Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts.
Aims: Erythropoiesis-stimulating agents used to treat anaemia in patients with chronic kidney disease (CKD) have been associated with cardiovascular adverse events. Hepcidin production, controlled by bone morphogenic protein 6 (BMP6), regulates iron homeostasis via interactions with the iron transporter, ferroportin. High hepcidin levels are thought to contribute to increased iron sequestration and subsequent anaemia in CKD patients. To investigate alternative therapies to erythropoiesis-stimulating agents for CKD patients, monoclonal antibodies, LY3113593 and LY2928057, targeting BMP6 and ferroportin respectively, were tested in CKD patients.Methods: Preclinical in vitro/vivo data and clinical data in healthy subjects and CKD patients were used to illustrate the translation of pharmacological properties of LY3113593 and LY2928057, highlighting the novelty of targeting these nodes within the hepcidin-ferroportin pathway.Results: LY2928057 bound ferroportin and blocked interactions with hepcidin, allowing iron efflux, leading to increased serum iron and transferrin saturation levels and increased hepcidin in monkeys and humans. In CKD patients, LY2928057 led to slower haemoglobin decline and reduction in ferritin (compared to placebo). Serum iron increase was (mean [90% confidence interval]) 1.98 [1.46-2.68] and 1.36 [1.22-1.51] fold-relative to baseline following LY2928057 600 mg and LY311593 150 mg respectively in CKD patients. LY3113593 specifically blocked BMP6 binding to its receptor and produced increases in iron and transferrin saturation and decreases in hepcidin preclinically and clinically. In CKD patients, LY3113593 produced an increase in haemoglobin and reduction in ferritin (compared to placebo). Conclusion: LY3113593 and LY2928057 pharmacological effects (serum iron and ferritin) were translated from preclinical-to-clinical development. Such interventions may lead to new CKD anaemia treatments.
B cells contribute to multiple aspects of autoimmune disorders, and B cell–targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti–human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell–dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell–targeting therapies in treatment of autoimmune conditions without causing B cell depletion.
e14624 Background: Thrombopoietin (TPO) interacts with its receptor (mpl) to initiate signal transduction and increase platelet production. Other mpl agonists have been developed for clinical use: MDGF (a recombinant protein) and romiplostim (a peptibody) are protein-based mpl agonists; eltrombopag (a small molecule) is a nonpeptide mpl agonist. Methods: Mpl agonists were compared for their ability to induce cellular responses and activate relevant signaling pathways in mpl-expressing cells. Proliferation was assessed by ATP bioluminescence in Baf3/mpl cells treated with mpl agonists for 2 days. Differentiation was assessed by flow cytometric-detection of CD41 and CD61 expression on human CD34+ cells cultured with mpl agonists for 7 days. Activation of signaling pathways was determined by Western blot analysis of proteins from serum-starved Baf3/mpl cells treated with mpl agonists for 20 minutes. Experiments were repeated 3 or more times. Results: The TPO concentration that produced a half-maximal (EC50) proliferative response of Baf3/mpl cells was comparable with that of romiplostim (Table). In contrast, a 30-fold greater concentration of MGDF and a 2,000-fold greater concentration of small molecule were required to produce the same degree of proliferation. In addition, a 1,000,000-fold greater concentration of small molecule compared with romiplostim was required to produce an EC50 for differentiation of CD34+ cells. Mpl agonists activated identical signaling pathways in Baf3/mpl cells: a similar dose-response for phosphorylation of JAK2, Stat3, Stat5, Erk1/2, and AKT was observed at their respective EC10, EC50, and EC100 concentrations. Conclusions: In Baf3/mpl cells, the small molecule was between 1,000- and 1,000,000-fold less potent than protein-based mpl agonists when corrected for molecular mass differences. Small molecule and protein-based mpl agonists were indistinguishable when mpl intracellular signaling was compared at similarly effective concentrations. [Table: see text] [Table: see text]
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