The ability to engineer and re-program the surfaces of cells has the potential to expand and enable the therapeutic use of cell based therapies for both tissue regeneration and cancer. To achieve this goal a number of cell surface engineering approaches have been devised. Clinically, genetic engineering of cells is by far the most commonly used approach for the modification of cell surfaces. Nevertheless, the preparation of the engineered cells is time consuming, requires several sophisticated steps and can lead to long term immune toxicities. Our group has employed the power of chemically induced protein dimerization to develop a method to produce chemically self-assembled nanorings (CSANs). We have shown that two E. coli dihydrofolate reductase molecules (DHFR2) fused to a single chain antibody (scFv) or targeting peptide can be engineered to spontaneously self-assemble, upon the addition of the chemical dimerizer, bis-methotrexate (Bis-MTX), into either highly stable bivalent or octavalent synthetic antibody CSANs. Recently, using a Bis-MTX phospholipid conjugate, we have prepared CSANs that rapidly (min) and stably (days) insert into cell membranes. In addition, when a scFv targeting EpCAM, a carcinoma and cancer stem cell marker, was fused to the DHFR2 building block, our first generation anti-EpCam chemically self-assembled chimeric antigen receptors (anti-EpCAM-CS-CARs) were formed and found to direct selective Tcell cell killing of EpCAM positive breast cancer cells. A unique feature of our approach is the ability to remove the anti-EpCAM-CS-CARs from the T-cells pharmacologically with a non-toxic drug. Therefore, we propose to prepare a small library of structurally different anti-EpCAM-CS-CARs and determine their ability to stably functionalize T-cell membranes and induce cytotoxicity toward EpCam positive tumor cells both in vitro and in vivo. This preliminary data will allow us to establish the potential for chemically self-assembled chimeric antigen receptors (CS-CARs) to serve as an alternative and complementary approach for the engineering of stable, yet pharmacologically reversible, therapeutic cell-cell interactions.
We are attempting to develop an easy and cost effective method for equipping cells with a molecular GPS system that will guide them to either damaged or diseased tissues so that they can be treated and repaired. In this proposal, this method is being tested as a way to guide immune cells to cancer cells and destroy them.
|Csizmar, Clifford M; Petersburg, Jacob R; Hendricks, Alex et al. (2018) Engineering Reversible Cell-Cell Interactions with Lipid Anchored Prosthetic Receptors. Bioconjug Chem 29:1291-1301|
|Stern, Lawrence A; Csizmar, Clifford M; Woldring, Daniel R et al. (2017) Titratable Avidity Reduction Enhances Affinity Discrimination in Mammalian Cellular Selections of Yeast-Displayed Ligands. ACS Comb Sci 19:315-323|
|Shah, Rachit; Petersburg, Jacob; Gangar, Amit C et al. (2016) In Vivo Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures. Mol Pharm 13:2193-203|