Structure-function studies of LAG3 interactions with antibodies and cellular ligands

PROJECT ABSTRACT In this proposal, we will determine how antibodies and cellular ligands regulate the immunosuppressive function of Lymphocyte-Activation Gene 3 (LAG3). LAG3 is an immune checkpoint protein that suppresses T cell activation upon engagement of the ligands Major Histocompatibility Complex Type II (MHCII) or Fibrinogen-like 1 (FGL1). Inhibitors of LAG3 have been widely investigated as potential immunotherapies given their ability to reverse LAG3-mediated immunosuppression in the tumor microenvironment. These studies eventually led to FDA approval of an antibody targeting LAG3, in combination with a PD1 inhibitor, for the treatment of metastatic melanoma. This breakthrough marked LAG3 as the “third checkpoint” to be successfully targeted in the clinic following PD1 and CTLA4, and a myriad of other LAG3-based therapies are now actively under clinical evaluation as cancer immunotherapies. The rational design of LAG3-targeting therapeutics has been challenging due to our incomplete understanding of LAG3 molecular biology. Recent, unexpected findings have shown that antibodies targeting all four domains of the LAG3 protein can function as effective in vitro inhibitors, and that a subset of these antibodies do not block LAG3-ligand interactions. Thus, it is unclear in which contexts ligand blockade is required for optimal LAG3 immunotherapy responses. Another major gap in our knowledge is that we have not been able to visualize how LAG3 engages MHCII and FGL1. This lack of structural information has prevented us from determining the molecular mechanisms used by ligands to control LAG3 function. Lastly, we do not know whether mechanisms of LAG3 inhibition, such as blockade of LAG3 dimerization or disruption of LAG3-ligand interactions, are associated with different immunotherapy responses in vivo. To address the issues above, we are taking a multidisciplinary approach that leverages our expertise in structural immunology, biologics development, and melanoma immunotherapy. In Aim 1, we will answer the question, “How do different ligands influence LAG3?” through a series of rigorous structure-function studies. In Aim 2, we will determine how the unique binding modes of LAG3 agonist and antagonist antibodies dictate their function. In Aim 3, we will use melanoma mouse models to assess the therapeutic potential of different classes of anti- LAG3 antibodies. Collectively, this research will provide fundamental insight into LAG3 immunobiology and will open new avenues for the development of optimal LAG3 immunotherapies for melanoma.