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105 4–1BB and optimized CD28 co-stimulation enhances function of human mono- and bi-specific third-generation CAR T cells
  1. Emiliano Roselli1,
  2. Justin Boucher1,
  3. Gongbo Li1,
  4. Hiroshi Kotani1,
  5. Kristen Spitler1,
  6. Kayla Reid1,
  7. Yannick Bulliard2,
  8. Nhan Tu1,
  9. Sae Bom Lee1,
  10. Bin Yu1,
  11. Frederick Locke1 and
  12. Marco Davila1
  1. 1H. Lee Moffitt Cancer Center, Tampa, FL, USA
  2. 2Atara Biotherapeutics, Thousand Oaks, USA


Background Co-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 NF-κB signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.

Methods We 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 4-1BB or mut06 together with the combination of both (BB06). We evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed LCK recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NSG mice. Additionally, we 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.

Results Co-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased polyfunctionality and LCK recruitment to the CAR (figure 1A), after repeated-antigen stimulation these cells expanded significantly better than second-generation CAR T cells (figure 1B). 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 (figure 1C). Bi-specific CAR T cells exhibited improved in vivo anti-tumor activity with increased persistence and decreased exhaustion (figure 1D).

Abstract 105 Figure 1

A. pLCK is increased in h19BB06z CAR T cells and associated with the CAR. CAR T cells were stimulated with irradiated 3T3-hCD19 cells at a 10:1 E:T ratio for 24hr. Cells were lysed and CAR bound and unbound fractions were western blotted. A single-cell measure of polyfunctional strength index (PSI) of CAR T cells. B. h19BB06z CAR T cells have the highest proliferation after repeated antigen stimulations. 5x105 CAR T cells were stimulated with 1x105 irradiated 3T3-hCD19 cells. After 1 week, half of the cells were enumerated by flow cytometry and the other half was re-stimulated with 1x105 fresh irradiated 3T3-hCD19 cells. This was repeated for a total of 4 weeks. C. 5x105 CAR T cells were co-cultured with 5x105 target cells (Raji-CD19High). After 1 week half the cells were harvested enumerated and stained by flow cytometry while the other half was re-stimulated with 5x105 fresh target cells (indicated by arrows). This was repeated for a total of 4 weeks. Frequency of PD1+CD39+ cells within CD8+ CAR T cells. D. Raji-FFLuc-bearing NSG mice were treated with 1x106 CAR T cells 5 days after initial tumor cell injection. Tumor burden (average luminescence) of mice treated with bi-specific or monospecific CAR T cells, UT and tumor control. Each line represents an individual mouse. (n = 7 mice per group).

Conclusions These 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- 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.

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