670. A Novel Approach to Expand Antigen Recognition in Chimeric Antigen Receptors

Abstract

Chimeric antigen receptor (CAR) technology, although very promising, is limited in its application by the availability of target antigens. We propose to expand the pool of potentially targetable cell-surface antigens using the variable lymphocyte receptor (VLR) of the sea lamprey as the antigen recognition region of the CAR. VLRs represent the functional region of the lamprey and hagfish adaptive immune system. They are single chained and variable length, crescent shaped proteins that are produced by assembly of leucine-rich repeat cassettes to form a gene encoding region capable of exceeding 1015 unique variations. Due to their difference in structure compared to Ig based antibodies, VLRs bind antigen in a geometrically dissimilar manner. This unique property of VLRs allows them the ability to bind antigen epitopes that may not typically be bound by scFvs, the result being a potentially expanded repertoire of tumor cell target antigens that may be used in CAR design and application. In our studies, VLRs have been successfully developed from immunized lampreys and target cell specific VLRs have been cloned for several different antigens including VLRs specific for cancer cell lines and purified proteins. This is accomplished using yeast surface display combined with flow sorting and results in monoclonal VLRs specific for the cell line with which the lampreys were immunized. The functionality of the VLR was initially demonstrated in Jurkat cells transduced with a previously generated VLR specific for the B-cell receptor of the mouse tumor line, BCL. CAR protein expression from whole cell lysates of transduced Jurkat cells showed expression of CAR protein. CAR cell surface expression was also confirmed in Jurkat cells using a construct co-expressing GFP preceding a P2A sequence with >90% cells GFP positive. Transduced CAR-Jurkat cells showed upwards of 85% activation in co-culture assays with target cells. In transduced but not co-cultured cells, activation was <5% and activation in naive cells co-cultured with BCL cells was <2%. These results establish the ability of these cells to effectively produce and express VLR-CAR protein. The BCL VLR-CAR construct was also shown to be functional in several different types of cytotoxic effector cells including gamma delta T-cells and NK-92 cells, where target cell killing was increased significantly over non-transduced cells, indicating that target cell specific toxicity is mediated through the VLR-CAR. From these results, showing that VLRs function effectively as the antigen recognition region of the CAR construct we have concluded that VLRs can serve as a unique alternative for directing CAR activity toward specific effector cells.