Therapeutic antibodies - natural and pathological barriers and strategies to overcome them

Abstract

Antibody-based therapeutics have become a major class of therapeutics with over 120 recombinant antibodies approved or under review in the EU or US. This therapeutic class has experienced a remarkable expansion with an expected acceleration in 2021-2022 due to the extraordinary global response to SARS-CoV2 pandemic and the public disclosure of over a hundred anti-SARS-CoV2 antibodies. Mainly delivered intravenously, alternative delivery routes have emerged to improve antibody therapeutic index and patient comfort. A major hurdle for antibody delivery and efficacy as well as the development of alternative administration routes, is to understand the different natural and pathological barriers that antibodies face as soon as they enter the body up to the moment they bind to their target antigen. In this review, we discuss the well-known and more under-investigated extracellular and cellular barriers faced by antibodies. We also discuss some of the strategies developed in the recent years to overcome these barriers and increase antibody delivery to its site of action. A better understanding of the biological barriers that antibodies have to face will allow the optimization of antibody delivery near its target. This opens the way to the development of improved therapy with less systemic side effects and increased patients' adherence to the treatment.

Keywords: Antibodies; Biological barriers; Pathological barriers; Therapeutic strategies.

Fig. 1

Schematic representation of the extracellular matrix in healthy condition and in the tumour microenvironment as a barrier to antibodies. A healthy ECM (left panel), located under the epithelium, consists in two distinct structures. The basement membrane (1) is composed of a very compact network of collagen fibrils and laminins that can self-associate to form a secondary network; perlecan further bridge these networks. The interstitial matrix is a looser matrix mainly composed of collagen fibrils assembled through the participation of fibronectin. Other components, including elastin, proteoglycans, and hyaluronan, contribute to the ECM organisation. Stromal cells present in the interstitial matrix interact with ECM components and growth factors. In cancers (right panel), tumour cells and cancer-associated fibroblast secrete a plethora of ECM proteins (such as collagens, fibronectin, elastin, or laminins), proteoglycans, cytokines, and growth factors. This ECM differs significantly in conformation and composition from that of normal tissues. The excessive accumulation of dense and rigid ECM results in the encapsulation of tumour cell clusters, acting as a physical barrier for antibody diffusion (3) (4). The acidic microenvironment associated with increased concentration of proteases within the ECM (5) represents a biochemical barrier degrading antibodies and reducing ADC efficacy. The increase interstitial pressure contributes to repelling antibodies (6). Finally, ECM-mediated hypoxia and low immune cell infiltrate (7) reduces antibody effectiveness and especially immunotherapy treatments. Strategies to overcome the ECM barrier (lower panel) and increase antibodies distribution and diffusion in the vicinity of the mAb target have been developed. Alternative administration routes have been considered (e.g. subcutaneous, intravitreal, or intra-articular) (8). Antibody fragments exhibit higher diffusion rate through the stroma as compared to full length antibodies (9). ADCs have been specifically designed to manipulate ECM acidic pH and high protease concentration for the efficient release of their payload (10). Combination of mAb with pH pump inhibitors or proteases inhibitors can also be considered to lower ECM-associated biochemical barrier (11). ECM remodelling strategies have been evaluated to increase antibodies diffusion and therapeutic efficacy (12). Finally, antiangiogenic mAbs can be used to restore interstitial fluid pressure and normalize anarchic blood vessels growth in order to enhance antibody delivery (13).

Fig. 2

Schematic representation of epithelial barriers to antibodies in healthy and disease conditions. At steady state (left panel), mAbs present in the lumen may encounter biological barriers at the mucosal surface limiting their bioavailability. Resident macrophages (1) patrolling the mucosa could phagocytose mAbs leading to their degradation or denaturation. The mucus (2) covering the epithelium can partly repel Abs. In homeostatic conditions, balance between proteases and anti-proteases is neutral (3), except in the gastro-intestinal tract where the acidic pH will activate specific proteases and alter mAb conformation. Once the epithelium is reached, tight junctions between epithelial cells (4) prevent paracellular transport of mAb. mAbs can cross the epithelial surface by transcytosis, mainly through their binding with FcRn (5) resulting in improvement or decrease in the mAb bioavailability depending on the location of its target. In inflammatory conditions (right panel), mucus structure and composition are altered (6) with thicker layer and tighter pores decreasing the diffusion of mAbs. The activation of resident immune cells and the recruitment of leukocytes generate a dysregulation of proteases / protease-inhibitors balance (7) leading to a proteolytic environment favoring mAb proteolysis. Local inflammation leads to cellular damage and epithelial apoptosis promoting para- and trans-cellular leakage of mAbs (8). Several strategies have been investigated trying to solve mucosal barrier problems. In order to circumvent epithelium impermeability, cell-penetrating or permeation enhancer strategies have been considered as well as reducing mAb size, using small fragments, in order to improve mAb trans-epithelial passage (9). The use of protease inhibitors, proton-pump inhibitors or rescuing mAb may protect from degradation and denaturation (10). When considering local target, mAb-pegylation will lower transcytosis and increase mAb retention within mucosa (11). In order to improve mAb diffusion through the mucus, the addition of mucolytics (12), the encapsulation of mAb in nanovectors or the use of small fragments have been investigated (13).

Fig. 3

Schematic representation of the blood-brain barrier (BBB) to antibodies in healthy and disease conditions, and the strategies developed to overcome this barrier. A healthy BBB (left panel) is composed of endothelial cells that are tightly sealed by tight junctions (1) and prevent paracellular transport, hindering antibodies from reaching the CNS. Pericytes (2), which share the basal lamina with endothelial cells, help maintain the BBB and regulate blood flow through the capillaries. Astrocytes interact with BECs through their end-feet (3) and contribute to the maintenance of the BBB integrity. Resting microglia (4) are motile and highly ramified surveillant cells that are constantly scanning the brain environment to maintain its homeostasis. In pathological conditions (middle panel), the BBB becomes hyper-permeable to blood-born cells and molecules, including antibodies. Tight junctions between endothelial cells are disrupted (5), leading to a loss of endothelial cell polarity and uncontrolled paracellular transport. Pericytes are lost and detach from endothelial cells (6). Activated microglia (7) and reactive astrocytes (8) undergo molecular and morphological changes and adopt a pro-inflammatory phenotype. Studies suggest that BBB permeability increases with age, therefore increasing the passage of antibodies from the periphery to the CNS. Strategies to overcome the BBB (right panel) and increase antibodies distribution into the CNS were developed, including brain-focused ultrasound (FUS) combined with administration of microbubbles that open reversibly the BBB (9); bispecific antibodies (10) that target endogenous receptors expressed in the BBB and facilitate transcytosis; invasive, direct injection to the brain (11) via intra-cerebroventricular or intra-cerebral administrations; and nose-to-brain delivery (12), where antibodies are delivered to the nose and cross the olfactory epithelium, reaching the brain.

Fig. 4

Therapeutic strategies to overcome barriers to antibodies. Depending on its location, different therapeutic strategies have been developed or are under consideration to help mAb reaching their target through the modulation of biological barriers. The choice of the mode of administration or specific tools and devices may allow a direct access to the target location. mAb engineering and/or formulation will help lowering the detrimental impact of barrier components. Finally, the administration of mAb in combination with a co-treatment will either destroy the barrier or improve mAb stability, favouring its access to its target.