to Optimize BBB Internalization and Uptake of Antibodies**

Research has revealed that the BBB is not only a substantial barrier for drug delivery to the CNS but also a complex, dynamic interface that adapts to the needs of the

CNS and responds to physiological changes [40]. Optimization of drug delivery across the BBB could be achieved by several approaches: (a) pharmacologically, to increase the passage of drugs through the BBB by optimizing the specific biochemical properties of a compound [11]; (b) by BBB modulation, which includes transient osmotic opening of the BBB; and (c) physiologically, exploiting the various transport mechanisms present at the BBB. Many biomolecules (e.g., antibodies, recombinant fusion proteins, and nanoparticles), however, cannot get through the BBB unless the permeability of the BBB is altered using modulation of the tight junctions of the cerebral endothelial cells, which can result in some serious complications [11]. Research has shown that BBB internalization and trans-barrier transport of biomolecules can be manipulated on the basis of their physicochemical characteristics [41]. As a result, it is evident that various biomolecules with different parameters and characteristics are able to transverse biological barriers dictated by the barrier's set of limitations and specific criteria for internalization. Hence, it is expected that at some point the BBB physiology and physicochemical characteristics of antibodies will allow for the control of the rate and extent of cellular uptake, as well as the delivery of the antibody intracellularly, which is imperative for drugs that require a specific cellular level to exert their effects at the targeted site in vivo. Designing antibodies that can overcome this BBB protection system and achieve optimal concentration at the desired therapeutic target in the brain is a specific and major challenge for scientists working in CNS discovery [42]. In recent years, some progress has been made in terms of enabling the development of pharmacokinetic and pharmacodynamic (PK/PD) relationships for antibodies as therapeutic agents as well as in understanding how these relationships are influenced by target antigens and molecular properties.

In order to enhance antibody delivery to the brain, the following strategies for delivery optimization have been explored: (i) development of BBB-crossing bispecific antibodies, which have been engineered to incorporate one specificity against a BBB RMT receptor (Table 2) and the second specificity against a CNS therapeutic target to produce a pharmacological effect; and (ii) protein engineering efforts, which allow for the customized design of antibody constructs with physicochemical, molecular, and binding properties better optimized for successful transport across the BBB. Notably, antibody uptake is highly influenced by factors such as their size, surface charge, structure, hydrophobicity, affinity, antigen internalization, and dual targeting with bispecific antibodies [40,41,43–45]. The previous sections discussed the different transport mechanisms for the internalization of antibodies. Taken together, this section discusses the ideal antibody characteristics when employing transport mechanisms to achieve optimal cellular uptake (i.e., achieve desirable concentration range) at the BBB. Thus, this section focuses on examining the physicochemical and functional parameters of antibodies in regard to their relations and interactions with the physiology of the BBB and how those relations and interactions both facilitate their development as outstanding therapeutics.

**Table 2.** Receptor-mediated targets (RMT) for transport at the blood–brain barrier. Part of this table is reproduced from Gao (2016) with permission of the copyright owner [9].


#### *4.1. Modification of BBB Permeability*

The BBB is the first barrier that restricts the transportation of drugs from the blood to the brain. Because of this, researchers have developed various strategies to overcome or bypass the BBB, including penetration of the BBB by temporarily enlarging the BBB pore size, which could allow molecules such as antibodies to diffuse directly into brain [9]. In essence, modulating the efficacy of the tight junctions between cerebral endothelial cells so that the paracellular route of access to the brain is accessible is an applicable approach that has been utilized to permeabilize the BBB to drugs and enhance brain uptake. For instance, Neuwelt et al. (1981) discovered that mannitol, a hypertonic solution, can be administered simultaneously with drugs to enhance their delivery to brain tumors [51]. Currently, researchers are still using this strategy to deliver drugs to the CNS. Hypertonic solutions are thought to osmotically remove water from the endothelial cells, causing the cell to shrink, which may cause cellular changes that can affect the tight junctions [11]. This method is transitory, as the barrier closes within 10–20 min following BBB disruption. Unfortunately, this method is not selective for a specific drug and may increase uptake of other blood-borne molecules, such as neurotransmitters, which could be potentially harmful. Similarly, solvents such as high dose ethanol or dimethylsulfide, alkylating agents such as etoposide, alkylglycerols, and vasoactive agents such as bradykinin and histamine, have all been used to open the BBB and facilitate the delivery of drugs to the brain [52]. Since these compounds must be of a certain concentration to open the BBB, the BBB returns to its intact status when the blood concentration of these compounds falls lower than the threshold. Therefore, the dose and administration schedule must be optimized. The opening of the BBB is again presumably nonselective; thus, the use of these agents to affect BBB permeability can be highly traumatic, and could potentially cause serious side effects, such as seizures, permanent neurological disorders, and brain edema [9,11].

