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A2940 - Engineering Lymphocytes to Block TGFbeta Pathway on Lung Fibroblasts Using Synthetic Notch Receptor
Author Block: J. C. Caraballo, N. Frankel, G. Allen, J. Willaims, W. Lim, D. Sheppard; UCSF, San Francisco, CA, United States.
Rationale: Treatment options for pulmonary fibrosis are extremely limited. Cell-based therapies have revolutionized the treatment of malignant disease and can be adapted to treat pulmonary disease, such as pulmonary fibrosis. In most cell-based therapies, lymphocytes are engineered with a chimeric receptor to target a malignant antigen and direct their response to the cancer. Using a synthetic receptor modeled on the Notch family (SynNotch), lymphocytes can be induced to trigger user-specified transcriptional activation in response to recognition of any user-specified targets. This approach could allow precise delivery of protein based anti-fibrotic agents directly in the disease microenvironment, while avoiding unwanted systemic effects. TGFbeta is a cytokine central to the development of tissue fibrosis but also has important physiologic roles in normal tissue. Hence, systemic delivery of TGFbeta inhibitors carries substantial risk of toxicity. As a prove of principle, we set out to engineer lymphocytes to locally deliver a recombinant pan-TGFbeta blocking antibody (1D11) in response to the activation of a SynNotch receptor targeting cell surface proteins on fibroblasts. Method: Using lentivirus, we created transgenic lymphocytes (Jurkat cells) to express 1D11 in its full form (IgG4) and two different single chain forms (scFV). One scFV with heavy chain variable region at the amino terminus followed by the light chain variable region (scFV H-L), and the other with light chain variable region at the amino terminus followed by the heavy chain variable region (scFV L-H). Cells express these proteins either constitutively or in response to Gal4 transcription factor. Lymphocytes were concomitantly transduced with a SynNotch receptor that releases Gal4 upon binding to CD19. Lymphocytes are then co-cultured with wild type or CD19-transfected fibroblasts (NIH/3T3) and then exposed to TGFbeta. Smad2/3 phosphorylation, an early step in the cascade of TGFbeta receptor activation, was measured by western blotting. Results: Lymphocytes produce and deliver 1D11 antibodies in IgG4 form and in scFV forms. Co-culture of fibroblasts with engineered lymphocytes resulted in prevention of TGFbeta-induced Smad2/3 phosphorylation in fibroblasts. 1D11 IgG4 and scFv L-H are more effective forms of the antibody when compared with scFV H-L. Conclusion: Lymphocytes can be engineered to secrete TGFbeta-blocking antibody. Co-culture of lymphocytes with fibroblasts successfully blocks TGFbeta signaling in vitro. These experiments set the stage for in vivo experiments and ultimately for the development of cell-based therapies to target fibrotic lung tissue and deliver any anti-fibrotic therapy that can be encoded in the genome directly to the fibrosis microenvironment.