Colorectal cysts as a validating tool for CAR therapy.

BACKGROUND: Treatment of cancers has largely benefited from the development of immunotherapy. In particular, Chimeric Antigen Receptor (CAR) redirected T cells have demonstrated impressive efficacy against B-cell malignancies and continuous efforts are made to adapt this new therapy to solid tumors, where the immunosuppressive tumor microenvironment is a barrier for delivery. CAR T-cell validation relies on in vitro functional assays using monolayer or suspension cells and in vivo xenograft models in immunodeficient animals. However, the efficacy of CAR therapies remains difficult to predict with these systems, in particular when challenged against 3D organized solid tumors with highly intricate microenvironment. An increasing number of reports have now included an additional step in the development process in which redirected T cells are tested against tumor spheres. RESULTS: Here, we report a method to produce 3D structures, or cysts, out of a colorectal cancer cell line, Caco-2, which has the ability to form polarized spheroids as a validation tool for adoptive cell therapy in general. We used CD19CAR T cells to explore this method and we show that it can be adapted to various platforms including high resolution microscopy, bioluminescence assays and high-throughput live cell imaging systems. CONCLUSION: We developed an affordable, reliable and practical method to produce cysts to validate therapeutic CAR T cells. The integration of this additional layer between in vitro and in vivo studies could be an important tool in the pre-clinical workflow of cell-based immunotherapy.

Chimeric antigen receptor preparation from hybridoma to T-cell expression.

The successful use of chimeric antigen receptor (CAR) for hematological cancer treatment has influenced the direction taken in translational research toward an increasing focus on personalized targeted immunotherapy. Thus, a growing number of labs worldwide are now interested in testing their old antibody collections in this format to broaden the spectrum of utility and improve safety and efficacy. We herein present a straightforward protocol for the identification of an antibody from a hybridoma and the design of the single chain fragment that will be placed on the extracellular part of the CAR construct. We further show how to test the expression and the activity of the construct in primary T cells. We illustrate our demonstration with two new CARs targeted against the B cell receptor, more precisely the light chains κ and λ, that represent potential alternatives to the CD19 CAR used in the treatment of B-cell malignancies.