Introduction

The targets of monoclonal antibodies can be classified into two groups: membrane-bound antigens and soluble antigens. Targets of food and drug adminsitration (FDA) approved mAbs for membrane boudn antigends include tumor surface markers (HER2, CD20, and CD19), tyrosine kinase receptors (EGFR and VEGFR) and a cytokine receptor (IL-6 receptor) and for soluble antigens include cytokines (TNFalpha, IL-6, IL12 and IL17), growth factor (VEGF) and other soluble diase mediators (IgE and C5). Previously, in terms of modulating the interaction between an antigen and an antibdoy, increases teh binding affinity to the antigen either by in vivo or in vitro affinity maturation has been the most general approach for improving the ptency of an antibody. In theory, even an antibody with infinite affinity would need to be at a concentraiton higher than that of the total antigens to neutralize the antigens in vivo. Antibody mediated antigen accumulation, which occurs when a conventional antibody targets a soluble antigen, increases teh total antigen concentraiton over the baseline by accumulating antibody-antigen complexes in plama. The accumulation occurs because the half-life of an IgG antibody is longer than the target antigen, so binding an antigen to an antibody increases the plasma antigen concentration by prolonging the half-life of the antigen. Some therapeutically important soluble antigens may require not only neutralization of a specific epitope of teh target molecule but also rather removal or elimination of the target molecule form circulation. In the natural system, the removal or elimination of some molecuels from cirulcation is acheived by several cell surface endocytic receptors, such as asialoglycoprotein receptor, high mannose receptor and low-density lipoprotein (LDL) recetpor, that deliver ligands to the lysome, but a conventional antibody accumulates soluble antigens in ciruclation. This problem can be overcome by engineering antibodies at both the variable region and constant region. Variable region engineering enables an antibody to bind to an antigen in plasma and dissociate form the antigen in endosome; constant region engineering increases cellular uptake of an antibody antigen complex into endosome, where the antigen is dissocaited.  A soluble antigen bound to a sweeping antibody can be actively taken up into the cell via Fc recetpor-mediated endocytosis and, because the antibody has a pH or calcium-dependent antigen bidning roperty, is dissocaited in an acidic endosome. (Igawa “Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens form circulation” Immunological Reviews 2016, 770, 132-151). 

For pH dependent antibodies see outline

Calcium-dependent binding to an antigen

While the techniques of taking advantage of the pH difference between plasma and endosomes are a known way to change antibody-antigen affinity, the calcium ion concentraiton between plasma and endosomes is also known to be different. Calcium ion concentraiton is reproted to be 1.2-2 mM in plasma, which drops to 3-30 uM in endosomes after endocytosis. Thus, an antibody with calcium ion-dependent bding, which binds to the antigen at the calcium ion concentration in plasma and dissocaites the antigen at the calcium ion concentration in endosomes, can be sued to generate calcium dependent antibodies. Accordingly, a human naive library was panned for IL-6 receptors in the presence of 2 mM calcium ion and the pahge was washed with a buffer containing calcium ion. Subsequently, populations that were dissocaited in the absence of calcium ion or in teh presence of EDTA were recvoered for teh next round. After multiple rounds of selection, a calcium dependent anti-IL-6 receptor antibody was isolated. (Igawa “Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens form circulation” Immunological Reviews 2016, 770, 132-151).

Engineering constant region to increase cellular uptake of an antibody-antigen complex

The purpsoe of constant region engineering is to incdrease the cellular uptake of an antibody-antigen complex into teh endosome. One approach is increasing the binding affinity to FcRn, and the other is increasing teh bidning affinity to FcyRIIb. FcRn is critical to the long half-life of IgG becasue it recylcles internalized IgG form endosomes to circulation. (Igawa “Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens form circulation” Immunological Reviews 2016, 770, 132-151).