pharmaceutics Review Antibodies for the Treatment of Brain Metastases, a Dream or a Reality? Marco Cavaco, Diana Gaspar, Miguel ARB Castanho * and Vera Neves * Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal * Correspondence: [email protected] (M.A.R.B.C.); [email protected] (V.N.) Received: 19 November 2019; Accepted: 28 December 2019; Published: 13 January 2020 Abstract: The incidence of brain metastases (BM) in cancer patients is increasing. After diagnosis, overall survival (OS) is poor, elicited by the lack of an effective treatment. Monoclonal antibody (mAb)-based therapy has achieved remarkable success in treating both hematologic and non-central-nervous system (CNS) tumors due to their inherent targeting specificity. However, the use of mAbs in the treatment of CNS tumors is restricted by the blood–brain barrier (BBB) that hinders the delivery of either small-molecules drugs (sMDs) or therapeutic proteins (TPs). To overcome this limitation, active research is focused on the development of strategies to deliver TPs and increase their concentration in the brain. Yet, their molecular weight and hydrophilic nature turn this task into a challenge. The use of BBB peptide shuttles is an elegant strategy. They explore either receptor-mediated transcytosis (RMT) or adsorptive-mediated transcytosis (AMT) to cross the BBB. The latter is preferable since it avoids enzymatic degradation, receptor saturation, and competition with natural receptor substrates, which reduces adverse events. Therefore, the combination of mAbs properties (e.g., selectivity and long half-life) with BBB peptide shuttles (e.g., BBB translocation and delivery into the brain) turns the therapeutic conjugate in a valid approach to safely overcome the BBB and efficiently eliminate metastatic brain cells. Keywords: adsorptive-mediated transcytosis; antibody fragments; blood–brain barrier; brain metastases; monoclonal antibodies; peptide shuttles 1. Brain Metastases Brain metastases (BM) account for significant morbidity and mortality. The exact incidence is unknown [1,2]. Based on various studies, investigators estimate that BM occurs in 10%–20% of adult patients with cancer [3]. Nevertheless, the incidence might be higher, and it is increasing due to prolonged life expectancy, increased resistance to cancer therapies, and improved imaging techniques. In addition, the increased patient survival by treating primary tumors may increase the number of patients that will develop more aggressive BM, or that are resistant to therapy. Among the different cancer types, lung cancer (19.9%), breast cancer (15.2%), and melanoma (6.9%) are the most common primary tumors developing BM [4]. After diagnosis, overall survival (OS) is poor. However, early diagnosis, improved systemic therapies, and multimodality treatments have significantly increased patients’ survival [5]. 1.1. BM Pathophysiology The pathophysiology of BM is complex and involves a multi-step process constituted of two major stages (Figure1)[ 6]. The first stage is tumor migration, which includes (i) metastatic clone progression, due to tumor cells’ ability to degrade extracellular matrix (ECM); (ii) intravasation (transendothelial migration of cancer cells into vessels); (iii) dissemination (spread of tumor cells via bloodstream); (iv) Pharmaceutics 2020, 12, 62; doi:10.3390/pharmaceutics12010062 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 62 2 of 22 Pharmaceutics 2020, 12, x FOR PEER REVIEW 2 of 25 extravasationbloodstream); (transendothelial (iv) extravasation migration (transendothelial of cancer migration cells into tissues). of cancer The cells second into stagetissues). corresponds The second to tumorstage corresponds colonization. to tumor colonization. Figure 1. Steps in the formation of brain metastases (BM). Metastases formation begins in the Figure 1. Steps in the formation of brain metastases (BM). Metastases formation begins in the microenvironment of thethe primaryprimary tumortumor withwith 1.1. metastatic clones developing, degrading the extracellular matrix (ECM), and susufferingffering anan epithelial–mesenchymalepithelial–mesenchymal transition (EMT) to further detach from thethe connectiveconnective tissue.tissue. 2. Subsequently, Subsequently, tumor tumor cells cells invade and enter the circulationcirculation (intravation).(intravation). 3. 3. The The dissemination dissemination within within the the vascular vascular system system drives drives tumor tumor cells cells to distant to distant sites, sites, like likethe thebrain. brain. 4. Then, 4. Then, they they extravasate extravasate across across the the blood–brain blood–brain barrier barrier (BBB) (BBB) and and enter enter the the brain parenchyma due toto thethe releaserelease ofof proteolyticproteolytic enzymesenzymes andand cellularcellular interactions.interactions. 