4-1BB and optimized CD28 co-stimulation enhances function of human mono-specific and bi-specific third-generation CAR T cells

Abstract

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to nuclear factor kappa B (NF-κB) signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with either 4-1BB or mut06 together with the combination of both and evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed lymphocyte-specific protein tyrosine kinase (LCK) recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NOD scid gamma (NSG) mice. We also engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased central memory phenotype, expansion, and LCK recruitment to the CAR. This enhanced function was dependent on the positioning of the two co-stimulatory domains. A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers. Bi-specific CAR T cells exhibited improved in vivo antitumor activity with increased persistence and decreased exhaustion.ConclusionThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono-specific and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

“…The superior cytotoxic activity of M28z CAR T cells following antigen exposure, is in concordance with the elevated effector functions associated with the TemRA phenotype. Previous reports have demonstrated skewness toward the effector phenotype in CD28-containing CAR T cells and central memory phenotype in 4–1BB containing CAR T cells 19 , 36 , 37 although this finding has not been universal. 38 …”

CAR-T Cell Therapy in Hematological Malignancies: Current Opportunities and Challenges

“…However, it has been demonstrated that 4-1BB co-stimulation could ameliorate CAR-T cell exhaustion compared with CD28 co-stimulation (191,192). Remarkably, combining CD28 and 4-1BB could simultaneously augment the anti-tumor effects and increase the persistence of CAR-T cells (193)(194)(195)(196). In addition, the CAR-T cell structure can be optimized by the fully humanized CARs.…”

Co-Stimulatory Receptor Signaling in CAR-T Cells

“…Due to the success of CD28 and 4-1BB containing second generation CAR-T cells, current research efforts are exploring attributes of additional co-stimulatory domains—including ICOS (CD278), OX40 (CD134) and CD27—or the combination of multiple co-stimulatory domains, as in the case of third generation CAR-T cells [ 59 , 60 , 61 , 62 , 63 ]. Third generation CAR-T cell products incorporate two co-stimulatory domains within the cytoplasmic signaling tail with the attempt of providing added benefits and overcoming limitations of any one co-stimulatory signal [ 64 ].…”

“…The choice of co-stimulatory domain included in the CAR can greatly impact several key aspects of CAR-T cell therapy such as the differentiation state of the infusion product, the kinetics of anti-tumor response, cytotoxic function and associated toxicities. Recent work has proposed that an optimized co-stimulatory signal can generate a more desirable CAR-T cell product with an extended in vivo life span [ 59 ]. This notion stems from prior evidence showing that excessive co-stimulation is detrimental to CAR-T cell performance [ 71 , 72 ], and that mutation of specific residues within the cytoplasmic tail of a CAR that disrupt downstream signaling enhanced anti-tumor efficacy and CAR-T persistence in vivo [ 60 , 73 , 74 ].…”