Cellular engineering to improve the efficacy and specificity of targeted immunotherapy

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Project Summary/Abstract Adoptive cell therapy (ACT) is a promising therapeutic approach for the treatment of cancer. However, the initial success of ACT has been limited to chimeric antigen receptor (CAR)-T cell therapies for hematological malignancies. Applying this cell therapy to solid tumors is challenged by the lack of targetable tumor antigens, the severe systemic toxicity and the suppressive tumor microenvironment. T cell receptor (TCR) gene therapy can overcome some of these challenges because it enables targeting of intracellular proteins presented as peptide antigens on the human leukocyte antigen (HLA) complex. However, the majority of naturally occurring TCRs are of low-affinity to their peptide-HLA targets. Engineering these TCRs via phage display or yeast display for higher affinity is complicated by the introduction of unwanted cross-reactivity and the poor association between affinity and function. This project seeks to tackle each of the major challenges of ACT in order to effectively reprogram the immune system to combat solid tumors. The F99 phase is focused on a TCR engineering platform for the creation, modification, and profiling of TCRs that can target tumor-associated self-proteins with minimal toxicity profiles. In this approach, I first raise T cells from the natural repertoire that recognize a related ‘foreign’ peptide that differs by one amino acid from the self-peptide. Then, I modulate the fine specificity of the TCR by directed evolution of the peptide binding region to switch its specificity towards the tumor self-antigen of interest. I demonstrate the value of this approach by the creation of libraries of viral-specific TCRs and the subsequent in vitro selection of TCRs that switched specificity to a closely related epitope. The engineered TCRs showed robust T-cell activation after ligand recognition and are of equal or higher efficiency than the parental receptor. Importantly, the engineered TCRs displayed no additional promiscuity or off-target specificities as compared to the parental TCRs. The goal for the remainder of my dissertation project is to apply this approach to the generation of cancer reactive TCRs. By controlling the fine specificity of TCRs, this approach will overcome two of the major challenges of ACT, namely increasing the breadth of antigens that can be used for ACT while also minimizing cross-reactivities. For the K00 phase, I will shift my focus to addressing the suppressive tumor microenvironment that surrounds solid tumors by developing novel synthetic receptors and testing them in mouse models. I plan to build upon my synthetic biology background to implement novel high-throughput screens, learn new statistical analysis methods, and gain experience working with in vivo mouse models of cancer. These new approaches, coupled with my already strong background in genetics, molecular biology and biochemistry will allow me to address the most pressing and challenging issues facing targeted immunotherapies. With the aid of this award, I intend to continue my research contribution to become a leader in the field of cancer immunotherapy.