Next Generation Sequencing of Immunized Mouse Splenocytes to Develop an anti-TIM3 Chimeric Antigen Receptor for Acute Myeloid Leukemia. Mus musculus musculus

The field of anti-cancer T cell therapy has had major advances in recent years with the development of the chimeric antigen receptor (CAR). Our group has developed and demonstrated that CARs against CD19 have complete remission rates of greater than 90% against B cell acute lymphoblastic leukemia (B-ALL). Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults, presenting greater than 20,000 new cases per annum and representing 80% of acute leukemia. Additionally, prognosis is poor with as much as 70% of patients, 65 years or older, dying within 1 year of diagnosis. A recent study examining AML has shown that a TIM3 monoclonal antibody (mAb) blocks AML engraftment and eliminates leukemic stem cells (LSC). Additionally, TIM3 mAbs have anti-tumor effects such as slowing tumor progression. Gene-expression analysis demonstrates an 8-fold increase of TIM3 in LSC compared to hematopoietic stem cells. Our own evaluation of TIM3 expression on AML blasts and LSC demonstrated that TIM3 is commonly co-expressed on CD33+ blasts. These results led us to hypothesize anti-TIM3 CAR T cells can target and kill AML, which we will evaluate by the development of novel anti-TIM3 CARs. Here, we describe a novel CAR design strategy by next generation sequencing (NGS) followed by CAR synthesis and functional validation against TIM3+ targets. NGS allows for direct identification of rearranged immunoglobulin genes, mutation and repertoire analysis, and comparison of multiple immunized mice. Furthermore, by eliminating the need for hybridoma production and screening we have developed a rapid, economical system for the development of novel CARs.