Generation and Characterization of Fully Human Monoclonal Antibodies Against Human Orai1 for Autoimmune Disease

Abstract

Calcium entry into T cells following antigen stimulation is crucial for nuclear factor of activated T cells (NFAT)-mediated T cell activation. The movement of calcium is mediated by calcium release-activated calcium (CRAC) channels. There are two key components of this channel: Orai1 is the pore-forming subunit located in the plasma membrane, and stromal interaction molecule 1 (STIM1) functions as a Ca 21 sensor in the endoplasmic reticulum. A subset of human patients carry mutations in either STIM1 or Orai1 that affect protein function or expression, resulting in defective store-operated Ca 21 influx and CRAC channel function, and impaired T cell activation. These patients suffer from a hereditary form of severe combined immune deficiency syndrome, highlighting the importance of the CRAC channel for T lymphocyte function in humans. Since autoreactive T cells play an important role in the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and organ transplantation, Orai1 becomes an attractive therapeutic target for ameliorating autoimmune disease. We developed a novel approach to inhibiting CRAC function by generating high-affinity fully human monoclonal antibodies to human Orai1. These antibodies inhibited I CRAC current, store-operated Ca 21 influx, NFAT transcription, and cytokine release. These fully human antibodies to human Orai1 may represent a novel therapeutic approach for the treatment of autoimmunity.

Expression of ORAI1, a Plasma Membrane Resident Subunit of the CRAC Channel, in Rodent and Non-rodent Species

“…After single-cell sorting and specificity characterization, the hybridoma line 266 was identified as a promising candidate as it did not bind to either ORAI2 or ORAI3 using FACS with cell lines expressing those family members. Using a chimera method more limited than the one previously described for mapping the binding epitope of the function-blocking antibody 2C1.1 (Lin et al 2013), we determined that mAb266 binds to a region of the second extracellular loop of ORAI1 (Fig 1B). Because of the binding of mAb 266.1 to rodent and human ORAI1, we used chimeras between ORAI2 and ORAI1 to perform the mapping.…”

“…Although the limited clinical presentation of Orai1-deficient individuals is in sharp contrast to its broad tissue expression, the clinical phenotype of loss-of-function of ORAI1, as well as the biological importance of SOC function in cell types outside the immune system, garners legitimate concern about target-related toxicity of any agent that inhibits CRAC function. Given our own interest in the pharmacological modulation of the CRAC channel (Lin et al 2013), we wanted to explore what nonclinical species would be relevant to investigate toxicity associated with CRAC channel inhibition. In this paper, we describe immunohistochemistry results using a mouse monoclonal antibody, mAb 266.1, which binds to an extracellular loop of ORAI1 and detects the rodent, cynomolgus monkey, and human proteins.…”

Inhibition of CRAC with a human anti-ORAI1 monoclonal antibody inhibits T-cell-derived cytokine production but fails to inhibit a T-cell-dependent antibody response in the cynomolgus monkey

“…The extrapolated serum concentration of the IC 50 for that assay is shown as a dotted line on Figure 4. ORAI1 is a key component of the CRAC channel, as shown in Figure 1, and the electrophysiology assay measures conductance of the CRAC channel (I CRAC ) (Lin et al, 2013). In addition, the 2C1.1 concentrations achieved during this study drove full occupancy of ORAI1 on T-cells throughout the study (see below).…”

“…''A5SU''. 2C1.1 was prepared at Amgen; its generation and characterization of its binding to human and cynomolgus monkey ORAI1 and functional properties have been previously reported (Lin et al, 2013).…”

“…While CsA shows good activity in vivo and can fully block T-cell responses in vitro, it was less clear how the 2C1.1 antibody, a partial inhibitor of CRAC channel function, would perform. 2C1.1, a fully human anti-ORAI1 monoclonal antibody (mAb), was previously characterized (Lin et al, 2013). 2C1.1 is a potent and partial antagonist of CRAC function in Ca 2+ flux and electrophysiology assays.…”