Success of T-cell based immunotherapy depends largely on exogenous modification and propagation of patient or donor derived T cells. The conventional paradigm is to stimulate primary T cells cross-linking endogenous CD3-complex with a monoclonal antibody (mAb) such as CD3ϵ targeting OKT3 alone or in combination with super-agonist mAb anti-CD28. The crosslinking via agonist mAb(s) lead to downstream activation signaling, phosphorylation of immunotyrosine activation motif and associated Ca2+ influx resulting in T-cell division and proliferation in vitro. We report an alternate approach to targeting T-cell signaling complex by cross-linking αβ-TCR of T cells through a novel TCR-specific single chain antibody. Towards this objective, first we generated several TCR targeted monoclonal antibodies. Next, we designed a panel of high affinity scFvs derived from a mAb clone A79 from which a specific mutant named SAYON-15 (Single chain AffinitY Optimized Novel mutant 15) possessing highest affinity towards TCR α-chain was selected. Affinity enhancement was achieved in silico via homology modeling and scanning mutations in the original scFv (A79). SAYON-15 in combination with either truncated human CD8 transmembrane or truncated human IgG1-Fc chain scaffold was expressed on K562 (HLA C-neg) cells. As activating and propagating feeder cells K562 HLA C-neg expressing SAYON-15 could drive expansion of αβ TCR+ T cells from donor derived peripheral or umbilical cord blood mononuclear cells in comparable fashion to gene modified K562 cells loaded with CD3ϵ specific mAb (OKT3). Ex vivo propagated T cells growth kinetics, phenotypic composition, TCR repertoire were studied and compared between two separate T-cell activation strategies described here. We analyzed ex vivo cultured T-cells immune repertoire with respect to TCRV gene rearrangement, clonal diversity, CDR3 composition by next generation sequencing. SAYON-15 expressing AaPc when compared with OKT3 expressing AaPC showed greater clonality, equivalent level of functional V-gene rearrangement, CDR3 chain length and V-J combinations. In summary, we report a novel TCR-specific scFv and harnessed its ability to numerically expand T cells ex vivo. This could potentially be used for clinical applications in the context of adoptive cell therapy.
Citation Format: Bipulendu Jena, Hiroaki Taguchi, Aleksandra Nowicka, Ana B. Korngold, Helen Huls, Laurence JN Cooper. Ex vivo generation of clinical grade T cells by using activating and propagating feeder cells to cross-link T cell receptor. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A179.
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