To circumvent these problems, focused ultrasound (FUS) and MRI are being employed as modulators of BBB function [53]. FUS has been used to enhance the delivery of various drugs to the brain, and it has been shown that the concentration of drugs in the brain hemisphere treated with FUS was approximately 3.5 times higher than the control hemisphere [53]. Combining FUS with other targeting methods could further elevate the accumulation of drugs in the brain. As an example, combining FUS with MRI targeting could improve the brain accumulation of drugs by 16-fold [54]. Although the toxicity of FUS on the brain is considered minor, and neurotoxicity was not observed, the clinical application of this method still should be viewed cautiously [55]. An advantage of these methods is that they can be focused with some precision to a particular region of the brain, thus modulating the BBB at a preferred site in order to release the drug locally. These modifications in BBB function and integrity appear to be transient and reversible, increasing the apparent safety of this method.

#### *4.2. Physiological Approach to Transport Antibodies Across the BBB*

Although the BBB is intact, mechanisms described in detail in Section 2 can be used to overcome this barrier. These strategies have been explored extensively over the past several decades when designing therapeutic antibodies for neurological disorders. Many of these strategies rely on receptors and carriers that are overexpressed on the BBB (Table 2), which can mediate the transport of specific ligands and their cargo.

Large molecules necessary for the brain's normal function are delivered to the brain by specific receptors that are highly expressed on the endothelial cells that form the BBB. This mechanism is described in the previous section as receptor-mediated transport (RMT). Additionally, the intercapillary distance in the brain is very small (on average 40 μm), and every neuron is virtually perfused by its own blood vessel, making these receptors abundant at the BBB [33]. Antibodies can be modified to be able to passage the BBB by conjugation to ligands that recognize receptors expressed at the BBB. This strategy in fact is the most effective way of delivering antibodies through the BBB and into the brain. This physiological approach targets IR, TfR, LRP-1 and 2, and other receptors. Overall, therapeutic compounds are able to cross the BBB after association with these specific ligands, forming "molecular Trojan horses" (Table 3). Proof of concept studies have demonstrated that TfR-specific antibodies bind to the receptor on the endothelial cells and allow the associated therapeutic agent to cross the BBB via receptor-mediated transcytosis, making TfR particularly promising in brain-targeted delivery [56]. Modifications are still being made in the use of TfR as a delivery system after studies showed that antibodies bound to the TfR were retained in the brain endothelium and did not penetrate into the CNS. To address this problem, a "brain shuttle" approach has been developed that fuses the C-terminus of a monoclonal antibody against Aβ, the peptide that accumulates in the brain of AD patients, to an anti-TfR Fab, which facilitates the BBB transcytosis of an attached immunoglobulin [57]. This differs from current approaches where the TfR antibody carries a therapeutic cargo or a bispecific antibody with optimized binding to TfR that targets the enzyme β-secretase (BACE1) associated with AD [58,59]. Compared with the monospecific anti-BACE1 antibody, the bispecific antibody had increased accumulation in the brain and led to an increased reduction in Aβ levels [60].

**Table 3.** Selected new peptides and antibodies with specific ability to cross the blood–brain barrier.


Multiple studies have extensively documented the use of the insulin receptor (IR) for the targeted delivery of drugs to the brain using specific antibodies directed against IR [46,66]. Animal studies have shown that total brain uptake of the anti-human IR is 4% of injected dose at 3 h post injection and confirmed that it is able to transport an associated molecule across the BBB. Furthermore, applications of the TfR and IR antibodies to a molecular Trojan horse for the delivery of therapeutics have been documented where different forms of conjugated and fusion proteins have been generated [33]. LRP-1 and 2 expressed on neuronal cells have also been exploited to deliver drugs to the brain in a similar fashion as TfR and IR [67]. For now, these receptor antibodies described above may not be the only answer to the biologics brain targeting question [68]. Regardless, the substantial research performed with these available antibodies has provided invaluable insight on the mechanisms of action of receptors at the BBB and has also helped to highlight protein engineering issues that must be addressed (as presented below) in order to develop a successful approach for transporting therapeutic antibodies across the BBB.
