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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]

Background: Regulation of gene expression by signal transducer and activator of transcription 1 (STAT1) within host tissues mediates the antitumor effects of interferon alfa (IFN α). We used a novel flow cytometric assay to examine phosphorylation-mediated activation of STAT1 within immune effector cell subsets following in vitro or in vivo IFN α treatments. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 17) or melanoma patients (n = 19) were treated in vitro with interferon alfa-2b (IFN α-2b) or phosphate-buffered saline (PBS) and subjected to multiparametric flow cytometry to measure the levels of phosphorylated STAT1 (P-STAT1) within immune cell subsets. We similarly analyzed PBMCs isolated from melanoma patients before and 1 hour after immunotherapy with IFN α-2b. All statistical tests were two-sided. Results: P-STAT1 levels in all major immune cell subsets increased within 15 minutes of in vitro IFN α-2b treatment of PBMCs; the increase was most pronounced in T lymphocytes and monocytes. Relatively low doses of IFN α-2b (i.e., 10²-10³ IU/mL) induced maximal STAT1 activation in vitro. Compared with melanoma patients, healthy donors had higher basal levels of P-STAT1 (specific fluorescence [Fsp]; i.e., FspPBS, the level of P-STAT1 in PBS-treated cells) in total PBMCs, natural killer (NK) cells, and T cells (mean FspPBS in total PBMCs: 5.5 in healthy donors versus 1.6 in patients, difference = 3.9, 95% confidence interval [CI] = 1.4 to 6.5, P = .004; mean FspPBS in NK cells: 4.6 in healthy donors versus 0.9 in patients, difference = 3.7, 95% CI = 1.7 to 5.7, P = .001; mean FspPBS in T cells: 6.8 in healthy donors versus 0.9 in patients, difference = 5.9, 95% CI = 2.5 to 9.3, P = .002). P-STAT1 was detected in the NK and T cells of two patients who received IFN α-2b immunotherapy (20 MU/m² [MU = million units], administered by intravenous injection). P-STAT1 levels in the PBMCs of a patient treated sequentially with 5 MU/m² and 10 MU/m² IFN α-2b (administered by subcutaneous injection) also increased in response to treatments with IFN α-2b but did not increase further with the increased dosage of IFN α-2b. Conclusion: This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN α immunotherapy. [ABSTRACT FROM AUTHOR]

This phase Ib clinical trial evaluated whether pretargeting of CD20 + clonogenic myeloma precursor cells (CMPCs) with anti-CD3 × anti-CD20 bispecific antibody-armed T cells (BATs) before autologous stem cell transplantation (SCT) in patients with standard-risk and high-risk multiple myeloma would induce antimyeloma immunity that could be detected and boosted after SCT. All 12 patients enrolled in this study received 2 BATs infusions before SCT, and 4 patients received a booster infusion of BATs after SCT. Pretargeting CD138 − /CD20 + CMPCs with BATs before SCT was safe and reduced levels of CMPCs by up to 58% in the postinfusion bone marrow in patients who remained in remission. Four of 5 patients who remained in remission had a >5-fold increase in IFN-γ enzyme-linked immunospot responses. SOX2 antibody increased after BATs infusions and persisted after SCT. The median anti-SOX2 level at 3 months after SCT was 28.1 ng/mL (range, 4.6 to 256 ng/mL) in patients who relapsed and 46 ng/mL (range, 28.3 to 73.3 ng/mL) in patients who remained in remission. The immune correlates suggest that infusions of targeted T cells given before SCT were able to reduce CMPC levels and induced cellular and humoral antimyeloma immunity that could be transferred and boosted after SCT. [ABSTRACT FROM AUTHOR]

