Target Antigen Density Governs the Efficacy of Anti–CD20-CD28-CD3 ζ Chimeric Antigen Receptor–Modified Effector CD8+ T Cells

Abstract

The effectiveness of chimeric Ag receptor (CAR)–transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR–T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR–T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR–T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.

A Tet-On Inducible System for Controlling CD19-Chimeric Antigen Receptor Expression upon Drug Administration

“…This activity has been described as the "ontarget/off-tumor" effect. Indeed, CAR T-cell therapy targeting ERBB2 and CAIX reportedly has involved lethal toxic accidents in clinical trials that were due to cytokine release syndrome triggered by the off-target recognition of low amounts of antigen (13)(14)(15)(16)(17). Clinical trials of CD19CAR T-cell therapy also revealed toxicities such as hypogammaglobulinemia, due to B-cell aplasia (18).…”

Introduction of Genetically Modified CD3ζ Improves Proliferation and Persistence of Antigen-Specific CTLs

“…By inserting the T2A sequence between the ATAM and tEGFR, the two proteins were 174 co-expressed at equimolar levels from a single transcript (18,19 …”

Programming CAR-T cells to kill cancer

“…In the context of CD22-CARs, a comparison between two scFvs targeting the same epitope but with varying affinities did not reveal a significant impact of affinity on CAR function, whereas an alternative CD22 binder (m971) with lower affinity proved most efficacious, possibly because of better accessibility of the targeted epitope 34,35 . Thus, for a given antigen density on a target cell, there is likely to be an optimal range of scFv affinities and CAR expression levels required for a specific and effective antitumor response [36][37][38] .…”