5.5. Once inside the brain, cancer cells colonize the tissuetissue andand developdevelop secondarysecondary tumors.tumors. The cellscells presentedpresented inin thethe primaryprimary tumortumor areare heterogeneous.heterogeneous. Among others, the tumor microenvironment is composed of cancer stem cells (CSCs), partially differen differentiatedtiated progenitor cells, and fullyfully di ffdifferentiatederentiated end-stage end-stage cells [cells6]. Recent [6]. Recent findings findings attribute toattribute CSCs the to primary CSCs responsibilitythe primary forresponsibility enhanced malignancy for enhanced since malignancy they can complete since th theey two can stages complete of metastases the two formation stages of (Figure metastases1)[ 7]. However,formation during(Figure cancer 1) [7]. progression, However, otherduring cells cancer undergo progression, an epithelial–mesenchymal other cells undergo transition an epithelial– (EMT), changingmesenchymal their plasticitytransition by (EMT), morphological changing and their phenotypical plasticity conversions by morphological [8,9]. EMT and enables phenotypical non-CSCs toconversions resemble a[8,9]. CSC EMT state. enables Thus, theynon-CSCs acquire to the resemble ability a to CSC invade state. and Thus, colonize they distantacquire sites, the ability creating to secondaryinvade and niches colonize that distant may progress sites, creating to a secondary secondary tumor niches [10 ].that Therefore, may progress in the end,to a secondary within the tumor microenvironment,[10]. Therefore, in the all end, cells within are malignant. the tumor Nevertheless, microenvironment, the development all cells are malignant. of distal metastases Nevertheless, only occursthe development in <0.1% of of disseminated distal metastases cancer cells.only Thus,occurs although in <0.1% the of formation disseminated of metastases cancer representscells. Thus, a majoralthough threat, the itformation is considered of metastas highly inees ffirepresentscient [8,11 a]. major threat, it is considered highly inefficient [8,11]. 1.2. BBB Physiology 1.2. BBB Physiology BBB is a complex system composed of a structurally distinct and continuous endothelial cell layer separatingBBB is two a complex brain compartments, system composed namely, of thea structural blood andly extracellular distinct and fluid. continuous Its components endothelial include cell anlayer endothelial separating cell two layer, brain adjoined compartments, by tight cell-to-cell namely, th junctione blood proteins,and extracellular and pinocytic fluid. vesicles Its components [12]. All together,include an they endothelial contribute cell to layer, the selective adjoined permeability by tight cell-to-cell of the barrier, junction allowing proteins, brain and homeostasis.pinocytic vesicles The BBB[12]. isAll also together, dynamic. they It responds contribute to regulatoryto the select signalsive permeability from both the of blood the andbarrier, the brainallowing [13], beingbrain thehomeostasis. main portal The into BBB the is brainalso dynamic. of gaseous It responds molecules, to such regulatory as O2 andsignals CO 2from, ions, both nutrients, the blood hormones, and the andbrain water [13], (Figurebeing 2the). Hydrophobicmain portal into compounds the brain ( < of500 gaseous Da) diff usemolecules, across thesuch endothelium as O2 and membrane.CO2, ions, nutrients, hormones, and water (Figure 2). Hydrophobic compounds (<500 Da) diffuse across the endothelium membrane. Carrier-mediated transport (CMT) is responsible for the transport of glucose Pharmaceutics 2020, 12, 62 3 of 22 Pharmaceutics 2020, 12, x FOR PEER REVIEW 3 of 25 Carrier-mediated transport (CMT) is responsible for the transport of glucose and amino acid residues. and amino acid residues. While water-soluble molecules (e.g., ions) cross the BBB through ion Whilechannels. water-soluble On the other molecules hand, macromolecules (e.g., ions) cross (pro theteins BBB and through peptides) ion channels. transport Onrely the on other endocytic hand, macromoleculesvesicles, which involve (proteins either and receptor-mediated peptides) transport transport rely on (RMT) endocytic or adsorptive-mediated vesicles, which involve transport either receptor-mediated(AMT) [14,15]. transport (RMT) or adsorptive-mediated transport (AMT) [14,15]. FigureFigure 2. 2.Pathways Pathways acrossacross thethe blood–brainblood–brain barrier (BBB) (BBB).. Representation of of the the BBB BBB formed formed by by the the endothelialendothelial cellscells and their interaction interaction with with astrocyt astrocytes.es. Different