Purpose: Cryotherapy of localized prostate, renal, and hepatic primary tumors and metastases is considered a minimally invasive treatment demonstrating a low complication rate in comparison with conventional surgery. The main drawback of cryotherapy is that it has no systemic effect on distant metastases. We investigated whether intratumoral injections of dendritic cells following cryotherapy of local tumors (cryoimmunotherapy) provides an improved approach to cancer treatment, combining local tumor destruction and systemic anticancer immunity. Experimental Designs: The 3LL murine Lewis lung carcinoma clone D122 and the ovalbumintransfected B16 melanoma clone MO5 served as models for spontaneous metastasis. The antimetastatic effect of cryoimmunotherapy was assessed in the lung carcinoma model by monitoring mouse survival, lung weight, and induction of tumor-specific CTLs. The mechanism of cryoimmunotherapy was elucidated in the melanoma model using adoptive transfer of T cell receptor transgenic OT-I CTLs into the tumor-bearing mice, and analysis of Th1/Th2 responses by intracellular cytokine staining in CD4 and CD8 cells. Results: Cryoimmunotherapy caused robust and tumor-specific CTL responses, increased Th1 responses, significantly prolonged survival and dramatically reduced lung metastasis. Although intratumor administration of dendritic cells alone increased the proliferation rate of CD8 cells, only cryoimmunotherapy resulted in the generation of effector memory cells. Furthermore, cryoimmunotherapy protected mice that had survived primary MO5 tumors from rechallenge with parental tumors. Conclusions: These results present cryoimmunotherapy as a novel approach for systemic treatment of cancer. We envisage that cryotherapy of tumors combined with subsequent in situ immunotherapy by autologous unmodified immature dendritic cells can be applied in practice.Minimally invasive therapies are an alternative approach to surgical intervention in the treatment of malignant diseases. Cryoablation, i.e., tissue destruction by repeated deep freezing and thawing, is under the larger category of thermal therapy and, during the past decade, it has become an acceptable clinical tool for the management of dermatologic tumors, hepatocellular carcinoma, renal and prostate tumors, and hepatic colorectal metastases (1, 2). Compared with surgical excision, the main advantages are the potential for less invasiveness resulting in reduced mortality and morbidity, and the ability to perform ablative procedures on outpatients, which decreases the treatment cost. In the case of hepatic colorectal metastases, the use of cryosurgery improves the percentages of resectability (2). A comparative study on domestic pigs showed that the cryoablation of renal parenchyma is beneficial over other necrosis-inducing ablations such as microwave thermoablation, radiofrequency energy, and chemoablation by ethanol, hypertonic saline, and acetic acid gels, in terms of reproducibility, consistency in size and shape, and the ability to monitor b...

Endoglin (CD105), a co-receptor in the TGF-beta receptor complex, is over-expressed on proliferating endothelial cells in the breast tumor neovasculature and thus offers an attractive target for anti-angiogenic therapy. Here we report the anti-angiogenic/anti-tumor effects achieved in a prophylactic setting with an oral DNA vaccine encoding murine endoglin, carried by double attenuated Salmonella typhimurium (dam-, AroA-) to a secondary lymphoid organ, i.e., Peyer's patches . We demonstrate that an endoglin vaccine elicited activation of antigen-presenting dendritic cells, coupled with immune responses mediated by CD8+ T cells against endoglin-positive target cells. Moreover, we observed suppression of angiogenesis only in mice administered with the endoglin vaccine as compared to controls. These data suggest that a CD8+ T cell-mediated immune response induced by this vaccine effectively suppressed dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature. It is anticipated that vaccine strategies such as this may contribute to future therapies for breast cancer.

