ABSTRACT There is an unmet need for more selective and sustainable therapeutics to treat a growing number of immune- related diseases. For example, autoimmune disorders affect more than 50 million patients in the United States alone and there was a 3.4% annual increase in type 1 diabetes among European children between 1989 and 2013. While conventional treatments may alleviate symptoms, they are often less specific and require long-term medication. There is a growing interest in developing “living drugs” with regulatory T (Treg) cells for treating various immunity-related diseases, given better understanding of immunological homeostasis and recent promising clinical outcomes from applying adoptive cell therapies. Many early-phase clinical trials with Treg cell products have demonstrated the feasibility and safety of this approach. Standardizing Treg cell manufacturing has been a substantial challenge, involving choice of cell source, methods for purifying, engineering, and expanding Treg cells, product specification, and release criteria. Over the past several years, we have been developing targeted microbubble-based methods for streamlining the manufacturing processes for therapeutic cell production, including T cell selection, activation, and engineering. One of the major hurdles for Treg cell manufacturing is isolating high purity cells that can be greatly expanded at scale for clinical therapy. To isolate Treg cells in high purity, multiparametric sorting using a set of surface markers (e.g. CD4, CD25, CD127) is needed. The two current major techniques, fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS), alone cannot meet the demand for isolating large-scale GMP grade Treg cells of high purity. We have recently invented an iterative, targeted microbubble-based platform for multiple parametric cell sorting at scale from apheresis blood samples. In addition, we have also demonstrated that anti-CD3/CD28 conjugated microbubbles are very efficient for bead-free T cell activation and long-term expansion. With this foundation, we will build an innovative platform for Treg cell processing, and will demonstrate the feasibility of producing adequate functional Treg cells of high purity from apheresis blood samples in phase I. Subsequently, we will generate antigen-specific CAR (chimeric antigen receptor) Treg cells that meet clinical specifications in phase II. Once successful, this could accelerate the adoption of this promising therapy to accomplish durable responses of suppressing rejection following solid organ or hematopoietic stem cell transplantation, as well as combating other immune-related disorders.

Project Narrative There is an unmet need for more selective and sustainable therapeutics to treat a growing number of immune- related diseases, and a growing interest in developing “living drugs” with regulatory immune cells for treating those diseases. However, standardized manufacturing for cell and gene therapy presents a substantial challenge. Here, we will apply our innovative technologies to overcome this hurdle, which could potentially lead to accelerating the adoption of this promising therapy.