Browsing by Subject "T cell receptor"

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Determining the relationship between TCR affinity and T cell response
(2018-08-16) Stevens, Christopher A.; Maynard, Jennifer Ann, 1974-; Georgiou, George; Alper, Hal; Ehrlich, Lauren; Jiang, Ning; Upton, Jason
The T-cell response is a critical component of controlling many cancers and viral diseases and recently developed T-cell based therapies have become very popular and effective treatment methods. Adoptive cell therapy involves the isolation of a patient’s or donor’s T-cells, expansion or modification ex vivo, and then administration into the patient. The recent FDA approval of an adoptive cell therapy involving an engineering antigen receptor has solidified this technique from a passing fad to a legitimate and effective technique for treating cancer. T-cells are able to recognize a vast range of target antigens through the use of a T-cell receptor (TCR) which targets small peptide fragments presented on the surface of diseased cells in the form of major histocompatibility complexes (pMHC). Further improvement of these therapies and methods requires a deeper understanding of how these TCR-pMHC interactions relate to functionality. Cytomegalovirus is a persistent viral infection that can reactivate periodically during periods of immunodepression. Viral control is mediated by a strong T-cell response and it can cause life-threatening complications in immunosuppressed individuals.Additionally, the T-cell response after chronic CMV reactivation becomes focused to a few individual clonotypes bearing ‘public’ TCRs that have shared sequences between individuals and are often high affinity. This makes CMV an excellent model for studying TCR-pMHC interactions. Several attempts to improve functionality of natural TCRs by engineering for higher affinity have resulted loss of specificity or reduced activation. Methods for direct selection of TCRs with improved activity are currently being developed, however they are very limited, whereas there are a variety of methods for affinity maturation of TCRs. A firm understanding of how TCR-pMHC binding affinity relates to activation would greatly benefit all T cell therapies. This work attempts to provide a better understanding of the relationship between TCR-pMHC affinity and T-cell activity by characterizing TCRs engineered in multiple platforms to obtain a range of affinities while maintaining antigen specificity. Additionally, we observe the relationship between affinity and activity of in vivo derived TCRs against CMV to help aid adoptive cell therapies, as well as the development of a vaccine to elicit potent T-cell responses.
Development of a model system to characterize antigen-specific T cell receptors based on TCR affinity and T cell function
(2021) Xia, Amanda; Jiang, Ning
Modern T cell cancer immunotherapies are based on the ability of cytotoxic T cells to recognize and attack infected cells. This process is activated by the T cell receptor (TCR) recognizing a peptide-bound major histocompatibility complex (pMHC) on the surface of target cells. It is of particular interest to target neoantigens, antigens derived from tumor-specific DNA mutations, to create a tumor-specific immune response. One method of broadening the T cell response is to genetically engineer the patient’s T cells to express neoantigen-specific TCRs isolated from healthy donors. It is important to understand how the affinity of the TCR-pMHC interaction relates to T cell function in the context of T cells expressing foreign TCR. Here, I created a model system to study human TCRs specific for cytomegalovirus (CMV) pp65, a commonly recognized CMV antigen. By transducing primary T cells with pp65-specific TCRs of known TCR affinity, I investigated how differences in TCR affinity related to T cell function. My model system demonstrated that transduced T cells could maintain high TCR expression over a sustained period and that the affinity of the TCR expressed positively correlated with cytokine production. This sets the foundation of a promising approach to study neoantigen-specific TCRs for the development of melanoma immunotherapy.
Engineering antibody and T cell receptor fragments : from specificity design to optimization of stability and affinity
(2014-12) Entzminger, Kevin Clifford; Maynard, Jennifer Anne, 1974-
B and T cells comprise the two major arms of the adaptive immune response tasked with clearing and preventing infection; molecular recognition in these cells occurs through antibodies and T cell receptors (TCRs), respectively. Highly successful therapeutics, clinical diagnostics and laboratory tools have been engineered from fragments of these parent molecules. The binding specificity, affinity and biophysical characteristics of these fragments determine their potential applications and resulting efficacies. Thus engineering desired properties into antibody and TCR fragments is a major concern of the multi-billion dollar biopharmaceutical industry. Toward this goal, we (1) designed antibody specificity using a novel computational method, (2) engineered thermoresistant Fabs by phage-based selection and (3) modulated binding kinetics for a single-chain TCR. In the first study, de novo modeling was used to generate libraries of FLAG peptide-binding single-chain antibodies. Phage-based screening identified a dominant design, and activity was confirmed after conversion to soluble Fab format. Bioinformatics analysis revealed potential areas for design process improvement. We present the first experimental validation of this in silico design method, which can be used to guide future antibody specificity engineering efforts. In the second study, the variable heavy chain of a moderately stable EE peptide-binding Fab was subjected to random mutagenesis, and variants were selected for resistance to heat inactivation. Thermoresistant clones where biophysically characterized, and structural analysis of selected mutations suggested general mechanisms of stabilization. Framework mutations conferring thermoresistance can be grafted to other antibodies in future Fab stabilization work. In the third study, TCR fragment binding kinetics for a clonotypic antibody were modulated by varying valence during phage-based selection. Binding affinity and kinetics for representative variants depended on the display format used during selection, and all TCR fragments retained binding to native pMHC antigen. This work demonstrates a general engineering platform for tuning protein-protein interactions. Using a combination of computational design and phage-based screening, we have identified antibodies and TCR fragments with improved binding properties or biophysical characteristics. The optimized variants possess a wider range of potential applications compared to their parent molecules, and we detail engineering methods likely to be useful in the engineering of many other protein-based therapeutics.
Engineering human autoimmune T cell receptors towards the development of a cellular therapy for multiple sclerosis
(2018-05) Leonard, Elissa Kathleen; Maynard, Jennifer Anne, 1974-; Georgiou, George; Ehrlich, Lauren I; Peppas, Nicholas A; Jiang, Ning J
Multiple sclerosis (MS) is a debilitating neurodegenerative autoimmune disorder caused by autoreactive T cells that recognize and initiate immune attack of the myelin sheath that protects neurons of the central nervous system. Over time, this results in scarring or destruction of the affected tissue, and cerebellar atrophy. Existing therapies are mostly non-specifically immunosuppressive or immunomodulatory, and all fail to fully prevent progression of the disease. T regulatory cells, whose function is impaired in MS patients, show great promise as a therapeutic target and as a cellular therapy, as their immunosuppressive activity is specifically targeted based on the identity of their T cell receptor (TCR). The one remaining obstacle in developing Treg cellular therapies for MS and other autoimmune diseases is engineering the TCR to give these cells optimal specificity and suppressive activity. Few, if any, human autoimmune TCRs have been successfully engineered, largely because most autoimmune TCRs, especially those observed in MS, show atypical and unstable binding of antigen compared to more common pathogen-specific TCRs, and thus are very challenging to work with. A unique yeast display system was developed in order to generate variants of an MS TCR with more stable binding of their cognate antigen. These variants showed enhanced avidity on both yeast and T cells, but lost all T cell reactivity to that antigen. This result, though unintended, contributes to our understanding of how these MS TCRs function, and indicates that the instability may somehow be essential to their interaction, or is the most stable interaction that also allows for T cell activation. As traditional receptor engineering techniques rely heavily on selection of variants with more stable or stronger binding of ligand, and this work demonstrated unreliable translation of that selection technique to improved T cell function, an alternative T cell-based strategy for direct screening and selection for TCR variants that endow improved T cell activity has been developed. Together, the work presented here provides insights into the molecular mechanisms involved in the function of human autoimmune TCRs, and provides novel techniques that can be employed to better engineer them.