SUMMARY The complement system comprises over 30 proteins, mostly plasmatic, forming the oldest branch of the human immune system. When activated by a pathogen, the complement system amplifies a loop that leads to the destruction of the invading organism. Plasma and surface inhibitors tightly regulate complement activation to prevent or limit self-damage. Unfortunately, gain-of-function (GOF) mutations in the complement activator factor B (FB) found in some rare patients cause permanent complement activation that can lead to autoinflammation. GOF-FB is more efficient at activating complement and is resistant to natural complement inhibitors. Given the enhanced activity of these mutants, we hypothesize that we can redirect GOF-FB to target specific malignant cells or tissues for therapeutic purposes and with minimal side effects. In this application, we propose generating and testing in vitro molecules to direct GOF complement activating proteins specifically to cancer cells and solid tumors. In Aim 1, we will fuse GOF-FB to single-chain variable antibody fragments (scFV) to direct complement activation to specific cells or antigenic targets. This engineered biologic approach to target immunotherapy will lead to less expensive and off-the-shelf alternatives to chimeric antigen receptor T cells (CAR-T) and be immediately relevant for hematologic malignancies. These molecules could also overcome some of the current issues with antibody therapies, including complement consumption and inhibition by the tumor cells. In Aim 2, we will couple GOF-FB to proteins that bind components of the solid tumor or its microenvironment. This approach will promote inflammation in regions in and around solid tumors and enhance local immune responses that can ultimately eliminate tumors or resolve tissue damage. Due to the nature of GOF-FB, these molecules will be resistant to tumor microenvironmental immune suppression mechanisms. In aim 3, we will generate murine GOF-FB that will pave the way to translate our findings to animal models allowing for in vivo proof of concept experiments, preclinical studies, and assessments of safety and efficacy. Our proposed work is highly innovative and yet supported by solid literature evidence. We propose to prove in vitro that GOF-FB can have potential as an anti-cancer therapy. These newly engineered molecules based on human complement immune disorders could pinpoint a new class of immune modulators with greater specificity, reach, and activity than current treatments. It represents a different direction from previous research performed in our lab, but the expertise required to carry out the proposed work and the collaborators are already in place. Furthermore, the proven efficacy of some FDA-approved complement inhibitors used to treat diseases caused by GOF mutations in complement activation assures that our approach will have minimal side effects that can be readily addressed. The work proposed here to leverage GOF mutations to...