Ipilimumab augments antitumor activity of bispecific antibody-armed T cells

BACKGROUND: Ipilimumab is an antagonistic monoclonal antibody against cytotoxic T-lymphocyte antigen-4 (CTLA-4) that enhances antitumor immunity by inhibiting immunosuppressive activity of regulatory T cells (Treg). In this study, we investigated whether inhibiting Treg activity with ipilimumab during ex vivo T cell expansion could augment anti-CD3-driven T cell proliferation and enhance bispecific antibody (BiAb)-redirected antitumor cytotoxicity of activated T cells (ATC). METHODS: PBMC from healthy individuals were stimulated with anti-CD3 monoclonal antibody with or without ipilimumab and expanded for 10-14 days. ATC were harvested and armed with anti-CD3 x anti-EGFR BiAb (EGFRBi) or anti-CD3 x anti-CD20 BiAb (CD20Bi) to test for redirected cytotoxicity against COLO356/FG pancreatic cancer cell line or Burkitt’s lymphoma cell line (Daudi). RESULTS: In PBMC from healthy individuals, the addition of ipilimumab at the initiation of culture significantly enhanced T cell proliferation (p = 0.0029). ATC grown in the presence of ipilimumab showed significantly increased mean tumor-specific cytotoxicity at effector:target (E:T) ratio of 25:1 directed at COLO356/FG and Daudi by 37.71% (p < 0.0004) and 27.5% (p < 0.0004), respectively, and increased the secretion of chemokines (CCL2, CCL3, CCL4,CCL5, CXCL9, and granulocyte-macrophage colony stimulating factor(GM-CSF)) and cytokines (IFN-γ, IL-2R, IL-12, and IL-13), while reducing IL-10 secretion. CONCLUSIONS: Expansion of ATC in the presence of ipilimumab significantly improves not only the T cell proliferation but it also enhances cytokine secretion and the specific cytotoxicity of T cells armed with bispecific antibodies

Microenvironment generated during EGFR targeted killing of pancreatic tumor cells by ATC inhibits myeloid-derived suppressor cells through COX2 and PGE\u3csub\u3e2\u3c/sub\u3e dependent pathway

Abstract 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\u3c0.005; MiaM, p\u3c0.05) 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\u3c0.0048), PGE2 (p\u3c0.03), and their downstream effector molecule Arginase-1 (p\u3c0.01), 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

Catalogue of Stone ScuIpture from the Area of Kula Atlagića

U radu se obrađuje problematika atribucije pojedinih kamenih ulomaka s lokaliteta Sv. Petar i Sv. Nikola u Kuli Atlagića. Većina se ulomaka čuva u Muzeju hrvatskih arheoloških spomenika u Splitu, a onamo su dospjeli u nekoliko etapa. Osim činjenice da pripadaju različitim stilskim epohama, postavlja se problem kako odrediti pripadnost ulomaka određenim objektima. U članku je priložen katalog kamenih ulomaka te njihova interpretacija. Rezultati rekognosciranja ponudili su nekoliko mogućih lokacija na kojima bi mogli biti objekti kojima pripadaju ulomci skulpture priloženi u katalogu, no tek sustavna arheološka iskopavanja mogu ponuditi sigurniju atribuciju.The village of Kula Atlagića is located 4km north-west of Benkovac in the direction of the medieval road via Magna which passed through the Zadar hinterland. The name of the village was first mentioned as late as the 17th century, while older documents mention two Croatian villages in that area: TihIići and Bojište (Bojišće). In today\u27s village stand three churches: two Orthodox dedicated to St Nicholas and one Catholic dedicated to St Peter. The two churches are mentioned in the archaeological literature at the beginning of the 20lh century. The church of St Nicholas in the Orthodox graveyard was completed in 1447 as witnessed by the inscription on the transom of the side portal, while the church of St Peter bears early Romanesque features, although it was destroyed several times during its turbulent history. The stone fragments which are presented in the catalogue were gathered over a long period of time. Some were brought to Knin on the prompting of Fra Lujo Marun while most of the sculptures were gathered after the Homeland War from the ruins of the church of St Peter, into which they had been incorporated as building material, and brought to the Museum of Croatian Archaeological Monuments in Split. According to their stylistic characteristics, the sculptures are divided into different groups: ancient, early medieval and early Romanesque. The problem which arose during the work on the sculptures was the attribution of individual groups to a particular object. The ancient sculptures could have belonged to a temple or shrine to the Roman god Liber which was probably located in the area around the church of St Nicholas as evidenced by entries in the diary of Fra Lujo Marun, who on several occasions made notes on both churches and the areas around them. The early medieval sculpture shows different characteristics and we could suppose that the earlier sculptures in this group were replaced by new furniture donated by some worthy benefactor. The question remains open as to where the early medieval church to which this furniture belonged was located. Numerous authors suppose that an early medieval church, which was in the 15\u27h century or earlier replaced by a new one, was situated on the site of today\u27s church of St Nicholas in the Orthodox graveyard. Because the investigations on this site have not been completed, we are left only with suppositions which could be confirmed by archaeological work. The fragments of early Romanesque sculpture are very specific and it is not possible to find close parallel s for them. The sculpture could be connected to the church of St Peter which was founded prior to the 12\u27b century, but investigations carried out in 1997 showed that this building had only one building phase and we cannot therefore talk of the possibility of the existence of an earlier church, at least not in the investigated area inside the church and in its immediate area. Translation: Nicholas Philip Saywel

