Bispecific Antibody Armed Metabolically Enhanced Headless CAR T Cells.

Adoptive T cell therapies for solid tumors is challenging. We generated metabolically enhanced co-activated-T cells by transducing intracellular co-stimulatory (41BB, ICOS or ICOS-27) and CD3ζ T cell receptor signaling domains followed by arming with bispecific antibodies (BiAbs) to produce armed "Headless CAR T cells" (hCART). Various hCART armed with BiAb directed at CD3ϵ and various tumor associated antigens were tested for: 1) specific cytotoxicity against solid tumors targets; 2) repeated and dual sequential cytotoxicity; 3) survival and cytotoxicity under in vitro hypoxic condition; and 4) cytokine secretion. The 41BBζ transduced hCART (hCART41BBζ) armed with HER2 BiAb (HER2 hCART41BBζ) or armed with EGFR BiAb (EGFR hCART41BBζ) killed multiple tumor lines significantly better than control T cells and secreted Th1 cytokines/chemokines upon tumor engagement at effector to target ratio (E:T) of 2:1 or 1:1. HER2 hCART serially killed tumor targets up to 14 days. Sequential targeting of EGFR or HER2 positive tumors with HER2 hCART41BBζ followed by EGFR hCART41BBζ showed significantly increased cytotoxicity compared single antigen targeting and continue to kill under in vitro hypoxic conditions. In summary, metabolically enhanced headless CAR T cells are effective serial killers of tumor targets, secrete cytokines and chemokines, and continue to kill under in vitro hypoxic condition.

Anti-tumor and immune modulating activity of T cell induced tumor-targeting effectors (TITE).

Adoptive transfer of Bispecific antibody Armed activated T cells (BATs) showed promising anti-tumor activity in clinical trials in solid tumors. The cytotoxic activity of BATs occurs upon engagement with tumor cells via the bispecific antibody (BiAb) bridge, which stimulates BATs to release cytotoxic molecules, cytokines, chemokines, and other signaling molecules extracellularly. We hypothesized that the release of BATs Induced Tumor-Targeting Effectors (TITE) by this complex interaction of T cells, bispecific antibody, and tumor cells may serve as a potent anti-tumor and immune-activating immunotherapeutic approach. In a 3D tumorsphere model, TITE showed potent cytotoxic activity against multiple breast cancer cell lines compared to control conditioned media (CM): Tumor-CM (T-CM), BATs-CM (B-CM), BiAb Armed PBMC-CM (BAP-CM) or PBMC-CM (P-CM). Multiplex cytokine analysis showed high levels of Th1 cytokines and chemokines; phospho-protein signaling array data suggest that the prominent JAK1/STAT1 pathway may be responsible for the induction and release of Th1 cytokines/chemokines in TITE. In xenograft breast cancer models, IV injections of 10× concentrated TITE (3×/week for 3 weeks; 150 µl TITE/injection) was able to inhibit tumor growth significantly (ICR/scid, p < 0.003; NSG p < 0.008) compared to the control mice. We tested the key components of the TITE for immune activating and anti-tumor activity individually and in combinations, the combination of IFN-γ, TNF-α and MIP-1ß recapitulates the key activities of the TITE. In summary, master mix of active components of BATs-Tumor complex-derived TITE can provide a clinically controllable cell-free platform to target various tumor types regardless of the heterogeneous nature of the tumor cells and mutational tumor.