This proof-of-concept study investigates the immune effects in metastatic breast cancer (MBC) patients after “vaccination” with activated T cells (ATC) armed with anti-CD3 x anti-HER2 bispecific antibody (HER2 BATs) followed by immune consolidation with immune ATC “boost” after high dose chemotherapy (HDC) and autologous stem cell transplant (SCT). Approximately 2 weeks after completion of vaccination portion of the study, immune T cells were obtained by leukopheresis, activated and expanded ex vivo and re-infused after HDC and SCT to test the hypothesis that transfer of immune unarmed ATC would accelerate reconstitution of anti-tumor activity after SCT. Eight metastatic breast cancer (MBC) patients received 8 infusions of HER2 BATs, low dose IL-2, and GM-CSF in the first part of the protocol to induce adaptive cellular and humoral responses. In the “boost” portion of the protocol, 6 of 8 patients received multiple infusions of unarmed ATC post SCT. There were no dose-limiting toxicities or delays in engraftment. Four of 6 patients tested for the immune correlative studies exhibited increases in anti-breast cancer (BrCa) cytotoxicity, antigen specific IFN-γ Elispots, anti-BrCa antibodies and increased IL-12 and Th1 serum cytokine levels after HER2 BATs infusions. Anti-BrCa tumor responses were seen as early as 2 weeks after SCT and persisted up to 2 years post-SCT. One out of 6 patients’ rapidly progressed and showed poor immune responses and high Th2 cytokine levels. There was a significant correlation (p < 0.002) between time to progression (TTP) and anti-BrCa cytotoxicity by immune T cells. This is the first study to show that adoptive transfer of immune T cells after SCT accelerates reconstitution of anti-BrCa specific immunity and correlates with delay TTP. [ABSTRACT FROM AUTHOR]

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STATI and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR. and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 nig sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-a, and evaluated for phosphorylation of STATI and STAT5. Pretreatment of PBMCs with 10 µM sorafenib decreased STATI and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 µ,M), IL-2 (2-24 nM), and IFN-a (10¹-106 U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MlPl-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells. [ABSTRACT FROM AUTHOR]

Background: Myeloid-derived suppressor cells (MDSCs) are one of the major components of the immune-suppressive network, play key roles in tumor progression and limit therapeutic responses. Recently, we reported that tumor spheres formed by breast cancer cell lines were visibly smaller in a Th1 enriched microenvironment with significantly reduced differentiation of MDSC populations in 3D culture. In this study, we investigated the mechanism(s) of bispecific antibody armed ATC mediated inhibition of MDSC in the presence or absence of Th1 microenvironment. Methods: We used 3D co-culture model of peripheral blood mononuclear cells (PBMC) with pancreatic cancer cells MiaPaCa-2 [MiaE] and gemcitabine resistant MiaPaCa-GR [MiaM] cells to generate MDSC in the presence or absence of Th1 cytokines and EGFRBi armed ATC (aATC). Results: We show significantly decreased differentiation of MDSC (MiaE, p<0.005; MiaM, p<0.05) in the presence of aATC with or without Th1 cytokines. MDSC recovered from control cultures (without aATC) showed potent ability to suppress T cell functions compared to those recovered from aATC containing co-cultures. Reduced accumulation of MDSC was accompanied by significantly lower levels of COX2 (p<0.0048), PGE2 (p<0.03), and their downstream effector molecule Arginase-1 (p<0.01), and significantly higher levels of TNF-a, IL-12 and chemokines CCL3, CCL4, CCL5, CXCL9 and CXCL10 under aATC induced Th1 cytokine enriched microenvironment Conclusions: These data suggest aATC can suppress MDSC differentiation and attenuation of their suppressive activity through down regulation of COX2, PGE2 and ARG1 pathway that is potentiated in presence of Th1 cytokines, suggesting that Th1 enriching immunotherapy may be beneficial in pancreatic cancer treatment. [ABSTRACT FROM AUTHOR]

