Preclinical Development of Bispecific Chimeric Antigen Receptor Targeting Both CD19 and CD22

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Despite advances in upfront therapy, relapsed and refractory ALL remains a primary cause of cancer-related mortality in children. Immunotherapy using genetically engineered T cells expressing chimeric antigen receptors (CARs) targeting CD19 has demonstrated dramatic clinical results in children and young adults with pre-B cell ALL with a 70-90% complete remission rate in multiple clinical trials.  However, not all patients respond and at least 10% of patients will relapse due to loss of the targeted CD19 epitope. CD22 is another pan-B cell surface antigen that is broadly expressed on ALL blasts and has been targeted successfully using immunoconjugates.  We have recently developed a highly active CD22 CAR currently being tested in a clinical trial. Conceptually, dual targeting both CD19 and CD22 on Pre-B ALL with a single bispecific CAR may result in enhanced avidity and increased T cell activation which may result in additive or synergistic responses compared to targeting a single antigen.  In addition, since there are heterogeneous expression levels of CD19 and CD22 on leukemic blasts, dual targeting may also provide a more broadly active therapy. Finally, simultaneously targeting of both CD19 and CD22 on the surface of pre-B cell ALL may reduce the likelihood of antigen escape due to downregulation or deletion. We describe the preclinical development of two novel CD19 and CD22 bispecific CARs assembled with CD19 and CD22 single chain fragment variable regions (scFv) binding domains on a single construct. T cells transduced the CD19/CD22 bispecific CARs have comparable CAR surface expression when compared to T cells expressing either the CD19 or CD22 CAR. T cells expressing the bispecific CAR are active in vitro against ALL cell lines expressing both CD19 and CD22 as measured by interferon gamma production and chromium release.  In vitro activity is maintained against K562 cells expressing either CD19 or CD22 alone.  Interestingly, the order and the linker between the CD19 and CD22 scFvs in the CAR construct has a major impact on the efficacy of the dual CAR with a membrane-distal CD22 binding scFv demonstrating improved efficacy. Finally, in xenograft models both bi-specific CARs efficiently eradicate ALL cell lines and patient derived xenografts. In vivo treatment of a Crisper-knockdown-CD19-negative leukemia line and a CD19-negative patient-derived xenograft is ongoing.  In summary, CD19/CD22 bispecific CARs demonstrate robust pre-clinical activity against pre-B cell ALL and provides a strategy to improve the clinical efficacy of CAR therapy.