Priming of pancreatic cancer cells with bispecific antibody armed activated T cells sensitizes tumors for enhanced chemoresponsiveness

In this study, we investigated the ability of bispecific antibody armed activated T cells to target drug resistant pancreatic cancer cells and whether or not “priming” these resistant cancer cells with bispecific antibody armed activated T cells could enhance subsequent responsiveness to chemotherapeutic drugs. Chemotherapeutic responses for pancreatic cancer are either limited or the tumors develop resistance to chemotherapy regimens. The impetus for this study was the remarkable clinical response seen in our earlier phase I/II clinical trial: a pancreatic cancer patient with drug resistant tumors who showed progression of disease following three infusions of anti-CD3 x anti-EGFR bispecific antibody armed activated T cells (EGFR BATs) was restarted on the initial low dose of 5-fluorouracil showed complete response, suggesting that BATs infusions may have sensitized patient’s tumor for chemoresponsiveness. In the current study, we tested the hypothesis that BATs can sensitize tumors for chemoresponsiveness. Gemcitabine or cisplatin-resistant MiaPaCa-2 and L3.6 cell lines were effectively targeted by EGFR BATs. Priming of drug sensitive or resistant cells with EGFR BATs followed by retargeting with lower concentrations of 50% inhibitory concentration of gemcitabine or cisplatin showed enhanced cytotoxicity. Gemcitabine or cisplatin-resistant cell lines show an increased proportion of CD44+/CD24+/EpCAM+ cancer stem like cells as well as an increased number of ABC transporter ABCG2 positive cells compared to the parental cell lines. These data suggest that bispecific antibody armed activated T cells can target and kill chemo-resistant tumor cells and also markedly augment subsequent chemotherapeutic responsiveness, possibly by modulating the expression of ABC transporters

CD20-Targeted T Cells after Stem Cell Transplantation for High Risk and Refractory Non-Hodgkin's Lymphoma

AbstractA phase I trial of infusing anti-CD3 × anti-CD20 bispecific antibody (CD20Bi) armed activated T cells (aATC) was conducted in high-risk/refractory non-Hodgkin's lymphoma patients to determine whether aATC infusions are safe, affect immune recovery, and induce an antilymphoma effect. Ex vivo expanded ATC from 12 patients were armed with anti-CD20 bispecific antibody, cryopreserved, and infused after autologous stem cell transplantation (SCT). Patients underwent SCT after high-dose chemotherapy, and aATC infusions were started on day +4. The patients received 1 infusion of aATC per week for 4 weeks after SCT with doses of 5, 10, 15, and 20 × 109. aATC infusions were safe and did not impair engraftment. The major side effects were chills, fever, hypotension, and fatigue. The mean number of IFN-γ Enzyme-linked Immunosorbent Spots (ElSpots) directed at CD20 positive lymphoma cells (DAUDI, P = .0098) and natural killer cell targets (K562, P < .0051) and the mean specific cytotoxicity directed at DAUDI (P = .037) and K562 (P = .002) from pre-SCT to post-SCT were significantly higher. The increase in IFN-γ EliSpots from pre-SCT to post-SCT in patients who received armed ATC after SCT were significantly higher than those in patients who received SCT alone (P = .02). Serum IL-7, IL-15, Macrophage inflammatory protein (MIP)-1 beta, IP-10, MIP-1α, and Monokine induced by gamma interferone increased within hours after infusion. Polyclonal and specific antibodies were near normal 3 months after SCT. aATC infusions were safe and increased innate and specific antilymphoma cell immunity without impairing antibody recovery after SCT