Objective. Engagement of Fc␥ receptor

CD38 is highly expressed on plasma cells and is an attractive target for multiple myeloma (MM) therapies. Several anti-CD38 antibodies including daratumumab and SAR650984 show promising results in clinical development, though such antibodies are not able to stimulate T cell-mediated killing of myeloma cells. To exploit a T cell immunotherapy mechanism while retaining the favorable drug properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via redirected T cell-cytotoxicity (RTCC) to stimulate T cell-mediated target cell killing regardless of T cell receptor antigen specificity. These anti-CD38 × anti-CD3 antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain lacks binding to Fcγ receptors to minimize nonselective T cell activation, yet retains binding to FcRn to maintain long serum half-life. We have previously reported that XmAb13551, a humanized and affinity-optimized anti-CD38 × anti-CD3 antibody, stimulates killing of the CD38+ MM cell line RPMI8226 by human T cells and suppresses human Ig levels in SCID mice engrafted with human PBMCs, showing much greater efficacy than daratumumab in these models (Blood 2014 124:4727). We also investigated efficacy of XmAb13551 in monkeys given a single dose of 2, 5, and 20 μg/kg. Within 1 hr after dosing, CD25 and CD69 activation markers were upregulated on T cells and within 8 hr, circulating CD38+ cells were depleted by > 95% at the 20 μg/kg dose. However, depletion of peripheral CD38+ cells was not sustained, suggesting that a large antigen sink was limiting drug exposure. Although higher dosing might overcome an antigen sink, higher doses of XmAb13551 (0.2 mg/kg or higher) resulted in a T cell-mediated cytokine release syndrome (CRS) in monkeys. We reasoned that an anti-CD38 × anti-CD3 antibody with reduced CD3 affinity would stimulate sufficient RTCC to deplete MM cells, yet would attenuate the acute T cell activation (and associated CRS) induced by high-affinity coengagement of T cells with CD38+ target cells. Using XmAb13551 as a starting point, we engineered a series of bispecifics retaining the same high-affinity (0.2 nM) binding to CD38, but with reduced affinity to CD3. We selected two antibodies - XmAb15426 and XmAb14702 - that have significantly reduced CD3 affinity. As expected, these molecules showed reduced potency in RTCC assays using T cells to kill RPMI8226 cells, with potency correlating with CD3 affinity (XmAb13551 > XmAb15426 >> XmAb14702). We next tested XmAb15426 and XmAb14702 at single doses of 0.5 mg/kg and 3 mg/kg, respectively, in cynomolgus monkeys. Both antibodies were well-tolerated at these higher doses, consistent with the moderate levels of IL6 observed in serum from the treated monkeys. Moreover, XmAb15426, with intermediate CD3 affinity, more effectively depletes CD38+ cells at 0.5 mg/kg compared to the original high-affinity XmAb13551 dosed at 2, 5 or 20 µg/kg. Depletion by XmAb15426 was more sustained compared to the highest dose of XmAb13551 in the previous study (7 vs. 2 days, respectively). Notably, although target cell depletion was greater for XmAb15426, T cell activation (CD69, CD25 and PD1 induction) was much lower in monkeys treated with XmAb15426 even dosed 25-fold higher than the 20 µg/kg XmAb13551 group. XmAb14702, with very low CD3 affinity, had little effect on CD38+ cells and T cell activation. Our results demonstrate that modulating T cell activation by attenuating CD3 affinity is a promising method to improve the therapeutic window of T cell-engaging bispecific antibodies. This strategy has potential to expand the set of antigens amenable to targeted T cell immunotherapy by improving tolerability and enabling higher dosing to overcome antigen sink clearance with targets such as CD38. We have shown that by reducing affinity for CD3, XmAb15426 effectively depletes CD38+ cells while minimizing the CRS effects seen with comparable doses of its high-affinity counterpart XmAb13551. Our preclinical data for XmAb15426 provide a rationale for clinical testing of this bispecific antibody in patients with multiple myeloma and other CD38+ malignancies. Disclosures Moore: Xencor, Inc.: Employment, Equity Ownership. Lee:Xencor, Inc.: Employment, Equity Ownership. Schubbert:Xencor, Inc.: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Ardila:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Chu:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Perforin/granzyme B-and Fas/FasL-mediated killing pathways are the main effector mechanisms of CTL and NK cells in antitumor immune responses. In this study, we investigated the relative role of these two lytic mechanisms in protection of the host from tumor progression, as well as spontaneous metastasis, using the D122 Lewis lung carcinoma and its gene-modified cells. Utilizing perforin knockout mice (B6-PKO) and Fas and FasL mutant (B6-MRL and B6-Smn) mice, we found that perforin expression in the host plays a crucial function in the prevention of metastasis. However, local tumor rejection of an H-2K b and B7-1 transfectant, 39.5-B7 cells, was not dependent either on perforin or Fas/FasL expression in vivo. In addition, CTL lysis of 39.5-B7 cells was independent of perforin and Fas/FasL interactions in 18-hour in vitro assays. We also confirmed that CD8 T-cells were responsible for rejecting 39.5-B7 local tumors, yet cytokines, TNF-a and gIFN were not involved in tumor rejection in vivo. Furthermore, blocking assays using caspase inhibitors (zVAD-fmk, zIETD-fmk and zLEHD-fmk) showed that, whereas caspase activation was partially required to induce 39.5-B7 lysis mediated by the perforin-dependent pathway, 39.5-B7 lysis by CTLs through the perforin-independent mechanism required caspase activation. Thus, these results suggested that perforin, Fas/FasL, gIFN and TNF-a independent lytic mechanisms, mediated by CD8 T cells, have a crucial role in rejection of 39.5-B7 cells in vivo. Caspase activation is a pre requisite for apoptosis of targets by CTLs

