<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Use of TCR antibody fusion proteins as bispecific agents for NK and T cell-mediated immunotherapy"^^ . "Bispecific agents are a promising approach for cancer immunotherapy as they enable the \r\nredirection of cytotoxic lymphocytes towards tumor cells by targeting different structures on the \r\ntumor cell surface and triggering cytotoxic lymphocytes through agonistic binding to activating \r\nreceptors. Targeted tumor antigens can be roughly divided into peptide antigens presented by \r\nmajor histocompatibility complex (MHC) class I molecules to antigen-specific T cell receptors\r\nand cell surface antigens recognized by antibodies. Peptides presented in the context of an \r\nMHC-I molecule mostly result from intracellular proteins, which make up the majority of the \r\nproteome, and thus serve as an highly attractive target for immunotherapeutic strategies. In \r\ncontrast to TCR-transgenic T cells that express novel TCR specificities in the natural cellular \r\ncontext, the development of soluble TCR-based therapies is hampered due to the poor stability \r\nof recombinant TCRs and a generally significantly lower affinity in comparison to therapeutic \r\nantibodies. In the past years progress has been made to address these problems, which\r\ninclude different modifications improving construct stability and methods for TCR affinity \r\nmaturation enabling the development and clinical application of first TCR-based bispecific \r\nrecombinant fusion proteins that retarget T cells to tumors displaying intracellular antigens \r\nthrough MHC molecules. \r\nThis study aimed to develop novel soluble bispecific TCR-based agents for the \r\nredirection of NK and T cells. To achieve potent bispecific mediators a bivalent immunoglobulin \r\nG (IgG)-like TCR-Fc fusion format was adapted. The ectodomain of the TCR V alpha/C beta chain was \r\nfused to the hinge/Fc part of human IgG1 and the ectodomain of the TCR V alpha/C beta chain was \r\nexpressed as a second soluble protein in cis using a ribosomal skipping sequence. Efficient \r\nassembly of TCR alpha and beta chains was facilitated by an additional artificial intermolecular \r\ndisulfide bridge in the TCR constant domains. The natural intermolecular disulfide bonds of the \r\nhuman IgG1 hinge region enabled assembly of TCR-Fc fusion proteins to stable homodimers \r\nwhich could be successfully expressed by transient transfection of CHO-S producer cells. \r\nTo enable NK cell redirection, specific mutations known to enhance Fc gamma RIIIa binding \r\nwere introduced in the Fc fragment. Other investigated formats made use of single chain \r\nvariable fragments (scFv) recognizing CD16 or NKp46 for NK cell redirection or binding CD3 epsilon\r\nfor the redirection and activation of T cells. NK- and T-cell-binding scFv antibodies were \r\nanalyzed after insertion at C-terminal end of TCR C alpha or C beta, respectively. A cytomegalovirus \r\n(CMV) pp65 peptide/HLA-A*02:01-specific TCR sequence was used as a model system. To \r\nanalyze the influence of TCR affinity, the wildtype TCR was compared to an affinity-maturated \r\nvariant. The soluble IgG1-like TCRs showed a target-specific, affinity- and concentration-dependent binding as well as NK and T cell redirection and activation upon co-culture with \r\npeptide-pulsed or transfected cells. In particular, the TCRalpha-alphaNKp46-Fc enhanced and TCRalpha-alphaCD3epsilon-Fc aglycan constructs were found to be highly potent in redirecting NK and T cells, \r\nrespectively. TCR-scFv-Ig fusion proteins efficiently elicited peptide antigen-specific activation \r\nof purified NK cells and T cells and induced cytotoxicity against different tumor targets.\r\nThese results were further confirmed using low- and and high-affinity variants of three \r\nTCRs recognizing HLA-A*02:01-restricted gp100, MART-1 and NY-ESO-1 peptides, \r\nrespectively, for the redirection of NK cells. TCRhigh aff.\r\n-alphaNKp46-Fc enh constructs, however, \r\nfailed to facilitate NK cytotoxicity against HLA-A2+ melanoma cell lines expressing the antigens \r\nof choice unless the cell lines were incubated with an excess of the cognate HLA-A2-binding \r\npeptide. Thus, the NK cell-engaging TCR-Fc fusion proteins constructs apparently were not \r\nsensitive enough to redirect and activate NK cells against melanoma cells presenting low \r\nquantities of naturally processed specific peptide/MHC-I complexes. \r\nIn sum, in this work a panel of novel recombinant bispecific TCR-Fc and TCR-scFv-Fc \r\nfusion proteins were genetically engineered, produced and demonstrated to facilitate the \r\nactivation and cytotoxicity of NK and T cells towards tumor cells expressing a model peptide-MHC-I complex in sufficient quantities. Further investigations are required to investigate if the \r\navidity of soluble TCR-Fc constructs can be sufficiently increased by multimerization \r\napproaches in order to overcome the sensitivity issues resulting from low abundance of \r\npeptide-MHC-I complexes and low-affinity TCRs."^^ . "2023" . . . . . . . "Annkathrin Chiara"^^ . "Teschner"^^ . "Annkathrin Chiara Teschner"^^ . . . . . . "Use of TCR antibody fusion proteins as bispecific agents for NK and T cell-mediated immunotherapy (PDF)"^^ . . . "Doktorarbeit_Teschner.pdf"^^ . . . "Use of TCR antibody fusion proteins as bispecific agents for NK and T cell-mediated immunotherapy (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Use of TCR antibody fusion proteins as bispecific agents for NK and T cell-mediated immunotherapy (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Use of TCR antibody fusion proteins as bispecific agents for NK and T cell-mediated immunotherapy (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Use of TCR antibody fusion proteins as bispecific agents for NK and T cell-mediated immunotherapy (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Use of TCR antibody fusion proteins as bispecific agents for NK and T cell-mediated immunotherapy (Other)"^^ . . . . . . "small.jpg"^^ . . 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