659 T cell receptor exchange by zygote engineering results in physiological T cell responses for therapeutic use in pancreatic ductal adenocarcinoma

Background Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy characterized by highly suppressive tumor microenvironment. Despite this, engineered T cell therapy has promise for effectively targeting PDA. To identify the underlying mechanisms of antigen-specific immunosuppression in PDA, we create novel TCR knock-in mouse models robust and standardized source naïve mesothelin (Msln)-specific cells. Methods Specifically, integrate two murine mesothelin-specific TCRs into physiologic Trac locus primary cells zygotes using CRISPR/Cas9 rAAV expressing DNA. Simultaneously CRISPR/Cas9, Msln was disrupted to circumvent tolerance. Results This strategy resulted rapid generation homozygous mice with null mutations Msln. In these TCR-exchanged (TRex) mice, most expressed 1045 (high affinity) or 7431 (low as determined tetramer staining. TRex exhibit phenotype rapidly differentiate effector upon antigenic stimulation. While high affinity elicits function CD4 cells, lower higher functional avidity less downregulation when antigen limiting. Historical transgenic which randomly integrated genome, increased PD1, CD25, CD69, decreased functionality, bias CD25-Foxp3+ Treg compared from mice. Further, integration retain superior following repetitive stimulations retrovirally transduced Adoptive transfer significantly prolongs survival bearing autochthonous When combined vaccine, cause involution fibroinflammatory stroma. Conclusions sum, generate that physiologically express desired TCR, circumventing shortcomings standard engineering strategies models. Ethics Approval University Minnesota Institutional Animal Care Use Committee approved all animal studies Dr. Ingunn Stromnes (2005-38115A.) Generation knockin (KI) animals performed Mouse Genetic Laboratory at Minnesota.