CAR T Cell Relapse CD19 Negative ALL Cells Express Integrins and Integrin Blockade Can Prolong Survival In Vivo

Relapsed and refractory B-cell precursor acute lymphoblastic leukemia (BCP ALL) remains difficult to treat, despite advances in upfront therapy and improved survival for de novo ALL. Chimeric antigen receptor T (CART) cell therapy is a major chemotherapy-independent approach, achieving disease-free remission that functions through an immunologic mechanism. However, even with this novel therapy, it is becoming evident that relapses occur with appreciable frequency. In some cases, anti-CD19 CART cell treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Based on our in-vivo relapse model using relapse ALL post-CART therapy lacking CD19 surface expression, we found that relapse occurs in the bone marrow. Therefore, we hypothesized that, similarly to cell adhesion mediated chemotherapeutic drug resistance (CAM-DR), cell adhesion mediated CART-cell resistance (CAM-CART-R) can occur resulting in relapse of ALL. To test our hypothesis, primary ALL cells were co-cultured with murine calvaria-derived bone marrow stromal cells, OP9, or cultured only with media in short term cultures, and anti-CD19 targeting CAR T cells were added to the culture. We observed that CAR T cells had less activity against ALL cells when in OP9-coculture, compared to media only co-cultures, supporting the notion of CAM-CART-R. Interestingly, three cases of primary ALL with CD19 knocked out using CAS9-CRISPR, showed significantly more adhesion to OP9 cells compared to CD19 competent ALL cells. Phenotypic analysis revealed high expression of integrins after knockout of CD19 in all three primary ALL cells. Phenotypic profiling of three cases of primary ALL relapse after anti-CD19 CAR T cell therapy also showed high expression of adhesion molecules including integrin alpha4 and alpha6. We showed that blocking integrins can de-adhere these primary ALL relapses from their respective counter-ligands in vitro. As a proof of principle that CD19-negative ALL cells post-CAR T cell relapse can benefit from integrin blocking therapy in vivo, we used the humanized anti-human integrin alpha4 antibody, Natalizumab. NSG mice were injected with one case of CD19 negative ALL cells post-CART cell relapse and treated intraperitoneally (n=6/group): with control IgG or Natalizumab. Natalizumab monotherapy significantly prolonged survival of leukemic mice compared to control IgG group (MST =85 days vs. MST= 66 days, p<0.005). Further combination treatments with chemotherapy are in progress. In summary, our data indicates that targeting adhesion molecules may be a new approach to treat anti-CD19 CAR T cell relapsed ALL.