The constructs were then transfected into Chinese hamster ovary (CHO) cells and purified from supernatants with Protein A-conjugated Sepharose columns (ProteNova). Furthermore, we successfully established a novel cynomolgus monkey ITP model and exhibited that the anti-Hp antibody exerted its effect in this model with only a single dose. Isoimperatorin This Fc-modified anti-Hp monoclonal antibody could be a useful therapeutic replacement for IVIG for the treatment of ITP. Keywords: FcRs, ITP, IVIG, platelets, cynomolgus monkey model We generated fragment crystallizable (Fc)-altered anti-haptoglobin (Hp) monoclonal antibodies with non-inferior efficacy compared to IVIG at considerably lower doses than IVIG and Isoimperatorin evaluated binding activity of anti-Hp antibodies to Fc gamma receptors with ELISA and inhibitory activity against the ADCC reaction. Furthermore, we successfully established a novel cynomolgus monkey ITP model and exhibited that the anti-Hp antibody exerted its effect in this model with only a single dose. This Fc-modified anti-Hp monoclonal antibody could be a useful therapeutic replacement for IVIG for the treatment of ITP. Rabbit Polyclonal to GNB5 Graphical Abstract Open in a separate windows Graphical Abstract Introduction Immune thrombocytopenic purpura (ITP) is an autoimmune disease that is primarily caused by macrophage-mediated phagocytosis of autoantibody-opsonized platelets in the reticuloendothelial system. Intravenous immunoglobulin (IVIG) exerts its immunomodulatory effects by blocking fragment crystallizable (Fc) gamma receptors (FcRs), including FcRIIa (CD32a) and FcRIIIa (CD16a), in ITP [1C4]. Although the mechanisms of action of IVIG have not been completely elucidated, the results from the clinical trial of GMA161, an anti-CD16 antibody that has shown efficacy on thrombocytopenia improvement in ITP patients [5], suggest that blockade of FcRs is a possible mechanism of IVIG action. Low-affinity FcRs, including CD16a and CD32a, Isoimperatorin bind immune complexes (ICs) with amazingly higher affinity than immunoglobulin G (IgG) monomers [1]. In this context, inhibition of FcRs by IVIG has been proposed to be further enhanced by the formation of ICs between the antibodies contained in IVIG and some components in the serum and by IgG dimers in the formulation [1, 6]. Indeed, Lemieux and others studies have shown that autoreactive IgGs isolated from IVIG form soluble ICs when mixed with serum [7, 8]. In addition, soluble ICs prepared had more potent inhibitory activity than IVIG in a mouse ITP model [9]. Furthermore, an Fc trimer with an artificially designed structure was shown to have enhanced potency over IVIG [10]. Although IVIG is a well-established therapeutic option for the treatment of numerous autoimmune and inflammatory diseases, the standard dose prescribed for autoimmune diseases, including ITP, is usually 2 g/kg, which is markedly high. To minimize the risk for thromboembolic events, slow IVIG administration has been recommended, as follows: 2 g/kg over 5 days, i.e. 0.4 g/kg/day in no less than 8 h each day [11]. The long time required to administer IVIG is usually burdensome to the medical staff and patients. Moreover, the use of IVIG has continued to increase in recent years, creating a space between the demand and supply of IVIG [12]. Thus, there is a high unmet need for developing substitution therapies for IVIG with non-inferior efficacy at a reduced dose. Recombinant Fc multimers such as CSL730, a recombinant Fc trimer, and PF-06755347, a recombinant Fc multimer, have demonstrated high potency in preclinical studies and are under phase I development [13]. One of the major challenges in evaluating therapeutic candidates with a FcR blockade mechanism is the development of animal models that can predict drug efficacy in humans. Although mouse ITP models have been used to investigate the causes of human ITP and mechanisms of action of IVIG, there are crucial differences in the FcRs of mice and humans [14, 15]. Previously reported mouse ITP models are induced by anti-platelet antibodies of either mouse IgG2a/IgG2b or IgG1 isotype, resulting in murine FcRIV or FcRIII dependent pathogenesis [14]. While murine FcRIV is usually reported to be the ortholog of human CD16a, there are no reports of human ortholog of murine FcRIII. Furthermore, the ortholog of human CD32a has not been recognized in mice [14]. In humans, not only CD16a but also CD32a have been suggested to play.