Engineering a CD19-Based Bispecific Molecule for CAR T Cell Therapy

Abstract

Cancer is a profoundly devastating disease both globally and within the United States. Current standards of care for treating cancer often includes surgical resection, chemotherapy, and radiation, each of which associates with its own set of adversities. An emerging class of treatment, immunotherapy, aims to utilize a patient’s own functional immune system as the therapeutic agent. Adoptive T cell therapy, but more specifically, chimeric antigen receptor (CAR) expressing T cell transfer, has had notable clinical success particularly against hematological malignancies. Chimeric antigen receptors are synthetic immunoreceptors which can redirect T cells towards varying tumor associated antigens, and, as a living cell, have the aptitude to develop sustainable memory and anti-tumor efficacy. However, conventional CAR T cells lack clinical modularity afforded by other treatments because, once transfused into a patient, the modified immune cell cannot be further altered. This nuance has resulted in several adverse side-effects which can be lethal to a subset of patients. Several resolutions have been posed to solve these reported complications, one of which is genetic encoding CAR specificity towards a secondary, bispecific molecule. This split-CAR approach has the propensity to improve antigen specificity, resolve antigen loss, afford dose-able T cell activation, and more. However, while many bispecific molecules have been developed, many lack both tunability and developability, both of which are important for the complexities and ever-changing nature of cancer. To meet this demand for engineered ligands, several high-throughput ligand selection methods have been developed for discovering ligands with a desired specificity. Furthermore, the associated CAR T cells may have poor aptitude for activation and expansion due to insufficient antigen availability. Conversely, conventional anti-CD19 CAR T cells can harness both healthy or malignant CD19-positive B cells for activation and expansion and thus have an abundance of available antigen. To these points, we utilized yeast-surface display and directed evolution as a pipeline for developing an CD19-based bispecific molecule capable of harnessing the proliferative aptitude of anti-CD19 CAR T cells to target antigens conventionally associated with solid tumors. Human CD19 was evolved for improved structural integrity through conformational selections using anti-CD19 monoclonal antibodies. Improved mutants were sequenced and provided input for designing a stably expressing, generation 2 CD19 library (termed Frame2). The second-generation diversity applied experimentally determined, beneficial mutations in multi-site fashion to drive the enhanced CD19 framework towards a higher stability and/or functionality. The Frame2 CD19 library was constructed as several fusion constructs containing either an anti-EGFR fibronectin domain or an anti-HER2 scFv in both N-terminal and C-terminal orientations and selectively evolved with anti-CD19 antibodies and the ligand-respective antigen. A set of functional bispecific CD19-ligand fusions were successfully developed. In theory, because the anti-CD19 antibodies used for fusion development have an identical binding domain to several anti-CD19 CAR constructs, these fusions should be detectable by CD19-targetted CAR T cells. Moreover, if the ligand domain also retains specificity, the CD19-ligand bispecific molecule should be capable of redirecting anti-CD19 CAR T cells to EGFR or HER2 expressing tumors.