Our group and others have determined that immune effector cells from patients with advanced cancers exhibit reduced activation of IFN signaling pathways. We hypothesized that increases in immune regulatory cells termed myeloid-derived suppressor cells (MDSC) could interfere with the host immune response to tumors by inhibiting immune cell responsiveness to IFNs. The C26 murine adenocarcinoma model was employed to study immune function in advanced malignancy. C26-bearing mice had significantly elevated levels of GR1+D11b+ MDSC as compared with control mice, and splenocytes from tumor-bearing mice exhibited reduced phosphorylation of STAT1 (P-STAT1) on Tyr701 in response to IFN-α or IFN-γ. This inhibition was seen in splenic CD4+ and CD8+ T cells as well as natural killer cells. In vitro coculture experiments revealed that MDSC inhibited the IFN responsiveness of splenocytes from normal mice. Treatment of C26-bearing mice with gemcitabine or an anti-GR1 antibody led to depletion of MDSC and restored splenocyte IFN responsiveness. Spleens from C26-bearing animals displayed elevated levels of iNOS protein and nitric oxide. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. The elevation in nitric oxide in C26-bearing mice was associated with increased levels of nitration on STAT1. Finally, splenocytes from iNOS knockout mice bearing C26 tumors exhibited a significantly elevated IFN response as compared with control C26 tumor-bearing mice. These data suggest that nitric oxide produced by MDSC can lead to reduced IFN responsiveness in immune cells. Cancer Res; 71(15); 5101-10. ©2011 AACR. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are innate immune effector cells that play a crucial role in immune surveillance and the destruction of cancer cells. NK cells express a low-affinity receptor for the Fc or constant region of immunoglobulin G (FcγRIIIa) and multiple cytokine receptors that respond to antibody-coated targets and cytokines in the tumor microenvironment. In the present work, microarray gene expression analysis revealed that the IL-21 receptor (IL-21R) was strongly upregulated following FcR stimulation. The IL-21R was found to be upregulated on FcR-stimulated NK cells at the transcript level as determined by reverse transcription polymerase chain reaction (RT-PCR). Immunoblot analysis revealed that protein expression of the IL-21R peaked at 8 h post-stimulation of the FcR. Inhibition of the mitogen-activated protein kinase (MAPK) pathway downstream of the FcR blocked the induction of IL-21R expression. Increased expression of the IL-21R sensitized NK cells to IL-21 stimulation, as treatment of FcR-stimulated NK cells led to significantly increased phosphorylation of STAT1 and STAT3, as measured by intracellular flow cytometry and immunoblot analysis. Following FcR-stimulation, IL-21-activated NK cells were better able to mediate the lysis of trastuzumab-coated human epidermal growth factor receptor 2 (HER2+) SK-BR-3 tumor cells as compared to control-treated cells. Likewise, IL-21-induced NK cell secretion of IFNγ following exposure to antibody-coated tumor cells was enhanced following FcR-stimulation. The analysis of NK cells from patients receiving trastuzumab therapy for HER2+cancer exhibited increased levels of the IL-21R following the administration of antibody suggesting that the presence of monoclonal antibody-coated tumor cellsin vivocan stimulate the increased expression of IL-21R on NK cells. [ABSTRACT FROM PUBLISHER]

Background: Myeloid-derived suppressor cells (MDSCs) are one of the major components of the immune-suppressive network, play key roles in tumor progression and limit therapeutic responses. Recently, we reported that tumor spheres formed by breast cancer cell lines were visibly smaller in a Th1 enriched microenvironment with significantly reduced differentiation of MDSC populations in 3D culture. In this study, we investigated the mechanism(s) of bispecific antibody armed ATC mediated inhibition of MDSC in the presence or absence of Th1 microenvironment.Methods: We used 3D co-culture model of peripheral blood mononuclear cells (PBMC) with pancreatic cancer cells MiaPaCa-2 [MiaE] and gemcitabine resistant MiaPaCa-GR [MiaM] cells to generate MDSC in the presence or absence of Th1 cytokines and EGFRBi armed ATC (aATC).Results: We show significantly decreased differentiation of MDSC (MiaE, p<0.005; MiaM, p<0.05) in the presence of aATC with or without Th1 cytokines. MDSC recovered from control cultures (without aATC) showed potent ability to suppress T cell functions compared to those recovered from aATC containing co-cultures. Reduced accumulation of MDSC was accompanied by significantly lower levels of COX2 (p<0.0048), PGE2 (p<0.03), and their downstream effector molecule Arginase-1 (p<0.01), and significantly higher levels of TNF-α, IL-12 and chemokines CCL3, CCL4, CCL5, CXCL9 and CXCL10 under aATC induced Th1 cytokine enriched microenvironment.Conclusions: These data suggest aATC can suppress MDSC differentiation and attenuation of their suppressive activity through down regulation of COX2, PGE2 and ARG1 pathway that is potentiated in presence of Th1 cytokines, suggesting that Th1 enriching immunotherapy may be beneficial in pancreatic cancer treatment. [ABSTRACT FROM AUTHOR]