ABSTRACT We have discovered a new mechanism for inflammation on the ocular surface. First, we discovered the presence of neutrophil extracellular traps (NETs) on the ocular surface of dry eye disease (DED) patients and subsequently, the presence of citrullinated proteins and anti- citrullinated protein antibodies (ACPAs). ACPAs not only cause ocular surface disease, but also stimulate formation of NETs, thus creating a self-perpetuating cycle of chronic inflammation on the ocular surface. We present an innovative pathophysiological concept: DED is characterized by breach of self-tolerance towards citrullinated antigens with generation of autoantibodies (ACPAs) and NETs could represent a source of citrullinated antigens fueling the ACPA autoimmune response over the ocular surface. We performed a first-in-human pilot clinical trial and showed that ocular surface immune globulin (OSIG) eye drops, formulated from pooled human immune globulin products (IVIG), were safe and efficacious in treating DED patients. Our findings shift the current paradigm that focuses on T-cell mediated inflammation as central to the pathophysiology of DED to also include autoimmune inflammation that is driven by post- translational modifications in self-proteins (citrullination) and autoantibodies (ACPAs). The purpose of this R24 application is to produce preclinical data that supports a commercial Investigational New Drug (IND) application to the U.S. Food and Drug Administration (FDA). The longer-term goal is for testing the efficacy of OSIG eye drops in clinical trials and positioning OSIG eye drops as a new topical biologic immunotherapy for DED patients. Therefore, in Aim A, we seek to develop an optimum OSIG eye drop formulation using Quality by Design (QbD) principles, test critical quality attributes (CQAs) and GMP manufacture clinical supplies. In order to use OSIG ophthalmic formulation in human, an FDA IND is a prerequisite, therefore, in Aim B, we propose to perform pre-clinical in vitro and in vivo toxicology and efficacy studies using the OSIG ophthalmic formulation to meet regulatory requirements for IND studies. In Aim C, we propose to perform a clinical study to identify DED subtypes, clinical presentations and patient characteristics that are most associated with ACPAs or NETs, hence most likely to show therapeutic benefit with OSIG therapy. To successfully achieve these three Aims, we have established a highly qualified, multi-disciplinary team that is experienced in ophthalmic drug development, and FDA processes and regulations. If the aims of this grant proposal are successfully achieved, we will be one-step closer to introducing the first immunotherapy for ocular surface diseases into clinical practice.