Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers

Abstract

Chimeric antigen receptor (CAR)–modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.

Bispecific Antibody Format and the Organization of Immunological Synapses in T Cell-Redirecting Strategies for Cancer Immunotherapy

“…The TCE led to a similar CD3ε and F-actin organization at the IS compared to that observed in TCR-stimulated Jukat cells while actin clearance and CD3ε distribution in CAR-T Jurkat cells were not properly organized [ 63 ]. Importantly, similar results were also observed when primary cells were used [ 61 ]. Therefore, this analysis indicates a higher quality of the IS assembled by TCEs in comparison to CAR-T cells.…”

“…We also have previously shown efficient activating signaling in addition to the polarization of F-actin and CD3ε to the IS induced by an anti-epidermal growth factor receptor (EGFR) and anti-CD19 TCE. The organized IS showed a similar topology to antigen-stimulated IS and correlated with efficient T cell activation in vitro and in vivo [ 61 , 62 , 63 ]. Therefore, TCE-mediated T cell redirection seems to induce the assembly of a canonical IS, as well as effective T cell activating signaling and function similar to events induced during antigen stimulation of T cells.…”

Efficient preclinical treatment of cortical T cell acute lymphoblastic leukemia with T lymphocytes secreting anti-CD1a T cell engagers

“… 34–40 Our previous work in B-ALL showed that CD19-STAb T cell therapy could prevent CD19 downregulation and subsequent tumor escape more efficiently and at lower E:T ratios than CD19-CAR T cells. 30 41 In contrast, loss of CD1a expression was not detected on cell surface of target cells co-cultured with either CD1a-CAR or CD1a-STAb T cells. These differences may be attributable to the impact that the density and biology of the targeted antigen plays on T cell activation.…”

“…STAb T cells represent a next-generation T cell-redirecting immunotherapy for B-ALL 30 and coT-ALL, being easily applicable to other cancers for which a suitable immunotherapy target is available. 23 A major advantage for STAb T cells over CAR T cells lies in the fact that an effective treatment with STAb T cells might require lower T cell doses, which could be of particular relevance when an adequate number of mature effector T cells cannot be engineered due to either the lymphopenic status of many multi-treated patients or manufacturing constrains in patients with aggressive and hyperleukocytic relapses.…”

“…In fact, several groups have shown promising therapeutic effects of STAb T cells in CD19 + B cell malignancies. [28][29][30] Here, we report for the first time the generation of STAb T cells secreting an anti-CD1a x anti-CD3 TCE (CD1a-STAb T cells) and demonstrate their efficacy in several in vitro and in vivo cutting-edge models of coT-ALL. Our results indicate that STAb T therapy controls tumor progression similar to CD1a-CAR T therapy and exhibits slightly higher persistence of T cells in vivo.…”