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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-1β 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. [ABSTRACT FROM AUTHOR]

Interleukin-2 (IL-2) activates extracellular signal-regulated protein kinase (ERK) within immune cells. To examine the profile of phosphorylated ERK (p-ERK) in IL-2 stimulated immune cells of normal donors and patients receiving IL-2 therapy, we developed a dual parameter flow-cytometric assay. An analysis of PBMCs stimulated with IL-2 indicated that IL-2 exposure induced p-ERK in CD56bright NK cells and CD14+ monocytes, but not in CD3+ T cells or CD21+ B cells. CD3+ T cells that were induced to express functional high-affinity IL-2R did not exhibit enhanced p-ERK following IL-2 treatment. Measurement of p-ERK within PBMCs from cancer patients 1 h following their first dose of IL-2 revealed a complete absence of circulating NK cells, consistent with earlier observations. However, the total number of circulating CD14+ monocytes increased in these samples and 97% of these cells exhibited ERK activation. p-ERK was not observed in T cells post-IL-2 therapy. Analysis of PBMCs obtained 3 weeks post-IL-2 therapy revealed high-p-ERK levels in CD56bright NK cells in a subset of patients, while levels of p-ERK returned to baseline in monocytes. These studies reveal an effective method to detect ERK activation in immune cells and demonstrate that IL-2 activates ERK in a subset of NK cells and monocytes but not T cells. [ABSTRACT FROM AUTHOR]