Heterogeneous network (HetNet) is an opportunistic technology that improves network throughput, extends cell coverage, and offloads the network traffic for 5G mobile communication network. HetNet involves a mix of radio technologies and various cell types working together seamlessly. In a HetNet, the coordination between macro cells (MCs) and small cells (SCs) has a positive impact onthe performance of the overall networks, and consequently on the overall user experience. Therefore, the improvement of user-perceived service quality requires high-efficiency network protocol as well as enhanced radio technologies. In this article, we introduce a 5G HetNet comprised of MCs, fixed and mobile SCs. Mobile small cells (mSCs) can be mounted on a car, bus, and train. They have different aspect from the fixed small cells (fSCs), and hence we address the technical challenges for mSCs in this article. We also analyze the network performance under two HetNet scenarios, such as MCs and fSCs, and MCs and mSCs.

IntroductionT cells that target tumor-associated antigens (TAAs) are readily detectable in patients with cancer, including those who received cancer vaccines. However, in most cases, such T cells fail to eradicate tumors in these patients. Thus, it is apparent that established tumors can induce immune tolerance through yet poorly defined mechanisms. 1 We hypothesized that immunization strategies that use different arms of the immune system may overcome this immune tolerance. The NKG2D receptor-ligand interaction is a good candidate to test this hypothesis because it occurs at the crossroad between innate and adaptive immunity. 2 NKG2D, a stimulatory lectinlike receptor, is expressed on natural killer (NK) cells, activated CD8 ϩ T cells, ␥␦ T cells, and activated macrophages. 3 It mediates costimulatory signals for CD8 ϩ T cells and stimulatory signals for NK cells and macrophages. 4,5 NKG2D ligands are related to major histocompatibility complex (MHC) class I molecules. In mice, such ligands include products of the retinoic acid early inducible-1 (Rae1) gene, H60, 3,6 and the UL16 binding protein-like transcript 1 (MULT1). 7 Importantly, in syngeneic mice, ectopic expression of NKG2D ligands causes NK cell-mediated rejection of transfected tumor cells. 8,9 It also primes cytotoxic T cells (CTLs), which are responsible for rejecting subsequent challenges with tumor cells lacking NKG2D ligand expression. 8 In our earlier study, 10 we demonstrated that engagement of the murine NKG2D receptor (by using a DNA vaccine encoding one of its ligands, H60) enhanced innate and adaptive immune responses induced by a survivin-based DNA vaccine. This, in turn, augmented the vaccine's antitumor efficacy in prophylactic and therapeutic settings against tumors of different origin and NKG2D expression levels. 10 Survivin, a 16.5-kDa inhibitor of apoptosis protein, represents an almost ideal target for cancer vaccines because it is overexpressed by essentially all solid tumor cells and by proliferating endothelial cells in the tumor microenvironment. In contrast, survivin is poorly, or only transiently, expressed by normal adult tissues. 11 Furthermore, overexpression of survivin in tumors is linked to decreased patient survival, increased tumor recurrence, and resistance to therapy. 11 In addition, spontaneous immune responses against survivin were recently demonstrated in a variety of patients with cancer. 12 Survivinbased DNA vaccines were also shown to induce T cell-mediated antitumor responses without severe toxicities in preclinical 13 and clinical trials. 14 Here, we demonstrate that the enhanced vaccine efficacy of NKG2D ligand H60 plus survivin is in part caused by increased lymphocyte cross talk, thus proving that activation of the innate and adaptive arms of the immune system provides an attractive strategy to overcome tumor-induced peripheral immune tolerance. Materials and methods Animals and cell linesFemale BALB/c mice, 6 to 8 weeks of age, were purchased from the Jackson Laboratory (Bar Harbor, ME). All animal expe...

Persistent infections caused by pathogens such as hepatitis C virus are major human diseases with limited or suboptimal prophylactic and therapeutic options. Given the critical role of dendritic cell (DC) in inducing immune responses, DC vaccination is an attractive means to prevent and control the occurrence and persistence of the infections. However, DCs are built-in with inherent negative regulation mechanisms which attenuate their immune stimulatory activity and lead to their ineffectiveness in clinical application. In this study, we developed a super DC stimulant that consists of a modified, secretory Toll-like Receptor (TLR)-5 ligand and an inhibitor of the negative regulator, suppressor of cytokine sinaling-1 (SOCS1). We found that expressing the super stimulant in DCs is drastically more potent and persistent than using the commonly used DC stimuli to enhance the level and duration of inflammatory cytokine production by both murine and human DCs. Moreover, the DCs expressing the super stimulant are more potent to provoke both cellular and humoral immune responses against hepatitis C virus (HCV) antigen in vivo. Thus, the strategy capable of triggering and sustaining proinflammatory status of DCs may be used to boost efficiency of DC vaccine in preventing and combating the persistent infection of HCV or other chronic viruses.